Copper and the Skin

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DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES

COPPER and the SKIN

9532_series 5/24/06 12:31 PM Page 1

DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES Series Editor Howard I. Maibach, M.D.

Published Titles: Bioengineering of the Skin: Cutaneous Blood Flow and Erythema Enzo Berardesca, Peter Elsner, and Howard I. Maibach Bioengineering of the Skin: Methods and Instrumentation Enzo Berardesca, Peter Elsner, Klaus P. Wilhelm, and Howard I. Maibach Bioengineering of the Skin: Skin Biomechanics Peter Elsner, Enzo Berardesca, Klaus-P. Wilhelm, and Howard I. Maibach Bioengineering of the Skin: Skin Surface, Imaging, and Analysis Klaus P. Wilhelm, Peter Elsner, Enzo Berardesca, and Howard I. Maibach Bioengineering of the Skin: Water and the Stratum Corneum, Second Edition Joachim W. Fluhr, Peter Elsner, Enzo Berardesca, and Howard I. Maibach Contact Urticaria Syndrome Smita Amin, Arto Lahti, and Howard I. Maibach Copper and the Skin Jurij J. Hostýnek and Howard I. Maibach Cutaneous T-Cell Lymphoma: Mycosis Fungoides and Sezary Syndrome Herschel S. Zackheim and Howard I. Maibach Dermatologic Botany Javier Avalos and Howard I. Maibach Dermatologic Research Techniques Howard I. Maibach Dry Skin and Moisturizers: Chemistry and Function, Second Edition Marie Lodén and Howard I. Maibach The Epidermis in Wound Healing David T. Rovee and Howard I. Maibach Hand Eczema, Second Edition Torkil Menné and Howard I. Maibach Human Papillomavirus Infections in Dermatovenereology Gerd Gross and Geo von Krogh The Irritant Contact Dermatitis Syndrome Pieter van der Valk, Pieter Coenrads, and Howard I. Maibach

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Latex Intolerance: Basic Science, Epidemiology, and Clinical Management Mahbub M. V. Chowdhry and Howard I. Maibach Nickel and the Skin: Absorption, Immunology, Epidemiology, and Metallurgy Jurij J. Hostýnek and Howard I. Maibach Pesticide Dermatoses Homero Penagos, Michael O’Malley, and Howard I. Maibach Protective Gloves for Occupational Use, Second Edition Anders Boman, Tuula Estlander, Jan E. Wahlberg, and Howard I. Maibach Sensitive Skin Syndrome Enzo Berardesca, Joachim W. Fluhr, and Howard I. Maibach Skin Cancer: Mechanisms and Human Relevance Hasan Mukhtar Skin Reactions to Drugs Kirsti Kauppinen, Kristiina Alanko, Matti Hannuksela, and Howard I. Maibach

DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES

COPPER and the SKIN Edited by

Jurij J. Hosty´ nek University of California at San Francisco School of Medicine San Francisco, California, U.S.A.

Howard I. Maibach University of California at San Francisco School of Medicine San Francisco, California, U.S.A.

New York London

Informa Healthcare USA, Inc. 270 Madison Avenue New York, NY 10016 © 2006 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid‑free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number‑10: 0‑8493‑9532‑1 (Hardcover) International Standard Book Number‑13: 978‑0‑8493‑9532‑1 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright. com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978‑750‑8400. CCC is a not‑for‑profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

This monograph on copper is dedicated to Dr. Roberto Milanino of the University of Verona, Italy. A scientist and teacher, he spent the past 30 years of his academic career investigating the biological function and importance of copper in the mammalian organism while navigating the narrows of Italy’s budget allocated to science. Among the leading authorities on this subject, he was a guiding light, set the proper accents, and kept things in perspective over the period of much of our work and writing in assembling this monograph. The whole is a clear reflection of his qualities as teacher. J. J. Hostyńek H. I. Maibach

Preface Preface

Metals have long interested the dermatologic community in terms of their toxicity and efficacy. A century ago, mercury enjoyed widespread usage in the treatment of syphilis. As the dermatologic sciences evolved, sufficient knowledge also evolved, leading to the clear assertion that metals may be toxic when applied to the skin. Nickel has enjoyed the greatest amount of study, especially because of its frequent induction of clinical allergic contact dermatitis in humans. Cobalt is also a common contact allergen, but its clinical significance is less clearly explored. Chromate in cement, leather, and other applications also enjoys considerable study. Most recently, gold salts have been recognized as a common inducer of cell-mediated immunity; however, this clinical significance is currently being investigated in terms of dermatitis and even restenosis. These data have led to several textbooks dedicated to individual metals. The first was on chromate (by Desmond Burrows), many metals and the skin (by R. Guy and J. J. Hostyńek), and most recently nickel (by J. J. Hostyńek and H. I. Maibach). The current volume on copper presents sufficient information to place it among the pantheon of the metallic gods and dermatology. Our aim was to mold contributions from individuals widely spread over several disparate disciplines into a cohesive, readily digestible text. The individual disciplines include basic chemistry (metallurgy), dermatotoxicology (irritant and allergic contact dermatitis), and membrane transport (percutaneous penetration).

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Our specific objective is to allow scientists in many fields to more efficiently focus their attention on this essential element (copper) and the skin. Hopefully, the parts will simplify understanding the whole. This volume differs from the others, not only in its extensive dermatotoxicologic profile of copper and its salts, but also as an equally impressive data on copper’s possible anti-inflammatory actions in man. The editors welcome suggestions for the next edition. Jurij J. Hostyńek Howard I. Maibach

Acknowledgment

We gratefully acknowledge the partial financial support by the International Copper Association, Ltd. (ICA) towards publication of this book.

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Contents

Preface . . . . v Acknowledgment . . . . vii Contributors . . . . xv 1. Copper and Copper Alloys . . . . . . . . . . . . . . . . . . . . . . . . 1 Harold T. Michels Introduction . . . . 1 Copper: Properties of the Element . . . . 1 Pure Copper . . . . 2 Copper Alloys . . . . 2 Properties of Copper Alloys . . . . 2 Copper Alloy Families . . . . 4 The High Coppers . . . . 4 Conclusions . . . . 6 2. Corrosion Chemistry of Copper: Formation of Potentially Skin-Diffusible Compounds . . . . . . . . . . . . . . . . . . . . . . . . 7 Jurij J. Hostyńek Introduction . . . . 7 Electron Configuration and Reactivity of Copper . . . . 8 Corrosion of Copper in the Environment . . . . 8 Corrosion of Copper in Physiologic Media . . . . 9 Conclusions . . . . 15 Glossary . . . . 16 Abbreviations . . . . 16 References . . . . 16 ix

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3. Basics of Metal Skin Penetration: Scope and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . 21 Jurij J. Hostyńek and Howard I. Maibach Introduction . . . . 21 Structure of Skin and Its Function as Diffusion Barrier . . . . 23 Descriptors of Dermal Absorption . . . . 25 Permeant Categories and Paths of Diffusion . . . . 28 Compounds Formed by Metals in Contact with the Skin . . . . 31 Variables Determining Skin Diffusion of Metal Compounds . . . . 35 Methods for Measuring Percutaneous Absorption . . . . 45 Analytical Methods for Metal Detection . . . . 53 Summary and Conclusions . . . . 56 Abbreviations . . . . 57 References . . . . 58 4. Percutaneous Absorption of Copper Compounds . . . . . . . . 67 Jurij J. Hostyńek and Howard I. Maibach Introduction . . . . 67 Qualitative Diffusion Data . . . . 68 Semiquantitative Data . . . . 70 Quantitative Data . . . . 71 Discussion and Conclusions . . . . 73 Limitations in Measuring Copper Absorption In Vivo . . . . 74 Interdependence of Systemic Copper and Zinc Levels . . . . 75 Recommendations for Research to Fill Existing Data Gaps . . . . 76 Conclusions . . . . 77 Glossary . . . . 78 Abbreviations . . . . 78 References . . . . 79 5. Diffusion of Copper Through Human Skin In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jurij J. Hostyńek, Howard I. Maibach, and Frank Dreher Introduction . . . . 81 Experimental . . . . 84 Results . . . . 85

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Discussion . . . . 88 Conclusions . . . . 92 Glossary . . . . 93 Abbreviations . . . . 93 References . . . . 94 6. Irritation Potential of Copper Compounds . . . . . . . . . . . . Jurij J. Hostyńek and Howard I. Maibach Introduction . . . . 97 Exposure to Copper . . . . 97 Solubilization of Copper Metal . . . . 98 Incidence and Epidemiology of Irritation Due to Copper . . . . 100 Pharmacology of Copper . . . . 101 Copper Irritancy in Skin and Mucosa . . . . 103 Conclusions . . . . 111 Abbreviations . . . . 112 References . . . . 112 7. Copper Hypersensitivity: Dermatologic Aspects—Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . Jurij J. Hostyńek and Howard I. Maibach Introduction . . . . 115 Metallurgy of Copper and Its Alloys, and Its Role as Sensitizer . . . . 117 Predictive Immunology Test Results for Copper . . . . 119 Diagnostic Tests for Hypersensitivity . . . . 119 Test Concentrations for Copper ACD . . . . 123 Immunogenic Potential of Copper . . . . 123 Summaries of Population-Based Studies . . . . 134 Summary of Selected Case Reports of Immune Reactions to Copper . . . . 138 Selection of Individual Reports of Immune Reactions to Copper . . . . 138 Comments . . . . 140 Conclusions . . . . 140 Abbreviations . . . . 141 References . . . . 141

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8. Copper in Medicine and Personal Care: A Historical Overview . . . . . . . . . . . . . . . . . . . . . . . . . 149 Roberto Milanino Introduction . . . . 149 The Sumeric Culture: Circa 4000–2300 B.C. . . . . 150 The Ancient Egyptian Culture . . . . 150 The Babylonian–Assyrian Culture: Circa 1750–539 B.C. . . . . 152 The Ancient Indian Culture: Circa 2800–1000 B.C. . . . . 152 The Ancient Chinese Culture: Circa 3000 B.C. to 1100 A.D. . . . . 152 The Pre-Columbian Meso- and South-American Cultures: Circa 600 B.C. to 1500 A.D. . . . . 153 The Ancient Greek Culture . . . . 153 The Ancient Roman Culture: Circa 600 B.C. to 476 A.D. . . . . 155 From the High-Medieval Age to the Early 20th Century . . . . 156 Beginning of the Scientific Age for Copper: 1928–1976 . . . . 157 Conclusions . . . . 158 Abbreviations . . . . 159 References . . . . 159 9. The Role of Copper in Onset, Development, and Control of Acute and Chronic Inflammation . . . . . . . . . . 161 Roberto Milanino Introduction . . . . 161 Studies on Copper-Deficient, Experimentally Inflamed Animals . . . . 163 Laboratory Animals: Studies on ‘‘Endogenous’’ Copper Metabolism in Acute and Chronic Inflammation . . . . 170 Human Subjects: Studies on ‘‘Endogenous’’ Copper Metabolism in Acute and Chronic Inflammations, with a Particular Reference to Rheumatoid Arthritis . . . . 179 Effects of ‘‘Exogenous’’ Copper Administration on the Inflammatory Process . . . . 184 Copper Anti-inflammatory Activity: Hypotheses Explaining the Possible Mechanisms of Action . . . . 203 Conclusions . . . . 216

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Abbreviations . . . . 219 References . . . . 220 10. Copper Jewelry and Arthritis . . . . . . . . . . . . . . . . . . . . 237 Brenda J. Harrison Introduction . . . . 237 The Copper Bracelet ‘‘Myth’’ and Hypothesis . . . . 239 The Copper Bracelet Trial . . . . 243 The Present State of the Copper Bracelets ‘‘Issue’’ . . . . 251 Is There Likely to Be a Future for Copper Bracelets in Arthritis Care? . . . . 256 Appendix A: Position Statements of Support Organizations, Government Agencies, Etc. . . . . 257 Appendix B: Miscellany . . . . 259 References . . . . 261 11. Role of Copper in Anti-inflammatory Therapy and the Potential for Its Transdermal Application . . . . . . . . . . . . Jurij J. Hostyńek and Roberto Milanino Introduction . . . . 267 Traditional and Modern Therapies for RA and Related Disorders . . . . 268 Drug Therapy . . . . 271 Precedents in Topical Delivery of Anti-inflammatory Agents . . . . 275 Role of Copper in AI Activity . . . . 275 Past Use of Copper Chelates in the Treatment of Rheumatoid Arthritis . . . . 278 Transdermal Delivery of Anti-inflammatory Copper Chelates vs. Conventional (Systemic) Anti-inflammatory Therapy . . . . 278 Conclusions . . . . 286 Outlook . . . . 288 Abbreviations . . . . 288 References . . . . 289 Index . . . . 295

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Contributors

Frank Dreher Neocutis, Inc., San Francisco, California, U.S.A. Brenda J. Harrison Department of Earth and Ocean Sciences, Copper Research Information Flow Project, University of British Columbia, Vancouver, British Columbia, Canada Jurij J. Hostyńek Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A. Howard I. Maibach Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A. Harold T. Michels Copper Development Association, Inc., New York, New York, U.S.A. Roberto Milanino Facolta` di Medicina e Chirurgia, Sezione di Farmacologia, Dipartimento di Medicina e Salute Pubblica, Universita` di Verona, Verona, Italy

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1 Copper and Copper Alloys Harold T. Michels Copper Development Association, Inc., New York, New York, U.S.A.

INTRODUCTION This first chapter describes copper, its properties and characteristics, and where it is used, both in its pure form and as alloys. The emphasis is on materials that come into contact with human skin. This chapter provides the background for the second chapter, which gives a detailed discussion of the corrosion resistance of these materials and how that relates to their interaction with humans by sweat. Common items made of copper and copper alloys that are touched by humans every day include copper and copper–nickel coins, copper–nickel–zinc door keys, and brass door knobs, door push plates, and sink faucet handles. COPPER: PROPERTIES OF THE ELEMENT Copper, atomic number 29, is classified as a metal in the periodic table of elements. It is the first element in the group containing silver and gold, and thus it is considered to be a semiprecious metal. Some of the properties of copper include:

melting point of 1083 C metallic luster and reddish color high electrical and thermal conductivity nonmagnetic alloys readily as both a solute and a solvent 1

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good corrosion resistance and durability forms a protective oxide in air and water face-centered crystal structure high malleability, formability, and ductility good machinability readily electroplated essential nutrient for life highly recyclable

These unique properties of copper account for its widespread and long-term use as an industrial material. PURE COPPER Copper is refined from ore shipped to fabricators, mainly as cathode, wire rod, billet, cake (slab), or ingot. Through extrusion, drawing, rolling, forging, melting, or atomization, fabricators form wire, rod, tube, sheet, plate, strip, castings, powder, and other shapes. These copper and copper alloys are then shipped to manufacturing plants where they are used to make products to meet society’s needs. COPPER ALLOYS Copper alloys are widely used in many applications, ranging from electrical wiring and connectors to musical instruments, from household plumbing tube and fixtures to keys, locks, doorknobs, and handrails. The applications are almost endless. The wide use of copper alloys is attributable to a long history of successful use, ready availability from a multitude of sources, the attainability of a wide range of physical and mechanical properties, and amenability to subsequent processing, such as machining, brazing, soldering, polishing, and plating. The properties of copper alloys, which occur in unique combinations found in no other alloy system, include high thermal and electrical conductivity, a wide range of attainable strength properties and excellent ductility and toughness, as well as superior corrosion resistance in many different environments. Nevertheless, to the uninitiated, copper alloys appear to be confusing and complex. They are generically described by such terms as brass, bronze, copper–nickel, and copper–nickel–zincs, which are called nickel silvers because of their shiny white color, even though they contain no silver. PROPERTIES OF COPPER ALLOYS Copper alloys provide important properties and characteristics including: Good corrosion resistance—which contributes to durability, and leads to long-term cost effectiveness.

Copper and Copper Alloys

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Favorable mechanical properties—ranging from pure copper that is soft and ductile to other alloys, such as the manganese bronzes that can rival the mechanical properties of quenched and tempered steel. Furthermore, almost all copper alloys retain their mechanical properties, including impact toughness, at very low temperatures. High thermal and electrical conductivity—copper has higher conductivity than any other metal except silver. Conductivity drops when copper is alloyed. However, even the copper alloys with relatively low conductivity transfer both heat and electricity far better than other corrosion-resistant materials, such as titanium, aluminum, and stainless steel. Biofouling resistance—copper inhibits the growth of marine organisms including algae and barnacles. This property, unique to copper, decreases when alloyed. However, it is retained at a useful level in copper alloys, such as the copper–nickels, which are routinely found in marine applications. Antimicrobial action—copper chemicals have been historically used as bactericides, algicides, and fungicides. However, recent studies indicate that bacteria, including certain harmful strains of Escherichia coli, and MRSA, or methicillin-resistant Staphylococcus aureus (a serious nosocomial or hospital-acquired infection) simply die in a few hours when placed on copper alloy surfaces at room temperature. Low friction and wear rates—copper alloys, such as the high-leaded tin bronzes, are cast into sleeve bearings and exhibit low wear rates against steel. Both the nickel bronze and the tin bronze are the industry standards for worm gears, an application in which low wear rates are important. Good castability—all are sand castable and almost all can be centrifugally and continuously cast. Many copper alloys can be permanently molded and precision or die cast. Wrought copper alloys are initially cast and subsequently hot and cold rolled. High fabricability—copper alloys are readily hot rolled extruded or forged. They then may be cold rolled to the desired thickness. Sheet, plate, strip, and bar products are readily forged, stamped, and bent into desired shapes. High machinability—good surface finish and high tolerance control is readily achieved. While the leaded copper alloys are free-cutting at high machining speeds, many unleaded alloys such as nickel– aluminum bronze are readily machinable at recommended feeds and speeds with proper tooling. Ease of subsequent processing—many copper alloys are routinely polished to a high luster, especially those with an esthetically pleasant color, such as the yellow brasses. Plating, soldering, brazing, and welding are also routinely performed.

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Availability of a range of alloys—in a given application, any one of several alloys may be a suitable candidate, depending on design loads and corrosivity of the environment. Reasonable cost—high processing yield and low machining costs make copper alloy very economical. Gates and risers from castings and chips from machining are also recycled, which leads to additional overall cost reductions. In addition, copper alloys do not require surface coatings, such as paints. The avoidance of surface coatings further reduces initial costs and provides additional maintenance savings. In addition, when the component reaches the end of its useful life, it too is readily and routinely recycled. COPPER ALLOY FAMILIES From a metallurgical viewpoint, many copper alloys are single-phase solid solutions in which the alloying elements, such as zinc, tin, and nickel, are substituted for copper in the copper matrix. Examples of single-phase solution alloys include the brasses that contain up to 35% zinc, and copper– nickels that contain up to 33% nickel. As alloy content is increased, a second phase may form. In the case of brass, when the zinc content is increased, a hard second phase called beta forms within the alpha copper-rich matrix. This second phase is found in yellow brass that contains up to 41% zinc. Beta, which slightly impairs room temperature ductility, markedly increases ductility at elevated temperatures. One of the most common systems used to designate specific copper alloys is the UNS system. Copper alloys are either wrought or cast. Wrought alloys range in UNS number from C10100 through C79999. They are subjected to hot and usually cold work after initial melting and solidification, and are generally available as wire, rod, bar, sheet, strip, and plate. Cast alloys range in UNS number from C80000 through C99999. They are typically cast into a mold in a variety of specific shapes and then machined without any hot or cold working. The Coppers The coppers (wrought: C10100–C15999, and cast: C80000–C81399) are essentially pure copper (99.7% min) with traces of silver or phosphorus. The presence of silver imparts annealing resistance, while phosphorus, a deoxidizer, aids in welding. These alloys, which have relatively low strength, are used in applications where high thermal and electrical conductivity are desirable, such as in electrical connectors and in hot metal handling. Copper is used in low denomination coinage. Some individuals wear copper wrist bracelets. THE HIGH COPPERS High copper alloys (wrought: C16000–C19999, and cast: C81400–C83299), which contain 95.1% copper (min), are unique in that they combine high

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strength with high thermal and electrical conductivity, two properties that are seldom found together in the same material. Chromium coppers are alloys containing up to 1.2% chromium. The strength of chromium copper is approximately twice that of pure copper, but its electrical conductivity remains high (80% of pure copper). Applications for chromium copper include welding clamps and high-strength electrical connectors. The Brasses The brasses (wrought: C21000–C49999, and cast: C83300–C89999) are the most commonly used casting alloys, and are essentially alloys of copper and zinc. The red brasses are alloys of up to 15% zinc and may contain varying amounts of tin if they are cast alloys. Lead may be present in various amounts to promote pressure tightness in castings in service and to facilitate free machining during the manufacturing process. The color of red brass is attributable to its relatively low zinc content. The largest-volume cast red brass alloy, C83600 (commonly known as 85-5-5-5), contains 85% copper, 5% tin, 5% lead, and 5% zinc. It has been used commercially for several hundred years and accounts for more tonnage than any other cast alloy. The yellow brasses are even lower in cost than the red brasses because their zinc content is higher, at 20–39% Zn. Yellow brass has a pleasant yellow color, which can be polished to a high luster. This accounts in part for its selection as decorative hardware. The Bronzes Bronze (wrought: C50000–C69999, and cast: C90000–C95999) is a very imprecise term. Strictly speaking, it originally referred to alloys in which tin was the major alloying element. Today, the term bronze applies to a broader class of alloys, which may contain little, if any, tin. The silicon bronzes and silicon brasses are essentially alloys of up to 20% zinc and up to 5% silicon. They have low melting points and high fluidity, which favor permanent molding and pressure die casting. Modern day cast tin bronzes are very similar to the alloys found in many relics from the ‘‘Bronze Age,’’ over 3500 years ago. They are basically alloys of copper and tin, where tin content can be up to 20%. The good aqueous corrosion resistance of tin bronzes accounts, in part, for the survival of these Bronze Age relics to this day. Additional attributes of tin bronzes include reasonably high strength, good wear resistance, and a low coefficient of friction versus steel, making them very useful for bearings, piston rings, and gear parts. Aluminum bronzes have complex metallurgical structures. Alloying elements always include aluminum and varying amounts of manganese,

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iron, and, in some versions, nickel. Aluminum imparts both strength and oxidization resistance by virtue of the formation of alumina (Al2O3)-rich protective films. These alloy are very wear resistant, and exhibit good casting and welding characteristics. Their corrosion resistance is superior in seawater, chloride, and in dilute acids. Applications are varied and include propellers and valves, pickling hooks, pickling baskets, and wear rings. Aluminum bronzes and especially nickel–aluminum bronzes are desirable alloys for fluid moving applications such as pump impellers, because of superior erosion-corrosion and cavitation resistance. Copper Nickels The copper–nickel alloys (wrought: C70000–C73499, and cast: C96000– C96999) are simple solid solutions of nickel in copper. Nickel content varies from 9% to 33%. A small amount of manganese (0.05–1.5%) and iron (0.4–1.8%) is present. Their excellent corrosion resistance in seawater, combined with their high strengths and good fabricability, accounts for their wide use in piping, heat exchangers, valves, ship tail-shaft sleeves, and other marine applications. They are also used in coins. Nickel Silvers Nickel silver alloys (wrought: C73500–C79999, and cast: C97000–C97999) contain nickel (11–27%) and zinc (1–25%). The presence of nickel primarily accounts for their pleasant silver luster but, in contrast to their name, the nickel silvers do not contain silver. In spite of their high degree of alloying, the nickel silvers are simple solid solution alloys. They offer good corrosion resistance, ease of castability, and good machinability. Major uses include hardware for food processing, seals, architectural trim, musical instrument valves, and door keys. Other Copper Alloys In the interest of completeness, two other types of copper alloys, which are both cast alloys, should be mentioned. They are the leaded coppers (C89000–C98000), which are typically employed where their high lead provides lubricity, and the special alloys (C99000–C99999), which have unique specific properties needed for specialized applications. CONCLUSIONS The copper alloys offer a wide range of properties to meet the needs of many applications that humans touch. The list is almost endless. End-use applications are limited only by the knowledge, creativity, and imagination of those who specify, design, produce, and process these copper alloy products.

2 Corrosion Chemistry of Copper: Formation of Potentially Skin-Diffusible Compounds Jurij J. Hostynek Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

INTRODUCTION In the background of this review lies the question, what effect, if any, does copper metal have when kept in contact with the skin, on inflammation, and arthritis in particular—i.e., concerning the unsettled issue of the ‘‘copper bangle.’’ Copper complexes given systemically have a well-documented therapeutic effect on inflammatory conditions (1), and anti-inflammatory agents (NSAIDs) have also proven successful by dermal delivery (2,3). Investigations demonstrating the efficacy of dermal assimilation of copper in arthritic and rheumatoid conditions by skin contact with the metal or its derivatives in humans have been few, poorly conducted, and have only brought qualitative, mostly subjective, evidence of benefits to be expected from the ‘‘mere’’ skin contact with the metal (4). The review of the microenvironment prevailing on the skin surface and the chemical agents present there aims to delineate acceptable chemical arguments that support the contention that copper, solubilized in that environment, is apt to be assimilated by the mammalian organism.

This chapter, in part, was reprinted from Hostyńek JJ. Factors determining percutaneous metal absorption. Food Chem Toxicol 2003; 41:327–345, with permission of Elsevier.

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ELECTRON CONFIGURATION AND REACTIVITY OF COPPER The transition metals in the periodic table have partially filled d (or f) electron shells, in which an inner shell is being filled with electrons, two in each of five orbitals, while the outer s-electron shell of slightly lower energy is complete. Characteristic for transition metals is a low ionization potential (electropositivity), i.e., the ease with which they can lose electrons to yield a variety of positive ionic oxidation states, and the ability to form complexes. This occurs mainly with sulfur, (e.g., in histidine), oxygen, or nitrogen groups (e.g., in cysteine), particularly in biological systems and chelates (cyclic coordination compounds) that are stable and likely to remain unchanged in physical processes, such as membrane transport. Copper has a single s electron outside the filled 3d shell, but the d electrons also are involved in metallic bonding, and the (most common) Cu(II) ion has the configuration 3d9, having lost the single s electron from the outermost shell and one d electron. While further oxidation to Cu(III) is difficult, the lower oxidation states can interchange easily. For instance, the equilibrium 2Cu(I) D Cu(0)þ Cu(II) can be readily displaced in either direction. Copper complexes with proteins—molecules composed entirely of alpha-amino acid residues covalently united head to tail by peptide bonds—to form unbranched polymers. Examples are copper metallothionein (a small, ubiquitous protein in the organism fulfilling a multifunctional role in absorption and reversible storage, transport and detoxification of the highly toxic free copper ion) ceruloplasmin (the enzyme involved in the antiinflammatory action of copper), blood clotting factors V and VIII, and copper glycyl-L-histidyl-L-lysine, Cu(II)-GHK, which may prevent the inhibition of mitochondrial glucose oxidation (5–8).

CORROSION OF COPPER IN THE ENVIRONMENT For metals and their alloys exposed to the environment, the major reactions are oxidation to the ionic form and liberation of electrons: M ! Mxþ þ xe and the reduction of the (obligatory) oxygen present to form water: O2 þ 4Hþ þ 4e ! 2H2 O In presence of oxygen, on the surface of pure copper metal cuprous oxide is formed, whereby copper ion persists in the monovalent Cu(I) state, due to the reducing action of the metal. In the presence of saline (water), oxygen dissolved in the medium can oxidize Cu(I) further to yield the cupric ion, Cu(II), with formation of free radicals (9). Although copper and its alloys have been known and widely used since prehistoric times, appropriately named the Bronze Age, corrosion of the

Corrosion Chemistry of Copper

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pure metal has hardly been investigated and is poorly documented. The corrosive action of the environment (e.g., seawater) has focused on its alloys, the bronzes and brasses, but not on pure metal itself (10). Well established is the corrosive action of saline, a prime factor in the dissolution of the metal (11), due to which copperware used in cooking is usually lined with tin. While main sources of exposure to copper occurs through the diet (animal organs, seafood, certain vegetables, and nuts), unsuspected exposure may be due to release of copper corrosion products from copper cooking and tableware (12), or the metal released into the plumbing system of water conduits (13). Low hardness, low alkalinity, chlorinated tap water in municipal water supplies, particularly of low pH and at elevated temperatures (as in hot water conduits), increases the rate of corrosion. The effects of temperature, chlorine, and organic matter on copper alloys have been investigated in a number of studies (14). Corrosion products of copper, brass, or bronze that are formed on exposure to air, of different hues of blue and green, are collectively referred to as verdigris, a term possibly derived from the term ‘‘vert de Grece,’’ mentioned in ancient texts. A mixture of cupric acetate Cu(CH3COO)2, basic cupric acetate Cu(OH) (CH3COO), and carbonate CuCO3, verdigris salts occurring naturally are mostly noticeable as patina on weathering copper objects, such as roofs and statues, described in Egyptian records dating back to 1500 B.C. In post-Renaissance Europe, verdigris was used in oil-based paint pigments to decorate interiors of homes, and as wood preservative. Formed by the action of acetic acid vapors on metallic copper since the Middle Ages and until the early 20th century, verdigris was produced in the wine-growing regions of France as a cottage industry. Flat copper strips were rubbed with verdigris, then layered with fermented grape husks for several weeks until crystals formed on the metal surface. Dripping wine on the surface promoted the formation of spongy green crystals that, scraped from the copper, were dissolved in vinegar, recrystallized on wooden sticks, crushed, and sold as pigment (15). CORROSION OF COPPER IN PHYSIOLOGIC MEDIA Copper and its alloys are present in numerous articles of everyday use, and thus come in regular, sometimes extended and intimate contact with the skin, or through systemic exposure by implanted prostheses or contraceptive devices [intrauterine devices (IUDs)]. Heat, moisture, sweating, and friction promote chemical reactions on the surface of objects in contact with the skin. Skin: The Action of Sweat and Sebum To gain insight onto the phenomenon of skin penetration by metals in general, it is important to account for the factors involved in that process

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stemming from the chemical microenvironment prevailing on the skin surface, as the oxidative action encountered there is a determinant factor in the formation of skin-diffusible derivatives. By the action of salts and acids present in sweat and sebum, metals in their elemental state are converted to the hydrophilic or lipophilic derivatives, respectively. Only as such do metals become diffusible via the transcellular, intercellular, or transappendageal route. Leaching or release of copper ion from metal objects (alloys) in contact with sweat is a multifactorial event that defeats prediction. Aside from immediate environmental factors, the microenvironment within the particular alloy in contact with a conducting medium (electrolyte, e.g., sweat) is a principal determinant, due to the action of electromotive forces prevailing at the interface of atoms with differing electromotive potential (16). Such metals alloyed with copper form a galvanic element (or pile), whereby an electron current flows from the more electronegative to the more electropositive one, resulting in oxidation or solution (‘‘corrosion’’) of the more electronegative metal. These effects, unpredictable quantitatively, vary in function of actual metals present and their ratios in alloy composition. A recent example with potential impact on public health is the recently minted, bimetallic (cupronickel) 1- and 2-Euro coins, consisting of 25% nickel. That bimetallic structure, which generates a potential difference of 30–40 mV between the components, increases nickel release, due to galvanic corrosion, facilitated in the medium of conducting sweat. As a consequence, reports of nickel sensitizations from dermatology clinics are on the increase in a number of countries where the new currency is in circulation, as nickel release in handling the coins may elicit contact dermatitis in those allergic to nickel (17–22). While amino acids, such as glycine or histidine, or proteins that can complex with cupric ions enhance the rate of copper released, the presence of saline and oxygen are prime factors in the dissolution of the metal. In their absence no copper is released (9,23). As formulated for the general reaction of a metal with amino acids or fatty acids present in sweat, the process can be presented as: 2M0 þ O2 þ 4HL ! 2ML2 þ 2H2 O where M0 is the metal in elemental form and HL the endogenous ligand. The Major Sweat Components Electrolytes The considerable variation in electrolyte composition occurring naturally in sweat became evident in a number of studies conducted on the subject. In one study on normal subjects, in Palmar sweat both sodium and chloride levels fell below 50 milliequivalents/L (mEq/L) for 99% of subjects, and below 65 mEq/L in all 649 volunteers tested (24).


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Using direct reading ion-selective electrodes, mean sodium concentration in forearm transudate was 1.7 mEq/L 0.7, and chloride concentration 2.8 mEq/L 3.5 (n ¼ 6) (25). Mean body sweat induced through exercise contained 24.2 2.2 mEq/L chloride ion in men, and 26.0 4.6 in women (26). Sodium and potassium content of pharmacologically stimulated sweat in men (pilocarpine, methylcholine, and acetylcholine) was seen to be higher than that measured in thermal sweat: mean values (in mEq/L) are 8.3 0.66 and 4.9 0.17, respectively (27). Sweat osmolality values in normal adults were seen to increase with increasing age. Range/mean (SD) values for men are 49–151/117 mmol/kg (33.4) and for women 66–187/134 mmol/kg (38.6). An increase in osmolality was observed to increase in tandem with the (normal) increase in sodium concentration in sweat among an aging population (28). The main cause for corrosion of metal surfaces from skin contact in individuals referred to as ‘‘rusters’’ is not due to elevated electrolyte concentration, as generally assumed, but rather seems to coincide with palmar hyperhydrosis. When the sodium concentration measured in normal subjects was compared to that of ‘‘rusters,’’ in fact no significant difference could be observed (mean values of 49.6 vs. 49.1 mEq/L, respectively) (29). Amino Acids Proteins and AAs are normal components of mammalian sweat. The data documented for humans differ significantly, probably due to differences in the stimulation methods applied, to regional differences in anatomic site, or the sampling methods used. Substantial variations were observed in relative concentrations of AAs in sweat collected from various areas of the skin, and their concentrations increased markedly in blood and urine on oral protein intake (30). No differences in AA patterns were seen between young and middle-aged adults, or between men and women (31). In contrast to essential elements, AAs are neither selectively excreted nor reabsorbed (32). Large individual differences occur in AA composition between eccrine forearm sweat from men under controlled exercise conditions. Comparison of AA excretions analyzed in sweat and urine by ion exchange chromatography showed comparable losses in those two media. In general, AA concentrations were considerably higher in the exercise sweat of untrained men than in the sweat of trained men determined by thermal and physiological stimulation. Total average AA values collected from 20 trained men and 20 untrained men were 12,797 and 24,855 mmol/L, respectively. For trained versus untrained men, highest values were seen for serine (3954/7782), glycine (2239/4392), alanine (1556/3028) and threonine (1057/1856), respectively (33).

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Lactate and Pyruvate Lactate is the major organic compound secreted in sweat. At rest and low sweating rates, the range in lactate concentrations is 30–40 mmol/L, at higher sweat rate levels 10–15 mmol/L. Under thermal stimulation and also during exercise, lactate concentration in sweat decreases with increasing sweating rate, but remains constant in blood. Differences in the excretion of lactate were also observed in function of physical fitness of three male volunteers: mean values for the sedentary individual was 21.71 0.85, for the fit individual 16.75 0.99, and the very fit individual was 12.75 0.50 mmol, respectively (34). In contrast, pyruvic acid (pyruvate) concentrations in sweat are low, found to vary between 0.1 and 1.2 mmol/L. The ratio of the two metabolites, lactate and pyruvate, was observed to increase with rising heart rate (35). Sebum Human skin features an acid mantle of pH 4–6 at the surface of the SC in normal, healthy subjects, which increases with depth to pH 7 at the juncture with live tissue (36). Determinants of this pH are protons originating in the epidermis or as products of sebaceous gland activity, which gradually reach the surface of the skin. They stem from three classes of compounds: amino acids (e.g., urocanic acid, pyrrolidone carboxylic acid) alpha-hydroxy acids (e.g., lactic and butyric acid, also present in sweat) acidic lipids (e.g., cholesteryl sulfate and free fatty acids, primarily oleic, linoleic, and behenic) (37–40) Sebum as secreted by the sebaceous glands is a complex mixture of lipids consisting of glycerides, but no free fatty acids. The occurrence of free acids in the SC and on the skin surface is the result of hydrolysis of phospholipids and glycerides by lipolytic enzymes occurring in the sebaceous ducts and on the skin surface, and of bacterial decomposition. On the skin surface, lipids of epidermal origin contain up to 20% free fatty acids, those originating in the pilosebaceous glands, 16% (40). They consist for the greater part of C16 and C18 acids, but their full range reaches from C5 to C22, with an average length of C16 (39,41). Such an acid milieu plays a regulating role for SC homeostasis, with relevance to the integrity of the skin’s barrier function and regeneration of the SC (42). It is believed that the acid environment on the skin surface both controls moisture loss from the epidermis and protects the skin from fungal and bacterial infection. These acid components making up the sebum also play an important role in solubilizing (‘‘corroding’’) metal surfaces in measurable amounts.


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The Release of Copper Ion in Artificial Sweat The definition and thus reconstitution of human sweat for experimental purposes fluctuate in function of numerous factors, which is why experimental results are not consistent. Most striking are changes in sweat composition, due to the rate of sweat secretion. Sodium and chloride content, one decisive factor in the corrosion of copper and metals in general, is as low as 5 mEq/L under quiescent conditions, due to a reabsorption (conservation) mechanism (43,44). As sweating rate increases, that control mechanism is overwhelmed and the sodium concentration can rise to approximate that occurring in plasma. Also other significant components of sweat such as urea, lactic acid, and potassium ions, increase at high rates of secretion and their concentrations can also reach the levels of plasma. Release of copper ions from various copper alloys in synthetic sweat was investigated in model experiments. Over a period of 24 hours at 35 C and a pH of 5.1, the range of Cu2þ liberated in artificial sweat was 80–100 mg/mL sweat (45). Walker and Keats performed a number of experiments to determine the solubility of copper metal in artificial sweat. In samples where initial copper concentration was of the order of 2 105 M, after 24 hours the samples turned blue and the concentration had increased to 2 103 mol Cu (46). Oxidation of Copper in Contact with the Skin Walker and Keats also measured the loss from copper bracelets worn by volunteers. Bracelets measuring 22 1.3 0.1 cm lost 80 mg in 50 days when worn around the ankles, and 90 mg when worn around the wrist (4). Weight losses of 0.1% to 0.8% were observed for copper bracelets weighing 14 g used in a trial involving volunteers and lasting one month (47). Such variation can be explained by differential mechanical wear, but a more likely alternative explanation is the variability in subjective sweat composition and sweating rate. Skin Penetration Data Although limited in number, experiments have demonstrated that copper compounds will penetrate the integument in humans and animals, mostly in a qualitative manner. Such information is based on clinical observations of remedial action in inflammatory diseases, describing therapeutic effects, and qualitative observation by spectroscopic methods (Warner RR, 1993, personal communication) (48–51). One experiment only measured diffusivity of a skin-identical complex, bis(glycinato) copper(II), in vitro on cat skin (52). A radioactive (64Cu) 0.05 M solution in physiological saline applied to excised cat skin revealed,

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after a lag time of nine hours, a steady-state transport rate described by a Kp¼ 24 104 cm/hr. Electron micrographs of skin sections stained for copper revealed the metal in all layers of the skin in exposed samples. In 24 hours, 3.3% of the applied copper(II) complex had completely penetrated the skin. This is equivalent to 3.0 mg of the complex. Atomic absorption analysis found 47 ppm copper in the washing solution beneath the skin (20 mL isotonic saline). This corresponds to 0.94 mg, which, in agreement with the radioactivity, is equivalent to 3.1 mg of the complex. To examine whether skin exudates will oxidize the surface of copper metal in contact with skin, forming potentially diffusible compounds, copper powder was applied under occlusion on the volar forearm of human volunteers at UCSF. When the application areas were sequentially stripped with adhesive tape and the strips then analyzed for metal content by inductively coupled plasma-mass spectroscopy, a concentration gradient became evident from the skin surface to the subcutis, characteristic of a passive diffusion process. The results indicate that in contact with skin compounds are formed, which can penetrate the intact stratum corneum to the level of live epidermis (unpublished study). Release of Copper in the Human Organism and Its Contraceptive Effects In the presence of saline, amino acids (e.g., glycine or histidine) or proteins that complex with cupric ions accelerate the release of copper by removing free copper ions from equilibrium. Copper incubated in a saline medium will cleave the S–S bond, and serum albumin will undergo a conformational change (11). Incubation with copper metal was seen to be spermatocidal at cupric ion concentrations below 103 mol (53), and enzymes important in the implantation process were also inactivated on incubation in the presence of copper metal in a saline medium. Copper metal incubated in saline in the presence of oxygen produces free radicals (9), and it appears probable that free radicals thus formed are responsible for the contraceptive action of copper in IUDs. The ‘‘copper IUD,’’ a plastic T-shaped device with copper wire wound around its stem, has an increased contraceptive effect in women as compared to the T insert without copper, and it continues to be a popular method of temporary contraception. With the aim of obtaining insight into the mode of action of these devices, studies were conducted to investigate the effect of copper IUDs on the uterine milieu in women. The chemical process involved on the surface of the metal in the uterus appears responsible for the contraceptive activity (i.e., inhibition of implantation) (54). In solutions of saline and serum albumin, a strip of 200 mm2 copper dissolved at a rate comparable to that of copper IUDs when measured in vivo (23). Such losses of copper metal were determined on a number of


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copper IUDs in the actual use environment, by weighing before their insertion into the uterus and after their removal a year later. Following one year’s use, the loss from two sets of IUDs (120 and 135 mm2 surface area) amounted to 10.0 2.2 and 11.0 2.8 mg, respectively, the average release per day being calculated at 28.7 mg (55). Increases in systemic copper levels via parenteral exposure from a copper IUD can also lead to adverse effects, even though the amounts liberated from such a device are relatively low. Frentz and Teilum (54) traced induction of copper hypersensitivity in patients to the action of a copper IUD. They measured copper released from the device at 90 mg/day. A fertility-related phenomenon due to the local increase in systemic copper ion was described by Vesce et al., who noted that the use of the copper intrauterine device was effective in the management of secondary amenorrhea. In 40 of 48 volunteers with functional secondary amenorrhea, regular menses were restored after insertion of an IUD, and normal menses were maintained as long as the IUD remained in place. After removal of the device, the effect persisted for one year. The authors of that study ascribe the mechanism of action to the copper ion-mediated release of prostaglandins from the endometrium (56). CONCLUSIONS Copper and its alloys are subject to chemical reactions on exposure to environmental or physiological factors, whereby products are potentially generated, which become diffusible through mammalian skin. The chemistry of oxidation is reviewed as well as the factors contributing to corrosion. Skin exudates (sweat and sebum) can react with metal surfaces that they come in contact with, but even in the healthy organism their composition is variable, in function of physical, pharmacological and environmental conditions, gender, age, sweat rate, or body site. This overview addresses sweat and sebum composition, and discusses components that determine the skin’s corrosive action: chloride ion, low molecular weight acids and amino acids (AAs) in sweat, and fatty acids in sebum, which hold the potential to solubilize copper-containing metal objects. These components can form copper salts and soaps whose molecular characteristics (size and polarity) will determine rate and route of cutaneous penetration. Rate and degree of copper corrosion is subject to a number of variable environmental and biological factors that make predictions unlikely. Oxygen and saline solutions are the main corrosive factors. Elemental copper in the mammalian organism is highly reactive and the resulting ions have biological effects. In contact with the skin, metallic copper is measurably oxidized by exudates under formation of derivatives that, at least in animals, penetrate the integument as measured by the resulting anti-inflammatory activity.

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In the living organism, corrosion becomes manifest through biological evidence of contraceptive and spermatocidal action, resulting from the oxidation process and products of copper IUDs. Conversely, evidence was presented that in amenorrheic women the copper IUD can restore regular menses. GLOSSARY Corrosion

Electromotive force

Ionization potential

Metallic elements

Oxidation

Oxidation potential

Penetration

Electrochemical process involving movement of electrons, through a metal from anodic to cathodic areas, and corresponding movement of ions in the electrolyte medium. Difference in driving force toward electron loss between two metals causing a flow of current, expressed in volt. Energy (electron volts, ev) required to remove an electron from its atomic orbit, with the value for the standard hydrogen electrode set at 0.00 ev as an arbitrarily selected standard. Characterized by luster, malleability, conductivity (thermal and electrical), and ability to form positive ions. Process of electron removal from an atom or ion, or the increase in the proportion of oxygen in a compound. Electrical driving force toward electron loss, expressed as a potential value (in electron volts, ev). Process of an exogenous agent entering one skin layer.

ABBREVIATIONS AA L mEq/L SC

amino acids ligand milliequivalents per liter stratum corneum

REFERENCES 1. Sorenson JRJ. In: Siegel A, ed. The anti-inflammatory activities of copper complexes. New York: Marcel Dekker, 1982. 2. Yano T, Nakagawa A, Tsuji M, Noda K. Skin permeability of various nonsteroidal anti-inflammatory drugs in man. Life Sci 1986; 39:1043–1050.


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3. Hadgraft J, du Plessis J, Goosen C. The selection of non-steroidal antiinflammatory agents for dermal delivery. Int J Pharm 2000; 207:31–37. 4. Walker WR, Keats DM. An investigation of the therapeutic value of the ‘‘copper bracelet’’—dermal assimilation of copper in arthritic/rheumatoid conditions. Agents Actions 1976; 6:454–458. 5. Cousins RJ. Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol Rev 1985; 65: 238–309. 6. Powanda MC. Systemic alterations in metal metabolism during inflammation as part of an integrated response to inflammation. Agents Actions 1981(suppl 8): 121–136. 7. Linder MC, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutr 1996; 63:797S–811S. 8. Vinci C, Caltabiano V, Santoro AM, et al. Copper addition prevents the inhibitory effects of interleukin 1-b on rat pancreatic islets. Diabetologia 1995; 38:39–45. 9. Oster G, Oster GK. Free radical production by metallic copper. Contraception 1974; 10:273–280. 10. Leidheiser HJ. The corrosion of copper, tin and their alloys. New York: John Wiley and Sons, 1971. 11. Oster GK. Reaction of metallic copper with biological substrates. Nature 1971; 234:153–154. 12. Li S, Miao X, Zhu D, Ni L, Sun C, Wang L. Copper release from copper tableware. Bull Environ Contam Toxicol 2003; 70:905–912. 13. Broo AE, Berghult B, Hedberg T. Copper corrosion in water distribution systems—the influence of natural organic matter (nom) on the solubility of copper corrosion products. Corrosion Sci 1998; 40:1479–1489. 14. Boulay N, Edwards M. Role of temperature, chlorine, and organic matter in copper corrosion by-product release in soft water. Water Res 2001; 35: 683–690. 15. Reese KM. Verdigris. Chem Eng News 2002; 80:72. 16. Cavelier C, Foussereau J, Massin M. Nickel allergy: analysis of metal clothing objects and patch testing to metal samples. Contact Dermatitis 1985; 12: 65–75. 17. Lideń C, Carter S. Nickel release from coins. Contact Dermatitis 2001; 44:160–165. 18. Aberer W. Platitudes in allergy-based on the example of the Euro. Contact Dermatitis 2001; 45:254–255. 19. Aberer W, Kranke B. The new EURO releases nickel and elicits contact eczema. Br J Dermatol 2002; 146:155–6. 20. Nestle FO, Speidel H, Speidel MO. High nickel release from 1- and 2-Euro coins. Nature 2002; 419:419. 21. Fournier P-G, Govers TR. Contamination by nickel, copper and zinc during the handling of Euro coins. Contact Dermatitis 2003; 48:181–8. 22. Lachapelle J-M, Marot L. High nickel release from 1- and 2-Euro coins: are there practical implications? Dermatology 2004; 209:288–290. 23. Oster GK. Chemical reactions of the copper intrauterine device. Fertil Steril 1972; 23:18–23.

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24. Sekelj P, Rasmussen K, McDougall D, Baggs J. Survey of electrolytes of unstimulated sweat from the hand in normal and diseased adults. Am Rev Respir Dis 1973; 108:603–609. 25. Grice K, Sattar H, Casey T, Baker H. An evaluation of Na, Cl and pH ionspecific electrodes in the study of the electrolyte contents of epidermal transudate and sweat. Br J Dermatol 1975; 92:511–518. 26. Yousef MK, Dill DB. Sweat rate and concentration of chloride in hand and body sweat in desert walks: male and female. J Appl Physiol 1974; 36:82–85. 27. Sato K, Feibleman C, Dobson RL. The electrolyte composition of pharmacologically and thermally stimulated sweat: a comparative study. J Invest Dermatol 1970; 55:433–438. 28. Willing SK, Gamlen TR. Sweat osmolality values in normal adults. Clin Chem 1987; 33:612–613. 29. Jensen O. ‘‘Rusters’’. The corrosive action of palmar sweat: II. Physical and chemical factors in palmar hyperhidrosis. Acta Derm Venereol (Stockh) 1979; 59:139–143. 30. Hier SW, Cornbleet T, Bergheim O. The amino acids of human sweat. J Biol Chem 1946; 166:327. 31. Coltman CA, Rowe NJ, Atwell RJ. The amino acid content of sweat in normal adults. J Clin Nutr 1966; 18:373. 32. Gitlitz PH, Sunderman FW, Hohnadel DC. Ion-exchange chromatography of amino acids in sweat collected from healthy subjects during sauna bathing. Clin Chem 1974; 20:1305–1312. 33. Liappis N, Kelderbacher S-D, Kesseler K, Bantzer P. Quantitative study of free amino acids in human eccrine sweat excreted from the forearms of healthy trained and untried men during exercise. Eur J Appl Physiol 1979; 42:227–234. 34. Fellmann N, Grizard G, Coudert J. Human frontal sweat rate and lactate concentration during heat exposure and exercise. J Appl Physiol 1983; 54:355–360. 35. Pilardeau PA, Lavie F, Vayasse J, et al. Effect of different work-loads on sweat production and composition in man. J Sports Med Phys Fitness 1988; 28:247–252. ¨ hman H, Vahlquist A. The pH gradient over the stratum corneum differs in 36. O X-linked recessive and autosomal dominant ichthyosis: a clue to the molecular origin of the ‘‘acid skin mantle’’. J Invest Dermatol 1998; 111:674–677. 37. Elias P. Epidermal lipids, barrier function, and desquamation. J Invest Dermatol 1983; 80:44s–49s. 38. Lampe MA, Burlingame AL, Whitney J, et al. Human stratum corneum lipids: characterization and regional variations. J Lipid Res 1983; 24:120–130. 39. Wertz PW, Swartzendruber DC, Kathi C, Downing DT. Composition and morphology of epidermal cyst lipids. J Invest Dermatol 1987; 89:419–425. 40. Schurer NY, Elias PM. The biochemistry and function of stratum corneum lipids. Adv Lipid Res 1991; 24:27–56. 41. Stillman MA, Maibach HI, Shalita AR. Relative irritancy of free fatty acids of different chain length. Contact Dermatitis 1975; 1:65–69. 42. Feingold KR. The regulation of epidermal lipid synthesis by permeability barrier requirements. Crit Rev Ther Drug Carrier Syst 1991; 193–210.


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43. Cage GW, Dobson RL. Sodium secretion and reabsorption in the human eccrine sweat gland. J Clin Invest 1965; 44:1270–1276. 44. Guyton AC. Textbook of Medical Physiology. Philadelphia: W.B. Saunders Co., 1991. 45. Boman A, Karlberg AT, Einarsson O, Wahlberg JE. Dissolving of copper by synthetic sweat. Contact Dermatitis 1983; 9:159–160. 46. Walker WR, Griffin BJ. The solubility of copper in human sweat. Search 1976; 7:100–101. 47. Walker WR, Beveridge SJ, Whitehouse MW. Dermal copper drugs: the copper bracelet and Cu(II) salicylate complexes. Agents Actions 1981(suppl 8): 359–367. ¨ ber die Kutane Kupferresorption aus einer Kupfer 48. Schmid R, Winkler J. U Enthaltenden Salbe. Klin Wochenschr 1938; 17:559–561. 49. Walker WR, Beveridge SJ, Whitehouse MW. Antiinflammatory activity of a dermally applied copper salicylate preparation (Alcusal). Agents Actions 1980; 10:1–10. 50. Beveridge SJ, Whitehouse MW, Walker WR. Lipophilic copper(II) formulations: some correlations between their composition and anti-inflammatory/ anti-arthritic activity when applied to the skin of rats. Agents Actions 1982; 12: 225–231. 51. Odintsova NA. Permeability of human skin to potassium and copper ions and their ultrastructural localization. Chem Abs 1978; 89:360. 52. Walker WR, Reeves RR, Brosnan M, Coleman GD. Perfusion of intact skin by a saline solution of bis(glycinato) copper(II). Bioinorg Chem 1977; 7:271–276. 53. Jecht EW, Bernstein GS. The influence of copper on the motility of human spermatozoa. Contraception 1973; 7:381–401. 54. Frentz G, Teilum D. Cutaneous eruptions and intrauterine contraceptive copper device. Acta Derm Venereol (Stockh) 1980; 60:69–71. 55. Hagenfeldt K. Studies on the mode of action of the copper-T device. Acta Endocrinol Suppl (Copenh) 1972; 169:3–37. 56. Vesce F, Jorizzo G, Bianciotto A, Gotti G. Use of the copper intrauterine device in the management of secondary amenorrhea. Fertil Steril 2000; 73: 162–165.

3 Basics of Metal Skin Penetration: Scope and Limitations Jurij J. Hostyńek and Howard I. Maibach Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

INTRODUCTION The skin as target organ presents imponderable and wide margins of variability. In vivo, permeability is subject to homeostasis regulating the overall organism; in vitro, the sections of skin used for diffusion experiments are likely to present artifacts. To further complicate the matter, diffusion of metals appears to defy laws empirically derived for passive diffusion across biological membranes. Endeavors to define rules governing skin penetration by metals toward derivation of predictive quantitative structure–diffusion relationships for risk assessment, thus, have been unsuccessful, and penetration of the skin still needs to be determined separately for each metal compound, by in vitro or in vivo assays. Because diffusion through biological membranes is highly metal specific, and, in addition, metal ions’ valence is mutable during the process of diffusion, molecular physicochemical parameters alone do not suffice to model migration of electrolytes into and through the strata of the skin. Certain factors are closely interrelated, and their combined effects are neither entirely understood nor predictable. For example, unless the dynamics This chapter, in part, was reprinted from Hostyńek JJ, Maibach HI. Skin penetration by metal compounds with special reference to copper. Tox Mech Meth 2006; 16:245–265, with permission of Informa Healthcare.

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of in situ changes in speciation (oxidation state), or electrophilic reactivity, among others, can be factored in, metal diffusivity will elude modeling. Experimental data available so far from in vitro and in vivo experimentation have been acquired under disparate conditions, and the base is too thin to allow development of a predictive algorithm considering the number of metals and metalloids of variable valence existing as free ions or forming chelates, coordination compounds, or complexes with electron donors, such as oxygen, sulfur, or phosphorus active in biological systems. The number of metals discussed in this review may appear limited; this is due to the circumstance that most scientists and government agencies so far have given priority to the limited number of industrial materials, which comport special risks from work place exposure in their investigations. Healthy and intact skin was formerly considered to be an impermeable barrier designed to shield the living organism from environmental injury, also affording protection from chemical agents. More recently, however, the skin is being recognized as a membrane, which can act as filter, impenetrable to microorganisms but selectively pervious to chemicals within limits of size and polarity, allowing molecular passage in as well as out of the skin, and acting as a permanent depot or as dynamic reservoir for essential trace elements (ETEs) and xenobiotics. Only larger structures (e.g., microorganisms, proteins, and polymers) or highly polar compounds, such as sugars, will not diffuse measurably through that barrier. Although exposure to exogenous agents due to skin absorption is usually less than through inhalation or ingestion, the large surface area of the body is now recognized as a pharmacologically relevant port of entry for both nutrients and xenobiotics. Dermal absorption can present substantial risks in case of exposure to toxins, be they of natural or of anthropogenic origin. Dermal exposure presenting the potential for percutaneous absorption has become an integral part of toxicological risk assessment, especially after incidents of significant morbidity and mortality following exposure to agents of facile skin diffusivity. Thus, 36 infants died in France upon exposure to baby talcum powder erroneously formulated with a high dose of the neurotoxic antimicrobial hexachlorophene (1–3) or, more recently, to dimethyl mercury, which proved lethal upon skin contact in trace amounts (4). Conversely, the skin is also an important secretory organ, which plays a vital part in the process of detoxification and the maintenance of homeostatic balance. By that token, on the one hand it is an important factor in the organism’s clearing mechanism, because it allows elimination of toxics, such as certain heavy metals, but, on the other hand, it can also lead to significant elimination of ETEs, such as the detrimental loss of copper through excessive sweating due to elevated temperature or physical strain. Excretion levels of six trace metals examined in exercise-induced sweat placed copper in first place (5,6).

Basics of Metal Skin Penetration: Scope and Limitations

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Assessment of cutaneous absorption of metal compounds by the use of new, refined analytical tools is reviewed here. This route of absorption may be of pharmacological interest in order to compensate for shortages of ETEs, due to nutritionally caused shortages or genetically caused defects in intestinal absorption of metals, such as copper. Such shortages in nutrients may be corrected through cutaneous dosing, as could be shown in the case of zinc in humans (7–9) and in the rat (10). It is the objective of this review to identify and describe the numerous factors that need to be weighed critically when evaluating in vivo or in vitro data stemming from various sources, and also in planning new such experiments. Here the term ‘‘metal’’ refers to metals in all its forms of occurrence: metallic state, electrolytic (ionized) form, and organometallics, complexes, coordination compounds, or chelates. The term absorption is used to describe metal penetration of one or several skin strata; penetration signifies metal becoming available systemically. This review addresses skin physiology and the various aspects of metals’ skin penetration according to the current scientific knowledge. This includes discussion of: 1. 2. 3. 4. 5. 6. 7.

Structure of the skin and its function as diffusion barrier Descriptors of dermal absorption Permeant categories and paths for their diffusion Metals derivatives formed in contact with skin Variables in skin diffusion by metal compounds Methods for measuring skin permeation Analytical methods for metal detection

STRUCTURE OF SKIN AND ITS FUNCTION AS DIFFUSION BARRIER Skin is a major organ involved in the maintenance of the body’s homeostasis: its composition, heat regulation, blood pressure control, and excretory functions. It is a multilayered organ, which is a living envelope consisting of dermis and epidermis, with the stratum corneum (SC) as the outermost layer. In men, the latter is a 10- to 40-mm thick layer of keratinized epithelial cells, variable by anatomical site, held together in a ‘‘tongue-and-groove’’ arrangement. It represents the rate-limiting diffusion barrier for most chemical compounds (11,12), particularly limiting the loss of water (13). SC thickness, seemingly a simple measurement, remains at best an estimate. Schwindt et al. summarize previous efforts, adding indirect measurements in vivo in men, utilizing transepidermal water loss (TEWL) measurements based on Kalia’s diffusion equation (14,15). The SC is composed of layers of proteinaceous cells separated by layers of lipid material. In humans it consists of about 40% protein, 15% to 20% lipids, and 40% water (16). Likened to a

24


brick-and-mortar structure, the SC consists of corneocytes (bricks) embedded in an intercellular matrix of lipids (mortar), rendering the membrane poorly permeable to water and other polar compounds (17). Interspersed in this envelope are appendageal shunts, openings for hair follicles, and sweat ducts. Underlying the SC are the strata of the avascular viable epidermis, consisting of keratinocytes, the source of corneocytes. The epidermis is also transversed by shunts: the hair follicles and sebaceous and sweat glands. In ascending order, the human epidermis consists of the stratum basale, responsible for the maintenance of epidermal–dermal adhesion. As cells move outward from the basement membrane, in the process of differentiation they undergo keratinization, transition to the stratum spinosum, then further to the stratum granulosum. The latter consists of electron-dense keratohyalin granules, which are irregularly shaped, nonmembrane-bounded, electron-dense granules that contain profilaggrin, a structural protein, and a precursor of filaggrin that plays a role in keratinization and barrier function. The uppermost layers of the stratum spinosum and stratum granulosum contain small membrane-bounded organelles known as lamellar granules, Odland bodies, lamellated bodies, or membrane-coating granules. These are the granules that contain lipids that can fuse to the cell membrane to release its lipid content that will fill the intercellular space (18). The stratum granulosum is also source of corneocytes, which, keratinized, form the SC, the outermost layer of dead cells constituting an efficient barrier against transcutaneous water loss. Besides generating the SC, other important functions of the epidermis are metabolism of xenobiotics, synthesis of melanin by melanocytes, and provision of a first-line immune defense by means of dendritic Langerhans’ cells. In its entirety, the epidermis with the intercellular lipids between the SC layers constitutes the rate-limiting barrier to the absorption of both hydrophilic and lipophilic xenobiotics. Transition from the lipophilic SC to the hydrophilic medium of the epidermis constitutes a discontinuity that forms an important second barrier, largely inhibiting further passage of lipophilic compounds once they have transited through the SC. In its entirety, from skin surface to circulatory system the epidermis consists of aqueous and lipid barriers. Removal of the hydrophilic epidermal layer in in vitro diffusion experiments enhanced the percutaneous absorption of lipophilic compounds across the SC, because it no longer functions as that rate-limiting secondary barrier for the diffusion of lipophilic compounds across the skin (19). The support system for the epidermis is the underlying dermis, carrier of nutrition through its vascular network; it is also involved in the immune function through the lymphatic system, macrophages, and the mast cells responsible for immune and inflammatory responses. It produces elastic connective tissue consisting of collagen, laminin, and fibronectin, among other components, and is the origin of sebaceous glands, eccrine and apocrine sweat glands, and hair follicles.


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DESCRIPTORS OF DERMAL ABSORPTION Mostly, but not exclusively, description of dermal absorption is based on two approaches:

The permeability coefficient, Kp, usually obtained through in vitro experiments for compounds from water where it can be measured directly Percent absorbed as fraction of dose applied in vivo

The Permeability Coefficient Kp The permeability coefficient Kp—a flux value, normalized for concentration— is a widely applied benchmark, representing the rate at which chemicals penetrate the skin. It is derived by Fick’s law of diffusion (20) from flux measurements through a biological barrier under conditions of steady state (Jss) from an infinite reservoir of the compound, through a biological barrier, and normalized for the concentration (C) applied: Jss ¼ Kp DC, e.g., expressed as cm/hr. Formulated to characterize passive diffusion of compounds across membranes in general, Fick’s law also applies to passive diffusion of xenobiotics through the SC of the skin. The law states that if the permeation process reaches the point of steady-state equilibrium (i.e., the concentrations in the donor and receptor phases remain constant over time), the steady-state penetration flux Jss per unit path length (here in cm) is proportional to the concentration gradient dC and to the penetrant’s permeation constant. In practice, in a typical experiment the donor solution is scaled in such a fashion that over the term of the experiment the concentration remains ‘‘practically’’ constant (a.k.a. ‘‘infinite’’ dose); because the receptor phase is constantly removed, receptor concentration equals zero. Thereby, the expression DC becomes equal to C, the concentration in the donor solution, and the equation is simplified as Jss ¼ Kp C or Kp ¼ Jss =C The technique, easily standardized, allows the determination of Kp through skin or other membranes as long as the barrier properties are not affected by either permeant or carrier solvents. This implies that the permeant may not react with the barrier material (i.e., change barrier permeability with time). Because several heavy metals react with epidermal protein to some degree, however this somewhat prejudices the general validity of Kp values (see section on variables). Expressing depth of diffusion per unit of time, the Kp provides a basis for comparison of the relative absorption rates of diverse chemicals. When the permeant is applied from an aqueous solution, as is the case for most metal

26


compounds, it also represents the method by which most compound-specific penetration data currently available have been generated, for drugs as well as hazardous pollutants (21,22). The Kp, here expressed in cm/hr, is the most convenient parameter for comparison of percutaneous penetration for purposes of dermatopharmacokinetics and dermatotoxicology. Values for metal derivatives range from 7 107 cm/hr for nickel dichloride (23) to 2.1 103 cm/hr for sodium dichromate (24) through human skin. Discrepancies in Kp values reported by different investigators often depend on whether mass balance was part of the study (i.e., whether the results included material remaining in the barrier material at the end of the study). For both dermatotoxicological and dermatopharmacological purposes, such materials should be considered part of overall absorption unless it is ascertained that the permeant does not diffuse further to reach the systemic circulation. Transition metals especially tend to react with SC protein and are retained in the outer strata, sometime to a degree significantly exceeding amounts reaching the receptor phase. Percent of Dose Absorbed Relevant for purposes of risk assessment is the total amount of chemical systemically absorbed. By that token it appears more important to determine the amount which has disappeared from the site of application (surface of the skin) and which eventually will flux through the skin and into the organism and not the amount which is collected in the receptor phase in vitro over the (necessarily) limited amount of time of the experiment. It thus appears important that, whenever possible, absorption data be determined in vivo, in men or primates, for as accurate a risk assessment as possible, avoiding the uncertainties of cadaver skin or adjustments for species variation. One principal in vivo methodology, using human volunteers or monkeys, is the one developed by Feldmann and Maibach (25–27) and is the most applied in vivo method for the determination of skin absorption, yielding much of the data so far available on skin penetration by drugs and pesticides. In human studies, following topical application, plasma levels of test compounds are low, often falling below assay detection, and so it becomes necessary to use radiolabeled chemicals. The compound labeled with carbon-14 or tritium, or a metal isotope, is applied to the skin in a minimal volume of a volatile solvent that is left to evaporate, and the total amount of radioactivity (RA) excreted is then determined. The amount retained in the body is corrected for by determining the amount of RA excreted following parenteral administration. The resulting RA value is then expressed as the percent of the applied dose absorbed. Fick’s postulates for membrane diffusion are not met there because a concentration of the penetrant cannot be defined and neither is a steady-state equilibrium reached with this method;


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thus a permeability coefficient characterizing the compound can only be calculated through prior conversion as given below, yielding an ‘‘apparent’’ Kp. When diffusion experiments are reported as the percent of the applied dose, disappearance over a time interval t is given either as [% lost/(time)], or as a first-order disappearance constant, k (min1), for that time interval. Assuming steady-state conditions, either of these parameters can then be converted to the disappearance over a time interval t and is given either as [% lost/(time)], or as a first-order disappearance constant, k (min1), for that time interval. Assuming steady-state conditions, either of these parameters can then be converted to the permeability coefficient, Kp. That is Kp ¼

J ð% lossÞ CA VA ð% lossÞ VA ¼ ¼ DC ½timeðhrÞ 100 A CA ½time 100 A

ð1Þ

where J (mol/cm2/hr) is the chemical flux, C (mol/L) is the chemical concentration gradient across the skin (which is reasonably assumed to be equal to the applied concentration CA), VA (mL) is the volume of chemical solution applied, and A (cm2) is the area of application. It follows, therefore, that, for a five-hour application of 1 mL of solution to a 3.14 cm2 area of skin Kp;5 ¼

Kp ¼

ð% lossÞ5 1570

J ðfraction lostÞ CA VA ¼ DC ½timeðhrÞ A CA

ð2Þ

where, assuming first-order disappearance kinetics (characterized by rate constant, k, min1), ðfraction lostÞ ¼ 1 expf60 k ½timeðhrÞg Thus, for a one-hour application of 1 mL of solution to 3.14 cm2 of skin, Kp is calculated as Kp ¼ ð1 expð60 kÞÞ=3:14 Together with the toxicity of a chemical the percent of a substance absorbed is the second most critical factor for risk assessment; it gives a measure for human exposure and an indication of the amount potentially absorbed in (the worst) case of total body immersion. For a chemical applied at 100 mg/cm2, at 1% absorption, for instance, the extent of systemic exposure to the compound applied on the entire body area of an average human adult (18,000 cm2) would be in the milligram range in the course of 24 hours.

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PERMEANT CATEGORIES AND PATHS OF DIFFUSION Several routes are available for the diffusion of compounds into and through the matrix of the skin. In order to facilitate a closer discussion of the alternatives, it is useful to associate them with the different categories of permeants as they transit alternate domains of the skin. Organic Compounds (Nonelectrolytes) The lipid matrix, which cements the corneocytes of the SC, is believed to be the principal route of transport for nonelectrolytes and lipophilic (organic) compounds into the deeper layers of the integument. Most extensively investigated so far is the process of intercellular diffusion by compounds from the categories of drugs, pesticides, organometals, and cosmetic materials, including fragrances. On first approximation, rate of penetration appears commensurate with compound polarity, measured as the octanol/water partition coefficient Poct, or its logarithm, log P (vide infra). Molecular transport of compounds has been described by mathematical models derived through multiple regression analysis of percutaneous absorption data and physicochemical constants of a large number of structures. Several mathematical models descriptive of molecular transport through human skin, in particular, have been developed more recently (21,28–32). Scheuplein et al. were among the first to describe an anatomically accurate and physicochemically reasonable model of the skin, and went on to demonstrate the correlation between lipophilicity (polarity) and experimental permeability coefficient values Kp of permeants in vitro (12,18,33,34). Polarity is most often expressed as the compound’s partition coefficient in equilibrium between n-octanol and water, Poct. Thanks to these modeling efforts, it is now possible to predict diffusion of nonelectrolytes through the skin as a model membrane with some accuracy, simply based on a few physicochemical parameters that characterize the permeant. Such models are useful in predicting the activity of chemicals in the absence of experimental data on a particular compound, without the need for its actual synthesis. Set in relation to the corresponding in vivo data, the predictive power of these models, known as quantitative structure–activity relationships (or QSAR), demonstrates that (i) three descriptors (size, polarity, and hydrogen bonding) are the principal determinants for cutaneous transport, and (ii) the properties of the intercellular SC lipids alone are sufficient to characterize the barrier properties of the skin as the route of permeation for that category of chemical structures (32). Rougier et al. developed a brief experimental method to predict skin absorption of lipophilic compounds. By that method, the systemic uptake of permeants can be derived from the amount that diffuses into the SC in vivo after a limited time of exposure (35). The chemical is dosed on the skin of animals or human volunteers, and after 30-minute-contact the surface of


29

the SC wiped clean of residual compound, and the SC removed by successive application of adhesive tape and stripping. The strippings are analyzed for chemical adhering to tissue removed, and from that value the amount may be estimated that eventually will be systemically absorbed over a longer period of time. Certain metal compounds (organometallics) also represent a lipophilic category (36) that can penetrate the SC with relative ease. Such derivatives of the more toxic heavy metals, therefore, represent a major toxicological risk, particularly in the work place, due to their ready skin penetration. Representative Kps are 2.9 103 cm/hr for methyl 203mercury dicyanamide50 and 2.3 103 cm/hr for lead naphthenate51. Electrolytes In our present understanding of structure and function of the skin, its permeation is determined by the physicochemical parameters of permeants. Intuitively, and also based on the therapeutic action of dermatologicals and cosmeceuticals, or the toxic effect of skin exposure to pesticides, we anticipate easy penetration by lipophilic compounds there, as is achieved in the process of inunction. But for polar structures, such as water or electrolytes (e.g., salts in aqueous solution), skin penetration to any significant degree is often dismissed because the skin appears designed as a barrier against external disturbance of a natural state of hydration. Still, for water and a number of hydrophilic-charged molecules, including a number of metal complexes, diffusion is also demonstrated, albeit the process proceeds at an average of two to three orders of magnitude more slowly than is the penetration of small-molecular-weight, lipophilic nonelectrolytes, where Kp values are on the order of 1 cm/hr (e.g., for the solvent toluene). Skin appendages or shunts (the hair follicles and sweat ducts which transit through all those layers) are considered the predominant pathway for the diffusion of large polar molecules or electrolytes across the skin as an early stage event. They constitute relatively large openings through which diffusion can occur if two principal conditions are met: 1. Sweat is present to serve as an aqueous diffusion medium 2. the outflow of sweat is significant (18,37) Measuring electric resistance to analyze for the diffusion pathways taken by charged molecules, Mali et al. calculated that 70% of the strong electrolytes permeate through sweat ducts (38). High current flow in iontophoresis confirmed that observation (12,30,39–43), as did use of autoradiography or microparticle-induced X-ray emission (PIXE) analysis (44,45). Considerable experimental data have indicated that the depth of sweat duct penetration is significantly dependent on ionic mobility (i.e., size) and electronic charge of the metal (46,47).

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Direct or indirect observations speak to the ease of such electrolyte diffusion through skin shunts. Penetration through sweat ducts can occur within one to five minutes following exposure, with no comparable transport occurring via the transcellular path in that time span (48–50). Poral transit by nickel salts, for instance, can also become manifest clinically; follicular inflammation or punctate erythema were observed in the context of skin patch testing with nickel salts (51). The relatively rapid flux across the appendageal pathway is followed by slower but continuous, potentially more important intercellular diffusion. It is reasonable to postulate that metal ion pairs (soaps) formed with fatty acids on the skin surface will preferably partition into the lipophilic environment. The intercellular lipid domains in the SC seem to present a ready pathway for diffusion of such compounds, because the relatively slower, transcellular rate of penetration does not explain such phenomena as provocation of allergic reactions due to casual contact with metallic objects, such as coins. Occupational health statistics on untoward health effects are a prime indication for the skin diffusivity of noxious electrolytes. With respect to specific (allergic) and nonspecific (irritant) contact dermatitis that may constitute up to 90% of workers’ compensation claims for skin diseases, percutaneous absorption of potential allergens and irritants is a key determinant of the risk of skin sensitization and irritation, and may be enhanced if protective clothing entraps or occludes the irritant against the skin, by increased hydration of the SC, and by contact with anatomical sites where skin permeability is greater. Thus, exposure to nickel, chromium, cobalt, and mercury compounds are premier causes for allergic and irritant reactions in industrialized countries (52). The route of transcellular diffusion may be of marginal importance, especially for electrophilic transition metals that tend to form permanent deposits on the skin surface. Absorption would be limited to the outermost layers of the SC, and, possibly, the epithelium of appendages. Such adsorption may also be terminal, resulting in the depot formation repeatedly described in the literature for a number of electrophilic metals (53,54). Such non-Fickian behavior clearly puts that class of compounds beyond the scope of theoretical algorithms predictive of percutaneous penetration, and the fate of chemicals thus retained in the SC remains uncertain, prompting the question: Will the compounds continue to diffuse slowly to be ultimately absorbed systemically, or will they be shed with the corneocytes in the process of desquamation? Studies with lipophilic compounds in vivo and in vitro show that within weeks most of the xenobiotics initially retained in the SC have been absorbed systemically or have reached the receptor fluid, respectively. Rougier’s predictive method is applicable only to compounds of Fickian behavior, but not to electrophilic metals that react with barrier tissue, rendering in-depth diffusion unpredictable (55). In addition, certain


31

metals, such as copper, zinc, and chromium, are essential nutrients subject to homeostatic controls, which appear to determine deposition or mobilization in response to fluctuating body burdens. Highly protein-reactive, electrophilic metals, such as mercury, to a large extent were seen to remain at the site of initial absorption. Hursh et al. measured the uptake of mercury vapor by the skin on human arms in vivo (56). The rate determined corresponded to a high permeability coefficient (Kp of 1–2 cm/hr). As much as half of the absorbed mercury, however, appeared to have reacted with SC protein and was eliminated through the process of desquamation in the following weeks. In that experiment, the forearm was exposed to mercury vapor enclosed within a saran bag. On average, 6.8% (range: 3.0–10.6%) of the Hg vapor originally in the exposure chamber was absorbed by the arm. Up to half the mercury initially in the forearm was shed by desquamation of epidermal cells during several weeks. The remainder diffused into the general circulation and could be measured as systemic mercury. For two subjects on the day following exposure, the SC was collected from a 35 cm2 area of the forearm by stripping off the superficial layers with adhesive cellophane tape. The 203Hg measured on the tape, normalized for the total exposed area, corresponded to only 0.3% and 1.3% of the Hg on the arm at that time. When the process was repeated for one subject 14 days later, and again 23 days after exposure, the additional recoveries normalized to the entire exposed area were 24% and 10.7%, respectively, reflecting gradual outward migration of keratinocyte and corneocyte carriers of the irreversibly bound mercury. The authors concluded that, due to protein reactivity, absorption of mercury vapor by the skin poses a minor occupational hazard when compared with inhalation. The three modes of diffusion available to xenobiotics also illustrate the characteristics of the skin, which can function to varying degrees as a barrier, a reservoir, and/or a filter, depending on the polarity and chemical reactivity of the solute. Topically applied compounds can penetrate the SC along more than one pathway simultaneously, albeit at different rates. A molecule can follow any of these routes—follicular, transcorneal, or intercellular—but it is difficult to assess the relative importance of each, as this will vary with the physicochemical nature of the molecule. COMPOUNDS FORMED BY METALS IN CONTACT WITH THE SKIN Direct and prolonged contact of metals and alloys with the skin may result in electrochemical reactions that release metal ions. Indicative of the process of skin penetration by certain metals is the onset of skin reactions, mostly immunological: contact with coinage, tools, jewelry, or articles of daily use. The majority of allergic reactions to metals involve nickel (57–64) and chromium (65–73).


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Leaching or release of metals from alloys in contact with body fluids, such as sweat or sebum, is difficult to predict with accuracy. Aside from immediate environmental factors, the microenvironment within the particular alloy is a principal determinant, due to the action of electromotive forces generated by the presence of other metals (37). Review of published data on corrosion shows that release rates, as determined through leaching tests, do not correlate with metal content in an alloy (74). Associated metals in immediate proximity form a galvanic element (or pile), whereby an electron current flows from the more electronegative (e.g., nickel) to the nobler, more electropositive one (e.g., copper), resulting in oxidation or solution (‘‘corrosion’’) of the more electronegative metal. Corrosion is an electrochemical process involving movement of electrons (e) through the metal from anodic to cathodic areas and related movement of ions in the electrolyte (sweat). At metal potentials and pH values that occur in sweat, anodic reactions for some metal constituents in alloys include Ni ! Ni2þ þ 2e ; Cu ! Cu2þ þ 2e ; Fe ! Fe2þ þ 2e ; Cr ! Cr3þ þ 3e The electrons generated are then consumed at the cathode, most commonly by reaction with oxygen: O þ H2 O þ 2e ! 2ðOHÞ Whether and how much nickel is mobilized will depend on the immediate surrounding material (e.g., copper) present in the alloy. The products of corrosion may then enter the organism, taken up through the skin, through the oral mucosa, or by the gastrointestinal (GI) tract (75). Transported by blood throughout the organism, copper, in particular, will be sequestered by metallothionein (MT) or coeruloplasmin, and deposited in various organs and tissues (76). In order to discuss the phenomenon of skin penetration by metal compounds as ionized salts, complexes, or organometallic compounds, and to correctly interpret the meaning of experimental results and clinical observations attributed to the action of metals, it is important to visualize the structure and function of the skin as a membrane, which can act as barrier, filter, reservoir, or an outright port of entry, and the microenvironment prevailing on its surface. Most direct evidence for formation of skin-diffusible oxidation products on skin contact is the facile elicitation of allergic reactions by elements, such as nickel (77), and detection of that metal deep in the SC upon skin contact (78). The chemical environment encountered on the skin surface explains the process of solubilization and, subsequently, diffusion upon contact of metals in their elemental state. By the action of salts and acids present in sweat and sebum, metals can be converted to a hydrophilic or lipophilic derivative, respectively. Only then do they become diffusible via the transcellular, intercellular, and transappendageal route. Skin exudates are secretions


33

of varying composition, covering the epidermis with a film that complements the skin’s barrier function. That film consists of two components of endogenous origin, sweat and sebum, secreted by the respective glands, which have the ability to corrode (oxidize and dissolve) metal surfaces on contact. Their composition varies in function of physical, pharmacological and environmental conditions, gender, age, sweat rate, body site, and method of collection. An abundance of eccrine sweat glands occurs widely distributed over all exposed skin areas, to dissipate body heat through evaporation. Besides these occur sebaceous sweat glands, closely associated with hair follicles, which produce an oily secretion, the sebum (79). The pathways that oxidation products follow in the process of diffusion through the skin will depend on the polarity of the salts formed with exudates, and can be predicted in light of earlier investigations of skin penetration by xenobiotics. Metal salts in dissociated ionic form do not penetrate the skin as readily as their unionized (e.g., organometallic) form. Being unionized, they are more lipid soluble and that seems to be the decisive factor for intercellular, relatively facile diffusion. Organic salts may penetrate in the form of ion pairs, and also exhibit relatively high fluxes. For a molecule that is dissociable in water, the dissociation constant and pH of the immediate environment will determine degree of ionization and, thus, permeability. Some organometallic compounds, such as derivatives of lead (80), mercury (81), or nickel (unpublished data), have actually been characterized as relatively good skin penetrants. Copper metal reacting with fatty acids may thus also form lipophilic compounds of marked diffusivity. Sweat Values for the main components of sweat have repeatedly been investigated over time, yielding increasingly accurate data reflecting improvements in analytical techniques. The main categories of solutes are discussed here. Listed in Table 1 are the prevalent ranges of eccrine sweat; they are approximations, as values are unavoidably subject to variation even in normal Table 1 Mean Levels of Eccrine Sweat Components Sodium Potassium Chloride Urea Lactic acid Amino acids Ammonia Source: From Ref. 82.

Men Women Men Women

51.9 mEq/L 36.5 mEq/L 7.5 mEq/L 10.0 mEq/L 29.7 mEq/L 260–1220 mg/L 360–3600 mg/L 270–2590 mg/L 60–110 mg/L


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subjects, due to the type of stress applied to sweat stimulation, ambient and body temperature, environmental humidity, diet and nutritional status, age, gender, sweat rate, skin area of collection, local skin temperature, muscular activity, etc. The validity of these values in adults is often questioned, however, due to differences in methods of analysis and sweat stimulation (physiological, physical, or pharmacological). The main cause for corrosion of metal surfaces from skin contact in individuals referred to as ‘‘rusters’’ is not due to elevated electrolyte concentration, as generally assumed, but rather seems to coincide with palmar hyperhydrosis in those individuals. ‘‘Rusters’’ exude palmar sweat at inordinately high rate and a low pH, with the pronounced corrosive action on metal surfaces observed. When the sodium concentration measured in normal subjects was compared to that of ‘‘rusters,’’ no significant difference could be observed (mean values of 49.6 mEq/L vs. 49.1 mEq/L, respectively) (83). However, the pH of sweat was measured to range between 2.1 and 6.9 (57). Both proteins and amino acids are normal components of mammalian sweat (84). Quantitative analysis of amino acids has become routinely possible, thanks to ion-exchange chromatography; however, the data documented for men by authors differ significantly, probably due to differences in the stimulation methods applied, to regional differences in anatomical site, or to the methods used for sampling. Substantial variations were observed in relative concentrations of amino acids in sweat collected from various body parts. Amino acids excreted in sweat are independent of dietary intake (85), although their concentrations increase markedly in blood and urine on oral protein intake. No differences in amino acid patterns were seen between young and middle-aged adults, or between men and women (86). Sebum Acid components making up the sebum also play an important role in solubilizing (‘‘corroding’’) metal surfaces. Human skin features an acid mantle of pH 4 to 6 at the surface of the SC, which increases with depth to pH 7 at its juncture with live tissue (87). Determinants of this pH are protons that originate in the epidermis or as products of sebaceous gland activity, gradually reaching the surface of the skin. They stem from three classes of compounds: Amino acids (e.g., urocanic acid and pyrrolidone carboxylic acid) Alpha-hydroxy acids (e.g., lactic and butyric acid), also present in sweat Acidic lipids (e.g., cholesteryl sulfate and free fatty acids, primarily oleic and linoleic) (17,58,88) At its point of origin in the live epidermis, sebum as secreted by the sebaceous glands is a complex mixture of lipids consisting of glycerides, but no free fatty acids.


35

The occurrence of free acids in the SC and on the skin surface is the result of hydrolysis of phospholipids and glycerides by lipolytic enzymes occurring in the sebaceous ducts and on the skin surface, and of bacterial decomposition. On the skin surface, lipids of epidermal origin contain up to 20% free fatty acids, with 16% originating in the pilosebaceous glands (58,88). They consist, to the greater part, of C16 and C18 acids, but their full range reaches from C5 to C22, with an average length of C16. Such an acid environment plays a regulating role for SC homeostasis with relevance to the integrity of the barrier function and regeneration of the SC barrier (89). It is now accepted that the acid environment on the skin surface supports a number of SC functions (90), some of which are

control of moisture loss from the epidermis and permeability barrier homeostasis, providing resistance to fungal and bacterial infection, SC integrity and cohesion, and enzymatic processes regulating corneocyte desquamation.

VARIABLES DETERMINING SKIN DIFFUSION OF METAL COMPOUNDS Skin penetration by metals is a multifactorial process, which is not fully understood, and even less predictable. Contributing to the uncertainty are the effects of chemical speciation of metallic elements, particularly that of the transition metals. Also, the skin as target organ presents imponderable and wide variability. Particularly with ETEs, numerous factors come to bear (e.g., in vivo, permeability can be subject to homeostasis regulating the overall organism; in vitro, the sections of skin used for diffusion experiments can present artifacts). Mathematically derived (mechanistic) models predictive of percutaneous penetration of organic molecules have been developed based on in vitro and in vivo data through regression analysis of experimental results, making the estimation of diffusivity of untested structures possible. However, similar reduction to a few molecular physicochemical parameters is not feasible when modeling electrolytes, because movement through biological membranes is highly element specific. A number of factors are closely interrelated, and their combined effects are not predictable. A detailed, in-depth review of parameters determining skin diffusivity of a large number of metals is given by Potts and Guy (32). Exogenous Factors Dose Rate of diffusion of certain transition metals is not commensurate with applied concentration. With increasing doses, absorption rates of certain transition metals can reach a plateau value, then decrease with a further


36

increase in applied concentration. For others, absorption steadily decreases with increasing dose. This is likely due to the buildup of a secondary diffusion barrier as a consequence of electrophilic metals forming stable bonds with proteins of the skin. Thereby, a depot accumulates in the SC, retarding further penetration in inverse proportion to metal concentration. In vitro, this leads to lag times that can be as long as days before any permeant is collected in the receptor compartment, an observation made mainly with salts of nickel, chromium, and mercury (54,91,92). An example of non-Fickian behavior is the diffusivity of nickel salts. Fluxes of NiCl2 and NiSO4 through full-thickness human skin were compared by Fullerton et al. (93). After lag times of about 50 hours, in experiments lasting 144 to 239 hours, occluded NiCl2 entered the receptor fluid about 5 to 40 times more rapidly than (i) NiSO4, (ii) NiCl2 with added Na2SO4, or (iii) NiSO4 with added NaCl. Without occlusion, the permeation of nickel was reduced by more than 90%, an indication of permeability increasing with hydration. Application of chromate with increasing concentrations does not consistently lead to higher penetration rates. With chromate doses at concentrations of between 0.00048 and 0.73 M in guinea pig skin in vivo, absorption of chromate increased to a maximum Kp of 2.64 103 at 0.26 M dose, reached a plateau, and then gradually decreased below detectable levels (94). Determined with human abdominal epidermis in vitro, the diffusion coefficient for chromate ion consistently decreased with increasing donor concentration (95). With human full-thickness abdominal skin in vitro, permeability coefficients for dichromate were also inversely proportional to the concentrations applied (96). In guinea pigs, absorption of mercuric chloride applied in vivo reached a maximum at 16 mg Hg/mL, then decreased to nondetectable levels with increasing concentrations (59). Vehicle The solvent can have a profound effect on permeant solubility and on the skin membrane, and thus on its barrier properties. Petrolatum, for instance, is a poor solvent for metal salts, where the permeant remains suspended in fine particles, affording less-than-ideal uniformity in skin contact; on the other hand, it has an occlusive effect, which increases skin hydration and thus promotes diffusion of a hydrophilic compound. Quantitative absorption of zinc in human skin in vitro showed a distinct dependence on vehicle. The permeability coefficients comparing fluxes from petrolatum and those from a hydrogel containing zinc chloride were based upon 72-hour periods. The permeability coefficients for 2.4% zinc formulated in petrolatum was 0.082 104 cm/hr. From the hydrogel containing the chloride salt, the permeability coefficient was 0.29 104 cm/hr (i.e., more than


37

three times higher) (60,61). Zinc oxide was applied on intact human skin in gum rosin versus a hydrocolloid carrier; after 48 hours penetration of zinc in human epidermis was more than twice the level as compared to the hydrocolloid vehicle (62). Dimethyl sulfoxide is a solvent that enhances skin penetration, as it causes swelling of basal SC cells and disrupts the normal keratin pattern. Dimethyl formamide and dimethyl acetamide applied in that solvent were seen to diffuse at a significantly accelerated rate (63). Of practical importance also is the choice of vehicle in standard diagnostic skin patch testing for sensitization, with the aim of optimum release of allergen into the viable epidermis while avoiding allergic or irritant contact dermatitis, leading to false-positive reactions caused by the vehicle itself. A method for determining the optimal solvent for diagnostic skin patch testing of allergens, with the intent of minimizing false-negative reactions, is their application in different solvents on hypersensitive patients. By recording the ratio of positive elicitation, the solvent can be identified that better promotes skin diffusion of the xenobiotic. The reaction threshold to nickel sulfate was tested in 53 sensitized patients in both water and petrolatum at equal concentrations (390 ppm) (64). The mean reaction threshold for nickel sulfate in water was significantly lower (0.43%) than in petrolatum (0.51%). Also the irritation potential of a chemical can be assessed by applying it in different solvents in dermatological diagnostics, aiming to minimize the chemical’s potential for irritation and, thereby, false-positive reactions. Thus, the irritant reactivity of nickel salts in petrolatum was greater than in water (97). Such differences in skin reaction in functions of solvent serve to demonstrate the effect of the solvent carrier on dermal structures and, thus, the diffusivity of permeants. Counterion and Molecular Volume Diffusion of a cation is necessarily tied to the diffusion of its counterion (i.e., diffusion of a metal will also proceed as an ion pair); otherwise, an electrical potential builds up, which will inhibit further diffusion. Thus, the overall volume is composed of two factors: the ionic radius of the elemental species [for example, chromium(III) as cations or chromium(IV) as chromate, the oxo-complex] and the variable size of the counterion (e.g., chloride versus sulfate versus nitrate, etc). Barrier diffusion of charged ions as ion pairs has been characterized as one criterion in skin permeation (98). Their obvious effect on permeant size, polarizability, and polarity ultimately find expression in respective diffusion constants (99,100). As the water content of the SC progressively increases in the deeper layers, degree of ion (complex) hydration increases further, to become fully hydrated toward the deeper regions of the membrane. The resulting increased steric hindrance also leads to a further decrease in ion mobility.


38

Experimental data also suggest that diffusivity of metal compounds through skin appears to correlate with their size and, for a given metal species, the counterion, thus, represents a determining variable. Own experimental data from depth analysis of nickel levels in the SC following in vivo application of a number of its salts confirm such effects of counterion size on their diffusion (101), an effect further amplified by hydration spheres. Polarity The polarity of nickel salts as measured by their solubility in the nonpolar solvent n-octanol at 22 C increases in highly significant intervals (p < 0.0005) in the following sequence: nitrate < chloride < acetate < sulfate (101). This increase in polarity determines the same sequential hierarchy for skin diffusivity and irritancy (93,97,102). Nature of Chemical Bond Going from inorganic, mostly water-soluble ionic mineral salts to lipophilic, organometallic compounds, bonds increasingly assume covalent character, and their penetration characteristics approach those of nonelectrolytes. With lead as an example, the role of ionic bond versus covalent bond is an obvious codeterminant for diffusion rates, the Kp ranging over four orders of magnitude, from 107 for the acetate to 103 for the lipophilic naphthenate (103). The range of penetration constants determined for a set of lead compounds illustrates the point of polarity as a determinant for skin penetration. That ranking of skin absorption in function of polarity was obtained for lead compounds by Bress and Bidanset, in vitro on human skin at equal concentrations, expressed as quantity absorbed (Table 2) (103). The lipophilic category, mainly alkyl and aryl derivatives of the more toxic metals, thus may represent a major health risk in chemical manufacture and industrial use, due to their ease of skin penetration. As an example, minute quantities of the highly toxic organometal dimethylmercury of previously unknown skin diffusivity were apparently absorbed Table 2 Skin Absorption of Lead Compounds Through Human Skin In Vitro in Function of Polarity Compound (as 10 mg Pb) Tetrabutyl lead Lead nuolate Lead naphthenate Lead acetate Lead oxide Source: From Ref. 103.

Amount absorbed (mg)

Percentage of dose absorbed

632 56 130 15 30 3 5.0 0.9 forehead > postauricular > abdomen > forearm > leg > back (115–117). Apart from regional SC thickness and shunt density, this appears to be mainly due to intercellular lipid weight percentage and composition (118). Because penetration of electrolytes appears to occur mainly through the skin’s appendages (12,18,30,42), diffusion in hairy areas may be enhanced, although absorption was also observed through the palm of the hands devoid of hair follicles; the route of diffusion there was probably via the sweat ducts (119). When investigating site dependence of percutaneous absorption for a number of organic compounds in vivo, Lotte et al. noted a linear relationship between penetration and TEWL (i.e., the relationship between the permeability of the skin to the outward movement of water and inward movement of permeants) (117). Following application of nickel chloride on the arm and back of human volunteers, based on tape strip analysis of the SC, the depth/concentrations of the two sites were steep though at differing gradients, declining toward deeper SC layers, with significantly different areas under the curve: 30.8 for skin on the back versus 62.4 on the arm (p < 0.0005), an indication of heightened diffusivity in arm skin (101). Age of Skin The incomplete barrier function observed in infants and young children gradually increases to values seen in the skin of mature individuals. Neonatal or infant skin has been found to be more permeable to lipophilic compounds than is adult skin. Feldmann and Maibach studied percutaneous penetration of taurocholic acid in vitro through male thigh skin as related to age; after 24 hours, while skin from a 32-year-old absorbed 8.2%, the skin of a 53-year-old absorbed only 0.3% of the dose (119). One theory associates such decreased permeability with age as function of diminishing blood supply (120). Our own in vitro investigation of permeability to a nickel salt (chloride) and a nickel soap (di-octanoate), using dermatomed skin under identical experimental conditions, confirms the decreasing trend in skin diffusivity with age, specifically between skin from a young (age 16 years) versus an older source (age 64 years) (unpublished data). While the ratios of diffusivity values for salt and soap remained the same, advanced age brought a reduction in rates in excess of two orders of magnitude (121). Homeostatic Controls Essential elements such as sodium, potassium, calcium, copper, or zinc present in the SC, epidermis, and dermis are kept in equilibrium by homeostatic control mechanisms. These mechanisms play a part in the overall physiological dynamics of hydration, and also appear to keep the body burden of


43

certain xenobiotics under control. Homeostasis ascertains maintenance of equilibria necessary for optimal functioning of the organism (e.g., by preventing undue loss due to perspiration or desquamation through a process of reabsorption, as has been described for sodium and calcium, in particular) (122,123). An important part of such controls are metal-binding MTs, single-chain polypeptides, present in several organs, including the skin. They have an unusual amino acid composition consisting of one-third cysteine (20 cysteines in mammalian MTs) and no histidine, aromatic, or heterocyclic components. This abundance of thiol groups imparts both reversible metal-binding capacity and the ability to scavenge free radicals. At least three functions have been attributed to MTs: metal sequestration, temporary (reversible) binding as well as long-term storage, and both intra- and extra-cellular metal transport. They bind the metabolically essential zinc and copper, as well as cytotoxic metals such as mercury, lead, or nickel. Levels of free ionic copper, a relatively toxic metal, are moderated to the minimum levels sufficient for physiologic needs; 1019 mol/L are estimated in blood plasma via binding to MT as well as the copper carrier protein ceruloplasmin (124). An excess of free ionic copper in cells would lead to oxidative damage, and the affinity of MTs for copper is second only to that of mercury. The dynamic equilibrium between ceruloplasmin and MT contribute to the prevention of both toxic accumulation and deficiency of copper in mammals (125). When present above specific threshold levels, even ETEs will exhibit acute toxicity; and by immobilizing excess amounts, MTs fulfill a critical detoxifying role. MTs thus remove, store, or release metals on environmental exposure or physiological demand, and are an important factor in homeostasis, regulating their uptake (diffusion) and release. Measurements of the rate of skin penetration by zinc have been contradictory, possibly due to such endogenous controls. Apparently zinc absorption does not occur by simple diffusion, but seems to be regulated by MTs (126). MTs thus provide a buffering capacity maintaining intracellular steady-state kinetics for both copper and zinc, and ensuring a supply of these metals for other metabolic functions. As homeostasis is responsible for a rapid exchange between skinapplied zinc and the large pool of endogenous metal, dermal absorption experiments conducted in vivo thus should account for natural processes that may counteract passive diffusion, adding a degree of uncertainty to permeability constants measured. Skin Sections Data are limited on the effect of individual skin layers (SC, epidermis, and dermis) on percutaneous penetration by metals, and on metal uptake in the individual strata. An example of the variability observed is the different


44

penetration rates measured in vitro for nickel chloride. Comparison of Kps determined through different skin strata by different authors shows that penetration by nickel chloride is slowest through the SC (37,53,91,93). From a Kp of 107 cm/hr in the SC, the values progressively increase toward fullthickness skin, with a maximum of 9.8 103 cm/hr in dermatomed skin. Skin sections prepared for in vitro experiments, other than dermatomed skin, are likely to introduce artifacts to varying degrees. One possible explanation for low diffusion values through the SC is that shunts in isolated SC (and to some degree in epidermis) swell shut upon hydration (127). In vitro diffusion data, which come closest to in vivo conditions, are likely to be those measured through split-thickness skin. That tissue can be standardized using the dermatome to a desired thickness (200–400 mm). Unlike other methods of skin sectioning, use of the dermatome is the best way of preparing skin for percutaneous absorption studies. A dermatome can be used with hairless or haired skin, without adversely affecting the viability of the membrane. Dermatomed tissue includes the layer of dermis where the permeant is taken up in the capillary system, without adding the variable thickness of dermal and subdermal tissue and fat. Skin Metabolism (Red/Ox) There is evidence of cutaneous metabolic effects on metals of exogenous or endogenous sources; both oxidation and reduction can occur, manifest in changes of oxidation state in situ during the process of permeation. The result can be altered immunogenicity of the metals; examples are known where oxidation can lead to enhanced immunogenicity of certain metals [e.g., of Ni(II) to Ni(III) or Ni(IV)] (128), or reduction to lesser immunogenicity [e.g., Cr(VI) to Cr(III)] (129). Animal experiments conducted by Artik et al. showed that biooxidation of Ni(II) to Ni(III) or Ni(IV) occurs through endogenous-reactive oxygen in the form of hydrogen peroxide or hypochlorite present in inflamed skin. In animal and cell line tests Artik did find that Ni(II) sensitizes only na€ve T cells following bio-oxidation to its higher valence, but not the form of Ni(II) itself (128). Chromium applied on the skin as chromate or dichromate [Cr(VI)] is reduced to chromic ion (Cr3þ) by tissue proteins containing sulfhydryl groups (130). In in vitro diffusion experiments with Cr(VI) salts, only chromic ion is found initially in the receptor phase. On sustained application, however, Cr(VI) as chromate or dichromate is seen to pass through the skin unchanged, an indication that a given tissue mass has only a limited capacity to reduce the chromate ion (105). For some metals, reduction can lead to discoloration of the skin due to accumulation of the element in the metallic state (e.g., systemic silver preferentially accumulates in the skin, where it is reduced to the metallic state). Such impregnation of dermal tissues with silver deposits results in permanent graying of the skin, a condition termed


45

argyria (131). Chronic dermal application of mercurials used as skin bleaches (mercurous chloride, ammoniated mercury, and mercurous oxide) can lead to tissue accumulation of metallic mercury, characterized by slategray pigmentation or hydrargyrosis cutis (132). The skin is a target organ for arsenic and is critically sensitive to arsenic toxicity, regardless of the route of exposure. This is due to the attraction of arsenic to the skin’s sulfhydryl-group containing proteins. Such accumulation of arsenic in the skin is characterized by hyperpigmentation, keratoses of the palms of the hands and soles of the feet, and diffuse macular pigmentation with the characteristic appearance of ‘‘raindrops’’ or diffuse darkening of the skin on the limbs and trunk, attributed to the reduction and deposition of the element in the metallic state (127). METHODS FOR MEASURING PERCUTANEOUS ABSORPTION In this context, the terms absorption, diffusion, and penetration are used interchangeably as they apply to the process of penetrating the outermost skin layer, the SC, and to all the associated and subsequent events, including distribution to the different strata and appendages of the skin. In Vitro Methods Preferably skin diffusivity of metals is measured in vitro using excised (animal or human) skin, especially in the investigation of materials of unknown or obvious toxicity. Reasons for such preference are: 1. Formation of depots in the SC by electrophilic metal ions, establishing a secondary barrier to further diffusion, which results in substantial lag times or failure to penetrate the epidermis altogether (93,105) 2. The need to use radiolabeled materials for the detection of exceedingly low levels of permeant 3. The pronounced toxicity of some (transition) metals, too hazardous to apply on the human organism in vivo Widely used methods for the exploration of percutaneous absorption, in vitro designs allow a preliminary and early evaluation of safety for chemicals in the developmental stage, as well as of those too toxic to test in living models. These methods offer the advantage of yielding data that reflect the process occurring in selected domains of the skin (SC, epidermis, and dermis) without involvement of other factors, such as secondary absorption, deposition, or metabolism in the body’s tissues. The most commonly used in vitro technique for measuring percutaneous absorption involves placing a piece of excised skin in a two-chamber diffusion cell. It consists of a top chamber to receive an adequate volume of

46


penetrant in solution, an O-ring to secure the skin in place, a temperaturecontrolled bottom chamber with continually circulating solution removing the penetrating amounts on the receptor side of the membrane, and a sampling port to withdraw fractions at specific time intervals for analysis (133). The solute diffuses from the fixed higher concentration medium in the donor chamber into the less concentrated solution in the receptor chamber. The solutions in both chambers are stirred continuously to maintain uniform concentrations. The advantage of diffusion experiments thus conducted lies in the applicability of Fick’s first law of diffusion. In the two-chambered system the two chambers are separated by the skin membrane. To satisfy the requirements of the Fickian diffusion, useful for the study of diffusion through biological membranes, an (ideally) infinite dose is placed in the donor chamber, and appearance of the permeant continually monitored in the receptor chamber. The solutions in both chambers are stirred to maintain uniform concentration. Experimental details are given with an example of steady-state flux measured through the SC (23). In the one-chambered system, the skin is placed on the chamber and is open to the environment above, simulating conditions prevailing in real-life application of drugs and cosmetic products to the skin. As permeation proceeds, steady-state (Fickian) conditions are not attained (20). The chamber beneath the skin serves as a container for the receptor fluid that is continually stirred. Sampling occurs through a side arm for analysis to determine rates of absorption (134,135). Automatic sample collection is also possible from a one-chambered cell (136). The receptor volume is small (0.13–0.26 mL) and allows complete and rapid flushing of the receptor chamber. Special provisions are necessary to avoid evaporation of volatile compounds from the surface of the skin. They can be collected through appropriate cell design (137,138). Limitations and Problems in Measuring Percutaneous Absorption of Metal Compounds In Vitro A limitation in the validity of percutaneous absorption values determined for electrophilic metals in vitro is the formation of depots in the skin. Such protein reactivity can result in depot formation in the SC before permeant diffusion continues into the deeper layers of the skin, and in vitro it can cause considerable delays (lag times) before the compound emerges in the receptor phase. For some metals, lag times of several hours or days have been recorded before any permeant appears in the receptor compartment, or as in some cases such as chromium, fails to emerge at all. This phenomenon has been reported for the more extensively investigated metals, such as nickel, cadmium, and chromium (93,105,139). Due to the electrophilicity of transition metals, in particular, it can be safely assumed that latency or retardation in the diffusion process is a predictable phenomenon. As permeability constants and bioavailability are calculated only from amounts of permeant collected


47

in the receptor phase at steady-state rates, the formation of depots is rarely part of in vitro diffusion studies, although it should be considered as part of the total-dose absorbed. The permeant retained in the different levels of the skin tissues may then eventually become available systemically by slow diffusion. For those metals where skin penetration was investigated, the data rarely are adequate for the calculation of flux or permeability coefficient, and values can only be derived through a number of assumptions. Results that are amenable to quantitative analysis often followed diverse experimental protocols that lead to results that are not necessarily comparable. While a permeability coefficient is ideally determined under steadystate conditions, the percutaneous absorption of metals rarely meets this criterion. A problem inherent in the in vitro approach is the lack of total-dose accountability, as results in the literature are often based on the quantity of permeant found in the receptor phase only. The amount of chemical collected in the receptor phase, expressed as percentage of dose, is then taken to be the amount becoming available systemically, and material retained in the diffusion membrane (SC, epidermis, and dermatomed or full-thickness skin) that may subsequently diffuse further, and thus become a significant factor for risk assessment purposes, is not determined. In Vivo Methods The purpose of measuring the percutaneous absorption in vivo is to study bioavailability of chemicals applied on the skin by analysis of dose moving into the skin, further into the systemic circulation, and beyond. Such kinetics measure diffusion by skin tape stripping, bioengineering techniques such the TEWL, or by traditional analysis of blood and excreta levels. Taken together, these components can define the dermatopharmacokinetics of a chemical. After topical application, the levels of xenobiotic in tissues and body fluids as a rule are below assay detection levels, and it is necessary to use tracer methods. The most relevant in vivo data on percutaneous absorption will be obtained from studies in humans themselves. However, animal models are needed for the development of basic pharmacokinetic principles and for the investigation of pharmacological mechanisms. On the basis of currently available data, the only animals in which permeation is consistent qualitatively and qualitatively with human permeation data are the weanling pig (138), the rhesus monkey (136), and the hairless rat (137). Extra body fat in the pig may affect drug distribution compared to humans, however, and must be considered. In the rhesus monkey, skin application should be limited to the nonhairy regions on the ventral surfaces of the animal.

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Overall, correlation of animal skin penetration with human absorption data made by different researchers can vary depending on the compound nature involved (22). Skin absorption rates determined in animals as a rule result in a conservative estimate as they overestimate the dose percutaneously absorbed in humans. Penetration and absorption studies must address toxicologic aspects and toxicokinetic aspects, as well as developmental and clinical issues. Also the benefits of using radiolabeled versus nonradiolabeled analytical methodology have to be considered. Studies can focus on drug in the skin, in venous blood draining the application site, in the systemic circulation, or in the excreta. For pharmacokinetics, drug concentrations are followed over time. For topical dermatological products, toxicokinetic and safety studies are preferably performed in the same species. An indirect method is the quantification of RA in excreta, where percutaneous absorption is determined by measuring the appearance of RA following topical application of a labeled compound. Measured is the total RA of parent compound and any labeled metabolites, which may occur in transit through skin and body. Label retained in the organism or excreted by another route (e.g., exhaled air) will not be counted by that method. Therefore, Feldmann and Maibach used the following expression to correct for RA unaccounted for: Percent absorbed ¼ 100 total RA from topical administration/total RA following parenteral administration Percutaneous absorption can be measured directly by including material exhaled and that remaining in tissues at the end of the experiment, besides excreted material in urine and feces. This sum then gives a direct measure of absorption (140,141). Because absorption is expressed as the percent of dose applied, no Kp becomes available using these methods. In the disappearance method the amount of drug absorbed is assessed as the difference between the amount applied and that recovered at a given interval. The method relies on the assumption that the difference between the quantity applied and that recovered corresponds to the amount absorbed; it can follow different approaches: 1. Following drug application for a fixed time, the residual formulation is washed from the skin surface, and the amount removed is analyzed. 2. The formulation is applied, and the drug content in the outer skin layers is followed in function of time by spectroscopic or radioisotopic monitoring techniques. 3. Disappearance (loss) of a radioactive compound from the surface of the skin and the appearance of RA in the excreta following the topical application of a labeled compound are followed over time.


49

The total RA in the excreta is a mixture of the parent compound and any labeled metabolites that may result from metabolism of the parent in the skin and in the body. The method can involve single-point measurement, or continuous or periodic monitoring of compound uptake. One limitation in this approach is the fact that once the applied compound has penetrated, its disposition in the organism eludes detection, especially because the application of radioactive material on men is limited for ethical reasons. Another problem inherent in this approach, particularly when involving electrophilic, protein-reactive ions, is that the rate measured may be a combination of absorption into the systemic circulation and the dose which is retained in the SC. Frederickson described in detail the problems arising when measuring absorption of compounds that permeate the skin slowly (142). As seen in in vitro experiments conducted with reservoirforming compounds, the portion retained in the surface strata of the SC is far more important than material reaching the receptor phase, if any (93,105,143). Also, depth of penetration through the epidermis remains unknown. Copper is subject to homeostatic control, and is known to be dynamically bound to MTs in the skin (93). Thereby, even recovery in excreta may not afford a relevant mass balance. Advantages of this method are that it requires small amounts of active formulation (at pharmacologically insignificant concentrations), it is inexpensive, relatively rapid, and applicable in clinical studies. The difficulties inherent in skin recovery, volatility of penetrant, and errors associated with using the difference between the amount of compound applied and amount remaining make this an inexact method for the quantitative determination of absorption rates. The disappearance method has been used extensively with radiolabeled metal salts, primarily by Wahlberg et al. To measure the loss of material from the skin surface over time, the guinea pig is the model mostly used in vitro and in vivo, with a limited number of experiments conducted on human skin in vitro (144). Consistently using that technique, Wahlberg and coworkers studied a dozen metal salts for their skin diffusivity. Although the data on skin disappearance were obtained over relatively short exposure times, the results are valuable as benchmarks, internally consistent yielding a scale of relative diffusivities allowing at least partial validation of other measurements for those metals. Zinc that is reversibly bound to sulfhydryl storage sites becomes available for immediate systemic absorption when deficiency develops (145). Skin conditions due to nutritional zinc deficiency, collectively described as zinc deficiency dermatoses, have been found to respond promptly to treatment, and dramatic improvement was seen within days of initiating transdermal zinc therapy (130,146). Occlusive dermal application of a

50


concentrated zinc ointment to healthy human volunteers on a normal diet on the other hand did not lead to a significant increase in serum zinc concentration. When a similar application was made to the skin of patients on total parenteral nutrition, a dietary routine that typically results in zinc deficiency, serum zinc levels were nevertheless maintained at surprisingly normal and constant values. Based on such observations the percutaneous absoption of zinc appears to depend on actual zinc status in the overall organism, and thereby is variable. The close association and interrelationship between zinc and copper, therefore, would indicate similar variability in trasdermal diffusion rates measured for the latter, making Fick’s law of diffusion inapplicable. These caveats notwithstanding, determined under observance of steady state and normalized for concentration, by default the Kp is still the most widely used descriptor of chemical diffusion through the skin. Based on a number of necessary assumptions, literature data can be transformed so as to put metal absorption values on a common scale, adequate for purposes of risk assessment. Skin Stripping—A Semiquantitative In Vivo Method Use of adhesive tape to sequentially remove layers of the SC in vivo following topical application of a compound was proven useful in dermato-pharmacokinetic and -toxicological research to investigate reservoir formation by drugs (147). Based on an absorption study of selected drugs in vivo, a relationship was defined between SC concentration and their eventual total (systemic) absorption (35). By partial stripping (six iterations) of the SC, the amount of chemical residing in the SC after 30 minutes exposure time highly correlates with the total amount of chemical penetration within four days, as determined by the standard urinary excretion method. It thus becomes possible to make a predictive assessment of total drug uptake from the analysis of superficial SC levels only. Although skin stripping can also be used in vitro, its use in vivo most of all permits drawing a more authentic profile of the SC, as it avoids problems otherwise encountered, such as the creation of artifacts and damage potentially associated with sectioning cadaver skin, storing conditions, or duration of the experiment (tissue integrity). Rougier’s tape stripping protocol obviates the need for urinary and fecal excretion analysis, and is applicable to nonradiolabeled determination of percutaneous absorption, because strippings remove permeant in quantities adequate for nonlabel assay, such as high pressure liquid chromatography (HPLC). Rougier’s correlation, however, cannot be expected to be applicable to investigation of highly electrophilic, protein-reactive compounds, such as transition metals, some of which appear to form permanent depots in the SC (e.g., nickel, lead, or mercury). For those metal compounds, the direct skin stripping


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technique followed by inductively coupled plasma–mass spectrometry analysis was useful in visualizing SC penetration by nickel, in particular (78,101). The outstanding biological characteristic of nickel, from a public health perspective, is its immunotoxicity. Nickel started out as a prime occupational hazard reported from the metalworking and refining industry in the late 19th century and the first part of the 20th century due to its allergenicity (139,148,149), but since the Second World War it has become a consumers’ affliction as well, because the metal is now part of most alloys used in the manufacture of common materials in tools and articles of daily contact. In a number of dermatotoxicity studies, the incidence of nickel hypersensitivity noted for women formerly ranged from 10% to 20%, that for men from 1% to 4%. In more recent studies from a number of countries, a striking increase in these numbers has been recorded, particularly among women and children of school age: to 31.9% among schoolgirls in Italy (150), to 43.7% among dermatological clinic patients in Poland (151), and to 30% among an unselected group of schoolgirls in Finland (152). To gain insight into the dermatopharmacokinetics of nickel as allergen, the protocol of sequential adhesive tape stripping was implemented to examine the penetration characteristics of both the nickel salts and nickel in its metallic state in human SC and its potential to reach the viable epidermis, following their application on the skin volunteers. Inductively coupled plasma-mass spectroscopy (ICP-MS) was used to analyze tape strips (78,101). For nickel chloride, sulfate, nitrate, and acetate, the depth-penetration profiles obtained by tape stripping and analysis led to a number of conclusions: 1. Up to 24 hours, most of the nickel dose applied remained on the SC surface 2. Nickel adsorbed accumulated in the uppermost SC layers, and the concentration gradient between the superficial and deeper layers increased commensurate with exposure time and concentration. 3. In a number of experiments, mass balance based on amounts retrieved from the skin surface and cumulative nickel analysis to the level of the glistening layer showed a deficit. Within 24 hours nickel salts thus appeared to penetrate beyond the SC to a minor degree, possibly via the skin shunts, to become systemically available. 4. While the concentration gradients of nickel adsorbed varied with counterion, anatomical site, dose, and exposure time, toward the level of the glistening layer for all variables tested, the depth profiles converged toward nondetectable levels ( 0.999) was used for quantitation for both Cu isotopes (63Cu and 65Cu), with 89Y being used as an internal standard. The ICP-MS was an Agilent 7500c spectrometer operated under He collision gas mode. The instrumental detection limit was 1000 ng/mL), a Perkin-Elmer 4300 dual view ICP optical emission spectrometer was used for analysis. Statistical Analysis Analyses of the AUC were performed using two-sided, paired Student t-test. The probability value, P < 0.05, was considered significant. RESULTS General Due to the passive nature of the diffusion process, for all subjects a decreasing concentration gradient from the horny layer to the subcutaneous tissue became evident (Figs. 1 and 2). This concentration gradient was steep in the outermost layers of the SC due to its barrier function (Schaefer and Redelmeier, 1996) (39) and became less so towards the viable layers of the epidermis.

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Hostyńek et al.

Figure 1 Plots of average values for copper removed by sequential tape stripping on the ventral forearm of three human volunteers after increasing periods of occlusion, as assessed by ICP-MS. Data points correspond to strip nos. 2, 3, 5, 10, 15, and 20.

Figures 1–2 show copper content for strips nos. 2, 3, 5, 10, 15, and 20 as a function of type and duration of occlusion, as resulting from ICP-MS analysis. Individual graphs are presented to illustrate the individually differing levels of copper naturally present in the skin of volunteers.

Diffusion of Copper Through Human Skin In Vivo

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Figure 2 Plots of average values for copper removed by sequential tape stripping on the ventral forearm of three human volunteers after increasing periods of semiocclusion, as assessed by ICP-MS. Data points correspond to strip nos. 2, 3, 5, 10, 15, and 20.

Copper Penetration Under Occlusion For purposes of range finding, occlusion was initially set at periods from minutes to 24 hours, as in earlier studies on nickel (23). However, over that

Hostyńek et al.

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period copper values did not exceed background values, possibly due to greater corrosion resistance of copper on skin contact. Figures 1A–C show copper values in sequential SC strips in terms of amount per unit area of tissue, presented for individual volunteers and increasing periods of occlusion. Notable in individual tracings are the differing baseline levels of copper naturally present in the skin of volunteers. Only the initial three strips were analyzed for baseline. For all periods of occlusion, initially profiles were slightly elevated above baseline. By the 10th strip, however, copper levels had reverted to baseline values. Copper Penetration Under Semiocclusion Figure 2 shows copper values in sequential SC strips in terms of amount per unit area of tissue, recorded for individual volunteers and increasing periods of occlusion. Again, the individual tracings show the differing baseline levels of copper (strips 2–20) naturally present in the skin of volunteers. Copper values decreased from the superficial SC layers to the deeper layers, the gradients increasing commensurately with occlusion time (24, 48, and 72 hours). After the 10th strip, the copper content in the SC approximated baseline levels, but at 72 hours occlusion continued above baseline values. The AUC values for the semioccluded experiments and their statistical significance are given in Table 1. DISCUSSION Results of this Study In our study of copper metal exposure, data obtained for the participating volunteers reveal a striking interindividual diversity in copper levels naturally occurring in the same anatomical area of the SC, determined before application of copper powder. Also the variable rate of SC penetration by Table 1 Copper Removed from Human Volar Lower Arm After Semiocclusion Occlusion time 0 24 48 72

Mean AUCa (ng/cm2)

SD

38,033 45,845 55,960 83,974

18,086

p-value 0.11 0.03b 0.08

a Average area under the curve (AUC) of triplicate stripping experiments on three volunteers removed by strips nos. 2–20. b Significant difference in copper removed after 48 hours versus background.


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the metal appears due to individual differences in metal oxidation in the microenvironment prevailing in the area of contact (chemical, i.e., exudates composition and sweating rate, and physical, i.e., ambient and skin temperature). A literature search for copper levels in the SC provided no information that could be used for direct comparison with our results. Only data on whole skin and hair were found. The normal copper concentration in healthy human skin tissue appears to vary according to anatomical site from 1 to 7 ppm dry weight, as determined by neutron activation analysis of biopsies from 15 individuals (24). The levels and distribution of copper in human skin (dermis and epidermis) was also investigated (25). The mean values determined by atomic absorption in dry tissue varied from 0.88 to 2.60 ppm, with a median value of 1.38 ppm. Using nuclear microscopy, hair from three normal subjects showed copper concentrations of 18 6 ppm in the cortex of hair, with marked increases in concentration at the periphery (approximately 100 ppm) (26). AA spectroscopy was used to analyze for copper content of hair in relation to age. Starting at 13 ppm at age 2, copper content increased to 60 ppm by the age of 12, and decreased again to levels of 10 ppm at age 80 (27). Discussion of natural levels of that essential trace element prevailing in the integument and, in particular, of those encountered in the volunteers participating in the present study lies outside the scope of this paper. Interindividual variability in the SC diffusion by copper, essentially going back to the rate of oxidation in the microenvironment of an individual’s skin, on the other hand, is readily explained, based on previous observations (28,29). Oxidation of a given metal in contact with the skin is a multifactorial process, as it will vary in function of prevailing environmental conditions, the skin’s pH, sweating rate, and the variability in the individual’s amount and composition of sebum, sweat, and salts, which is, in turn, a function of gender and age (30–36). The soluble metal complexes formed are capable of penetrating the corneocyte envelope, forming a reservoir of the metal in the outermost layers. The process of oxidation appears to be slower under occlusion, due to limited access of air (oxygen). As became evident from these in vivo experiments, availability of oxygen is one critical factor in the oxidation process of metals. In the presence of sweat as electrolyte, the electrochemical oxidation of copper leads to the formation of cupric ions (Cu2þ). This is coupled with the concurrent reduction of oxygen, which with water forms hydroxyl ions. In the absence of oxygen, the reaction cannot proceed and cupric ions are not liberated. One observation in this study points to the difference in ionization potential and thereby corrosivity of copper when compared to that of nickel described earlier (29). In general terms, oxidation of a metal to the ionic form and liberation of electrons proceeds according to Eq. (1): M ! Mxþ þ xe

ð1Þ

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and the reduction of the (obligatory) oxygen present to form water is represented in Eq. (2): O2 þ 4Hþ þ 4e ! 2H2 O

ð2Þ

The difference in corrosion rates between the two metals is expressed as the standard oxidation potential in the equations below, characterizing the oxidation for elementary nickel [Eq. (3)] and elementary copper [Eq. (4)], respectively. While nickel, brought in intimate contact with the skin under occlusion, reacted to form skin-diffusible derivatives within minutes of exposure, copper seems not to significantly diffuse for up to 24 hours. This may be explained by the differences in oxidation potential between nickel (þ0.23 ev) and copper (0.34 ev), nickel thus being more easily oxidizable. This should not come as a surprise since copper belongs to the select group of the so-called noble, i.e., nonreactive metals. Nið0Þ ! Niþ2 þ 2e þ 0:23 ev

ð3Þ

Cuð0Þ ! Cuþ2 þ 2e 0:34 ev

ð4Þ

Other metals besides copper, particularly when they are in intimate contact with other metals in alloys forming a galvanic element, are electrolytically oxidized by the skin’s sweat and sebum, forming skin-diffusible compounds: e.g., nickel and references therein; beryllium, cobalt, chromium, and mercury, and references therein (28,29). At the other end of the spectrum in the electromotive series, under specific circumstances skin reactions have also been attributed to gold in contact with skin, a metal even less reactive than copper (37). Positive patch test reactions were recorded in patients with facial and eyelid dermatitis that were ascribed to gold released from jewelry, apparently promoted by sweat and associated with the abrasive action of titanium dioxide in cosmetics and sunscreens. A contributing factor is presumed to be static absorption of loosened metal particles to titanium dioxide, which can act as adjuvant promoting penetration of skin strata (38). Nederost and Wagman concluded that a subset of gold-allergic patients would benefit from avoidance of gold jewelry coming in contact with skin to which cosmetics were applied containing titanium dioxide and becoming subject to substantial friction. The directionally increasing slope of copper concentration profiles with increasing time of exposure, traced up to 72 hours of occlusion, indicates a slow but gradual formation of a depot in the outer SC due to its barrier function (39). Such concentration gradients are typical for a passive diffusion process, as there are no active transport mechanisms involved through skin. Copper ions are highly electrophilic and readily complex with SC proteins, leading to such depot formation. Such depots in the SC merit consideration for purposes of risk assessment. In vitro diffusion experiments with several copper complexes showed that a substantial portion of the


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permeant is retained in the SC, epidermis, and dermatomed skin used for that purpose (Hostynek JJ, unpublished data). Such buildup in the SC is a fair indication that the exogenous agent (copper) eventually may become available systemically. For that reason also, measuring diffusion rates only presents part of the overall picture, as the chemical absorbed into the SC may continue to diffuse into the viable tissues, even after exposure has stopped. Further absorption of such material, appropriately referred to as the SC reservoir (40), is the result of a counter-current process that includes desquamation versus release. Results confirm that absorption of copper into the SC does occur. Because copper levels above baseline values, albeit approaching background and after prolonged semiocclusion, are still seen at the level of the 20th strip (0.4–1.4 mg/cm2), the level of the stratum lucidum. This is taken as an indication that the metal has penetrated beyond the deepest SC layers, and thus is likely to have reached the viable epidermis. Although extremely fine particles may penetrate the loosely packed superficial stratum disjunctum, they would not be expected to migrate further into the deeper, tightly packed cells of the stratum compactum. Literature on human skin penetration by particulates indicates that follicles will be penetrated up to a size of 50 mm, and that the SC by an optimal diameter of 5 mm, migrating to a depth of ca. 10 tape strips, as observed by optical methods (41–44). In the studies cited above, the intent was to promote depth penetration of a dermatological preparation applied, achieved by massage of the treated skin area. Particles lodged in hair follicles would not appear as artifacts since tape stripping of skin only removes SC without associated hair follicles (22). Toxicological Considerations and Pharmacological Evidence for the Effects of Exogenous Copper Toxicity of the ionized form of this essential trace element is kept in check by an efficient homeostatic mechanism involving metallothionein and ceruloplasmin (45). Data available indicate that copper may have noxious effects only following chronic oral (or parenteral) exposure to high amounts of the metallo-element, particularly upon chronic oral ingestion with food (e.g., water) that exposes the human organism to more than 5 mg/kg of copper per day (46). Sorenson (47) investigated the acute toxicity of AI copper complexes, partly listed in Table 2. Comparison with the respective chelating agent demonstrated that the copper complexes were less toxic and damaging to the organism than the parent drug. In a phase I trial to investigate the cutaneous absorption of copper in the skin, the metal was applied as copper (I) oxide and in its elemental form as an ointment (paraffin and Vaseline) under gauze. Based on daily application

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Table 2 Acute Toxicity and Therapeutic Indices of Copper Complexes TI Copper complex Cupric acetate Cupric anthranilate Cupric aspirinate

LD50 (sc) (mg/kg)

CFE

PA

350 750 106 760 100

70 50 150

750 760

Abbreviations: TI, therapeutic index; CFE, carrageenan foot edema; PA, polyarthritis; sc, subcutaneous. Source: From Ref. 47.

on the skin of volunteers over a four-week period, the authors concluded that dermal exposure to copper in concentrations of up to 20% does not present a toxic risk (48). The risk of copper toxicity may exist only by administering amounts of the metallo-element far greater than those that can be actually used for therapeutic purposes, by transdermal exposure in particular. CONCLUSIONS The present observations serve to confirm action of skin exudates (sweat and sebum) in reacting with copper metal on prolonged contact and in the presence of air, to form in situ diffusible salts, such as the chloride, pyruvate or lactate, and lipophilic derivatives (soaps) of likely diffusivity (8). The role of the skin as a toxicologically important route of exposure to environmental agents in general and metal compounds in particular is hereby underscored. Skin is an important port of entry not only to hazardous materials such as readily oxidized metals that harbor the potential for serious health effects, but also to essential elements such as copper, whose release and diffusivity through skin contact has the potential for beneficial and therapeutic action in the inflamed organism. These findings may contribute to the acceptance of the long-held belief in the AI effects of copper metal in direct contact with the skin in particular, but also may promote the concept of external AI therapy as alternative to systemic dosing, such as intravenous, intra-articular or intraperitoneal. Critical for AI activity may be the supplementation of endogenous copper with exogenous sources, irrespective of the agent’s nature: elemental, salt, complex, or as a covalent derivative, as endogenous formation of the chelate of maximum biological activity will occur by homeostasis. The mechanism of copper complexes acting as AI agents is not known in all its details, yet evidence so far points to the formation of a unique, as yet undefined metabolite that might be responsible for the suggested clinical AI effect.


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GLOSSARY Absorption Coinage metals

Corrosion Electromotive series Ionization potential

Metallic elements

Noble metals

Oxidation

Oxidation potential

Penetration Permeation Reduction

Uptake into the organism. Copper, silver, and gold, with negative oxidation potential relative to the hydrogen (standard) oxidation potential defined as 0.00 V. Their characteristic electronic arrangements permit facile oxidation. Possible oxidation states are I, II, and III. They are often stabilized through formation of characteristically covalent complexes. Electrochemical process involving redox reactions in the presence of electrolytes. Arrangement of metals by decreasing order in their ability to oxidize. Energy (electron volts, ev) required to remove an electron from its atomic orbit, with the value for the standard hydrogen electrode set at 0.00 ev as an arbitrarily selected standard reference. Characterized by the ability to form cations (positive ions), by luster, malleability, conductivity (thermal and electrical) and the formation of cations (positive ions). Descriptive term used to characterize electrochemically inert metals, mostly copper, silver, gold, palladium, and platinum. Process of electron removal from an atom or ion (e.g., the increase in the proportion of oxygen in a compound). Electrical driving force toward electron loss, expressed as a potential value (in electron volts, ev). Passive diffusion process of a solute through skin. Diffusion through one or several layers. Process of electron gain by an atom or an ion (e.g., the increase in the proportion of hydrogen in a compound).

ABBREVIATIONS AI AUC ev SC

anti-inflammatory area under the curve electron volt stratum corneum

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sc ICP-MS

subcutaneous inductively coupled plasma–mass spectroscopy

REFERENCES 1. Lideń C, Carter S. Nickel release from coins. Contact Dermatitis 2001; 44: 160–165. 2. Wass U, Wahlberg JE. Chromated steel and contact allergy. Contact Dermatitis 1991; 24:114–118. 3. Black H. Dermatitis from nickel and copper in coins. Contact Dermatitis Newslett 1972; 12:326–327. 4. Karlberg AT, Boman A, Wahlberg JE. Copper—a rare sensitizer. Contact Dermatitis 1983; 9:134–139. 5. Saltzer EI, Wilson JW. Allergic contact dermatitis due to copper. Arch Dermatol 1968; 98:375–376. 6. Fat L, Gyorffy L. Occupational dermatitis due to copper exposure. Stockholm: OEESC, 2005:51–52. 7. Hostynek JJ. Factors determining percutaneous metal absorption. Food Chem Toxicol 2003; 41:327–333. 8. Hostynek JJ. Flux of nickel salts versus a nickel soap across human skin. Exog Dermatol 2003; 2:216–222. 9. Walker WR, Griffin BJ. The solubility of copper in human sweat. Search 1976; 7:100–101. 10. Whitehouse MW, et al. Alternatives to aspirin, derived from biological sources. Agents Actions 1977; 7(Suppl 1):43–57. 11. Odintsova NA. Permeability of human skin to potassium and copper ions and their ultrastructural localization. Chem Abstr 1978; 89:360. 12. Bentur Y, et al. An unusual skin exposure to copper; clinical and pharmacokinetic evaluation. J Toxicol Clin Toxicol 1988; 26:371. 13. Dollwet HH, Schmidt SP, Seeman RE. Anti-inflammatory properties of copper implants in the rat paw edema: a preliminary study. Agents Actions 1981; 11:746–749. 14. Bommannan D, Potts RO, Guy RH. Examination of stratum corneum barrier function in vivo by infrared spectroscopy. J Invest Dermatol 1990; 95: 403–408. 15. Cullander C, et al. A quantitative minimally invasive assay for the detection of metals in the stratum corneum. J Pharm Biomed Anal 2000; 22:265–279. 16. Higo N, et al. Validation of reflectance infrared spectroscopy as a quantitative method to measure percutaneous absorption in vivo. Pharm Res 1993; 10: 1500–1506. 17. Loeffler H, Dreher F, Maibach HI. Stratum corneum adhesive tape stripping: influence of anatomical site, application pressure, duration and removal. Br J Dermatol 2004; 151:746–752. 18. Rougier A, Lotte C, Maibach HI. In vivo percutaneous penetration of some organic compounds related to anatomic site in humans: predictive assessment by the stripping method. J Pharm Sci 1987; 76:451–454.


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19. van der Molen RG, et al. Tape stripping of human stratum corneum yields cell layers that originate from various depths because of furrows in the skin. Arch Dermatol Res 1997; 289:514–518. 20. Parsons ML, Major S, Forster AR. Trace element determination by atomic spectroscopic methods-state of the art. Appl Spectrosc 1983; 37:411–418. 21. Schwindt DA, Wilhelm KP, Maibach HI. Water diffusion characteristics of human stratum corneum at different anatomical sites in vivo. J Invest Dermatol 1998; 111:385–389. 22. Finlay A, Marks R. Determination of corticosteroid concentration profiles in the stratum corneum using skin surface biopsy technique. Br J Dermatol 1982; 107:33–38. 23. Hostynek JJ, et al. Human stratum corneum penetration by nickel: in vivo study of depth distribution after occlusive application of the metal as powder. Acta Dermato-Venereol (Suppl) 2001; 212:5–10. 24. Molochia MM, Portnoy B. Neutron activation analysis of trace elements in skin IV. Regional variations in copper, manganese and zinc in normal skin. Br J Dermatol 1970; 82:254–255. 25. Meyer BJ, et al. Distribution of copper in the skin. South Afr Med J 1972; 46:907–912. 26. Watt F, et al. Analysis of copper and lead in hair using the nuclear microscope; results from normal subjects, and patients with Wilson’s disease and lead poisoning. Analyst 1995; 120:789–791. 27. Petering HG, Yaeger DW, Witherup SO. Trace metal content of hair. Arch Environ Health 1971; 23:202–207. 28. Flint GN. A metallurgical approach to metal contact dermatitis. Contact Dermatitis 1998; 39:213–221. 29. Hostynek JJ, Reagan KE, Maibach HI. Oxidative properties of skin exudates— a determinant for nickel diffusion: a review. Exogenous Dermatol 2002; 1:7–17. ¨ hman H, Vahlquist A. The pH gradient over the stratum corneum differs in 30. O X-linked recessive and autosomal dominant ichthyosis: a clue to the molecular origin of the ‘‘acid skin mantle.’’ J Invest Dermatol 1998; 111:674–677. 31. Guyton AC. In: Textbook of medical physiology. 8th ed. Philadelphia: W.B. Saunders Co., 1991:277; 801. 32. Yousef MK, Dill DB. Sweat rate and concentration of chloride in hand and body sweat in desert walks: male and female. J Appl Physiol 1974; 36:82–85. 33. Lampe MA, et al. Human stratum corneum lipids: characterization and regional variations. J Lipid Res 1983; 24:120–130. 34. Schurer NY, Elias PM. The biochemistry and function of stratum corneum lipids. Adv Lipid Res 1991; 24:27–56. 35. Wertz PW, et al. Composition and morphology of epidermal cyst lipids. J Invest Dermatol 1987; 89:419–425. 36. Willing SK, Gamlen TR. Sweat osmolality values in normal adults. Clin Chem 1987; 33:612–613. 37. Nedorost S, Wagman A. Positive patch-test reactions to gold: patients’ perception of relevance and the role of titanium dioxide in cosmetics. Dermatitis 2005; 16:67–70.

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38. Yongchao Q, Yiping H, Wanlaui R. Adsorption behavior of noble metal ions (Au, Ag, Pd) on nanometer-size titanium dioxide with ICP-AES. Anal Sci 2003; 19:1417–1420. 39. Schaefer H, Redelmeier TE. Skin barrier—principles of percutaneous absorption. Basel: Karger, 1996:162–163. 40. Schaefer H, Redelmeier TE. Skin barrier—principles of percutaneous absorption. Basel: Karger, 1996:129–152. 41. Illel B. Formulation for transfollicular drug administration: some recent advances. Crit Rev Therap Drug Carrier Systems 1997; 14:207–219. 42. Illel B, Schaefer H. Transfollicular percutaneous absorption. Acta DermatoVenereol 1988; 68:427–430. 43. Rolland A, et al. Site-specific drug delivery to pilosebaceous structures using polymeric microspheres. J Pharm Res 1993; 10:1738. 44. Schaefer H, et al. Follicular penetration. In: Scott RC, Guy RH, Hadgraft J, eds. Prediction of percutaneous penetration. London: IBC, 1990:163–166. 45. Hellman NE, Gitlin JD. Ceruloplasmin metabolism and function. Annu Rev Nutr 2002; 22:439–458. 46. Aggett PJ, Fairweather-Tait S. Adaptation to high and low copper intakes: its relevance to estimate safe and adequate daily dietary intakes. Am J Clin Nutr 1998; 67:1061S–1063S. 47. Sorenson JRJ. Copper chelates as possible active forms of the antiarthritic agents. J Med Chem 1976; 19:135–148. 48. Gorter RW, Butorac M, Cobian EP. Examination of the cutaneous absorption of copper after the use of copper-containing ointments. Am J Therap 2004; 11:453–458.

6 Irritation Potential of Copper Compounds Jurij J. Hostyńek and Howard I. Maibach Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

INTRODUCTION Following clarification of frequently encountered terms in dermatotoxicology we present a synopsis of irritant reactions of the skin to copper and its compounds: types of untoward skin reactions in general, aspects of human exposure to copper, and a description of predictive and diagnostic methods to assess irritancy through bioengineering methods, in vivo and in vitro, in humans and animals. The review discusses case studies, followed by critical examination of literature reports, with consideration given to a number of confounding factors in diagnosis. To a limited extent, the review also discusses immunotoxicity, copper pharmacology, and therapeutic benefits of exposure. EXPOSURE TO COPPER Natural sources of human copper exposure due to volcanic exhalations, weathering of mineral deposits, and runoff are a minor factor. The major release of copper stems from anthropogenic emissions, stemming from

This chapter, in part, was reprinted from Hostyńek JJ, Maibach HI. Review: skin irritation potential of copper compounds. Tox Mech Meth 2004; 14:205–213, with permission of Informa Healthcare.

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major industrial activities such as mining, smelting and refining, agricultural and industrial use of copper pesticides and preservatives, the burning of coal, waste incineration, and widespread consumer applications of copper (e.g., brake-pad releases). Thus, occupational exposure is preponderant and mainly through inhalation. Concentrations of copper in the occupational setting are rarely reported, as the focus there lies mainly on other elements of greater toxicity. It is thus difficult to relate health effects from those environments specifically to copper. Most countries limit copper-containing dust to a range of 0.5–1.0 mg Cu/m3, and copper in fumes to 0.1 and 0.2 mg Cu/m3 (1). For purposes of occupational hazards in the United States, a limited number of compounds are recognized as hazardous on cutaneous exposure, and are identified as such by a ‘‘skin’’ notation in the listing of hazardous chemicals by the American Conference of Governmental and Industrial Hygienists (ACGIH) in their listing of threshold limit values (TLVs). The purpose of such labeling is to raise attention to the fact that cutaneous absorption can present a significant risk of systemic toxicity. The criterion most frequently used for a ‘‘skin’’ listing is acute animal toxicity from skin absorption, i.e., a dermal LD50 below 1000 mg/kg. This may be an indication of either rapid skin penetration or extreme toxicity, or both. The TLV values applicable to copper as fume are 0.2 and 1 mg/m3 as respirable dust or mist, for purposes of irritation, gastrointestinal exposure, or metal fume fever (inhalation). In the 2001 edition of TLV guidelines, copper does not rate a skin notation (2). Exposure of the general population to this essential trace element is of minor importance, limited to normal dietary intake of copper naturally occurring in plants and meat, and the metal released into drinking water conveyed through copper tubing. Systemic exposure to copper occurs through its slow release from dental materials and intrauterine devices (IUDs). Topical exposure comes from the release of copper in alloys used in jewelry, as it is measurably released in contact with skin exudates. SOLUBILIZATION OF COPPER METAL Dermal Inflammatory skin reactions of different types are due mostly to exogenous factors, primarily chemical agents impacting the skin. To exert an irritant or inflammatory action they must penetrate the stratum corneum (SC), a layer of inert keratinized cells, before reaching the viable layers of the epidermis and dermis. Among the irritant chemicals are acids, bases, organic solvents, salts, soaps and detergents, and pharmacological agents. Copper and other elements in their metallic state have no effect on the skin. They become potential irritants or allergens only when they are corroded (oxidized) and thus become soluble through the action of exudates encountered on the skin surface, or in a relatively corrosive physiological environment such as the oral cavity or the uterus.

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By the action of salts and acids present in sweat and sebum on the skin, most base metals are converted to the hydrophilic (ionized salts) or lipophilic (soap) form, respectively. Sweat composition fluctuates considerably in function of the rate of sweat secretion (3). Besides sodium and chloride, other significant corrosive components of sweat are potassium, urea, lactate and pyruvate, amino acids, proteins, and acidic lipids. The formation of free acids in the SC and on the skin surface is the result of hydrolysis of those acidic lipids by lipolytic enzymes occurring in the sebaceous ducts and on the skin surface, and of bacterial decomposition (4,5). It is the oxidizing (corroding) action of such acids that results in the formation of soaps with copper (and metals in general) upon intimate and prolonged contact with articles of daily use, which potentially result in skin irritation or allergic reactions once they reach the viable structures of the skin, since these relatively lipophilic compounds penetrate the SC with relative ease as compared to ionized salts (electrolytes) (6). Metallic objects used in jewelry or drug-like devices (dental materials, orthopedic implants) as a rule are not made of copper alone, but the metal is incorporated in alloys that have corrosion (oxidation) characteristics quite different from those of the constituent metals. An exception is the wire used in IUDs, presumably made of high-grade copper only. The characteristics of alloys are determined by electrochemical characteristics of the elements in contact with each other; oxidation and formation of potentially allergenic ions will vary in function of alloy composition. The electrochemical potential (galvanic effect) between diverse elements in close proximity provide the driving force for such reactions resulting in enhanced corrosion (7). The more electropositive (baser) the element (e.g., nickel), the more stable it is in the ionized state, and will transfer electrons to the more electronegative, nobler metal (e.g., copper). The actual concentration of a metal in the alloy is thereby only of secondary importance. Ultimate biological activity of the alloy is determined by the rate at which metal ions are released, i.e., whether they reach a concentration sufficient to provoke a reaction in the adjacent tissues. Release of copper in synthetic sweat related to chloride ion concentration was determined by Boman et al. After 24 hours, copper dissolved from coins and copper thread in the range of 80–100 mg/mL sweat, with an inverse relationship between the concentration of copper and chloride ion (8). Lideń et al. determined the release of copper from gold-containing jewelry in artificial sweat. Amounts released over one week ranged between 0.11 and 0.66 mg/cm2, depending on alloy composition (9). Systemic Corrosion and solution of copper in the physiological environment may be considered as equivalent to systemic dosing. Human plasma is an aggressive physiological medium for dissolving metals. Corrosion of the foreign object in this microenvironment releases components into the organism, some of which can then act as irritants or allergens. Levels of free ionic copper,


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however, a relatively toxic metal, are moderated to the minimum levels required for physiological needs, 10 19 mol/L estimated in blood plasma, through binding to ceruloplasmin and metallothionein. The dynamic equilibrium between ceruloplasmin and metallothionein prevents toxic accumulation or deficiency of copper in mammals. Dental Alloys Amounts of copper released from commonly used dental casting alloys, measured in cell culture over 10 months, was 0.15 mg/cm2/day (10). Cytotoxicity of metals thus released from dental alloys could be considered a correlate of irritancy. Accordingly, Wataha et al. (11–14) investigated in vitro corrosion rates of dental casting alloys in various culture media to obtain a measure of biological risk to oral tissues in a number of investigations. Grimsdottir et al. (15) also studied the cytotoxic effect of orthodontic appliances in an attempt to obtain a measure of tissue irritation caused by corrosion. Such data do not allow derivation of an objective measure of copper irritancy; however, release of metal ion in a simulated environment is highly dependent on presence and concentration, and thus the (galvanic) interaction with other metals, resulting in variable and unpredictable concentrations/cytotoxicity of individual metal ions, e.g., copper, as most of the metal is protein bound. Intrauterine Devices Increases in systemic copper via parenteral entry from a contraceptive IUD can lead to adverse effects reported: systemic nonspecific contact dermatitis and immediate immunologic contact urticaria, even though the amounts liberated from such a device are relatively low. Copper levels determined in intrauterine fluids from women who had used the T-380 A device were 11.4 4.7 mg/mL after six months; 11.54 7.0 mg/mL after one year and 6.24 1.5 mg/mL after three years. Overall, concentrations over the entire period surveyed ranged from 3.9 to 19.1 mg/mL (16). It is inferred that the toxic effect of copper ions thus released in the uterus (present in the form of complexes with proteins) are responsible for cutaneous eruptions, although most of the reported cases appear to belong to the category of nonimmunologic systemic contact dermatitis (16). These investigations on the release of copper ion from alloys in the physiological environment in vivo and in vitro and the potential biological effects from exposure help to explain the cases of systemic irritative response to IUDs and dental materials (17,18). On close examination, the immunologic relevance of many of those reports is unclear. INCIDENCE AND EPIDEMIOLOGY OF IRRITATION DUE TO COPPER Incidence of irritant contact dermatitis (ICD) is difficult to establish, as often patients do not consult a doctor. ICD, especially of the hands, is reported to


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be more common in women than men, possibly due to greater exposure to irritants in ‘‘wet work’’ in the household; also, women are more likely to consult with a doctor than men are. Studies in twins indicate that heredity is a factor in susceptibility to irritants, but variability is too great for generalizations. Atopic dermatitis seems to bring greater risk for ICD (18,19). Based on 5839 dermatology patients patch tested by the North American Contact Dermatitis Group, in which the role of occupational exposure to allergens and irritants was evaluated, 19% were found to be occupationally related. Of those, 60% were of allergic and 32% of irritant origin. The hands were the predominant part of the body affected, 80% of those due to exposure to irritants (20). For copper specifically, the aspects of epidemiology, prevalence, or population studies cannot be addressed since, in contrast to other metals such as nickel or chromium, reports of untoward reactions, systemic as well as cutaneous, are extremely rare. The two geographical areas with the most complete databases are the National Office for Occupational Health (Helsinki, Finland) and the State of California. The figures emanating from these sources are skewed in that they probably represent a small portion of the actual frequency of disease due to inherent weaknesses in reporting systems. PHARMACOLOGY OF COPPER Beneficial as well as adverse health effects due to copper, an essential trace element, are well characterized. Two pathological conditions stand out due to their chronicity. Menkes’ syndrome is remarkable in that there is no known cure and homocygots usually die early in life. Wilson’s disease (WD) is an inherited copper metabolism disorder, impairing biliary tract copper excretion that leads to excessive levels of the element in tissue, particularly in the liver if left untreated. Left untreated, such copper accumulation leads to hemolytic anemia, which over the years can result in progressive hepatic failure and ultimately death (21). The characteristic, brown ‘‘Fleisher rings’’ that develop in the eyes of Wilson’s disease patients are caused by the deposition of metallic copper. However, WD is very much treatable, if not cured, by penicillamine therapy and dietary control. WD patients lead seemingly normal lives as long as they are on medication and restrict copper intake. Deficiency of copper is associated with characteristic integumentary and skeletal abnormalities, defects in growth and development, and abnormalities in sensory perception (22). Copper status of the organism is reflected in ceruloplasmin levels. Plasma levels below 125 mg/dL are generally considered as indicative of copper deficiency (23). Menkes’ Syndrome Albinism, the striking absence of pigmentation in the skin, hair, and eyes, is characterized by the absence of the copper enzyme tyrosinase, which

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converts tyrosine to melanin in the melanocyte (24). Menkes’ kinky hair syndrome, a hereditary defect in intestinal copper absorption that causes retardation in growth, focal cerebral and cerebellar degeneration, and hair to be abnormally sparse and brittle, becomes manifest in early infancy (25). Afflicted infants have low levels of copper and ceruloplasmin, dying usually within the first year of life. Although copper absorption and reabsorption are impaired, tissue copper levels of many epithelial tissues, including the skin fibroblasts, are elevated, and an increased production of metallothionein, the cysteine-rich protein that binds copper in cells, appears to be the cause of such accumulation. The biochemical defect underlying Menkes’ syndrome however is largely unknown. Copper as Antimicrobial Copper itself proved highly antimicrobial in plumbing and in lab tests with several bacterial strains and some viruses. A chlorophyllin copper complex, derived from chlorophyll by replacing the chelated magnesium with copper, has anti-inflammatory and antimicrobial properties, as well as a marked stimulating effect on epithelial cell growth rates and cell regeneration. First established in tissue culture studies, these findings were confirmed clinically through wound healing and deodorizing characteristics observed in animal and man (26). Administered orally, chlorophyllin copper complex is classified as a safe and effective internal deodorant by the U.S. Food and Drug Administration (27). Transdermal Anti-inflammatory Action of Copper Exogenous copper has demonstrable anti-inflammatory effect, as several copper complexes like Cupralene, Dicuprene, Alcuprin, or Permalon are successfully employed in treating human arthritis (28–31). The potential for copper’s activity as an anti-inflammatory agent by transdermal delivery is subject to controversy, however. This is because scientific studies designed to demonstrate therapeutic benefits for arthritic conditions through dermal contact with metallic copper so far have been inadequate. Quantitative data for percutaneous penetration of copper’s putative oxidation products, which may be generated in contact of the metal with skin in humans, are still outstanding. One missing, important factor for a convincing case of such potential benefits is the deficiency in systematic and adequate scientific research into the penetration of copper through human skin in any of its forms, as polar mineral salts or as the more lipophilic complexes, and thus a lack of solid scientific data documenting the therapeutic value of transdermal copper delivery. It can be safely assumed that endogenous copper has natural anti-inflammatory activity, and that such activity may also be reinforced by exogenous copper. In a review of anti-inflammatory activity of exogenous copper, Milanino et al. (32) concluded that copper, indeed, is active


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as an acute anti-inflammatory agent irrespective of chemical form, including inorganic copper salts. That there is a direct connection between copper and rheumatoid arthritis is supported by the fact that low molecular weight copper concentrations in plasma and synovial fluids increase in response to the disease, and when such increases are induced further by administration of exogenous copper, they are observed to have a definite anti-inflammatory effect in both laboratory animals and humans. COPPER IRRITANCY IN SKIN AND MUCOSA In Vivo Assays Kinetics and specificity of nickel hypersensitivity were assessed by Siller and Seymour in mice presensitized with nickel sulfate and challenged with Cu (II) sulfate, chromic chloride, cobaltous chloride, nickel chloride, and nickel sulfate. The challenge concentration for the metal salts was 0.0152 M, and for Cu (II) sulfate, 0.003 M. A reaction occurred at 24 hours, resolving at 48 hours, consistent with an irritant reaction. Cu (II) sulfate was found to be ‘‘profoundly more irritant than the other metals’’ (specific numbers not given) (33). The biocompatibility and metal release were investigated in vivo through implantation of representative specimen alloys in rats, and in vitro in a battery of cell culture tests (7). In addition, combinations of dissimilar alloys were investigated in relation to possible enhanced corrosion by galvanic effects. Implantation and cytotoxicity tests on epithelial cells, macrophages, and erythrocytes were performed, and the results compared. The severity of tissue response in implantation tests corresponded to the nobleness of the casting alloys joined to amalgam. The most severe reaction occurred in the tissue in proximity of the LG-1 alloy, probably due to its high copper content. Similar results were obtained in the in vitro macrophage test. All of the alloys except the high-gold alloy (LM-Hard) had a toxic effect on epithelial cells. The combination of the casting alloys with amalgam diminished such toxicity. For the study, limit ratios of the metals used in the alloy were evaluated in order to test the biological significance of the galvanic currents with respect to these materials. Implantation Test Twenty-eight Wistar rats, weighing 250–300 g, were implanted subcutaneously with the various alloy combinations for time periods of 7, 30, and 60 days (Table 1). Two rats in each group were allocated for each alloy combination. Polystyrene implants serving as controls were left in 10 rats for the same observation times. After the allotted time, the animals were sacrificed with ether. The specimens including the surrounding tissues, submandibular glands, liver, kidney, spleen, and part of the spinal cord were examined.


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Table 1 The Compositions of the Examined Alloys (wt%) Alloy

Au

Pt

LM-hard LG-1a Micro Midi ANA 68 Revalloy

76.9 50.0 5.7 44.9

1

a

1.0 0.1

Pd

Ag

Cu

1.4 9.0 25.0 3.0

9.6 4.0 67.2 39.0 67.6 69.6

11.1 34.0 12 5 2.8

Sn

Zn 2.9

1.0 1.0 26.2 26.9

0.26 0.96

Experimental alloy.

The connective tissue reactions to the implants were diagnosed as mild, moderate, or severe on the basis of the degree of infiltrate, vascularity, and fibrosis. In addition, special attention was paid to estimation of the giant cells and foreign bodies in the tissues removed. Whenever foreign bodies were histologically recognized, Energy dispersion X-ray (EDAX) analysis was performed to reveal their constituents. Polystyrene control: The response to polystyrene was an uncomplicated repair of the surgical wound. Collagen fibers were present at day 7, and a thin compact collagenous capsule enclosed the implant by day 30, followed by an acellular capsule detectable by day 60. LM-Hard gold alloy: At day 7, the tissue surrounding the implant showed a moderate inflammatory cell infiltration and proliferating fibroblasts. Giant cells and a well-defined capsule were detectable at day 30. The capsule matured to a dense connective tissue membrane by day 60. Foreign bodies were still detectable around the implants after 30 and 60 days. EDAX showed the presence of Au, Cu, and Fe in these bodies. LM-Hard/ANA 68 combination: The inflammatory response around the implant was extensive by day 7. Granulation tissue formation was delayed, and characterized by numerous macrophages and extensive capillary proliferation. Foreign body aggregates found around the implant were verified by EDAX to consist of Au, Ag, Hg, Cu, Sn, and Zn. A subacute inflammation with prominent vascularity still persisted at day 30. Foreign bodies were shown to contain Cu, Hg, Fe, Sn, and Zn. At day 60, the inflammation had resolved into the mild stage, and a collagenous capsule surrounded the implant. Micro/ANA 68 combination: The initial reaction of the tissue against the Micro/ANA 68 combination presented heavy inflammation due to granulocytes, plasma cells, and macrophages. The reaction subsided by day 30, but the vascularity still remained prominent. By day 60, the tissue outside the fibrous capsule contained a few accumulations of lymphocytes. EDAX analysis disclosed the presence of Au, Ag, Cu, Fe, Hg, Sn, and Zn in the foreign bodies adjacent to the implants.


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Midi/ANA 689 combination: After the seven-day observation period, the Midi/ANA 68 implant had induced a strong cellular reaction with pronounced vascularity. Macrophages were abundant. After 30 days, there was a fibrous inflammatory region adjacent to the implant, contiguous with a zone of granulation tissue composed mainly of fibroblasts, mononuclear cells, and small blood vessels. Foreign bodies around the implant contained Ag, Au, Cu, Hg, Pd, and Sn. On day 60, a capsule with well-oriented collagen fibers existed, with only a few inflammatory cells present. LG-1/ANA 68 combination: By day 60 there still was a subacute inflammatory infiltration with high cellularity, plasma cells, and lymphocytes in predominance. Giant cells, macrophages, and occasional granulocytes were also detected. Some collagen was apparent, but it was poorly orientated. At this stage, foreign bodies were seen containing Ag, Au, Cu, Hg, and Sn in abundance. Histopathology: None of the biopsies from different parenchymal organs showed any morphological changes due to implants. Occasionally, foreign bodies or blackish precipitates were present in liver, kidney, and spleen. EDAX analysis showed them to contain calcium, chloride, sulfur, silicon, potassium, and iron in varying proportions. In addition, occasional copper particles were found in the kidney following the implantation of the LG-1/ANA 68 combination. Also, a few particles containing Ag were detected in the spleen of the same animals. The results obtained in the investigations of alloy combinations showed that when implanted in living tissue they caused reactions different in character and intensity, which finally led to the formation of fibrous capsules. The authors conclude that the differences in the severity of the responses observed can, in most instances, be explained on the basis of electrochemical reactions due to the different electrical potentials responsible for the release of metal ions. In the present study, EDAX analysis showed the presence of alloy elements in the surrounding tissue in every case. The composition of the elements was not identical to that of the original alloys, which indicates in situ corrosion, rather than particles dislodged from the test alloy during the implantation procedure. The severity and duration of the inflammatory reaction around the implants fully corresponded to the nobleness of the alloys, and, surprisingly, not to the suggested electric potential difference generated between the combined alloys. Using EDAX analyses, the foreign bodies adjacent to the gold (LMHard) implant were always shown to contain both gold and copper, thus suggesting that gold may be complexed to copper within cells, or evoking a copper-like biological response that is also causing localized accumulation of copper. Whether such a possible gold–copper complex is related to the adverse effects of gold or is a normal pathway in gold metabolism is not known. The finding that capsules around the amalgam implants contained mercury and tin particles are in agreement with previous observations (34).


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The extensive reaction to the LG-1/ANA 68 combination is apparently related to the release of copper from the LG-1 alloy. In vitro studies on binary Cu–Pd alloys have shown that a preferential dissolution of Cu is followed by an enrichment of Pd on the alloy surface (35). Amalgam, on the other hand, corrodes continuously. The high copper content of LG-1 (34%) accounts for a continuous copper release with a slow rate of Pd enrichment, thus maintaining a persistent inflammation with high cellularity adjacent to the implant. These findings are consistent with recent reports dealing with tissue response to Ag–Pd–Cu–Au I alloys and pure copper implants (36,37). The abundance of macrophages and copper around the LG-1/ANA 68 implants supports the results of McNamara and Williams (37) who showed that the pigmented material found in connection with Cu implants was composed of Cu-containing macrophages. The cells had absorbed large amounts of copper and remained damaged in the area, attracting more macrophages to these sites. Cu particles could seldom be found in the liver after implantation of the LG-1/ANA combination. This is contradictory to the findings of Yli-Urpo and Parvinen (38), who always found elevated levels of Cu and Hg in the liver and kidney after implantation of different alloy combinations. This discrepancy can be explained by the different methods used. The disadvantage of EDAX analysis is that only the surface of the specimen to a depth of 2–3 mm can be analyzed. Agarose Overlay Test The effects of alloys and their combinations on cultured human epithelial cells were examined. The cytotoxic effect of the test alloy was evaluated by measuring the zone of cell lysis around the alloy. Midi produced the most prominent cytotoxicity, whereas LM-Hard had no effect. All of the alloy combinations were less cytotoxic than the constituent alloys when tested separately. The diminishing cytotoxicity was most prominent with the combination of Midi/ANA 68. The reaction between the alloy and the culture medium can result in the leakage of metal ions from the alloy into the culture medium, while the cells themselves have no detectable effect on the corrosion process. LG-l, Micro, Midi, and ANA 68 alloys showed a marked cytotoxic activity in the agarose overlay test. The release of copper could be the major factor responsible for the observed rapid cytotoxic effect of Midi. The minor degree of cytolysis caused by LG-1, despite its high copper content, might be due to the preferential release of the least noble metal, zinc, thus retarding the release of the more noble constituents, copper included. The equal degree of cytolysis caused by Micro and ANA 68 (both containing equal amounts of silver) substantiates the concept of the role of silver as a cytotoxic agent. Surprisingly, the degree of cytolysis diminished when the casting alloys were combined with ANA 68. This is probably due to the electrochemical passivation, which


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was most pronounced in the Midi/ANA 68 combination, and least in the LG-l/ANA 68 combination. Erythrocyte Lysis Assay When the hemolytic activities of the alloys were tested, Midi, Revalloy, and LM-Hard were shown to possess a slight hemolytic activity. Microscopy of the cell pellet did not show any hemagglutination, which otherwise might cause low hemolysis values. Incubation of Midi, Revalloy, and LM-Hard with erythrocytes resulted in a slight degree of hemolysis. The mechanisms by which the metal particles produce their biological effects are not known in detail. It has been proposed that interaction between the erythrocyte membrane and the particles would be the most important factor in hemolysis (29). Additional mechanisms conferring the hemolytic activity are the chemical nature at the metal surfaces, particle size, and their surface charge. Since LG-1 did not show any hemolytic activity, cytotoxicity cannot be attributed to copper release. Further studies are needed, however, in order to elaborate on the elements and membrane components involved in the hemolytic mechanisms of dental alloys. Toxicity Test Using Murine Macrophages Latex and Revalloy particles are phagocytized faster than the other alloy particles. In the cultures of macrophages, which had been in contact with LG-1, a phagocytosis rate of only 25% was detectable, as compared to 80% due to Revalloy. The number of alloy particles phagocytized per macrophage was significantly lower than that of the Latex particles. The proportion of nonviable macrophages after exposure to the alloys, except Microalloy, was slight. More pronounced cellular damage with pyknosis and vacuolization appeared after exposure to Microalloy. No difference in toxicity was observed after one day compared to one-hour exposure to the alloys except for LG-1, which showed cell damage comparable to that due to Microalloy. A considerable amount of lactic dehydrogenase (LDH) was released by Microalloy. In contrast, little LDH release occurred when the cultures were exposed to Revalloy or latex particles. Solubility of particulate alloys into the macrophage culture medium: The concentration of zinc in medium from alloy cultures was higher than in the controls. The solubility of Zn was most prominent from Midi alloy. In addition, copper release from LG-1 and Midi alloys was found. No release of Au, Ag, or Sn was detected in any of the culture media (Table 2). The corrosion of metal implants in human and mammalian organisms (due to body fluids) may lead to local reactions in the surrounding tissues. The tissue response will depend on the corrosive behavior of the metal, the rate of release of metal ions, and their physiological activity. Since each element will be released at a different rate from a complex alloy and many


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Table 2 Soluble Metals Concentration in Macrophage Culture After One Hour Particulate alloy LM-hard LG-1 Micro Midi Revalloy Control

Zn SD (mg/mL)

Cu SD (mg/mL)

0.62 0.15 0.71 0.09 0.70 0.14 1.45 0.61 0.83 0.21 0.56 0.13

0.21 0.10 0.71 0.30 0.14 0.04 0.52 0.44 0.13 0.17 0.17 0.10

have a different mechanism of toxicity, it is difficult to establish the biocompatibility of the alloy using a single test method. A combination of test methods was used in an attempt to assess the behavior of a variety of complex dental alloys in different bioenvironments. Amalgam particles were phagocytized faster than the other alloy particles. This might be due to the differences in particle size. The present results indicated that particulate Microalloy was the most toxic of the alloys tested, whereas particulate Revalloy was well tolerated by the cells. Analyses of the soluble elements in alloys revealed only relative low concentrations of copper and zinc. Gold, silver, and tin were not detectable in any of the supernatant determined from the experiments described. It seems that the alloys are not sufficiently soluble in tissue culture medium for their effects to be exerted with extracellular toxic levels. These findings are in agreement with previous reports where no definite correlations could be found between the solubility of the particles and toxicity. Thus, it seems more probable that the alloys exert their toxic effects directly intracellularly after being phagocytized. Copper has been shown to cause degenerative changes in macrophage morphology, which could explain the increased LDH values due to LG-1 and Micro, despite only mild and moderate changes in cell morphology. Comparison of the different tests: Some correlation was seen between the in vivo implantation test and the in vitro macrophage test. This can be explained by the central role of macrophages in the manifestation of inflammation. Furthermore, macrophages are the first cells with which foreign bodies come into contact in living tissue. The in vitro results obtained from the agarose overlay test and the erythrocyte lysis test did not correlate well with the in vivo results. The toxicity established by the agarose overlay test would indicate the toxicity of soluble silver and copper rather than that of the alloy in itself. In hemolysis, on the other hand, interaction of the alloy constituents with biomembranes is one of the likely mechanisms involved in the toxicity of particulate alloys. Some evidence exists that materials that have not been phagocytized but that come into contact with the cell surface can cause macrophage destruction comparable to hemolytic activity. The interpretation of the results obtained by the different test methods is difficult, and the dynamic state of cells and their possible metabolic alterations due to


109

the implants are not fully understood. The behavior of alloys in a biological environment and the precise effect of each constituent element in different tests need to be studied more extensively. The severity of tissue response in the implantation test corresponded with the nobleness of the alloys combined with ANA 68. The most severe reaction was seen in the area surrounding LG-l, probably due to its high copper content. Similar results were obtained in the in vitro macrophage test. The agarose overlay test showed a somewhat similar zone of lysis for all the alloys except for LM-Hard. The combination of the alloys with ANA 68 reduced the lytic zone, which could be accounted for by a surface passivation of the alloy. LM-Hard, Midi, and Revalloy showed a slight hemolytic activity. A poor correlation was established between the agarose overlay, the erythrocyte hemolysis, and implantation tests. In Vitro Assays Schmalz et al. evaluated the suitability of a commercially available model system based on a recombined coculture of human fibroblasts and human epithelial cells for assessing mucosal irritancy of metals used in dentistry, as no valid animal or in vitro model exists for this purpose (39). That model had been introduced for evaluating the time-dependent irritancy of cosmetic products, where cell viability and prostaglandin E2 (PGE2) release from the cells were used as markers for the irritative potential of test materials. The human fibroblast–keratinocyte cocultures were exposed to test specimens fabricated from copper, zinc, palladium, nickel, tin, cobalt, indium of high purity (99.98–99.99%), and from a dental ceramic. Cell survival rates decreased after exposure to copper (14–25%), cobalt (60%), zinc (63%), indium (85%), nickel (87%), and the nonoxidized/oxidized high noble cast alloy (87%/90%) compared to untreated control cultures. Dental ceramic, palladium, and tin did not influence cell viability. In parallel, the PGE2 release was continuously monitored up to 24 hours using a competitive displacement enzyme immunoassay. PGE2 release increased most highly in the cultures exposed to copper (6–25-fold), cobalt (seven-fold), indium (four-fold), and zinc (two-fold) compared to untreated control cultures. The PGE2 determination proved to be a nondestructive method for continuous monitoring of cell reactions in the same culture. The model used seems promising for evaluating the time-dependent mucosal irritancy of dental cast alloys. Cell viability of exposed cell cultures was determined by the MTT test after 24 hours. Survival rates were calculated relative to values obtained in untreated cultures. For PGE2 release, assay aliquots (100/mL) were taken from exposed media and the amount of PGE2 released from treated and untreated cell cultures was quantified against a standard curve of purified PGE2, using a competitive displacement enzyme immunoassay. Threedimensional fibroblast–keratinocyte cocultures were exposed to one high noble dental cast alloy and various metals frequently found in cast alloys.

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Identical levels of cell viability were found in untreated control cultures and in cultures exposed to a dental ceramic, which was used as a negative control material. Pure copper was the most toxic metal tested. In copper-exposed cultures, a time-dependent decrease of cell viability at a level of 14% to 25% of untreated cell cultures was observed. Because of the demonstrated high toxicity, copper was routinely included as a positive reference material in all subsequent experiments evaluating the effects of other test materials. Cobalt and zinc induced a moderate decrease of cell viability to a level of about 60% of untreated cell cultures. Pure nickel, indium, and oxidized and nonoxidized specimens of the high noble cast alloy, were weakly toxic. Similar to the dental ceramic, no cytotoxicity was observed after exposure of the cocultures to palladium and tin specimens. Survival rates after exposure to copper, zinc, indium, cobalt, nickel, and the high noble alloy (oxidized and nonoxidized) were significantly different from those of untreated control cultures. Total amounts of PGE2 released from cell cultures exposed to test materials and from untreated control cultures steadily increased during the exposure period. The spontaneous PGE2 release from untreated tissues was identical with values obtained from cultures exposed to specimens of the dental ceramic and nontoxic metals. The amounts of PGE2 released after exposure to copper were about 10-fold higher than those released from untreated cultures after a 24-hour exposure. After 30-minute exposure to copper specimens, significantly higher PGE2 levels were already found compared to untreated controls. In contrast, no differences were found between the PGE2 levels measured in media of untreated tissues and tissues treated with all other test materials. In repeated experiments, the amounts of PGE2 released from cultures exposed to copper varied, being 6–25-fold higher than those released spontaneously. Indium and cobalt in contrast produced increases that were considerably lower than those elicited by copper in the same experiments (4–7-fold). The induction of an increased PGE2 release from human fibroblast–keratinocyte cocultures was inversely related to cell viability measurements after exposure to copper. The dramatic effect of copper on cell viability is in accordance with data from other in vitro and in vivo studies (40,41). This is due to the oxidative potential of pure copper and the toxicity of copper ions in vitro (42,43). As a consequence of copper toxicity, the cell viability was reduced to about 15% to 25% of untreated control cultures. The increases of PGE1 levels by factors of 2 (zinc) to 25 (copper) are among the highest observed in vitro so far (44,45). The model system based on a recombined coculture of human fibroblasts and human epithelial cells seems promising for evaluation of the mucosal irritative potential of dental materials; however, further studies, particularly on interexperimental variations, are needed before it can be established as a routine test model candidate. Cell viability as measure of cytotoxic potential in HaCaT cells (a spontaneously immortalized human kertinocyte line) and, indirectly, of irritancy in vivo, was determined on human keratinocytes in vitro by Brosin


111

et al. (46) for five metal salts. The endpoint used to assess cellular viability was metabolism of the tetrazolium salt XTT [2,3-bis (2-methoxy-4-nitro5-sulfophenyl)-5-(phenylamino carbonyl)-2H-tetrazolium hydroxide]. The metal salts showed the following rank order in cytotoxicity at an exposure time of 24 hours: potassium bichromate > Cu (II) sulfate > cobalt chloride and palladium chloride > nickel sulfate. The authors found an excellent correlation to the rank order of the metals’ known irritative potency, as it was determined in vivo for purposes of contact allergy screening by the ICDRG, but recognized that such a test hardly applies to the complex pathomechanism of skin irritation. As such, the presented XTT assay on HaCaT cells would be well-suited for an initial screening of substances to establish a relative order of irritancy as part of a battery of tests targeting different aspects of skin irritation. This could be subsequently followed by irritation tests in humans. CONCLUSIONS Data on the dermal irritation by copper and its compounds is scant, and its irritancy has not determined, e.g., in terms of an irritant dose ID50. Irritancy of copper can only be comparatively characterized in relation to other metal salts. A rank order for the irritancy of metal compounds can be inferred from the patch test concentrations recommended as nonirritating for the purpose of cutaneous allergy testing: potassium dichromate 0.5% in petrolatum; copper sulfate, cobalt chloride, and palladium chloride ex equo: 1% in aqueous solution; and nickel sulfate: 5% in petrolatum. With the exception of its mineral salts, copper (II) compounds (complexes, soaps) exhibit low irritancy and several have been adapted as therapeutics for epicutaneous applications as antiseptics or deodorants (e.g., the chlorophyllin copper complex, gluconate, oleate, or citrate) or in transdermal drugs (copper salicylate, copper phenylbutazone). Because of the increasing need for reliable skin irritation tests and in order to reduce the number of animal experiments, in vitro alternatives have been developed. So far, in vitro studies show that different chemicals induce irritant inflammatory responses, which vary considerably in the time course of the response, and that there are differences in the components of the inflammatory response to different irritants. Although no single test can be considered as an indirect, though reliable, measure of skin irritation in vivo, a battery of tests, each addressing a different aspect of such multifactorial phenomena leading to skin irritation, may well be a critical step preparatory to in vivo testing in humans. Distinguishing between irritant and allergic contact dermatitis can be challenging; thus, copper cross-reactivity/concomitant sensitization with other transition metals and failure by practitioners to resort to patch testing for resolution of questionable skin reactions, in many cases, leads to questionable diagnosis of irritation.


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Cu (II) sulfate is clearly an irritant when applied in pet under occlusion for 48 hours. However, there are no currently available data that allow us to determine the threshold for induction of acute or cumulative irritancy dermatitis for copper or any of its salts. Fortunately, the technology to define this is readily available (cumulative irritancy testing). These are now being generated in this laboratory. ABBREVIATIONS ACD ARL ELISA ICD ICU IUD LDH MM MTT NICU SC TEWL TLV

allergic contact dermatitis adult rat lung enzyme-linked immunoassay irritant contact dermatitis immunologic contact urticaria intrauterine device lactate dihydrogenase mitochondrial membrane mitochondrial toxicity test nonimmunologic contact urticaria stratum corneum transepidermal water loss threshold limit value

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30. Hangarter W. Inflammatory diseases and copper. Clifton, NJ: Humana Press, 1982. 31. Sorenson JRJ, Berthon G. Copper potentiation of non-steroidal antiinflammatory drugs. New York: Marcel Dekker, 1995. 32. Milanino R, Marrella M, Gasperini R, Pasqualicchio M, Vela G. Copper and zinc body levels in inflammation: an overview of the data obtained from animal and human studies. Agents Actions 1993; 39:195–209. 33. Siller GM, Seymour GJ. Kinetics and specificity of nickel hypersensitivity in the murine model. Australasian J Dermatol 1994; 35:77–81. 34. Eley BM. Tissue reaction to implanted dental amalgam, including assessment by energy dispersive X-ray micro-analysis. J Pathol 1982; 138:251–272. 35. Gniewek J, Pezy J, Baker BG, Bokris JOM. The effect of noble metal addition upon the corrosion of copper. J Electrochem Soc 1978; 125:17–23. 36. McNamara A, Williams DF. The response to intramuscular implantation of pure metals. Biomaterials 1981; 2:33–40. 37. McNamara A, Williams DF. Enzyme histochemistry of the tissue response to pure metals implants. J Biomed Mater Res 1984; 18:184–206. 38. Yli-Urpo A, Parvinen T. Metal degradation and tissue accumulation following subcutaneous implantation of combinations of materials. Proc Finn Dent Soc 1980; 76:124–128. 39. Schmalz G, Arnholt-Bindsley D, Hiller K-A, Schweikl H. Epithelium-fibroblast co-culture for assessing mucosal irritancy of metals used in dentistry. Eur J Oral Sci 1997; 105:86–91. 40. Borenfreund E, Puerner JA. Cytotoxicity of metals, non-metal and metalchelator combinations assayed in vitro. Toxicology 1986; 39:121–134. 41. Romeu-Moreno A, Aguilar C, Arola L, Mas A. Respiratory toxicity of copper. Environ Health Perspect 1994; 102(suppl 3):339–340. 42. Schedle A, Samarapoompichit P, Rausch-Fan XH, et al. Response of L-929 fibriblasts, human gingival fibroblasts, and human tissue mast cells to various metal cations. J Dent Res 1995; 74:1513–1520. 43. Reuling N, Pohl-Reuling B, Keil M. Histomorphometrische Untersuchungen der Gewebevertraeglichkeit dentaler Legierungen. Deutsche Zahnaerztliche Zeitschrift 1991; 46:215–219. 44. Gate Y, Niisat N, Sakurai T, Furuyama S, Sugiya H. Comparison of the characteristics of human gingival fibriblasts and periodontal ligament cells. J Periodontol 1995; 66:1025–1031. 45. Ratkay LG, Waterfield JD, Tonzetich J. Stimulation of enzyme and cytokine production by methyl mercaptan in human gingival fibroblasts and monocyte cell cultures. Arch Oral Biol 1995; 40:337–344. 46. Brosin A, Wolf V, Mattheus A, Heise H. Use of XTT-assay to assess the cytotoxicity of different surfactants and metal salts in human keratinocytes (HaCaT). Acta Derm-Venereol (Stockh) 1997; 77:26–28.

7 Copper Hypersensitivity: Dermatologic Aspects—Overview Jurij J. Hostyńek and Howard I. Maibach Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

INTRODUCTION Reports of immune reactions of both the immediate and delayed types due to cutaneous or systemic exposure to copper have been reviewed, in the endeavor to draw a comprehensive profile of the immunogenic potential of that metal and its compounds. Also the metal’s immunotoxic potential is briefly reviewed. In principle, as noted for other transition metals, the electropositive copper ion is potentially immunogenic due to its ability to diffuse through biological membranes to form complexes in contact with tissue protein. Based on predictive guinea pig test and the local lymph node assay, copper has a low sensitization potential. Reports of immune reactions to copper include immunologic contact urticaria (ICU), allergic contact dermatitis, systemic allergic reactions, and contact stomatitis, but considering the widespread use of copper intrauterine devices (IUDs) and the importance of copper in coinage, items of personal adornment, and industry, unambiguous reports of sensitization to the metal are extremely rare, and even fewer are the cases that appear clinically relevant.

This chapter, in part, was reprinted from Hostyńek JJ, Maibach HI. Copper hypersensitivity: dermatological aspects—an overview. Rev Environ Health 2003: 18:153–185, with permission.

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Reports of immune reactions to copper mainly describe systemic exposure as cause to IUD, and to prosthetic materials in dentistry, implicitly excluding induction of hypersensitivity from contact with the skin as a risk factor. We provide a diagnostic algorithm that might clarify the frequency of copper hypersensitivity. Objective Intent of the overview is to establish a synopsis of dermatologic immune reactions ascribed to copper exposure, and to examine the criteria applied in such diagnosis, since not always has such causation been demonstrated unequivocally. The review discusses metallurgy of copper, predictive and diagnostic tests and describes types of immune reactions and the potential for the copper ion to act as sensitizer, followed by critical examination of literature reports applying strict diagnostic criteria, with consideration given to a number of confounding factors that may have led earlier investigators to erroneous interpretation of signs, symptoms, or test results. The last decade has seen a marked expansion in interest in metalallergic contact dermatitis—from a focus mainly on nickel and chromate to currently gold, cobalt, palladium, and others. Case report methodology now is much of the literature citations in this area. Here, we critically review the citations and suggest diagnostic criteria that might clarify how often copper hypersensitivity occurs in man. The skin is a target organ and indicator for allergy. While the stratum corneum (SC) is a partial barrier to the passive penetration of allergens, to electrophilic, protein-reactive metals in particular, live tissue of the epidermis and dermis actively process penetrants or systemically absorbed allergens that reach it. Such immune reactions to chemicals in the skin are broadly categorized into two distinct classes: 1. Allergic contact dermatitis (ACD) or delayed-type reactions mediated by allergen-specific T lymphocytes. It expresses as a wide range of cutaneous eruptions upon (a second) dermal contact or systemic exposure to haptens in individuals with preformed cellular immunity (type IV allergic reactions). 2. ICU or immediate-type hypersensitivity, which involves IgE antibody. The latter most notably results in respiratory allergy, but can also manifest in separate stages collectively described as ‘‘contact urticaria syndrome’’ (1), local or generalized urticaria, urticaria with extracutaneous reactions such as asthma, rhinoconjunctivitis, and gastrointestinal involvement, and ultimately anaphylaxis (type I reactions). Copper has been alleged to sensitize de novo on systemic exposure following inhalation or implantation. The resulting dermatosis thus induced is

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described as systemic contact dermatitis or urticaria (2–5). Copper complexes are also known to elicit skin reactions upon systemic challenge in the previously sensitized organism (6). METALLURGY OF COPPER AND ITS ALLOYS, AND ITS ROLE AS SENSITIZER Dissimilar metals, combined in alloys for the fabrication of medical devices such as dental materials, evoke currents in electrolytic media such as saliva and degrade, resulting in a steady release of metal ions. In immediate proximity of dental restorations or IUDs, this can lead to adverse (intraoral or intrauterine) reactions such as lichenoid lesions of the oral or genital mucosa. Beyond local effects at the implant site, ions can be transported into distal tissues such as the skin, giving rise to pathological processes such as manifest allergic reactions. Among the metals that commonly form allergenic ions are nickel, cobalt, chromium, and mercury. Exposure type, duration, and environmental conditions (sweat, oxygen supply) in proximity of the metal are critical for mobilization of ions leading to induction or elicitation of immune reactions. As most articles of common human contact are alloys and not made of the pure metal itself, electrochemical interaction between components are significant for the release of allergenic ions potentially leading to immune reactions (7). Reports of copper as immunogen are few, and rarely could the clinical relevance of copper sensitivity be demonstrated with certainty. Consequently, the question as to incidence or prevalence of copper sensitivity among the general population is moot, the number of cases too low to express as percentages. Nevertheless, two characteristics of copper in contact with tissues put the metal into a category that renders appropriate a discussion of its role in inducing reactions in the immune system. Copper belongs to the family of electrophilic transition metals, which makes the copper ion highly protein reactive, i.e., likely to be haptenized, thus recognizable by the immune system as non-self or foreign. Although belonging to the nobler metals highly resistant to corrosion (oxidation, dissolution), in the physiological environment (IUD, dental materials, implants) or in contact with skin exudates, elemental copper is converted to diffusible forms that can penetrate biological membranes. This latter factor merits detailed discussion, also to lay the groundwork for demonstrating how copper and other metals eventually become biologically available from contact with endothelial and epithelial barriers. The oxidation of copper (0) in body fluids has been investigated as a factor that may determine induction or elicitation of immune reactions. Release of metal ions experimentally determined in synthetic body fluids may not adequately mimic the degree of corrosion (oxidation, release) as it occurs in contact with live skin or in the physiological environment,

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however. This is due to the fact that composition of such media used for routine experimentation lacks important components that, in contact of foreign materials with a living organism, determine the nature of reaction product, the rate of reaction, and thus the path of diffusion of the end products through biological barriers. These formulas appear to omit important factors, for instance those present in skin exudates, which can play a determining role in metal oxidation: proteins, and, most importantly, free fatty acids in the sebum (8–13). Together with metal ions the latter are likely to form lipophilic soaps, presumably diffusible via the intercellular lipid matrix of the SC. Evidence for skin diffusivity in a model experiment was obtained by in vivo application of copper oleate over 24 hours on human back skin. Urinary copper levels were subsequently seen to increase significantly over several days (14). While it is a good indication of facilitated permeation, that result in itself does not indicate the actual path followed by the permeant, however. Evidence for an actual path of diffusion was obtained in a different experiment; localization of copper in the intercellular spaces was made visible through electron microscopy following application of copper acetate on human skin (15). Human plasma or serum are the most corrosive physiological media and can play a decisive role on the path towards systemic immunization. Comparative tests simulating corrosion of implant metals in vitro demonstrated that the electrochemical process of oxidation in the presence of enzymes, proteins, and other components of actual serum is accelerated in comparison to standard simulating media (16). Corrosion testing of implants thus becomes more relevant for in vivo conditions when it is conducted in a proteinaceous medium (whole blood, serum, saliva) (16,17). The present synopsis of hypersensitivity cases arising from contact with copper amply, albeit indirectly, confirms the diffusivity of copper derivatives through biological barriers. In addition, copper derivatives (abietate, naphthenate, oleate, sulfate, 8-quinolinolate) used as pesticides are reported to act as irritants when coming in contact with the skin—evidence for their diffusion beyond the SC, reaching the live strata of the skin (18–20). The practice of using copper compounds, including metallic copper, as patch test materials for diagnostic purposes in dermatology also is based on empirical evidence gathered for their diffusion to reach the live strata of the epidermis when applied under occlusion. Finally, conversion of copper metal to diffusible compounds has been demonstrated in our laboratory in a semiquantitative manner (unpublished data). The SC of human volunteers was analyzed in depth for copper content following application of finely distributed metal on the skin under semiocclusive conditions. After application of the metal as micronized powder on the volar forearm for periods up to 72 hours, inductively coupled plasma mass spectroscopy analysis of sequential tape strips showed that the gradients of copper distribution profiles increased proportionally with


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occlusion time, from 24 to 72 hours, rising to 10 ppm after the longest period, significantly above the initial background level of 2 ppm. PREDICTIVE IMMUNOLOGY TEST RESULTS FOR COPPER Thus far, copper has been tested for sensitization potential in two predictive tests: the guinea pig maximization test (GPMT), a standard method used as predictor of skin sensitization potential, and in the local lymph node assay (LLNA) (21,22). In the GPMT, on 20 guinea pigs Boman et al. (23) noted two positive reactions at 24 hours and seven at 48 hours after using 1% copper sulfate pentahydrate in pet. Karlberg et al. (24) later found no difference between copper-exposed and control animals at 1% to 0.1% CuSO4 in pet. Basketter et al. (25,26) obtained a 0% response in the same test, but later in the LLNA the result was positive. In the LLNA adapted to test for allergenicity of metal salts also, under modified conditions, cupric ion significantly increased lymph node cell proliferation. Testing of cupric ion as the chloride in dimethyl sulfoxide at 1%, 2.5%, and 5% concentrations showed significant increases in lymph node cells proliferation, with ratios of test to control lymphocyte proliferation of 8.1, 13.8, and 13.6, respectively. Also, mice could be sensitized in the LLNA by application of copper (II) sulfate (27,28). When the National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods tested cuprous chloride in the LLNA, that copper salt was also found to increase lymph node cells proliferation, resulting in a positive test reading (29). DIAGNOSTIC TESTS FOR HYPERSENSITIVITY A differential diagnosis of chemically induced urticaria (ICU), immediatetype irritant (non-immunologic contact urticaria) and ACD is sometimes difficult, particularly when dealing with strong irritants. In simplest terms, it is mainly based on concentration of the xenobiotic (agent) necessary to induce a skin reaction, and on the time course of reaction. The Open Test The material is applied to intact skin or slightly dermatotic skin, with wheal and flare developing in minutes, a positive indication of or ICU (see above). The SPT for Immediate-Type Allergy (Contact Urticaria) Sensitization is defined as a positive skin prick test (SPT) response with or without clinical symptomatology. One drop (20 mL) of putative allergen in an appropriate solvent (e.g., propylene glycol), vehicle (negative control),

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and histamine in physiological saline (positive control) is placed on three separate sites on the volar aspect of the forearm. Using a sterile prick device inserted through the drop, the underlying superficial epidermis is gently pricked. One needle is used per skin site and discarded. Immediately after pricking, each skin site is blotted dry. After 15 to 30 minutes the skin sites are evaluated for wheal and flare response. An edematous reaction (wheal) of at least 3 mm in diameter, surrounded by a flare, and at least half the size of the histamine control is considered positive in the absence of such a reaction in the vehicle control. SPT positives are retested to confirm the response. Ultimately, diagnosis should be based on clinical history and negative controls (30). Unlike with the open test, controls are mandatory. Radioallergosorbent Test Immediate allergic hypersensitivity can be diagnosed by radioallergosorbent test (RAST), an in vitro immunologic procedure designed to detect specific IgE antibodies in serum (31). Initially, a hapten–protein conjugate between a reactive compound and human serum albumin (HAS) has to be synthesized for the radioimmunoassay. The allergen (hapten–protein conjugate) is coupled to a paper disc. IgE antibodies in a serum sample, which are specific for the conjugate, bind to the conjugate epitopes on the disc, and the portion of bound IgE is detected by 125I-labeled anti-human IgE. Usually, the results are expressed as a percentage of the total activity, the ratio between the binding to the hapten-HAS disc and a disc onto which HAS had been coupled and run in the same experiment. The RAST Inhibition Test Also cross-reactivity of various haptens can be determined by the RAST method. Serial dilutions of conjugate are allowed to react with an individual’s serum. The mixture is then used for RAST determination. Degree of reduction of the serum RAST values after absorption are expressed as percent inhibition (32). The Patch Test for Delayed-Type Allergy (ACD) The key diagnostic tool for ACD is patch testing. Objective is to reproduce the skin reaction to a suspected allergen under controlled conditions, by dosing the substance (or a standard series of allergens) in a suitable vehicle at a nonirritant concentration on adhesive tape and placing it on the skin. Penetration through the SC is promoted by airtight occlusion. The test is left on for at least 48 hours. Some agents elicit reactions only after a substantial delay (late phase reactions), such as copper or gold, possibly due to their known anti-inflammatory activity (33–39). Compounds of poor skin diffusivity such as transition metal salts may produce reactions with considerable delays also (36,37).


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It is suggested that in order to resolve doubtful cases a more differentiated approach using dilution series is advisable for diagnostic purposes. This would include using a dilution series rather than the standard practice of applying the single 1% or 2% copper sulfate solution for patch test assessment of allergic response to the metal salt. Detection of allergens by patch testing with salts dissolved in water, dispersed in petrolatum, or in their elemental form, and subsequent removal of the allergen resulting in clinical improvement are the simplest and most direct connections between cause and effect. As described in greater detail below, a confounding factor in etiology and diagnosis for a number of transition elements, and particularly in the case of copper, is the well-documented cross-reactivity with other metal ions, primarily nickel, but also palladium. These are reactions occurring when haptens of similar size and electron shell configuration are transferred to the same carrier protein. In fact, in the majority of copper sensitivity cases reported, the patients, when tested for multiple metal allergies, were positive to two or more metals, nickel being the most frequent one (40). That is an allergen that can induce clinical manifestations in even minute amounts, and is ubiquitous in the normal environment (41). Thus, false-positive reactions to copper may be due to presence of trace contaminants in the putative cause for allergy, e.g., the IUD, or even in the diagnostic test material, e.g., the metal disc (also see later). Because of the inherent irritancy of copper sulfate under patch test occlusion, and the relatively small number of normal volunteer controls used, we estimate that the nonirritating dose for diagnostic testing approximates 1% to 2% in petrolatum. For verification of copper allergic hypersensitivity, application of 1% copper sulfate in water or petrolatum is recommended by the International Contact Dermatitis Research Group, or of an occluded copper (metal) disc over 2 to 4 days. Petrolatum is the recommended vehicle for copper sulfate, although uniform distribution of the crystalline salt is problematic and poor penetration from petrolatum makes that choice less than ideal. Any positive reactions warrant further evaluation to ascertain clinical relevance (42). One alternative method advocates the use of metals in the elemental state for diagnostic skin tests, and several authors have used copper discs or currency for patch testing (33,43–53). The diagnostic value of this approach is put in question by one investigation, however, where metallic copper was immersed in synthetic sweat to analyze for metal release. Over 24 hours, the final copper concentration was 0.01%, considered by the authors to be too low to elicit a reaction except in highly sensitized individuals (54). Role of Vehicle in Patch Testing In choosing a vehicle for percutaneous penetration, a factor for consideration is the effect it will have on the skin membrane and thus its barrier

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properties, since the solvent of a xenobiotic (metal) can significantly influence its diffusivity and thus bioavailability. Petrolatum, for instance, is a poor solvent for metal salts because the permeant remains suspended as fine particles affording less than ideal uniformity in skin contact, but on the other hand it has an occlusive effect that would increase skin hydration and thus promote diffusion of a hydrophilic compound. Another solvent that enhances penetration is dimethylsulfoxide (DMSO) (see later for mercury chloride). As an instance, Sharata and Burnette point to dimethyl formamide and dimethyl acetamide associated with DMSO, which cause swelling of basal SC cells and disrupt the normal keratin pattern. They located the electron-dense metal ions mercury and nickel in the intercellular spaces and corneocytes, whereas in control membranes those metals were seen almost exclusively in the intercellular space. Thus, certain solvents may modify intercellular solute diffusion to include the transcellular path (55). How the nature of the vehicle can either influence the rate of release of a compound or modify the barrier properties, thus determining the level of percutaneous absorption of xenobiotics, is illustrated by further examples from the literature. An instance of practical importance is the choice of vehicle in standard diagnostic skin patch testing for sensitization, with the aim of optimum release of allergen into the viable epidermis while avoiding allergic or irritant contact dermatitis, leading to false-positive reactions caused by the vehicle itself. The enhancing effect on skin penetration by DMSO was demonstrated by experimental results on guinea pigs in vivo. The use of neat DMSO as vehicle for 0.239 M HgCl2 significantly increased the skin penetration of the compound relative to water as vehicle, and thereby the percutaneous toxicity (56). Mortality for the test animals at three weeks post-treatment increased from 20% with water to 80% with DMSO. Permeability coefficients for mercury in DMSO averaged 2.7 103–4 103 cm/hr for the first five hours, as compared to 0.44 103–1.47 103 cm/hr when the carrier was water. Poorer penetration of salts formulated in petrolatum was demonstrated repeatedly. Fullerton et al. (57) explored the effect of water as vehicle for NiCl2 and of petrolatum for both NiCl2 and NiSO4 at l.32 mg Ni/mL through in vitro experiments with full-thickness human skin. Petrolatum was the poorest vehicle for NiC12 as less than 1 mg Ni/cm2 reached the receptor in 70 to 98 hours compared to 12.6 mg Ni/cm2 in water. For NiSO4 in petrolatum, a poorer penetrant than NiCl2, no detectable nickel reached the receptor phase over 163 hours. The permeability coefficients for 2.4% zinc chloride through human skin in vitro were compared when applied from petrolatum and from a hydrogel over 72-hour periods. The permeability coefficient from petrolatum was 0.082 104 cm/hr. Applied from the hydrogel base, the permeability coefficient was 0.29 104 cm/hr, more than three times higher (58,59).


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One method for determining the optimal solvent in diagnostic skin patch testing of allergens is its application on hypersensitive patients and recording the ratio of positive elicitation reactions. Thereby the solvent is identified, which better promotes diffusion of the xenobiotic. The reaction threshold to nickel sulfate was tested in 53 sensitized patients in both water and petrolatum at equal concentrations (390 ppm) (60): in petrolatum three patients were positive, and five to the aqueous solution, leading to the conclusion that the mean reaction threshold for nickel sulfate in water is lower (0.43%) than in petrolatum (51%) due to better diffusivity in the former. Also the irritation potential of a chemical can be assessed by application in different solvents in dermatological diagnostics aiming to minimize the chemical’s potential for irritation and thus false-positive reactions. The irritant reactivity of nickel salts in petrolatum was found to be greater than in that of water (61). These two concordant examples from dermatological practice serve to illustrate the differentiated promotion of diffusivity by different vehicles. From experience in dermatological practice, particularly in consideration of the clinical picture emerging from the few cases that document copper allergy, and because of the complexity of the irritant dermatitis syndrome, adhering to the criteria set out in the Operational Definition of ACD is recommended when a definition of clinical relevance is sought (42,62). TEST CONCENTRATIONS FOR COPPER ACD Since relatively few dermatotoxicological investigations have researched copper’s characteristics as allergen, no definite value has been assigned as to copper sulfate’s threshold-inducing sensitization, nor is an optimal concentration defined that would reliably elicit reactions in the sensitized organism. Thus, the patch test doses vary: 1% aq. (Gruppo Italiano di Ricerca Dermatiti da Contatto); 1% to 2% pet.; in a dental screening tray concentrations include 1% aq. and 2% aq. (63,64). IMMUNOGENIC POTENTIAL OF COPPER Systemic ACD Systemically induced allergic disease that can be caused by T-cell-mediated reactions to metals (copper) (65), potentially occur when copper or coppercontaining alloy materials used in IUDs, implants in replacement surgery or orthodontic appliances, are oxidized with release of free copper ions. These are absorbed through the epithelia and carried to the skin and the mucosa via blood and the lymphatic circulation. There the allergen is intercepted by antigen-presenting cells and recognized by T cells that migrate to the lymph nodes with blastic transformation, proliferation of cytotoxic lymphocytes, and production of cytokines. These in turn recall neutrophils and eosinophils to the reaction site, cause capillary dilation and increased


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permeability, resulting in cutaneous inflammation appearing as wheal and flare (Menne, 1996 membrane); lichen planus and asymptomatic contact hypersensitivity (dental alloy contact dermatitis) are increasingly being linked with oral exposure to materials used in dental fillings, orthodontic prostheses, cements and components of dentures, bridges, bands, and wires. Such reactions can be either immunologic contact stomatitis or systemic anaphylactic stomatitis (type I reactions), or delayed contact stomatitis (type II). In a few instances, copper was implicated as possible cause for the latter, as copper is commonly a part of alloys used in dental materials (Table 1) (33,66,67). In a study investigating the release of copper from a selection of orthodontic appliances in organic and inorganic solutions made up to different pH values to imitate the oral environment, Stoffolani et al. found that the levels of metal mobilized were well below those ingested with a normal daily diet. From that of result they concluded that the quantities released should be of no concern. The relevance of that conclusion, particularly for purposes of immunology, invite further discussion, however, to be pursued elsewhere (68). A study of professionals (dental technicians, orthodontists and their assistants) involved in making and handling such materials reveals that in Table 1 Copper (0) and Nickel (0) Content (wt%) of Dental Materials Alloy

Ni, wt% (other metals)

Cu, wt%

Copper (0) JSC—gold alloy

100 11.5

Degussa Training Metal Trindium

87.5

Duracast MS

81.6

Goldent

76.0

Modulay

77.0

ANA 68

5.0

87.0

DispersalloyTM

12.4

ValiantTM

20.0

Gallium alloy GF

15.0

N/R N/R (Au, Ag, Pt, Zn, Ir) N/R (Zn, Sn, Co) 1.0 (Al, Mn) 4.1 (Al, Fe, Mn) 0.5 (Al, Zn, Mn) N/R (Au, Ag, Pd) N/R (Ag, Sn, Hg) N/R (Ag, Sn, Zn) N/R (Ag, Sn, Pd) N/R (Ag, Sn, Pd)

Abbreviation: N/R, none reported.

Manufacturer AB JS Sjo¨ding, Kista, Sweden AB JS Sjo¨ding, Kista, Sweden Hereaus Kultzer GmbH, Hanau, Germany Trindium Corp. of America, Los Angeles, California, U.S.A. Duracast Inc., Brasilia, Sao Paulo, Brazil Goldent Inc., Brasilia, Sao Paulo, Brazil J. F. Jelenko, Armenac, NewYork, U.S.A. AB Nordiska Affineriet ANA, Helsingborg, Sweden J & J Dental Products Co., Tallaght, Dublin, Ireland L.D. Caulk Co., Dentsply Intl, Inc., Milford, Delaware (U.S.A.) Tokuriki Honten Co. Ltd., Tokyo, Japan


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handling dental devices they also run the risk of developing hypersensitivity to allergenic materials, metals among them, as in one study 40% of orthodontists and 43% of dental assistants reported work-related skin problems (69). Copper Intrauterine Devices Copper metal in contact with biological substrates (as in IUDs) is highly reactive and releases free copper ions. Release in vivo was determined at 0.71 I-1 micro mol/day (45 I-1 micro g) from a surface area of 200 mm2 and 1.29 I-1 micro mol/day (82 mg) in a culture medium in vitro (70). After it was discovered that copper metal placed in the uterus of animals had a contraceptive effect (71), the principle was applied to humans: a plastic T-shaped device with copper wire or a copper sleeve was introduced as a pharmacologic agent and became widely used as an IUD to regulate fertility. Research suggests that copper prevents fertilization rather than implantation (72). Reports of untoward reactions by women using the IUD (generalized eczema, edema) sometimes mention that the condition worsens during the perimenstrual period of the cycle, and patients often test positive to patches of copper as metal or as the sulfate (48). Upon removal of the device patients usually experienced complete remission. Dual Immune Response to Copper While organic compounds infrequently cause both types of reactions, dual immune response appears more common for metals and metallic compounds. Their reactivity towards protein results in a complete antigen that triggers both IgE antibody production (type I) and cellular (T cell, type IV) immune reactions. Immunogenic effects that result from exposure to metals can be attributed to the same factors that determine their toxicological and biological effects. Metal ions in general, and certainly those belonging to the transition group of elements, such as copper, have an ionic radius too small to be antigenic. Containing a partially filled d shell, these metals oxidize to highly electropositive cations that can act as haptens interacting with tissue protein. They form bonds that range from fully ionized to fully chelated complexes, and have the ability to modify the native protein configuration. These are recognized as non-self by hapten-specific T cells in the host immune system (73), leading to allergic reactions of the two different types. Copper is one of several metals causing more than one type hypersensitivity presenting with multiple symptoms in allergic responses, in part depending on type of exposure: immediate type, immunologic contact urticaria sometimes associated with respiratory hypersensitivity, delayed-type cutaneous hypersensitivity, systemic allergic reactions, and contact stomatitis (2–4,33,45,47,51,66,74,75). Concurrent occurrence of immediate- and delayed-type sensitivity has also been observed in the same individual (50,76).


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It should be noted, however, that, overall, human case reports of copper-induced immunologic reactions are rare. The Major Factors with the Potential to Induce Copper Sensitivity Dental Materials Over the past 15 years, there has been considerable effort to replace the use of traditional materials such as mercury in dental restorative work, and dental casting alloys contain increasing amounts of copper. This use of various substitute, metal-based materials has proceeded without the necessary corollary knowledge of their irritant and allergenic potential, however. In reports of contact stomatitis (contact allergy of the oral mucosa), no reports of rechallenge have been published which would strengthen the causal association with copper. Tables 2–4 list the different types of allergic reactions associated with exposure to copper. Recommended Patch Test Procedure in Suspected Copper Allergy of the Delayed Type Establish clinical history (anamnesis) determining nature of contact and physical form of putative allergen. Physical examination of the patient. Patch testing with 2% CuSO4 in petrolatum. In case of positive outcome follow up with serial dilution patch testing (1%, 0.5%, and 0.1%). Repeat open application test (ROAT) or provocative use test (PUT) with a dilution series of CuSO4: 2%, with at least 10 na€ve control subjects to demonstrate that positive reaction is not irritant in nature, then further at 1% and 0.5%. The substance is applied once or twice daily for 14 to 28 days (107,108). A positive reaction usually appears within four days, less frequently between five and seven days. Delayed reactions have been noted in patch testing with copper. To confirm positive patch test reactions and identify false-negative reactions on patch testing, intradermal tests may be considered (109). Herbst et al. (110) provide the scientific background of intradermal testing for ACD. As an ‘‘alternative’’ predictive test for ACD, the local lymph node assay was developed on mice for the detection of contact allergens (22). It has been adapted to test for allergenicity of metal salts also. Under modified conditions, cupric ion was seen to significantly increase lymph node cell proliferation, as mice could be sensitized by application of copper (II) sulfate (27,28).

Adhesive pads

Occup. exposure to copper

1

1

Diagnostic test

Urticaria

Pruritus and urticaria Rhinitis Urticaria, flushing, pruritus Urticaria, eczema

Copper metal 2% aq. CuSO4

Cu prick test Cu metal, 5% aq. CuSO4, actyl choline patch Open/closed patch 5% aq. CuSO4

Scratch, CuSO4

Generalized rash None Bronchial asthma Cu/NH3 inhal.n Generalized uricaria Scratch, 1% aq. CuSO4

Visible signs and symptoms

Abbreviations: CR, clinical relevance; IUD, intrauterine device.

2 1

1

Dental Occup., Cu–ammonia Dermatology patient (IUD) Dermatology patient (IUD) Patients (IUD) Patient (IUD; dental)

Cohort/etiology

1 1 1

Cases

Table 2 Immunologic Contact Urticaria Due to Copper

1–2 1?

2

1? 1 2

CR

Pos. to copper acetyl 3 acetonate/urticaria; not to Cu metal Positive at 20 min. Positive 3 at 24 hr

Positive Punctate wheals

Positive

None Dyspnea Erythema

Challenge reaction

50

76

80 47

79

77 78 2

References

Copper Hypersensitivity 127

Metal contact Dermatol. patients Teleph. lineman Jewelry

Metalworker

Furniture polishers Welder Dermatol. patients Agricultural Jewelry Dermatol. patients/jewelry Occupational Dental patient Dentistry students Enameller Jewelry Painter Agricultural Dermatol. patients Dermatol. patients

1 2 1 1

1

10 1 6 4 2 140

Abbreviation: CR, clinical relevance.

1 1 2 1 3 1 5 3 1

Dermatol. patients

Cohort/etiology

3

Cases

Itching, eczema Oral symptoms None Dermatitis Dermatitis Itching, edema Dermatitis Oral symptoms Dermatitis

Dermatitis Eczema Eczema Dermatitis Dermatitis Dermatitis

Eczema

Dermatitis Dermatitis Dermatitis Eczema

Hand eczema

Visible signs and symptoms Patch test

2% CuSO4 aq. 1% CuSO4 pet. 1% CuSO4 aq. 5% CuSO4, pet. 2% CuSO4 pet. 10% Cu pigment, in pet. 2% CuSO4, pet. 1% CuSO4 2% CuSO4, pet. Cu metal

Copper metal Brass metal 1% aq. CuSO4 10% aq. CuSO4 Copper metal Copper coins 1.25–5% aq. CuSO4 2% aq. CuSO4, multiple salts 5% aq. CuSO4 0.1–2% CuSO4 pet. 0.25–5% CuSO4 pet. 1% CuSO4, pet. 5% CuSO4 aq. Copper metal

Table 3 Allergic Contact Dermatitis Due to Copper

Necrosis at 24 hr Positive patch Positive patch Positive Positive to Cu and Ni Positive patch Positive at 48–96 hr Positive patch Positive at 72 hr Positive at 72 hr

Positive Positive Positive patch Positive patch Positive Positive at 72 hr Positive at 24 hr Multiple positives, including Cu(II) Positive–irritation? Positive at 72–96 hr Positive to Cu and Ni Positive at 24–72 hr Positive to Cu and Ni; irr? Positive

Challenge reaction

1 0–1 1 0–1 0–1 1–2 0–1 0–1 2–3

50 87 88 89 90 91 92 93 53

84 74 85 19 86 49

83

0–1 1? 2 0 0 1? 0–1

81 82 44 45

43

References

2–3 0–1 1 1–2

0–1

CR

128 Hostyńek and Maibach

1

1 2

3 1 2

1 1

Multiple metal sensitivities

Nephritis Oral lesions

Eczema, edema Edema, erythema None

Dermatol. patients (IUD) Generalized dermatitis, itching Dermatol. patient (IUD) Generalized dermatitis Dermatol. patient (IUD) Pruritus and dermatitis

3

Dermatol. patients (IUD) Dermatol. patient (IUD) Dentistry students, staff, patients Dermatol. patient (IUD) Dermatol. patients (dental) Dermatol. patient (dental)

Dermatol. patient (IUD) Generalized dermatitis Dermatol. patients (IUD) Generalized dermatitis

1

1

1 4


Dermatol. patient Lychen planus (dental) Dermatol. patient Rash (dental) Dermatol. patient (IUD) Generalized eczema

Cohort/etiology

1

Cases

Patch test

97 98 99

1? 1 0–1 1 1

Pos. to CuSO4 and NiSO4 Positive patch at 72 hr

Cu and other metal patches

Eczema

95 96

2 0–1

CuSO4 5% aq. CuSO4

48

0–1

(Continued )

51

100 67

94 4 1

1 2

3

2

33 46

2–3

Positive at 96 hr, negative

References

0–1

CR

Challenge reaction

Aerythema, vesicles, excoriation Positive, negative to other 5% aq. CuSO4 metals 5% CuSO4 10% NiSO4 Positive 2% aq. CuSO4 Positive patch 1 pos. to NiSO4 0.01% CuSO4 Cu metal Pos. patch at 24/48 h. Pos. to Cu metal at 24 hr Positive patch 1% aq. CuSO4 0.1%–1% CuSO4 Positive patch at 24 and 72 hr None CuSO4 1% aq. CuSO4 Vesicles and spongiosis 5% aq. CuSO4 Positive at 24 and 72 hr

Cu metal, CuO 1% aq. CuSO4 Cu metal

Table 4 Systemic Allergic Contact Dermatitis Due to Copper

Copper Hypersensitivity 129


Dermatol. patient (dental) Dermatol. patient (dental) Orodynia, lychen planus

Lychen planus

Dermatol. patient Gen. oral eruptions (dental) Dentistry students Metal allergies Dermatol. patient (IUD) Perimenstrual angioedema Dermatol. patient (IUD) Generalized skin reactions Dermatol. patient (IUD) Perimenstrual dermatitis

Cohort/etiology

2% aq. CuSO4, other metal salts 2% aq. CuSO4, other metal salts 2% aq. CuSO4

1% aq. CuSO4 0.01–1% aq. CuSO4 Cu metal Copper salt (?)

1% CuSO4 pet.

Patch test

Systemic Allergic Contact Dermatitis Due to Copper (Continued )

Abbreviations: CR, clinical relevance; IUD, intrauterine device.

3

1

1

1

40 1

1

Cases

Table 4

Positive to Cu and other salts at 4 days Positive at 48 hr

Positive to copper and silver salt Positive

Positive patch Positive patch, positive

Positive patch

Challenge reaction

68 106

0–1

105

104

102,103 52

101

References

0–1

1?

0–1

0 1

0–1

CR



131

Recommended Screening Procedure in Suspected Copper Urticaria

Open test: Application on healthy skin first and observation of the test area for 60 minutes. If reaction is negative, on previously affected skin (as suggested by patient’s anamnesis) spreading of 2% aq. CuSO4 on a 3 3 cm area. Immunologically mediated reactions usually appear within 15–20 minutes, nonimmunologic ones within 45–60 minutes after application (111). This difference in delay is a major distinction between specific and nonspecific contact urticaria. A positive reaction is seen as edema or erythema (wheal and flare). A minimum of 10 na€ve background controls with the test solution is suggested. A nonimmunologic reaction will appear in the controls due to release of inflammatory mediators from the cells without participation of specific IgE antibody (112). A use test is suggested, handling the suspected agent and recreating the original scenario inducing the reaction (108). When open application is negative, a prick test with 2% aq. CuSO4 is suggested. A group of more than 10 background controls is required in prick testing using physiologic saline solution to ascertain that copper does not produce such lesions in normal controls. The occluded application of a copper disk over 48 hours can also confirm suspected sensitization. In case of a positive test the open application may be repeated for verification.

Confounding Factors in Copper Allergy Test Results: Cross-Reactivity, Contaminants, Irritation, and Angry Back Syndrome In many cases where copper allergy is suspected, positive patch tests to copper (as metal or the sulfate) are equivocal, and assignment of clinical relevance can be difficult or impossible because case reports in the literature most often lack relevant details. One element of uncertainty in the diagnosis of copper allergy is its cross-reactivity with other (adjacent) transition metals in the periodic system of elements. Observations of multiple sensitivity to metals have been made frequently, attributed to cutaneous or systemic contact with alloys, and it is challenging for the investigator to ascribe the clinical observation either to concomitant sensitization or to cross-reactivity. Often patients react to compounds that are not the primary sensitizer. Originally, Epstein (82) had raised the question of nickel and copper cross-sensitization in 1955, and since then many cases of simultaneous sensitivity to nickel and copper in the same organism have been reported (Tables 2–5). The immunologic mechanism involved in hypersensitivity to multiple metals and crossreactivity between copper and other transition elements has been investigated in two independent in vitro studies and the event is well characterized now,

190 Enamellers and decorators 12 Dentistry students 60 Dentistry students 46 Agricultural workers 311 Dental patients 233 Metal workers 520 Dermatol. patients 2660 Dermatol. patients

60 Dermatol. patients

1 2 32 5 5 7 3 1

1

1

10 Furniture polishers 37 Patients 1190 Dermatol. patients 652 Agricultural workers 964 Dermatol. patients/jewelry 10,936 Dermatol. patients

Cohort/etiology

10 0 0 4 140

Cases

Dermatitis

Dermatitis None Metal allergies Dermatitis Allergies Metal allergies Oral symptoms Dermatitis

Allergy

Dermatitis Dermatitis Eczema Dermatitis Dermatitis


Table 5 Population Studies of Copper Hypersensitivity

2%–0.01% aq./5% pet. CuSO4 5% CuSO4, pet. 1% CuSO4 aq. 5% aq. CuSO4 2% CuSO4, pet. Metals (?) 1% aq. CuSO4 1% CuSO4 2% CuSO4, pet. Cu metal 1% aq. CuSO4 Copper alloy

5% aq. CuSO4 5% pet. CuSO4 2%–0.125% pet. CuSO4 1% CuSO4, pet. Copper metal

Patch test

89 88 102,103 92 115 116 93 52 40

2

114

84 113 24 19 49

References

0–1 1 0 0–1 0 0 0–1 2–3

1–2

Positive Positive Positive patch Positive patch Positive at 48–96 hr Metals (?) ? Positive patch Positive at 72 hr Positive at 72 hr Positive patch

1? – – 0 0–1

CR

Positive; irritation? Negative Negative Positive at 24–72 hr Positive

Challenge reaction



133

making it possible to put the numerous case reports on copper-induced allergy in better perspective. Specifically, nickel ion-specific T-cell clones appear to be recognized both by copper and palladium ions, but not by others such as cobalt. This reactivity is likely to be favored by their bivalency and proximity to nickel in the periodic table of elements. Investigations showed that among a large panel of nickel-specific T-cell clones four different types of reactivity can occur: reactivity to nickel only, cross-reactivity between nickel and palladium, cross-reactivity of nickel to copper, or to both palladium and copper ion, which both neighbor nickel in the periodic table of elements (117,118). In light of these results, copper-positive patients are now more often screened for allergy to other metals also, but only few among them are found to be truly copper-sensitive. To illustrate the possibility of cross-reactions due to dental materials in particular, a number of commercial alloys are tabulated, with special attention given to the listed presence of copper and nickel (Table 1). Purity of test materials can be a source of diagnostic equivocation with the potential for false-positive results. Copper patch test material may contain nickel as an impurity, as analytical grade copper sulfate was shown to contain up to 0.002% nickel; high-purity copper wire in IUDs, which is also used for skin testing, contain 0.0003% (3 ppm) nickel (24). Note that with metal ACD in humans, highly sensitized subjects can react down to a few parts per million of the hapten (119). A potential cause of false-positive, clinically nonrelevant reactions that can result in patch testing is hyper-reactive skin, also known as the excited skin syndrome or ‘‘angry back’’ (120–122). This condition can result from multiple inflammatory skin conditions or from strong positive patch-test reactions, magnifying adjacent patch test responses or inducing nonspecific reactions. This is a potential occurrence in testing for copper when several different metal patches are simultaneously applied on the patient. Multiple positive reactions may require separate, sequential tests with the involved substances. Finally, several studies, especially those involving retrospective reviews or large population groups, routinely examine skin reactions at 48 hours, missing potential late-phase (72 hr) reactions after patch application (Table 5) (33–39). They may result in false-negative diagnoses and under-reporting of hypersensitivity to copper. Determining Clinical Relevance The open literature has been critically reviewed for clinical relevance of the cases reported. A problem encountered often in the evaluation of diagnostic tests from patients reacting to chemical substances is understanding the clinical relevance of test results, because little or no data are reported to qualify positive results. This becomes particularly difficult in the interpretation of tests, which appear to indicate a compound as primary sensitizer that is known to have no or little sensitization potential, such as copper.


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Benezra et al. have addressed the problem of classification by suggesting a systematic analysis of available data to arrive at an expression of degree of confidence in the results reported by investigators, thus to better define morbidity of a putative allergen. A degree of confidence has been assigned to all cases listed in Tables 2–5, listing the literature reports. Although Benezra et al. (123) designed the system with skin contact sensitizers in mind, which lead to delayed-type reactions, the approach appears more generally valid and is applied to all cases reviewed here. Criteria for Assignment of Degree of Confidence Presence of vehicle-treated or untreated controls Concentration of test substance judged sufficient to elicit a response Use of an appropriate vehicle Purity of test reagent to exclude possible reaction to contaminants Sufficient number of cases for meaningful response The evidence provided in the reports is evaluated towards classification of the agent (copper) as allergen and a degree of confidence on a scale from 0 to 5 is assigned to indicate how well the test results demonstrate that the chemical does or does not induce the immune reaction: 5 ¼ results meet all of the criteria 4 ¼ all criteria met, but number of cases is marginal 3 ¼ parameters such as controls are missing but reports point to substance as sensitizer 2 ¼ controls are absent and there are no other details indicating substance as sensitizer 1 ¼ results not considered to be reliable 0 ¼ test fails all of the criteria Since evaluation of criteria is subjective, degree of confidence should be viewed within a range of 1 of the number assigned. Listed in the following are two categories of reports relative to copper hypersensitivity: populationbased studies (also listed in Table 5), selected from published reports of immune reactions to copper, which surveyed larger samples—random cross-sections of the population, cohorts of specific occupational exposure, wearers of IUDs, dermatological clinic data bases, or groups exposed to copper in dental materials. That section is followed by selected case reports of more anecdotal value. SUMMARIES OF POPULATION-BASED STUDIES Barranco, 1972 Upon review of the literature the author noted six cases of ACD to copper: three cases attributed to contact with brass, and one each to exposure to


135

copper sulfate, copper metal, and jewelry. The author also reported on a case of dermatitis attributed to the use of a copper IUD. Although of questionable clinical relevance due to patch testing with 5% CuSO4, it holds a somewhat historical interest as it is the first report of eczematous dermatitis to copper due to systemic exposure. Tested for the other frequent metal allergens: Ni, Cr, Co, and Hg besides Cu, all patch tests were negative except for a strong reaction to 5% CuSO4. Remission was noted after removal of the IUD (3). Dhir, 1977 A cohort of 10 furniture polishers who had developed skin reactions on handling ethyl alcohol tinted with 5% copper sulfate were tested with that solution and aq. 5% CuSO4. All 10 patients reacted to both materials; the test with the same materials were negative on 15 control subjects (84). Jouppila, 1979 Assessed were 37 patients wearing copper IUD and presenting with skin rashes. Epicutaneous tests for copper, nickel, and cobalt allergy showed reactions to nickel (four) and cobalt (one), but none to copper. The authors concluded that allergy to copper was not likely to be the cause of the side effects (124). Karlberg, 1983 Of 1190 eczema patients tested with serial dilutions (2–0.125%) CuSO4 in pet. over a three-year period, none had a reaction to copper only, 13 reacted to copper and other metals. Thus, no sensitization to copper specifically became evident, leading to the assumption that the (multiple) reactions noted were due to metals contaminating the test allergen. According to Karlberg, highest-grade copper metal contains 0.0003% nickel, analytical grade copper sulfate up to 0.002%. In the GPMT using dilution series of 0.1–0.01% CuSO4 for induction and 1–0.05% in pet. for elicitation, Karlberg determined that copper sulfate was a grade I allergen. In her review of the literature prior to 1982, Karlberg noted four relevant and 20 probably relevant cases of copper hypersensitivity. Over 90 cases were classified as uncertain or not relevant (24). Lisi, 1987 The authors studied the prevalence of irritant or ACD from pesticides by patch tests on 652 outpatients with skin disorders. Of 564 subjects tested with 1% CuSO4, four cases showed positive reactions, none of which were

136


irritant morphology. They presumed that allergic reactions could not be considered of definite relevance due to the scarcity of clinical details. In particular, data are missing on confirmatory retesting of positive tests conducted 2–3 months later (19). Romaguera, 1988 Nine hundred and sixty-four dermatology patients complaining of metal intolerance, experienced mostly in contact with jewelry, were patch tested with standard allergens and metal washers. Of 52% of patients giving positive reactions to nickel, 14% were also positive to copper (among other metals), none to CuSO4 (concentration not given). The relevance of copper sensitivity is uncertain due to the minimal experimental details given, and the probability of contamination of the metal washers used (48). Zabel, 1990 Records on 10,936 patch test reactions collected in a dermatology clinic over the period 1975–1985 were reviewed, in addition to patch tests conducted on 118 patients wearing IUDs. Besides the record of patients with positive reactions to multiple metals (mostly nickel), one eczematous IUD-wearing patient reacted to CuSO4 at 5% in pet. only. After removal of the IUD the eczema resolved. The causative role of copper is uncertain due to lack in supporting evidence in that case (116). Motolese, 1993 The authors reported on skin sensitization to metals encountered in a cohort of enamellers and decorators. Relevance of the only positive reaction to copper was uncertain due to the high concentration of 5% CuSO4 used in the test. Also, too few clinical details were given to establish a firm causeand-effect relationship in that case (89). Kawahara, 1993 The cause of occupational allergies was investigated in a dental technology school by testing a cohort of 12 students with 40 potential contact allergens occurring in the manufacture of prostheses and was determined to be dust, mist, and fumes in their environment. Two reacted to 1% aq. CuSO4; the reactions could not be assessed as to their clinical relevance due to lack of any further details (88). Tschernitschek, 1998 Over the period 1982–1997 in a dental clinic, of 311 patients who were patch tested for dental materials-induced hypersensitivity, 13% showed positive


137

reactions. Most frequent among the sensitizing materials were metals (77 of 107). Three among those reacted to copper and cadmium, two to copper only. Significance cannot be assigned due to total lack of experimental details (115). Candura, 1999 Of 233 ACD outpatients patch tested with the standard GIRDCA test series in a dermatology clinic, three had positive reactions to copper along with other metals, four to copper only. The importance of the causative role of copper cannot be assessed due to a total lack of experimental details (116). Vilaplana, 2000 A testing program including 520 patients with dental prostheses who presented with adverse oral mucous membrane reactions was conducted using a special metal test series that included 1% CuSO4 in pet. Of 289 patients with one or more positive reactions, one patient only reacted to copper, classified as a reaction of past relevance (sic) by the authors; two patients had reactions to copper with unknown relevance (93). Wo¨hrl, 2001 In the endeavor to assess the relevance and diagnostic value of positive reactions to copper, 2660 routine patch tests recorded in an allergy clinic over 2.5 years were screened for positive reactions to copper (2% CuSO4 in pet.) and the other metals in the immediate vicinity in the periodic system of elements: nickel, palladium, cobalt, and mercury. Of 94 cases that were copper-positive, 26 were enrolled in a retest program involving CuSO4 at 5%, 2%, 1%, 0.6%, 0.2%, and 0.05% aq. Testing with copper foil was also included. Of the original 26 inductees, 10 were positive to copper on retesting with 5% CuSO4 in pet., but eight of those also reacted to a nickel patch. Two of 10 showed unequivocally positive reactions to 2% CuSO4 in pet. Two were positive to copper foil. Only one case showed an isolated sensitivity to copper and not to any of the other test allergens, presenting with chronic eczema of the fingertips. That patient’s occupation as electrician would characterize the case as ACD to copper induced through cutaneous contact. One other patient with multiple metal sensitivities appeared to have clinically relevant sensitivity to copper. Presenting with eczema to a golden ring (test to gold negative), the condition resolved when the patient exchanged the gold ring with one made of silver. Although authors concluded on copper–nickel cross-reactivity on the T-cell level in 9 of the 10 cases, with a high statistical association, and copper sensitivity being of low clinical relevance, all reactions cleared at 96 hours, a delay that is typical for irritant reactions rather than ACD (52).

138


SUMMARY OF SELECTED CASE REPORTS OF IMMUNE REACTIONS TO COPPER The process of diagnosis and remediation of copper allergy recorded in the literature so far follows an overly direct and simple path, with little or no clinical data. Solidly worked-up cases are rare, which makes assignment of relevance difficult and is the reason for the low scores in clinical relevance assigned to the reported cases in Tables 2–5. In most cases the examining physician (dermatologist), having confirmed ACD, often by CuSO4 patch test only, without controls or tests with other metals, advises the patient to remove amalgam or antifertility device. As a rule, then, improvement of the condition or complete remission follows promptly. As the signs of allergy are no longer present, no follow-up testing is done. Cases where no improvement was noted have not been published. With few exceptions, sensitization to copper results mostly from two types of exposure: leaching of the metal from dental amalgams, and from copper containing IUDs, due to corrosion in the physiological environment. The majority of signs and symptoms of allergy reported point to the delayed type, ACD, or systemic allergic contact dermatitis (SACD). At first glance, the literature search produced eight records with nine cases of ICU (Table 2), 21 records with 160 cases of ACD (Table 3), and 21 records with 71 cases of SACD (Table 4). Many of the cases, diagnosed by using a 5% CuSO4 patch in pet., are classified as having borderline relevance, because patch tests at that strength are now considered to cause probable irritation—rather than ACD (3,89). A few of the cases listed in the tables merit detailed discussion due to their extraordinary characteristics. A number of them also show that sensitization originally thought to be due to copper actually resulted from exposure to other metal(s) or chemical agents.

SELECTION OF INDIVIDUAL REPORTS OF IMMUNE REACTIONS TO COPPER Frykholm, 1969 An example of SACD due to dental materials was reported by Frykholm et al. A patient with oral lichen planus-1ike reactions to dental restorative materials showed positive skin reactions to epicutaneous tests with copper metal, Cu (II) hydroxide, Cu (II) oxide, and Cu (I) oxide at 72 hours, while the test sites were unreactive at 48 hours. Tests carried out on 20 controls were negative. The patient’s oral lesions flared up in conjunction with the skin tests. On repeated retesting at month-long intervals with metallic copper and Cu (II) oxide, positive reactions to all test materials were until the fourth day (33).


139

The late appearance of the skin reactions (24–96 hr) is characteristic of a ‘‘delayed allergic reaction,’’ to gold in particular due to its anti-inflammatory activity (125,126). The delays in tissue involvement seen with copper may also be ascribed to that characteristic, which makes the metal an effective antirheumatic agent. An alternative explanation for the delayed reaction may involve the protein reactivity and depot formation by copper, resulting in delays in SC penetration as seen in other electrophilic metals, e.g., aluminum, silver, mercury, or chromium known for their retention in the SC (127–131). Shelley, 1983 A case of potential contact urticaria, which defies interpretation, was reported for a woman with occult sensitivity to copper. Wearing an IUD and dental fillings with copper amalgam, she developed urticaria and flushing with pruritus upon physical exercise, emotional stress, or overheating, more severe during the perimenstrual period. On testing, metallic copper and copper-containing coins elicited no response, but on the test sites she developed punctate wheals when challenged with exercise or on injection with acetylcholine in subthreshold concentration. Also challenge with 5% aq. CuSO4 induced the same response as the copper metal. Tests with nickel metal and the sulfate were negative. That cholinergic urticaria reaction observed is attributed to a collaborative effect of acetylcholine and copper on mast cell membranes inducing degranulation, a clinical response that an antibody–antigen reaction on the mast cell surface alone could not produce. No subsequent such patients have been reported (46). Hocher, 1992 A woman with IUD presented with interstitial nephritis and renal failure. Patch tests were positive for copper, nickel, and cobalt. An in vitro lymphocyte-stimulating test with copper was also positive. On removal of the contraceptive device renal function returned to normal. No similar cases have been published (100). Laubstein, 1990 A case of occupational exposure to copper metal presented with an itching eczematous condition, which healed on absence and returned on resumption of work. That highly sensitized individual reacted with necrosis on 24-hour testing with 2% aq. CuSO4, and also on contact with copper metal. Followup brought an urticarial reaction after 20-minute exposure to copper metal, and to serial dilution of aq. CuSO4 after 24 hours, reactions that extended to necrotic ulceration. Upon changing occupation the patient became free of symptoms. This description suggests two mechanisms—ACD documented with a positive response to a copper disk, and immunologic contact urticaria (49).


140

Sterry, 1985 A female patient reported with eczema and pruritus while wearing selfadhesive pads treated with the disinfecting agent copper-acetyl acetonate. Reactions to patches with copper-acetyl acetonate on 12 control subjects were weakly positive at 24 hours, disappearing after 48 hours. The test with metallic copper and CuSO4 on the patient was negative, but positive to the disinfectant at 50% pet. and 50% aq. On open patch testing, the patient was positive to both copper-acetyl acetonate 50% aq. and acetyl acetone (100%) alone. This suggests that allergy was probably due to acetyl acetonate and not copper (76). COMMENTS Many case reports of sensitization attributed to copper may be difficult to classify as such with certainty. Copper sensitivity may overlap with nickel hypersensitivity, or nickel alone may even be the only causative agent, as in dermatological or dental practice they can only be distinguished with difficulty when assessing exposure in the individual patient. As results from several in-depth investigations, patients with a positive test to copper also appear sensitized to nickel, and vice versa. This can be attributed to cellbiological and metallurgical factors: Investigations at the cellular level have established cross-reactivity between the two metals, which may account for the frequency of copper hypersensitivity reported (117,118). At the exposure level, often copper and nickel are associated in IUDs or orthodontic materials. Copper of highest purity still contains traces of nickel; thus, sensitization observed may be concomitant (24,124,132). In dermatological practice, diagnostic test materials copper sulfate or copper metal discs also contain low levels of nickel sufficient to elicit a reaction in an organism highly sensitive to nickel, leading to a false-positive diagnosis. There may be true allergic reactions to copper exposure, topical or systemic: to copper salts, to the metal, or to its alloys. Judging from the cases reviewed so far, such responses are rare. CONCLUSIONS Systemic as well as topical exposure to copper can cause both immediate and delayed-type sensitization. Contact dermatitis and urticaria attributed to copper metal or its compounds has been suggested, with effects from dental materials and IUDs as the main etiological factors. Immune reactions occurring in industry are few, considering the number of copper smelters


141

and refinery workers in daily contact with the metal. The majority of sensitization reports may be due to copper cross-reactivity with nickel and palladium. Thus, true allergic reactions to copper appear rare, particularly those induced by skin contact, which is consistent with copper’s rating as a grade I allergen in the guinea pig maximization test. Most cases of confirmed copper allergy result from its presence in orthodontic materials, and those reactions are mostly of the delayed type. Firmer chemical and epidemiologic judgments will be possible when: 1. Additional experimental data become available on the nonirritating dose(s) suitable for diagnostic patch testing (in petrolatum and water), and in water for prick testing. On the basis of Wo¨hrl’s data, 2% in petrolatum may be appropriate (52). 2. Authors describe their clinical experimental data with details of the several steps as documented in the Operational Definition of ACD (56), specifically re-patch testing upon indication, serial dilution patch testing, and use testing (PUT/ROAT). Those steps will help clarify clinical relevance.

ABBREVIATIONS ACD aq ICDRG ICU IUD LLNA pet. PUT ROAT GPMT

allergic contact dermatitis aqua International Contact Dermatitis Research Group immunologic contact urticaria intrauterine device local lymph node assay petrolatum provocative use test repeat open application test guinea pig maximization test

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5. Menne´ T, Veien N, Sjolin K-E, Maibach HI. Systemic contact dermatitis. Am J Contact Dermatitis 1994; 5:1–12. 6. Veien NK, Menne´ T, Maibach HI. Systemically induced allergic contact dermatitis. In: Menne´ T, Maibach HI, et al., eds. Exogenous Dermatoses: Environmental Dermatitis. Boca Raton: CRC Press, 1991:267–282. 7. Flint GN. A metallurgical approach to metal contact dermatitis. Contact Dermatitis 1998; 39:213–221. 8. Rubin RW, Penneys NS. Subpicogram analysis of sweat proteins using twodimensional polyacrylamide gel electrophoresis. Anal Biochem 1983; 131:520–524. 9. Sens DA, Simmons MA, Spicer SS. The analysis of human sweat proteins by isoelectric focusing. I. Sweat collection utilizing the Macroduct system demonstrates the presence of previously unrecognized sex-related proteins. Pediatr Res 1985; 19:873–878. 10. Marshall T. Analysis of human sweat proteins by two-dimensional electrophoresis and ultrasensitive silver staining. Anal Biochem 1984; 139:506–509. 11. Elias P. Epidermal lipids, barrier function, and desquamation. J Invest Dermatol 1983; 80:44s–49s. 12. Lampe MA, Burlingame AL, Whitney J, et al. Human stratum corneum lipids: characterization and regional variations. J Lipid Res 1983; 24:120–130. 13. Schurer NY, Elias PM. The biochemistry and function of stratum corneum lipids. Adv Lipid Res 1991; 24:27–56. ¨ ber die Kutane Kupferresorption aus einer Kupfer 14. Schmid R, Winkler J. U Enthaltenden Salbe. Klin Wochenschr 1938; 17:559–561. 15. Odintsova NA. Permeability of human skin to potassium and copper ions and their ultrastructural localization. Chem Abs 1978; 89:360. 16. Brown SA, Merritt K. Electrochemical corrosion in saline and serum. J Biomed Mater Res 1980; 14:173–179. 17. Samitz MH, Katz SA. Nickel dermatitis hazards from prostheses. Br J Dermatol 1975; 92:287–290. 18. Cronin E. Metals. Contact dermatitis. Edinburgh: Churchill Livingstone, 1980:328–335. 19. Lisi P, Caraffini S, Assalve D. Irritation and sensitization potential of pesticides. Contact Dermatitis 1987; 17:212–218. 20. Adams RM. Metals. Chromium. In: Adams RM, et al., eds. Occupational Skin Diseases. Philadelphia: Saunders, 1990:208–217. 21. Magnusson B, Kligman AM. The identification of contact allergens by animal assay. The guinea pig maximization test. J Invest Dermatol 1969; 52:568–576. 22. Kimber I. The murine local lymph node assay: principles and practice. Am J Contact Dermatitis 1993; 4:42–44. 23. Boman A, Wahlberg JE, Hagelthorn G. Sensitizing potential of beryllium, copper and molybdenum compounds studied by the guinea pig maximization method. Contact Dermatitis 1979; 5:332–333. 24. Karlberg AT, Boman A, Wahlberg JE. Copper—a rare sensitizer. Contact Dermatitis 1983; 9:134–139. 25. Basketter DA, Roberts DW, Cronin M, Scholes EW. The value of the local lymph node assay in quantitative structure-activity investigations. Contact Dermatitis 1992; 27:137–142.


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44. van der Meer DBJ, te Heerenveen H. Een geval van contactallergie voor koper en zink. Ned T Geneesk 1957; 101:2166–2167. 45. Saltzer EI, Wilson JW. Allergic contact dermatitis due to copper. Arch Dermatol 1968; 98:375–376. 46. Trachtenberg DI. Allergic response to copper. J Periodontol 1972; 705–707. 47. Shelley WB, Shelley ED, Ho AKS. Cholinergic urticaria: acetylcholinereceptor-dependent immediate-type hypersensitivity reaction to copper. Lancet 1983; 1:843–846. 48. Forck G. Allergie gegen Kupfer-T-Spirale. Allergologie 1984; 7:343. 49. Romaguera C, Grimalt F, Vilaplana J. Contact dermatitis from nickel: an investigation of its sources. Contact Dermatitis 1988; 19:52–57. 50. Laubstein B. Kupferallergie—eine seltene Sensibilisierung. Dermatol Monatsschr 1990; 176:421–425. 51. Bezzon OL. Allergic sensitivity to several base metals: a clinical report. J Prosthet Dent 1993; 69:243–244. 52. Purello d’Ambrosio F, Ricciardi L, Isola S, et al. Systemic contact dermatitis to copper-containing IUD. Allergy 1996; 51:658–659. 53. Wo¨hrl S, Hemmer W, Focke M, Go¨tz M, Jarisch R. Copper allergy revisited. J Am Acad Dermatol 2001; 45:863–870. 54. Boman A, Karlberg AT, Einarsson O, Wahlberg JE. Dissolving of copper by synthetic sweat. Contact Dermatitis 1983; 9:159–160. 55. Sharata HH, Burnette RR. Effect of dipolar aprotic permeability enhancers on the basal stratum corneum. J Pharm Sci 1988; 77:27–32. 56. Wahlberg JE, Skog E. The effect of dimethyl sulfoxide on the percutaneous absorption of mercuric chloride in the guinea pig. Acta Derm Venereol (Stockh) 1967; 47:209–215. 57. Fullerton A, Andersen JR, Hoelgaard A. Permeation of nickel through human skin in vitro—effect of vehicles. Br J Dermatol 1988; 118:509–516. 58. Pirot F, Panisset F, Agache P, Humbert P. Simultaneous absorption of Cu and Zn through human skin in vitro. Skin Pharmacol 1996; 9:43–52. 59. Pirot F, Millet J, Kalia YN, Humbert P. In vitro study of percutaneous absorption, cutaneous bioavailability and bioequivalence of zinc and copper from five topical formulations. Skin Pharmacol 1996; 9:259–269. 60. Wahlberg JE. Vehicle role of petrolatum. Absorption studies with metallic test compounds in guinea pigs. Acta Derm Venereol (Stockh) 1971; 51:129–134. 61. Wahlberg JE. Nickel: the search for alternative, optimal and non-irritant patch test preparations. Assessment based on laser Doppler flowmetry. Skin Res Technol 1996; 2:136–141. 62. Ale SI, Maibach HI. Clinical relevance in allergic contact dermatitis. Derm Beruf Umwelt 1995; 43:119–121. 63. de Groot AC. Patch testing concentrations and vehicles for testing contact allergens. In: Kanerva L, Elsner P, Wahlberg JE, Maibach HI, eds. Handbook of Occupational Dermatology. New York: Springer, 2000:1257–1276. 64. Kanerva L, Estlander T, Jolanki R. Occupational skin allergy in the dental profession. Dermatol Clin 1994; 12:517–532. 65. Veien NK. Systemically induced eczema in adults. Acta Dermato-Venereol (Stockh) 1989; 147(suppl):12–55.


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66. Nordlind K, Lideń S. Patch test reactions to metal salts in patients with oral mucosal lesions associated with amalgam restorations. Contact Dermatitis 1992; 27:157–160. 67. Hay IC, Ormerod AD. Severe oral and facial reaction to 6 metals in restorative dentistry. Contact Dermatitis 1998; 38:216. 68. Stoffolani N, Damiani F, Lilli C, et al. Ion release from orthodontic appliances. J Dent 1999; 27:449–454. 69. Jacobsen N, Hensten-Pettersen A. Occupational health problems and adverse patient reactions in orthodontics. Eur J Orthodont 1989; 11:254–260. 70. Chantler E, Critoph F, Elstein M. Release of copper from copper-bearing intrauterine contraceptive devices. BMJ 1977; 6062:288–291. 71. Zipper J, Medel M, Prager R. Suppression of fertility by intrauterine copper and zinc in rabbits: a new approach to intrauterine concepts. Am J Obstet Gynecol 1969; 105:529–534. 72. Alvarez F, Brache E, Fernandez B, Guerrero B, Guiloff R, Hess R. New insights on the mode of action of intrauterine contraceptive devices in women. Fertil Steril 1988; 49:768–773. 73. Sinigaglia F. The molecular basis of metal recognition by T cells. J Invest Dermatol 1994; 102:398–401. 74. Fo¨rstro¨m L, Kiistala R, Tarvainen K. Hypersensitivity to copper verified by test with 0.1% CuSO4. Contact Dermatitis 1977; 3:280–281. 75. van Joost TH, van Ulsen J, van Loon LA. Contact allergy to denture materials in the burning mouth syndrome. Contact Dermatitis 1988; 18:97–99. 76. Sterry W, Schmoll M. Contact urticaria and dermatitis from self-adhesive pads. Contact Dermatitis 1985; 13:284–285. 77. Reid DJ. Allergic reaction to copper cement. Br Dent J 1968; 124:92. 78. Popa V, Teculescu D, Stanescu D, Gavrilescu N. Bronchial asthma and asthmatic bronchitis determined by simple chemicals. Dis Chest 1969; 56:395–404. 79. Beckmann M, Wagner H. Allergische Reaktionen bei kupferhaltigen Intrauterinpessaren. Medizinische Welt 1979; 30:1855–1856. 80. Sabbah A, Drouet M, Hergon E. Rhinite par sensibilite au cuivre. Allergie et Immunologie 1983; 15:209–210. 81. Gaul LE. Incidence of sensitivity to chromium, nickel, gold, silver, and copper compared to reactions to their aqueous salts including cobalt sulfate. Ann Allergy 1954; 12:429–444. 82. Epstein S. Cross-sensitivity between nickel and copper. J Invest Dermatol 1955; 55:269–274. 83. Bockendahl H, Remy W, Masuch E. Untersuchungen zum Mechanismus des Kontaktekzems gegen Kupfer. Archiv fuer Dermatologische Forschung 1974; 250:167–171. 84. Dhir GG, Rao DS, Mehrotra MP. Contact dermatitis caused by copper sulfate used as coloring material in commercial alcohol. Ann Allergy 1977; 39:204. 85. Walton S. Investigation into patch testing with copper sulfate. Contact Dermatitis 1983; 9:89–90. 86. van Joost T, Habets JMW, Stolz E, Naafs B. The meaning of positive patch tests to copper sulphate in nickel allergy. Contact Dermatitis 1988; 18:101–102.

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87. Hackel H, Miller K, Elsner P, Burg G. Unusual combined sensitization to palladium and other metals. Contact Dermatitis 1991; 24:131–132. 88. Kawahara D, Oshima H, Kosugi H, Nakamura M, Sugai T, Tamaki T. Further epidemiologic study of occupational contact dermatitis in the dental clinic. Contact Dermatitis 1993; 28:114–115. 89. Motolese A, Truzzi M, Giannini A, Seidenari S. Contact dermatitis and contact sensitization among enamellers and decorators in the ceramics industry. Contact Dermatitis 1993; 28:59–62. 90. Giorgini S, Brusi C, Francalanci S, Acciai MC, Sertoli A. Prodotti alternativi e prevenzione della dermatite allergica da contatto. II. Prevenzione della dermatite allergica da contatto da orecchini di bigiotteria. Annali Italiani di Dermatologia Clinica e Sperimentale 1994; 46:151–158. 91. Raccagni AA, Baldari U, Righini MG. Airborne dermatitis in a painter. Contact Dermatitis 1996; 35:119–120. 92. Rademaker M. Occupational contact dermatitis among New Zealand farmers. Australasian J Dermatol 1998; 39:164–167. 93. Vilaplana J, Romaguera C. Contact dermatitis and adverse oral mucous membrane reactions related to the use of dental prostheses. Contact Dermatitis 2000; 43:183–185. 94. Dry J, Leynadier F, Bennani A, Piquet P, Salat J. Intrauterine copper contraceptive devices and allergy to a copper and nickel. Ann Allergy 1978; 41:1978. 95. Rongioletti F, Rivara G, Rebora A. Contact dermatitis to a copper-containing intra-uterine device. Contact Dermatitis 1985; 13:343. 96. Hausen BM, Hohlbaum W. Verursachen Kupfer-Intrauterinpessare eine Kontaktallergie? Dtsch Med Wochenschr 1986; 111:1016–1021. 97. Lauter H. Messingallergie? Allergologie 1987; 10:156–157. 98. Siliotti F, Caroti S, Caroti A, Alborino F. Considerazioni su di un caso di allergia a IUD medicato al rame. Ginecological Clinics 1987; 8:197–200. 99. Namikoshi T, Yoshimatsu T, Suga K, Fujii H, Yasuda K. The prevalence of sensitivity to constituents of dental alloys. J Oral Rehabil 1990; 17:377–381. 100. Hocher B, Keller F, Krause PH, Gollnick H, Oelkers W. Interstitial nephritis with reversible renal failure due to a copper-containing intrauterine contraceptive device. Nephron 1992; 61:111–113. 101. Vilaplana J, Romaguera C, Cornellana F. Contact dermatitis and adverse oral mucous membrane reactions related to the use of dental prostheses. Contact Dermatitis 1994; 30:80–84. 102. Kansu G, Aydin AK. Evaluation of the biocompatibility of various dental alloys: part 1—toxic potentials. Eur J Prosthodont Restorative Dent 1996; 4:129–136. 103. Kansu G, Aydin AK. Evaluation of the biocompatibility of various dental alloys: part 2—allergenical potentials. Eur J Prosthodont Restorative Dent 1996; 4:155–161. 104. Fedorov SM, Ado VA, Mokronosova MA, Seliskii GD, Perlamutrov YN, Samuilova TL. Allergic dermatitis due to metal sensitizers contained in jewellery and intrauterine devices. Vestnik Dermatologie i Venereologie 1997; 1:49–50. 105. Pujol RM, Randazzo L, Miralles J, Lomar A. Perimenstrual dermatitis secondary to a copper-containing intrauterine contraceptive device. Contact Dermatitis 1998; 38:288.


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8 Copper in Medicine and Personal Care: A Historical Overview Roberto Milanino Facolta` di Medicina e Chirurgia, Sezione di Farmacologia, Dipartimento di Medicina e Salute Pubblica, Universita` di Verona, Verona, Italy

Remember always that some ideas that seem dead and buried may at one time or another rise up to life again, more vital than ever before. —Louis Pasteur INTRODUCTION In the pre- and protohistoric ages, medicine and therapy were both strongly linked with religion and thus were mainly presented to communities and patients in magic-religious clothing, although empiricism actually lay at the core of these arts. Medicinal plants were widely employed as drugs, and, in some cases, their active principles are really valuable and still used clinically today, e.g., atropine (Atropa belladonna, Datura stramonium—L.), digitalin (Digitalis purpurea—L.), opium and derivatives (Papever somniferum—L.), quinine (Chincona offinicinalis—L.), reserpine (Rauwolfia serpentina—L.), salicin and derivatives (Salix alba—L.), tubocurarine (Chondrodendon tomentosum— R. & P.), etc. Drugs from the animal kingdom were numerous, such as the donkey fat, rhinoceros horns, and pig liver; the latter, for instance, was 149

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considered a good remedy for anemia (an empiric indication consistent with our modern medical knowledge). Minerals, for example those containing antimony, arsenic, copper, gold, iron, lead, mercury, sulfur, or zinc, were also used frequently. Many of the above ingredients were routinely mixed according to magic-empirical criteria in recipes, often kept secret, and usually administered accompanied by complex esoteric rituals. The aim of this review, however, is focused on the use of copper in therapy during the prescientific millennia preceding our modern medical culture. Humans have employed copper in medicine since before 3500 B.C., mostly referring to it as a ‘‘generic drug,’’ but also recognizing its antiseptic as well as anti-inflammatory potentials. In particular, attributing anti-inflammatory properties to copper appears to have been an extremely clever intuition. What is even more surprising is that this metallo-element was believed to be endowed with therapeutic properties by almost all major ancient cultures. In particular, while its diffusion among Mediterranean, near-, middle-, and far-Eastern civilizations may be explained on the basis of their frequent mutual contacts, the presence of copper in the ‘‘pharmacopoeia’’ of the pre-Columbian Meso- and South-American cultures seems to derive entirely from the autochthonous experience of these populations. THE SUMERIC CULTURE: CIRCA 4000–2300

B.C.

Present day knowledge ascribes to this culture the basic merit of having invented the near- and middle-Eastern ideographic writing, which is conventionally considered as the beginning of the historical age. This society developed recurrent commercial and cultural relations with the neighboring areas and cultures (1). Malachite (basic cupric carbonate) was one of the products that Sumerians frequently exported to Egypt as well as closer regions, mainly for making jewels and cosmetic products, and also for pharmaceutical preparations. It is documented, through indirect evidence (mostly Egyptians), that the utilization of pulverized malachite for generic medical purposes was a practice commonly used by the Sumerians themselves (2). THE ANCIENT EGYPTIAN CULTURE The Predynastic Age to the II Intermediate Period (XVII Dynasty): Circa 3900–1550 B.C. (3,4) Probably the first significant medical document known today is the Kahun papyrus, which was most likely written during the kingdom of Seosostris II (about 1880 B.C., XII dynasty), and is essentially focused on gynecological problems (4,5). The Kahun papyrus appears to make no specific mention of the use of copper as a drug. However, we know from other minor written fragments of those times, as well as numerous and much more exhaustive subsequent medical

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manuscripts, that the utilization of pulverized malachite was extremely common, since the beginning of the predynastic period, for preparing a typical blue-green eye makeup (5). In the middle and late period of this Egyptian age, this cosmetic became dark gray, as shown by several tomb pictures, probably because pulverized galena (lead sulfide) was already added to malachite (2,6). This eye makeup was used not only for decorative ritual purposes exclusively related to the Osiris cult but also for the prevention and cure of eye infections, widespread in the ancient as well as modern Egypt. These infections were mainly caused by the dryness of the climate and sandy winds characterizing the area (2,5–7). Actually, in this practice, it may be possible to recognize the first empiric attribution of antiseptic properties to copper preparations. The XVIII Dynasty to the Ptolemaic Dynasty: Circa 1550 B.C. to 30 A.D. The most famous and comprehensive documentation of Egyptian medical science are the Ebers’ and Smith’s papyri. They summarize medical information of those times based on, if not mainly, previous orally transmitted and written traditions. Both these treatises belong to the XVIII dynasty, the first of the New Kingdom (1550–1075 B.C.) (5,7). The papyrus of Ebers (circa 1500 B.C.) is a true medical encyclopedia, dealing with both generic medical and therapeutic problems. The pharmacological sections of the Ebers’ papyrus emphasize the importance of personal cleanliness, and reports the use of many drugs mainly coming from medicinal plants (castor oil, senna, tamarisk, thyme, etc.), but minerals are also not neglected. For example, this text clearly codifies, for the first time, the preparation of kohl, i.e., the mixture of malachite and galena. This compound is certainly identical to that used in previous centuries, and is still routinely applied as an eye cosmetic, for the same decorative–ritual and practical medical purposes as described earlier (2,5,6). The papyrus of Smith (about 1450 B.C.) mainly addresses surgical problems as Egyptian society (like the later Roman one) was frequently involved in wars and, consequently, was forced to develop efficient emergency medical procedures (5). This papyrus also mentions the use of pulverized malachite thought to be endowed with astringent, healing, and antiseptic properties for the treatment of postoperative wounds (2,5). Interestingly, these antiseptic and healing potentials of copper are presently fully recognized (8), although no longer routinely employed in modern medical practice. However, copper preparations, in particular cupric sulfate and basic cupric sulfate (9), are still widely used in agriculture; copper is also a potent antimycotic agent, especially effective in the treatments of plants, such as tomatoes and grapes, against fungal infections.

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THE BABYLONIAN–ASSYRIAN CULTURE: CIRCA 1750–539

B.C.

The medical and pharmacological knowledge of these societies is reported on approximately 800 medical clay tablets recovered from circa 100,000 found during the excavation of the Assyrian Library of Nineveh (about 650 B.C.) (2,10). Babylonian–Assyrian medicine particularly focused on studying the liver as a basic tool of its diagnostic. This organ, however, was not examined according to modern semeiotic procedures (directly on patient), but instead physicians examined the livers as well as the viscera of animals sacrificed during the propitiatory rituals performed in order to recognize the disease, then chose the appropriate therapy (2). This practice was clearly an esoteric superstition called Haruspecism. Most likely, Haruspecism reached its highest levels over 900 years later with the development of the Etrurian culture in Italy (11). In fact, the Etrurian haruspices not only ‘‘specialized’’ in this kind of ‘‘medical approach’’ but also transformed it into a more general divinatory procedure, which included the magically inspired interpretation of many other natural phenomena, such as the flight of birds (11). About 250 drugs of plant origin, and also 120 compounds derived from minerals, are cited in the Babylonian–Assyrian pharmacopoeia. Among the minerals, those containing copper are mentioned specifically, being qualified as generic therapeutic remedies (2). However, most likely for the first time, the use of a copper bracelet is quoted in these reports as useful, albeit nonspecific, pharmacological tool (10). THE ANCIENT INDIAN CULTURE: CIRCA 2800–1000

B.C.

The medical knowledge acquired since the remote origins of the ancient Indian culture and orally transmitted generation to generation was later summarized in two major medical books: the Samhita Charaka and the Susruta Samhita (written in Sanskrit during the Brahamanic age, approximately between 800 B.C. and 1000 A.D.) (1,2). Surgery was highly developed in the ancient Indian culture and, unsurprisingly, their pharmacopoeia made a wide use of medicinal plants, among which is mentioned Cannabis indica L. as an analgesic (cannabinoid derivatives). This drug was frequently employed, together with black henbane (Hyoscyamus niger—L.), which contains such analgesic–narcotic principles as hyoscyamine and scopolamine, as a general anesthetic in surgical operations (2). Although no other significant information is available, the use of copper (as sulfide or sulfate) for making preparations used for nonspecific medical purposes is reported in these texts (2). THE ANCIENT CHINESE CULTURE: CIRCA 3000 B.C. TO 1100 A.D. According to legend, the medical book Nei Ching (‘‘The Canon of Medicine’’) was written by the mythical king Huang Ti (the ‘‘Yellow Emperor’’) about 2700 years B.C. (2). Actually, this treatise was most likely edited much later


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and, moreover, appeared in two separate parts, about 10 centuries apart. In fact, the first part dates back to approximately the second century B.C., the second part to about the eighth century A.D. However, ‘‘The Canon of Medicine’’ is the first-known compendium in which millennia of Chinese medical and therapeutic traditions are summarized (2). The pharmacological use of copper sulfate (or sulfide) is undeniably documented in the above text, not only for the topical treatment of skin and eye diseases but also for ‘‘blood purification.’’ The latter is, probably, the first-known indication for oral administration of copper (2). THE PRE-COLUMBIAN MESO- AND SOUTH-AMERICAN CULTURES: CIRCA 600 B.C. TO 1500 A.D. The medical traditions of the better-known Meso- and South-American civilizations (i.e., Maya, Aztec, and Inca) are referred to in numerous documents, albeit those written exclusively by the Spanish invaders (2). In particular, the cranial trepanation procedure deserves an especial attention. Although known and occasionally practiced since at least the fifth to fourth millennium B.C. among most of the world’s prehistoric and historic cultures, including that of Egypt, this kind of surgery was chiefly practiced by Meso- and South-American societies between the fifth century B.C. and the fifth century A.D. (12,13). Craniotomy, however, reached an astonishing level of specialization among the Inca ‘‘surgeons,’’ who frequently made use of this peculiar technique and whose patients actually had an extraordinary survival rate, assessed far beyond 50%. In fact, archeologists discovered skeletons of a great number of Incas whose skulls underwent this procedure, and who survived the distressing experience (12). Interestingly, a human cranium found in the necropolis of Cuzco (Peru) clearly shows the holes resulting from two successive cranial trepanations; obvious signs of bone regeneration surrounding these holes testify to the individual’s long survival following the surgical procedures (12). The main purpose of this intervention was undoubtedly a ritual–magical one (2,12,13); nonetheless, the craniotomy also bears real clinical value in reducing intracranial pressure due to post-traumatic meningeal edemas or hemorrhages (10). Notably, ‘‘gauze’’ soaked in a copper sulfate solution was routinely used to ‘‘disinfect’’ the surgical wounds after the removal of the skull section (2,13). THE ANCIENT GREEK CULTURE It is relevant to note that the chronological classification of Ancient Greek society and culture is more conventional than factual. For instance, the dating of Archaic Greek, of the classic Greek, and, especially, of the official end of this unique civilization (which is made to coincide whit the Roman conquest, i.e., 323 B.C.) is merely symbolic. In fact, as we will see below,

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the Greek culture developed and survived much longer, and is still largely present in the roots of our contemporary occidental civilization. Archaic Greek: Circa 1300–500

B.C.

Although the ancient Greeks attributed the beginnings of their medicine to the probably mythical Thessalonian prince Asclepios (about 1200 B.C.), earlier documented Greek medical records came down from the MediterraneanGreek colonies, especially the Sicilian one (11,14). Actually, significant medical theories were derived from observations of ancient Greek scholars. For instance, Alcmeon (circa 560 B.C.) postulated that the brain was the center of human sensorial and intellectual life, and Empedocles (an Alcmeon’s contemporary) speculated that respiration occurred both at the pulmonary and skin pore levels (11). It was probably in those times that the use of copper preparations, most likely borrowed from the Mesopotamian, Egyptian, and Minoan medical traditions, was introduced as a nonspecific remedy (11,14). Classic Greek: Circa 500–323 B.C. About 100 years after Alcmeon’s and Empedocles’ age, when classic Greek culture developed under the influence of the thoughts of the great philosophers (such as Socrates and Plato), Hippocrates (460 B.C.) was probably the first physician to invoke the separation of medicine, religion, and myth, stating, ‘‘No disease is more divine or more human than any other, since every illness is due to a natural cause, in the absence of which it cannot take place’’ (11). The Hippocratic perspective on human brain functions was also very modern; redefining the previous hypothesis of Alcmeon, Hippocrates wrote, ‘‘I say that the brain is the most powerful organ of the human body . . . The eyes, ears, tongue, hands and feet all act under the control of the brain. The brain transmits its messages to the human conscience’’ (11,14). Nevertheless, this does not necessarily mean that Hippocrates’ attitude had been a sort of ‘‘revolutionary’’ one. In fact, Hippocrates was especially careful not to express open dispute with the contemporary religious opinions and traditions, and in the prologue to his famous ‘‘Hippocrates’ swearing,’’ Hippocrates wrote, ‘‘I swear in the name of Asclepios, the Physician, Igea, Panacea, and all the gods and goddesses, calling them to witness, that I will follow . . . ’’ (11). Hippocrates’ major work is the Corpus Hippocraticum, which consists of 72 volumes, of which only 17 are sure to have been written by Hippocrates himself. The other 55 are ‘‘contaminations of ’’ and ‘‘additions to’’ the original texts by numerous respectable physicians (but also by some quack doctors) in the course of following centuries (11,14). In Hippocrates’ original books, the use of copper preparations was clearly prescribed for the therapy of many such diseases as cutaneous and eye diseases, vaginal disorders (using an irrigation with copper solutions or suspensions), and hemorrhoids (using ‘‘suppositories’’ containing copper) (11,14).


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Finally, ancient Greek medicine was the first to suggest the wearing of the copper bracelet as an antiarthritic remedy, which brings us directly to our present debate on the effectiveness of topical copper preparations in the treatment of rheumatic conditions (14,15). THE ANCIENT ROMAN CULTURE: CIRCA 600

B.C.

TO 476

A.D.

From the beginning, ancient Romans were primarily engaged in bringing their ‘‘Pax Romana’’ (the archetype of our contemporary globalization process) to all societies living in the known world. Thus, the Roman age based both the origin and development of its knowledge, the medical one included, on Greek thought and tradition, although initially some Etrurian and later Arab contributions were also present (11). Therefore, in spite of the much older origin of Roman society and culture, the major works describing Roman medicine and pharmacology were all written between the end of the first century B.C. and the second century A.D., and are attributable to three prominent names, i.e., Pliny ‘‘the elder,’’ Celsus, and Galen (11). Caius Pliny ‘‘the elder’’ (23–79 A.D.) wrote the 37 volumes of Naturalis Historia, in which Pliny discourses not only upon the subjects of astronomy, geography, zoology, and botany, but also on medicine, physiology, and pharmacology. Pliny’s pharmacopoeia, along with medicinal plant and animal derivatives, devotes significant attention to minerals, copper in particular. This metal is frequently mentioned in many preparations in which it is present in the form of pharmacologically active compounds such as copper oxides, copper sulfide, copper sulfate, basic cupric carbonate, and basic cupric acetate (C.P.E., Nat. Hist. XXX and XXXI) (16). Their practical use was particularly recommended for the treatment of eye and skin diseases, tonsillitis, throat inflammation, etc. (C.P.E., Nat. Hist. XXX and XXXI) (16). Aulus Cornelius Celsus (probably, 30 B.C. to 38 A.D.) was the author of De Medicina, an eight-volume encyclopedia, in the third tome of which, treating on ‘‘the fevers,’’ Celsus codified his famous ‘‘four cardinal signs of inflammation,’’ rubor, tumor, calor et dolor, to which a fifth sign, the fuctio laesa, was then added by Galeno (17,18). It was again Celsus who, probably first, formally recognized copper as useful antiseptic and, especially, anti-inflammatory agent; notably, the antiinflammatory potential of basic cupric carbonate (i.e., malachite) has been confirmed by our contemporary research (19). Copper compounds were indeed frequently employed in Celsus’ very elaborate prescriptions such as those suggested for the treatment of anal rhagades, hemorrhoids, tonsillitis, wound disinfection, etc. We like to report as an example, Celsus’ recipe for curing anal rhagades:

Verdigris (basic cupric acetate): parts 2 Myrrh: parts 12 Antimony: parts 16

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Poppy ‘‘tears’’: parts 16 Acacia: parts 16 To be suspended in wine before local application (17) In the writings of Celsus, a great deal of attention is also dedicated to surgery. In fact, Celsus first introduced in orthopedics the application of copper plates (which was thought to be appropriate for those conditions, since they are also antiseptic and anti-inflammatory) to promote the recovery of disassembled fractures (17). Interestingly, this practice was still in use more than 1400 years later, as exemplified by a human skeleton recently discovered in Belgium (cathedral of Vrasene) (17). Claudius Galeno (129–200 A.D.) wrote many medical books such as the Ars magna and the Ars parva, as well as works of pharmacology collected in the treatises De Methodus Medendi and De simplicium medicamentorum temperamentis et facultatibus (11). Galeno, a Civis romanus (although fully Greek by origin, culture, and thought), was an eminent and innovative anatomist, physiologist, and pathologist, but a less pioneering therapist (18). In fact, while Galeno’s ‘‘general medicine’’ writings describe some sort of interesting experimental work, Galeno’s pharmacology books are mainly a summary of therapeutic experiences gathered by the ancient Mediterranean and Eastern cultures (the Greek one in particular), skillfully mixed with the pharmacological reports of Celsus as well as Pliny ‘‘the elder’’ (18). Thus, copper was also frequently used in Galeno’s therapeutics, but still on the basis of an empirical methodology, using essentially the same copper preparations, and being addressed to the same pharmacological targets as in the previous Mesopotamian, Egyptian, Chinese, Indian, Greek, and, of course, Pliny’s and Celsus’ heritages. Galeno’s pharmacopoeia is, therefore, devoid of any original ‘‘scientific’’ approach, perhaps with one exception: occasionally, Galeno’s ‘‘new’’ drug mixtures were tried to test in self-experimentation (11,17,18). Nevertheless, Galeno’s works were destined to play a significant role in the future trends of medical and pharmacological scientific ‘‘development’’ over the following 1500 years, especially in our European ‘‘world,’’ and also in the Arabian one (18).

FROM THE HIGH-MEDIEVAL AGE TO THE EARLY 20TH CENTURY The classic Greek and Roman ‘‘biomedical’’ background deeply affected biological, medical, and pharmacological thought at least up until the ‘‘Galilean revolution,’’ which generated a totally new way of seeing and doing science (second half of 16th, first half of 17th centuries) (11,18,20). Thus, different fragments of Hippocrates’, Pliny’s, Celsus’, and Galeno’s writings were blended according to current ideas and prejudices, mainly in


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consequence of the dogmatic and actually prevailing Catholic culture, which found its major justification in Scholastic philosophy (a re-reading of Aristotelian thought), as elaborated by Thomas Aquinas (13th century A.D.) (20,21). Notably, in this discouraging context, remarkable attention was dedicated to Galeno’s works, since Galeno possibly believed, and certainly stated, ‘‘the human body is merely an instrument of man’s soul’’ (11,18,20). Nevertheless, the pharmacological use of copper survived during those dark centuries, although essentially based more on the imaginative superstitions derived by the ‘‘innovative discoveries’’ of contemporary quack doctors and alchemists, than on the ‘‘good old’’ empiricism that characterized the progress in medicine and pharmacology made by scholars of the ancient cultures, particularly classic Greek–Roman. Later on, the ‘‘Galilean revolution’’ also began to strongly influence the world of biology and medicine and the related biomedical disciplines slowly evolved into our modern experimental development process of scientific learning. Pharmacology, and especially copper, however, were destined to wait still a while longer to see their ‘‘renaissance.’’ In fact, focusing on copper, it was only in the first half of the 19th century that Rademacher, introducing the so-called ‘‘medicine of experience,’’ deduced, after a clinical trial lasting 25 years, ‘‘the body has a strange predisposition for a great number of diseases which disappeared, or could be healed, in the presence of copper. Healthy subject did not show any response to copper’’ (18,22). At the end of the 19th century, copper was administered internally and was found to be effective against numerous illnesses such as skin diseases and the infections of tuberculosis and syphilis (22). The roots of these clinical uses of copper, however, were again to be found in a sort of ‘‘renewed empiricism,’’ which, according to Classic Age traditions, considered copper a valuable antiseptic and anti-inflammatory agent. Between the end of the 19th and the beginning of the 20th centuries, mounting consideration for the discoveries of modern microbiology, physiology, and biochemistry, as well as the appearance of chemistry as science in the field of pharmacology (e.g., the synthesis of arsenic derivatives, salicylates, sulfanilamides, etc.) (18,22), led to the abandonment of the use of copper in medicine. It is true that during the 1940s some copper complexes, such as Cupralene and Dicuprene, were still employed for treating arthritic diseases, but the advent of the therapy with exogenous cortisone extracts or derivatives rapidly led also to their disuse. BEGINNING OF THE SCIENTIFIC AGE FOR COPPER: 1928–1976 Curiously, it was only in the first half of the past century, while copper therapy was gradually being neglected by a majority of clinicians, that the importance of copper in biology and medicine finally found its experimental justification.

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In fact, in 1928 copper was first shown to be essential for life (23) since it is required for the synthesis of hemoglobin. Subsequently, its involvement in inflammation was demonstrated both in humans (1938) and laboratory animals (1953) (24,25). Almost 30 years after the scientific reports above, in 1967, a dramatic increase of the concentration in ceruloplasmin (the major multifunctional copper protein present in the serum, synthesized by the liver and deeply involved in iron metabolism and copper delivery to the extrahepatic tissues) was observed in the serum and periodontal tissue specimens of patients suffering from periodontal disease (an acute inflammatory process) (26,27). In 1968, a relevant increase of serum copper and ceruloplasmin was also reported in rheumatoid arthritic patients (28). Subsequently, the same evidence was found to characterize the adjuvant arthritis of the rat, an experimental systemic disease still the best existing model of human rheumatoid arthritis (29,30). All the above were only occasional reports, however. The leading breakthroughs that characterized the beginning of scientific development of ‘‘copper and inflammation’’ research came in 1976, when Sorenson’s (31) classic paper indicated many copper complexes as active acute and chronic anti-inflammatory agents, and when Whitehouse (32) posed the question about a possible ‘‘ambivalent,’’ i.e., pro- and anti-inflammatory, role of copper in the development and control of inflammation.

CONCLUSIONS Today, the fact that ‘‘endogenous’’ copper exerts a key role in the development and control of inflammation is a well-recognized biological phenomenon (33). Moreover, the anti-inflammatory/antiarthritic potential of ‘‘exogenous’’ copper in curing experimental models of inflammation has also been demonstrated repeatedly by many research groups (34,35). Recently, great progress has been made, at the molecular level, in understanding how copper ‘‘traffics’’ in the organism and within the cells. Although this basic topic of research is still in its infancy, in the near future it may yield essential information for the understanding of the mechanism(s) by which this metal, both ‘‘endogenous’’ and ‘‘exogenous,’’ can keep under appropriate control the development of the inflammatory process. Therefore, the present state of the art appears most encouraging for the continuation of research to ascertain whether copper could represent a truly innovative approach to the curing of human inflammatory diseases, rheumatoid arthritis in primis. Nevertheless, few serious attempts have been made, and none currently, to study the use of copper in the therapy of such diseases. There are several prejudicial barriers that keep copper out of both experimental laboratories and the clinic: copper is generally thought to be far more toxic


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than it actually is (36) and, especially, the marketing of copper compounds offers profits too low to be advantageous to business. Nonetheless, we would like to stress that the pharmacological exploitation of the anti-inflammatory and antiarthritic copper potential is anything but an obsolete idea. On the other hand, rheumatoid arthritis and related degenerative diseases that are found worldwide and are the cause of great disability with high social and economic costs, still wait for a pharmacological therapy that is both effective and safe. One can hope for the continuation of research in this field.

ABBREVIATIONS L R&P

taxonomic classification by Linne´ taxonomic classification by Ruiz and Pavon

REFERENCES 1. Carpanetto D, Bianchini P. l’Enciclopedia: Atlante Storico. Vol. 31. Chap. 1. Roma: Gruppo Editoriale l’Espresso, 2004. 2. Sterpellone L. Dagli dei al DNA. Vol. 1. Chapters 1–6. Roma: Antonio Delfino Editore, 1988. 3. Baines J, Ma´lek J. Atlante dell’Antico Egitto. Novara: Istituto Geografico De Agostini, 1985:36. ¨ S 1984; 20:158. 4. von Beckerath J. Handbuch der a¨gyptischen Ko¨nigsnamen. MA 5. David RA. The pyramid builders of ancient Egypt. In: Chapters V and VI. London: Routledge and Kegan, 1986. 6. Rossi Osmida G. La scoperta della vanita`. Archeo 1989; 58:62. 7. Guy E, Rachet MF. Dictionnaire de la Civilisation e´gyptienne. Paris: Librairie Larousse, 1972:100,192. 8. Sorenson JRJ. Pharmacological activities of copper compounds. In: Berthon G, ed. Handbook of metal–ligand interactions in biological fluids. Vol. 2. New York: Marcel Dekker Inc., 1995:1128. 9. Windholz M, ed. The Merk index. 10th ed. Rahway: Merck & Co. Inc., 1983:379. 10. Radicchi R. Civilta` sumero-akkadica. Chapters 4 and 5. Pisa: Giardini Editore, 1968. 11. Sterpellone L. Dagli dei al DNA. Vol. 2. Chapters 1–4. Roma: Antonio Delfino Editore, 1988. 12. Capasso L. Aria al cervello. Archeo 1990; 63:121. 13. Cuory C. Me`de`cine de l’Amerique precolombienne. Chapters 2,3, and 6. Paris: Roger Dacosta, 1969. 14. Pravega D. La terapia nella medicina greca. Chapters 1–7. Pisa: Giardini Editore, 1963. 15. Walker WR, Beveridge SJ, Whitehouse MW. Dermal copper drugs: the copper bracelet and Cu(II) salicylate complexes. In: Rainsford KD, Brune K,

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17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

34.

35.

36.

Milanino Whitehouse MW, eds. Trace elements in the pathogenesis and treatment of inflammation. Basel: Birkhaüser Verlag, 1981:359. Lotz L-O, Weser U. Biological chemistry of copper compounds. In: Rainsford KD, et al, eds. Copper and zinc in inflammatory and degenerative diseases. Chap. 3. Dordrecht: Kluwer Academic Publisher, 1998. Capasso L. I romani in farmacia. Archeo 1989; 57:54. Ackerknecht EH. Therapeutics from the primitives to the 20th century. Chapters I–VI. New York: Hafner Press, 1973. Bonta IL. Microvascular lesions as target of anti-inflammatory and certain other drugs. Acta Physiol Neerl 1969; 15:188. Bynum WF, Browne EJ, Porter R, eds. Macmillan dictionary of the history of science. New York: The Macmillan Press, 1981:449. Vattimo G, Ferraris M, Marconi D (consultant authors). Le Garzantine: Filosofia. 2nd ed. Milano: Garzanti Libri s.p.a., 1999:1036. Deuschle U, Weser U. Copper and inflammation. Prog Clin Biochem Med 1985; 2:97. Hart EB, Steembock H, Waddel J, et al. Iron in nutrition. Vol. VII. Copper as a supplement to iron for haemoglobin building in the rat. J Biol Chem 1928; 77:797. Heilmeyer L, Stuwe G. Der Eisen-kupferantagonismus im Blutplasma beim Infektionsgeschehen. Klin Wochenschr 1938; 17:925. Wintrobe MM, Cartwright CE, Gubler CJ. Studies on the function and metabolism of copper. J Nutr 1953; 50:395. Linder MC. Biochemistry of copper. Chap. 4. New York: Plenum Press, 1991. Sweeney SC. Alterations in tissues and serum ceruloplasmin concentration associated with inflammation. J Dent Res 1967; 46:1171. Lorber A, Cutler LS, Chang CC. Serum copper levels in rheumatoid arthritis: relationship of elevated copper to protein alteration. Arthritis Rheum 1968; 11:65. Karabelas DS. Copper metabolism in the adjuvant induced arthritic rat. Ph.D. thesis. Ann Arbor: Michigan State University, 1972. Rainsford KD. Adjuvant polyarthritis in rats: is this a satisfactory model for screening anti-arthritic drugs? Agents Actions 1982; 12:452. Sorenson JRJ. Copper chelates as possible active forms of the anti-arthritic agents. J Med Chem 1976; 19:135. Whitehouse MW. Ambivalent role of copper in inflammatory disorders. Agents Actions 1976; 6:201. Milanino R, Velo GP, Marrella M. Copper and zinc in the pathophysiology and treatment of inflammatory disorders. In: Ne`ve J, Chappuis P, Lamand M, eds. Therapeutic uses of trace elements. New York: Plenum Publishing Corporation, 1996:115. Milanino R, Moretti U, Marrella M, et al. Copper and zinc in the development and control of inflammation. In: Handbook of metal–ligand interactions in biological fluids. Berthon G, ed. Vol. 2. New York: Marcel Dekker Inc., 1995:886. Sorenson JRJ. Copper complexes offer a physiological approach to treatment of chronic diseases. In: Ellis GP, West GB, eds. Progress in medicinal chemistry. Vol. 26. Amsterdam: Elsevier, 1989:437. Medeiros DM, Wildman R, Liebes R. Metal metabolism and toxicities. In: Massaro EJ, ed. Handbook of human toxicology. Chap. 3. Boca Raton: CRC Press LLC, 1997.

9 The Role of Copper in Onset, Development, and Control of Acute and Chronic Inflammation Roberto Milanino Facolta` di Medicina e Chirurgia, Sezione di Farmacologia, Dipartimento di Medicina e Salute Pubblica, Universita` di Verona, Verona, Italy

INTRODUCTION The involvement of ‘‘endogenous’’ copper in inflammation was discovered in the second half of the last century when evidence was published that: (i) total serum copper markedly increased in aseptic and septic acute inflammation, such as turpentine edema, Staphylococcus aureus abscesses, and typhoid vaccine injection (1953); (ii) total serum copper and ceruloplasmin as well as the ceruloplasmin measured within the inflamed tissue significantly increased in patients suffering from acute periodontal disease (1967); (iii) total serum copper was found significantly elevated in rheumatoid arthritic patients (1968), and in adjuvant-induced arthritis (AA) in the rat (1972), still a very useful experimental model for that human disease (1–5). It was, however, only in 1976, i.e., about two millennia after the first This chapter, in part, was reprinted from Milanino R. Copper: an overview of the role of endogenous and exogenous metal in the development and control of the inflammatory process. Rev Environ Health 2006; 21:1–70, with permission.

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documented intuition by Celsus on this issue in pharmacology (6), that the researchers’ attention again addressed the role of ‘‘exogenous’’ copper as a potentially effective anti-inflammatory and antiarthritic drug. In fact, in that year, two major papers were published. The first one, due to the work of J.R.J. Sorenson (7), not only showed that many subcutaneously injected copper(II) complexes or salts were very active anti-inflammatory and antiarthritic agents, but also proposed that the complexes extemporaneously formed in vivo with ‘‘endogenous’’ copper were the real active forms of the most common and currently used antiarthritic drugs (such as salicylate, aspirin, D-penicillamine, etc.). The above theory has actually been the subject of a lively debate among medicinal chemists, coordination chemists, and pharmacologists, and it is still an unresolved question. The second, especially stimulating paper was published by M.W. Whitehouse (8) in the same year. In that article, the author speculated on the role of both ‘‘endogenous’’ and ‘‘exogenous’’ copper in inflammation, based on the somewhat contradictory evidence known at that time: In some circumstances, soluble copper preparations behave as acute or chronic inflammatory (or irritant) agents. Acute and chronic inflammations are both characterized by a significant increase of total serum copper, which, in light of its irritating potential, may be even regarded as a factor capable of worsening the pathological process. D-Penicillamine, a very well-known de-coppering agent that is still now successfully used in the therapy of Wilson’s disease (9), is also a valuable antiarthritic drug. Pathological (jaundice and other liver diseases) or physiological (pregnancy) events that cause total serum copper to remarkably increase above normal levels, on the other hand, often brought about spontaneous remission of rheumatoid arthritis. Supplementation with copper using a number of folk remedies such as Cu bangles, cider vinegar, shellfish, nuts, etc., as well as the treatment with copper complexes or salts reported by Sorenson (7), seem to be or actually are remarkably good anti-inflammatory/antiarthritic agents. Thus, Whitehouse proposed that copper, either ‘‘endogenous’’ or ‘‘exogenous,’’ can be both injurious and beneficial, even at one and the same time, acting in the organism as ‘‘inert,’’ ‘‘toxic,’’ or ‘‘pharmacoactive,’’ depending on the different physiological or pathological conditions characterizing the organism itself. The actual state of things was then clearly far from being settled, and this intriguing issue induced a number of research teams to begin studying in more detail the roles that ‘‘endogenous’’ and ‘‘exogenous’’ copper could really have on the onset, development, and control of either acute or chronic inflammation.

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STUDIES ON COPPER-DEFICIENT, EXPERIMENTALLY INFLAMED ANIMALS In 1977, our research group set out the idea to study the development of acute and chronic inflammatory processes in experimental animals made deficient of copper by feeding them with a diet containing very low amounts of this essential metallo-element. The rationale of the approach was based on the assumption that inflammation developing in a copper-deprived animal could perhaps give some valuable indication about what the prevailing effect of copper on this kind of diseases was, i.e., a pro- or an anti-inflammatory one. Before beginning the project, normal female and male rats were kept on diets that, according to Owen and Hazelrig (10), were either sufficient (10 ppm) or deficient (0.4 ppm) in copper for three months, and then a complete toxicological examination of the animals was performed. It resulted that the copper-deprived female rats had a reduced ponderal growth compared with control animals. However, the major blood cell and hematochemical serum markers were within normal range; moreover, normal was also the condition of the numerous tissues, organs, and apparatus, examined both macro- and microscopically (11). On the other hand, the same copper-deficient dietary regimen was severely impairing for male rats’ survival, and marked deviations from normality were found evaluating many of the parameters mentioned above (unpublished observation). This latter finding was subsequently confirmed by Fields and coworkers (12), although it is still an unexplained phenomenon. Acute Inflammation First, the effect of copper depletion on the development of an aseptic acute inflammatory process was examined in female rats kept on a 0.4 ppm Cucontaining diet for 30 or 90 days, using the carrageenan-induced paw edema (CPE) model (13). Between the 15th and the 20th day from the beginning of the copper-deficient feeding, the concentration of serum copper was found dramatically reduced, to about a mean 6% of the control values, and this level remained constant throughout the whole experiment (Table 1, zero time). The effects of the treatment on the development of the paw edema were, nonetheless, totally different. In fact, after 30 days of copper deprivation the mean swelling measured in the deficient group was comparable to that of the control, whereas 90 days after the beginning of the experiment, the metallo-elementdeficient animals developed an inflammatory reaction significantly greater than that measured in the normally fed controls, which reached its maximum difference (þ58%) about 20 hours after the subplantar irritant injection (Table 1). The above proinflammatory effect of copper depletion was confirmed in male rats by Denko and coworkers (14,15), who employed the same experimental model (maximum increase of the foot edema þ31%), although the deficient diet used was less severe in both copper content (0.6 ppm) and

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Table 1 Serum Copper Concentrations and Paw Edema Volumes After 30 and 90 Days of Normal (Cu ¼ 10 ppm) or Copper-Deficient (Cu ¼ 0.4 ppm) Feeding

Days of diet 30

90

Total serum copper (mg/dL SD)

Paw edema volume (plethysmographic units SD)

Hours after challenge

Normal

Deficient

Normal

Deficient

0 3 5 20 0 3 5 20

193.2 185.9 20.3 179.2 20.6 270.3 22.3a 190.4 21.0 197.1 20.4 187.6 19.1 276.2 22.1a

12.1 10.7 11.9 14.1 10.1 13.8 91.7 18.1a 12.9 11.4 10.5 15.9 26.1 19.2 102.2 19.0a

— 22.2 3.4 24.1 3.5 15.4 3.6 — 22.1 3.0 25.3 3.5 15.1 3.6

— 21.3 2.6 22.9 3.9 14.5 3.8 — 28.6 4.1b 30.8 5.1c 23.8 5.7b

Note: Statistics—Student’s t test (data standard deviations; N ¼ 13 rats per group and per hour). a P < 0.001; comparison versus the zero time values of either normally fed or deficient rats. b P < 0.001; comparison versus the time-matched, normally fed animals. c P < 0.010; comparison versus the time-matched, normally fed animals. Source: The data reported in this table are the mean values of those obtained in an unpublished preliminary experiment (N ¼ 3 rats per group) and those published in Ref. 13.

length of feeding (30 days). However, the data reported in Table 1 provided other, relevant evidence. Firstly, the proinflammatory effect observed was independent of the amount of total copper present in serum, but dependent on the metallo-element-deficient length of the dietary intake. Secondly, at the 20th hour and in all tested groups, the acute inflammatory challenge promoted a statistically significant increase of total serum copper concentration that, later on, we demonstrated to be very highly correlated to the serum ceruloplasmin level, as measured by the oxidase activity method (16). Interestingly, this total serum copper increase was found to be far more dramatic in the deficient rats (þ671% at day 30, and þ873% at day 90) when compared with that measured in the control animals (þ45% at day 30, and þ40% at day 90). Ceruloplasmin being a copper protein essentially synthesized and secreted by the liver as an acute-phase reactant, and considering that it was proposed to exert a protective role in inflammation, one could speculate that this increase of circulating ceruloplasmin may represent the first evidence of a main role for ‘‘endogenous’’ copper as a physiological agent acting in the control of the development and remission of inflammatory process (14,17). To better understand the soundness of the latter hypothesis, as well as the fact that the proinflammatory effect of copper deficiency was independent of total serum copper concentrations and, conversely, linked to the amount of copper contained in the depleted diet and to the length of time that the animals were exposed to it, further experiments were done. Two


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different copper-deficient diets were considered (containing either 0.2 or 0.7 ppm of copper), fed to female rats for two different periods of time (respectively, 30 and 150 days), in two models of aseptic carrageenaninduced acute inflammation, i.e., the relatively mild CPE, and the more distressing carrageenan-induced pleurisy (CP) (18). The results obtained in the CPE test following the 0.2 ppm copperdepleted diet regimen showed that the proinflammatory effect observed after 30 days of preliminary deficient feeding was not only very obvious (Table 2) but also remarkably higher than that obtained in the previous experiment (Table 1) (13). In fact, the average paw swellings measured three, five, and 20 hours after the challenge were of þ36% (0.4 ppm Cu diet for 90 days, Table 1) versus a þ51% (0.2 ppm Cu diet for 30 days, Table 2); moreover, the maximum enhancement of the local inflammatory reaction, which was gauged at the 20th hour also in the 0.2 ppm diet-treated group, was of about 95% (Table 2). A similar dramatic proinflammatory effect was promoted as well by the 0.2 ppm copper-deficient diet given for 30 days in the carrageenan-induced pleurisy model. In the CP experimental disease, the intrathoracic injection of a sterile carrageenan suspension caused the leakage, from the blood into the thoracic cavity itself, of an inflammatory exudate composed by a fluid fraction in which a large amount (tens of millions) of ‘‘inflammatory’’ cells (mainly Table 2 Serum Copper Concentrations and Paw Edema (CPE) or Exudate (CP) Volumes After 30 Days of 0.2 ppm Copper-Deficient Feeding (Control Diet: Cu ¼ 10 ppm)

Inflammatory model CPE

CP

Total serum copper (md/dL SD)

Paw edema or exudate volumes (plethysmografic units or mL SD)


Normal

Deficient

Normal

Deficient

0 3 5 22 0 6 22

171.2 22.5 167.2 29.6 163.5 30.1 291.3 29.4a 171.4 29.1 173.8 26.1 248.8 24.2a

4.6 7.2 7.3 7.6 5.8 4.3 24.9 26.8 7.9 5.9 6.7 3.7 12.1 9.4

0 18.2 1.6 24.3 2.1 10.1 3.3 0 0.83 0.3 —

0 25.4 3.7b 28.9 1.5b 19.7 3.1b 0 1.64 0.4b —

Note: Statistics—Student’s t test (data standard deviations; N ¼ 13 rats per group and per hour). a P < 0.001; comparison versus zero time values (CPE or CP), of either normally fed or deficient rats. b P < 0.001; comparison versus time-matched, normally fed animals. Source: The data reported in this table are the mean values of those obtained in an unpublished preliminary experiment (N ¼ 3 rats per group) and those published in Ref. 18.

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neutrophils) were present. This inflammatory reaction (that is routinely scored six hours after the challenge) was found to be remarkably higher in the copper-deprived rats compared with the response of the normally fed pleuritic animals (þ98%, Table 2). Nevertheless, the increase of the total exudate volume was accompanied by a proportional rise in its cellular component, which, in turn, seemed to suggest that the anti-inflammatory activity of ‘‘endogenous’’ copper might have little effect on the overall process of leukocyte migration, at least in conditions of copper deficiency and, in this particular model, of acute inflammation. However, another evidence needs to be stressed. The severity of the 0.2 ppm-induced copper deficiency seemed to have reduced the body stores of the metal to such an extent as to prevent the inflammatory process from triggering the statistically significant increase of total serum copper (Table 2). Such an increase is seen, as a rule, in normally fed rats, and was reported also in the copper-depleted, inflamed animals examined in the previous experiment (Tables 1 and 2) (13). When the above acute inflammations (i.e., CPE and CP) were induced in rats fed a 0.7 ppm Cu diet for about five months, no significant differences were measured in either the paw edema or the total exudate volumes between normally fed and copper-deficient animals (Table 3) (18).

Table 3 Serum Copper Concentrations and Paw Edema (CPE) or Exudate (CP) Volumes After 150 Days of 0.7 ppm Copper-Deficient Feeding (Control Diet: Cu ¼ 10 ppm)

Inflammatory model CPE

CP

Total serum copper (md/dL SD)

Paw edema or exudate volumes (plethysmografic units or mL SD)


Normal

Deficient

Normal

Deficient

0 3 5 22 0 6 22

172.3 27.9 — — 250.4 31.8a 171.4 29.1 — 285.3 33.9a

21.5 20.8 — — 70.1 89.9 21.3 29.1 — 89.6 86.4b

0 18.9 6.0 29.3 8.2 13.8 5.1 0 1.0 0.2 —

0 20.3 5.7 27.9 8.4 12.7 4.9 0 1.1 0.4 —

Note: Statistics—Student’s t test (data standard deviations; N ¼ 13 rats per group and per hour). a P < 0.001; comparison versus the zero time values (CPE or CP), of either normally fed or deficient rats. b P < 0.050; comparison versus the zero time values (CPE or CP), of either normally fed or deficient rats. Source: The data reported in this table are the mean values of those obtained in an unpublished preliminary experiment (N ¼ 3 rats per group) and those published in Ref. 18.


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The determination of total serum copper levels (made at zero time and 20 hours after the challenge) showed that this parameter increased, as expected, in the inflamed controls, but its rise was far less significant in the inflamed-depleted animals. However, this latter and unpredicted result is not considered to be a biologically significant one since the single means measured were characterized by extremely high standard deviation values (Table 3) (18). The above-summarized data lead to suggest the following proposals:

In rats, the induction of a copper-deficiency status is a condition that promotes a very significant enhancement of the acute inflammatory reaction, which, in turn, speaks in favor of an anti- rather than a proinflammatory role of ‘‘endogenous’’ copper. The observation that the amount of copper left in the diet and/ or the length of the deprived feeding are able to influence the reported phenomenon seems to indicate a sort of dose- and time-dependent effect of copper deficiency, testifying that this phenomenon is a real and not a casual event. It must be stressed that this conclusion has been confirmed in a subsequent study by Kishore and coworkers (19). The rats not dramatically depleted of copper (i.e., those that underwent feeding with a diet containing 0.4 ppm of the metallo-element) react to the challenge by secreting relatively striking amounts of ceruloplasmin into the blood from the liver. Considering that this protein appears to exert an anti-inflammatory action in vivo (14,20), this evidence seems to further support the hypothesis of a natural defensive role of ‘‘endogenous’’ copper in inflammation. Finally, the findings just mentioned led us to speculate that the appearance of the enhanced acute inflammatory reaction promoted by the deficiency of copper could strictly depend on the amount of copper still present in the liver (21). Later on, this hypothesis found its experimental demonstration in male rats fed a 0.5 ppm Cu for 20, 40, or 60 days and then tested in the CPE model (19). Interestingly, in the same paper the author showed that not only the paw edema in copper-deficient animals was highly and negatively correlated to the concentration of copper in the liver, but also that the correlation with liver Cu,Zn superoxide dismutase (SOD 1) activity was inconsistent (19). Conversely, the activity of SOD 1 in total blood cells of copper-deprived rats (fed a diet containing 0.2 ppm of copper, for one month) was found to be reduced to about 50% of the control values; similar results were also obtained in copper-deficient pigs (22,23). These two last observations directed the attention to the role that superoxide radicals play in the development of the inflammatory process in vivo, and, in turn, in

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the scavenging activity of many copper compounds other than the SOD 1 itself (24,25). Thus, the above information led us to speculate that ‘‘endogenous’’ copper could control the inflammation also by regulating the superoxide metabolism, and, as a consequence, its deficiency may, at least in part, justify the exacerbation of the acute inflammatory reaction observed in the laboratory animal (18). Chronic Inflammation The effects of dietary copper deficiency on experimental chronic inflammation were first studied in the adjuvant arthritis of the female rat, after 60 days of feeding the animals with either a copper-depleted (Cu 0.4 ppm) or a control (Cu 10 ppm) diet, before carrying out a tail-intradermal injection of heat-killed Mycobacterium butyricum finely suspended in liquid paraffin (complete adjuvant) (26). Note that the copper-deficient feeding was continued throughout the experiment (26). As expected, in the normally fed rats the systemic reaction to the challenge began to become evident macroscopically at about 12–14 days after the inoculum and reached its maximum development between the 18th and the 28th day (Fig. 1, upper panel). Together with a marked loss of body weight, the other major pathological signs [that represent the basis of the arthritic score routinely used to assess disease severity, the maximum theoretical value of which is 31 (5,26)] showed: (i) a dramatic swelling of the paws, particularly the hind ones; (ii) the appearance of numerous arthritic nodules randomly disseminated in the tail; and, especially, (iii) a very deep bone and cartilage degradation at the joint levels (chiefly the hind tibiotarsals), very clearly documented at both X-ray and histological examination, of which a severe joint stiffness and, in turn, a dramatic impairment of the deambulation ability were the consequence. In our experiment, in this group of normally fed animals, the highest arthritic score (29.4) was measured at day 21 (Fig. 1, upper panel) (26). At the same time, a progressive increase of total serum copper concentration was observed, which also reached its maximum (þ82%) 21 days after complete adjuvant injection (Fig. 1, lower panel) (26). On the contrary and rather surprisingly, the copper-deficient animals did not react to complete adjuvant injection, and the systemic response expected was almost totally abolished by dietary copper depletion. In fact, only a barely visible swelling of the hind paws was observed 21 days after the challenge (highest arthritic score 4.3; Fig. 1, upper panel), and the X-ray and histological examinations showed that the joint status of these copper-deficient and complete adjuvant-injected rats appeared to be almost fully normal (26). Moreover, throughout the experiment the total serum copper concentration, albeit extremely low, remained constant ranging between 10 and 15 mg/dL (Fig. 1, lower panel) (26). The fact that the diet-induced copper deficiency was a condition able to strongly


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35

*

Arthritic score

28

*

Cu-normal

*

21 14 7 Cu-deficient 0 0

7

14

21

28

Days after the inoculum

Serum Cu (µg/dL)

350

*

*

*

*

Cu-normal

280 210 140 70 Cu-deficient 0 0

7

14

21

28

Days after the inoculum Figure 1 Development of the adjuvant arthritis (arthritic score) and increases of total serum copper concentrations in normally fed and dietary Cu-depleted rats. Arthritic score (upper panel) and total serum copper (lower panel) in normally fed and copper-deficient adjuvant-arthritic rats. Source: From Refs. 11 and 26.

inhibit the normal development of the AA of the rat was confirmed by other authors (27). Previous reports have shown that copper is involved in the prostaglandin biosynthesis, and, in particular, that it seems to increase the conversion of arachidonic acid to prostaglandin F2a (an anti-inflammatory mediator), and to reduce the production of the prostaglandin E2 (a proinflammatory product of the arachidonic acid cascade) (28–31). In our experiment (26), we showed that the lung prostaglandin-synthetase activity was the same in

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normal and copper-depleted nonarthritic animals. Moreover, no difference was observed in the reactivity to exogenous prostaglandin E2 or F2a added to strips of rat stomach fundus or colon from nonarthritic copper-deficient, compared with nonarthritic normally fed animals. Thus, the above data seemed to exclude the influence of copper depletion on prostaglandin synthesis pathway as a possible mechanism explaining the observed phenomenon. On the other hand, at that time it was already known that a lower immunological reactivity (as measured by cytotoxic anaphylactic reaction, phagocytic, and serum bactericidial activities) was promoted by copper deficiency in the rat (32). Consequently, considering that in the development of the adjuvant-arthritis model a normal response of the immune system is strictly required, we proposed that the protective effect of copper deficiency on the ‘‘normal’’ development of the AA was an epiphenomenon secondary to an impairment of the immune function (5,26). This hypothesis was confirmed later on by Kishore and coworkers (33), who showed that the copper-deficient male rats (Cu in the diet 0.5 ppm; length of feeding 40 days) were actually in a state of apparent immunosuppression as demonstrated by impaired ex vivo responsiveness to the T-cell-dependent contact-sensitizing antigen oxazolone and diminished capacity to respond to the T-cellindependent antigen Type III pneumococcal polysaccharide stimulation. LABORATORY ANIMALS: STUDIES ON ‘‘ENDOGENOUS’’ COPPER METABOLISM IN ACUTE AND CHRONIC INFLAMMATION Illnesses always promote more or less remarkable, and sometimes dramatic, changes of the whole-body homeostatic mechanisms that come into play with the aim of self-defense, or may be ‘‘side effects’’ of the disease itself, which could even contribute to worsening the condition. This was, actually, the theoretical question posed by Whitehouse (8) speculating on the possible ‘‘inert,’’ ‘‘toxic,’’ or ‘‘pharmacoactive’’ role of ‘‘endogenous’’ copper in the development and control of inflammation. Thus, in light of the ascertained proinflammatory effect of nutritional copper deficiency on the development of the acute processes, it seemed reasonable to conduct more detailed studies on the changes in ‘‘endogenous’’ copper metabolism induced by the inflammatory process in laboratory animals. Some of the most significant results obtained are summarized in Tables 4 (and references therein; acute inflammation) and 5 (and references therein; chronic inflammation). Status of Copper in Blood and Urine Since the early reports, many different studies carried out in different animal species have shown that the acute inflammatory process, regardless of the noxa used to promote it, is characterized by a significant increase of total


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Table 4 Experimental Animals: Examples of the Changes in Copper Status Induced by Acute Inflammation in Some Relevant Body Compartments Species Rat

Disease

Turpentine edema Radiation injury Turpentine abscesses Femur fracture CP CP CPO Rabbit Turpentine edema Ocular inflammation Ocular inflammation Dog Turpentine abscesses Guinea pig Turpentine abscesses Mouse Collagenase injection

Total Blood Kid- Inflamed serum cells Liver ney area References I U I — I I I I I — I I I

U — — — I U U — — — — — —

— — I D U U U — — — — — —

— — — — U — — — — — — — —

— — — — — P I — I I — — —

1 34 35 36 37 38 38 39 40 41 42 43 2

Abbreviations: I, significantly increased; U, unchanged; D, significantly decreased; P, present in the inflammatory exudate.

serum (or plasma) copper concentration (Table 4) (2,3,34–43). An exception was apparently represented by the effect of radiation injury in the rat (34). However, this result failed to be confirmed by more recent work, which showed a remarkable increase of serum copper, even in that experimental model of inflammation (44,45). Also, chronic inflammation induced in the experimental animals typically brings forth a remarkable rise of total serum (or plasma) copper (Table 5), albeit an occasional report showed that, in dogs chronically suffering from nonexperimentally induced dermatitis or anal gland fistula, these diseases did not cause significant changes in metallo-element status in the serum (4,33,42,46–51). As a general rule, the inflammation-induced increase of total serum copper is accompanied by a parallel increase of serum ceruloplasmin concentration, and, as noted before, these two parameters characterizing serum copper status are highly and statistically significantly correlated in normal as well as acutely and chronically inflamed animals (4,16,52,53). Moreover, it has been shown in hamsters that transcription of ceruloplasmin mRNA increases within three hours of induction of inflammation by turpentine injection, reaching a peak 2.5-fold above normal at 12–18 hours (54). Thus, at least in animals fed a diet containing standard amounts of copper, the total serum (or plasma) copper measured during inflammation appears to represent an extremely reliable index of circulating ceruloplasmin levels, which, in turn, belies an old report according to which the human inflammatory process (rheumatoid arthritis) is distinguished by a selective rise of the

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Table 5 Experimental Animals: Examples of the Changes in Copper Status Induced by Chronic Inflammation in Some Relevant Body Compartments Species Rat

Dog

Disease AA AA AA Chronic inflammations AA AA AA S. aureus-induced arthritis Chronic otitis

Total Blood Inflamed serum cells Liver Kidney area References I I I I

— — — —

I I I I

D — — —

— — — —

4 33 46 47

I I I I

I U — —

I I I —

D — U —

I — — —

48 49 50 51

I

—

—

—

—

49

Abbreviations: AA, adjuvant-induced arthritis; I, significantly increased; U, unchanged; D, decreased.

non-ceruloplasmin-bound fraction of serum copper (1). This latter claim was subsequently further disproved by Freeman and O’Callaghan (55), who, directly measuring (following column fractionation) the non-ceruloplasmin-bound copper in the serum of normal and inflamed animals and humans, showed that this fraction of circulating metal undergoes only negligible variations during both adjuvant arthritis in the rat as well as human rheumatoid arthritis. Another issue is worth emphasizing in view of its relevance in the studies on ‘‘endogenous’’ copper involvement in human inflammatory diseases. Although the above-reported increases of circulating copper and/or ceruloplasmin are important markers in revealing the existence of an inflammatory state [unless, for example, pregnancy is present, or the subject is undergoing steroid therapy (56)], the data obtained during the past years in our laboratory, studying over 1200 inflamed rats, show that serum or plasma copper as well as ceruloplasmin concentrations are not directly correlated with the actual severity of the pathological condition examined, and cannot even discriminate between acute and chronic processes. In fact, it was found that different acute nonseptic inflammations (e.g., rat CPE and CP), which developed in very similar manner, caused remarkably variable increases in the above parameters (57,58). A great variation in increased total plasma copper concentration was also reported among adjuvantarthritic rats regardless of illness severity level (26,48). In particular, the average increase in total plasma copper in animals, which manifested experimental disease to a mild degree, was of about 54%, and that measured in rats bearing a fully expressed pathology was approximately 60% (46). Therefore, hypercupremia that characterizes any inflammatory reactions appears


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to be a sort of ‘‘all or nothing’’ phenomenon that has indeed little value as a marker of disease seriousness. To conclude the analysis on copper status in blood of acutely or chronically inflamed animals, the erythrocyte compartment remains to be considered. A number of contradictory data have been reported on this issue. In fact, according to some authors neither acute nor chronic inflammatory processes changed the concentration of copper in the erythrocytes, whereas an increase of this fraction of total blood copper was reported in rats with carrageenan-induced pleurisy and adjuvant arthritis (3,37,38, 48,49). However, the increase of erythrocyte copper measured in the arthritic rats (48), although statistically significant at day 3 and 21 after challenge, was small and probably did not have major biological relevance. Moreover, as ascertained later on studying the same parameter in human rheumatoid arthritis (59), the rise of copper in the red cells of chronically inflamed subjects was, most likely, a secondary phenomenon on which the significant decrease in erythrocyte volume had a not negligible influence. Finally, very few and contradictory data exist on the urinary copper excretion in acutely and chronically inflamed rats. For example, the metal concentration in the urine was found to increase in femur-fractured animals, whereas the challenge with complete adjuvant did not modify this parameter (36,49). Therefore, at least as the experimental models are concerned, it is not possible to draw any conclusion on this topic on the basis of existing evidence. Status of Copper in Solid Tissues and Inflamed Areas Before getting into a detailed discussion of inflammation-induced changes in copper status in some solid tissue involved in onset, development, and control of the inflammatory processes, a preliminary issue deserves attention. In most, if not all, papers dealing with copper and inflammation, the amount of the metallo-element measured in the different body compartments is exclusively reported as concentration values. Nevertheless, in some instances this could be a misleading index. As a matter of fact, we studied the concentration and total amount of liver copper during a daily ninehour period in normal rats, i.e., between nine hours and 30 minutes and 18 hours and 30 minutes (Table 6) (60). The results reported in Table 6 clearly show that during the period of time considered, an apparent statistically significant increase in liver copper concentration occurred. However, this phenomenon was essentially due to a parallel and progressive decrease of liver weight (possibly caused to different states of liver hydration and/or glycogen content); conversely, the total amount of the metal measured in this compartment remained constant throughout the experiment (60). This physiological phenomenon strongly suggests: (i) the evaluation of copper concentration and total amount, as well

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Table 6 Daily Changes in Hepatic Copper Concentration and Total Amount in Normal Female Rats Time (hours) 0 3 6 9

Total serum Cu (mg/dL)

Liver weight (g)

Liver copper Cu (mg/g)

Total liver Cu (mg)

151.3 þ8% þ0% þ3

6.6 4% 12%a 21%b

4.31 þ5% þ16%b þ25%b

33.03 þ2% þ5% þ3%

Note: Statistics—Student’s t test. a P < 0.05. b P < 0.01. Source: From Ref. 60.

as (ii) the routine use of a control group (‘‘time controls’’) should be utilized in every experiment, and these rats should be killed at the same time as those of each treated group, thus ensuring that the results obtained and their discussion could have a better chance of being fully reliable (60). Actually, we strictly followed this rule in all the experiments carried out in our laboratory. As shown in Tables 4 and 5, the liver is the tissue in which copper status has been most frequently studied in inflamed animals. We already know that the liver is the major organ responsible for ceruloplasmin synthesis and secretion into blood (17,56). Then, in the acute processes, the first striking evidence is that, in spite of the remarkably increased production and release of this protein induced by inflammation, the level of total hepatic copper does not decrease (37,38). The femur fracture in the rat is, perhaps, the only model of an acute inflammatory process in which a reduced liver copper concentration was reported (36); whether or not this isolated result depends on the peculiarity of this important traumatic condition is unknown. On the other hand, when chronically inflamed animals were studied, the hepatic level of copper was always found to be significantly increased, both as concentration and total amount values (4,33,46–50). Moreover, we observed in the adjuvant-arthritic rat that a rise of hepatic copper level preceded the appearance of any visible signs of the disease, and, afterwards was found to be directly proportional to the manifested severity of the pathology (46,61). In greater detail, liver copper total amounts were statistically significantly increased above control values three days after the challenge (þ15%), i.e., during the asymptomatic phase (46). Furthermore, when the disease reached its complete development (i.e., at day 14, 21, and 30), the average hepatic copper rise was of about þ61% in the severely arthritic rats, but only of about þ31% in those animals that manifested the pathology to a mild degree, these two data being significantly different in the cross Student’s t test evaluation (48). Thus, according to our results and in contrast with total serum copper and ceruloplasmin concentrations, the hepatic metal level


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status appears to be a consistent index of the overall importance of the inflammatory process, being able to clearly discriminate among acutely, and chronically weakly or seriously affected rats. The kidneys, together with the red blood cells, the liver, and to some extent the plasma, are compartments in which copper seems to be easily exchangeable and thus available to be moved to other tissues according to actual body needs (61). As a consequence, the study of copper status in the kidneys could provide useful information to better understand the effect of inflammation on this ‘‘endogenous’’ metal metabolism. Unfortunately, very few data are available, especially on the acutely inflamed animals, in which the renal copper level was found not to be influenced by the development of the carrageenan-induced pleurisy of the rat (37). On the contrary, the chronic inflammatory process appeared to cause a significant decrease of kidney copper levels, although other authors failed to confirm this evidence (4,48–50). One of the above papers described that, like hepatic copper increases, the decreases of both concentration and total amount of copper in kidneys were not only statistically significant during the asymptomatic phase of the rat AA (day 3), but also dependent on the seriousness of disease development, and reached their maximum level in the severely arthritic rats 30 days after complete-adjuvant injection (48% compared to the total metal amount measured in the time- and age-matched, healthy controls) (48). Whether or not the decrease in total copper amount observed in the kidneys (which, differently from the liver, cannot be promptly supplied by metallo-element from the digestive tract) may be the expression of the need for a physiological mobilization of copper towards extrarenal biological pathways more directly involved in the control of the inflammatory process is presently unknown. Occasionally, but limited to the rat AA model, the concentration of copper in compartments other than blood, liver, and kidney has been studied. Thus, copper levels were found to be increased in the brain, stomach, bone, and pancreas, while, according to the same paper, they were unchanged in the heart and skeletal muscle (50). Other authors, however, found the concentration of this metal unaffected in the femur and in the brain (49,62,63). An increase of copper concentration and total amount was measured in the spleen of the adjuvant-arthritic rat, being once again directly related to the seriousness in the development of pathology (46). However, these increases actually may bear, per se, an uncertain biological meaning, since they were found to be mainly dependent on the increases of spleen weight and total serum copper levels, both of which were induced by the experimental pathology itself (48). Although very rarely evaluated, the analysis of copper status within inflamed fluids and tissues certainly deserves special attention. The evidence so far available shows that copper concentration in acute inflammations (Table 4) was found to be higher, compared with the noninflamed fluids

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or tissues, in the inflamed and untreated aqueous humor of endotoxininjected rabbits, in the intrathoracic exudate caused by the injection of a sterile carrageenan suspension in the rat (in this instance, due to technical reasons, the comparison with the extremely small amount of fluid present in the thoracic cavity of noninjected animals was not done), and in the carrageenan-injected, inflamed rat paw (sectioned at the level of the tibiotarsal joint) (38,40,41); in the last case a significant inflammation-induced copper increase was observed, considering both the concentration and the total amount of metallo-element. Copper concentration was, instead, found unchanged in the colon of rats in which an experimental untreated colitis was induced (64). On the other hand, the intravenous (IV) injection of 67Culabeled porphyrins in male rats showed a very remarkable uptake of the radioactive compound not only in the liver and kidneys but also in neoplastic tissue, when present and wherever localized in the animal body (65). In addition, the subcutaneous (sc) treatment with 64Cu-labeled D-penicillamine of rats with an experimental sponge granuloma evidenced a selective accumulation of the radioactive metal within both the implanted sponge and the capsule that had surrounded it as a consequence of the organism’s reaction (66). Also in the AA rat model (Table 5), the inflamed hind paws (again sectioned at the tibio-tarsal joint) showed a statistically significant rise in copper concentration (and total amount), already evident during the asymptomatic phase (i.e., seven days after the challenge), and became dramatic when the disease reached its symptomatic stage (48,62,63). Like previously described for the liver and the kidneys, when the symptomatic phase was evaluated specifically, the copper status of the hind paws was clearly able to discriminate between slightly and severely affected animals (Table 7). Concerning the increased amounts of copper in the carrageenaninjected rat paw, a further point is worth emphasizing. The onset of the Table 7 Hind Paws Copper Status During the Symptomatic Phase of the Complete-Adjuvant-Induced Arthritis of the Rat Mild disease Days after the inoculum 14 21 30

Cu (mg/g) a

þ26% þ38%a þ32%a

Severe disease

Cu total (mg) a

þ92% þ50%a þ47%a

Cu (mg/g) a,b

þ39% þ136%a,b þ106%a,b

Cu total (mg) þ160%a,c þ183%a,c þ260%a,c

Note: Statistics—Student’s t test. a P < 0.01 versus time- and age-matched noninflamed controls. b P < 0.01 versus copper concentration of time- and age-matched mild-disease group. c P < 0.01 versus copper total micrograms of time- and age-matched mild-disease group. Source: The data reported in the table are converted to percentage increases from the original values published in Ref. 48.


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acute inflammation is characterized by progressive leakage from the local vascular bed, of an inflammatory exudate made by a plasma-proteins-rich fluid (containing also ceruloplasmin) in which a great number of inflammatory cells (mainly leukocytes) are present (67). This phenomenon induces the formation of an edema that, when carrageenan is used as inflammatory agent, is already measurable one hour after the challenge, and, in most instances, reaches its highest values between three and about 4–8 hours later (68,69). Subsequently, the liquid fraction of the exudate is rapidly reabsorbed, whereas the cell fraction remains in situ for several days to carry out the repair and remission processes (67). If the data reported in Table 8 are taken into account, it clearly emerges that the statistically significant increase of paw-copper concentration precedes the hours in which the characteristic rise of total plasma copper (and ceruloplasmin) is measured. Moreover, the paw copper is still significantly elevated above control values 96 and 144 hours after the challenge, i.e., when the edema has almost entirely disappeared and the total plasma copper returned to normal. The same is also observed if the total amounts of paw copper are considered (data not shown). In the chronic processes, the edema present in the affected animals is mainly formed by a solid tissue made of different inflammatory cell types (such as leukocytes, lymphocytes, macrophages, fibroblasts, epithelioid cells, etc.) together with local necrotic cells and newly formed fibrous as well as synovial tissues; but, in contrast with acute inflammation, the serum-plasmaderived fluid component is much less prominent (5,67). Evaluating the status of copper in the hind inflamed paws of the arthritic rats, a scenario is observed

Table 8 Percentages of Concentration Changes in Plasma and Paw Tissue Copper in Carrageenan-Inflamed Rats Time after the challenge (hours) 0 1 3 5 24 48 72 96 144

Edema weight (g)

Total plasma copper (mg/g)

Paw copper (mg/g)

0 0.38a 1.04b 1.19b 0.51b 0.44a 0.41a 0.23 0.16

0 4 2 3 þ74b þ63b þ55b þ8 2

0 þ12a þ20b þ19b þ21b þ26b þ25b þ25b þ21b

Note: Statistics—Student’s t test versus the time- and age-matched controls. a P < 0.05. b P < 0.01. Source: From Refs. 37, 38, and 60.

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similar to that described in the case of the acutely inflamed animals. In particular, as pointed out before, a significant increase of copper concentration and total amount are measured during the asymptomatic phase of the experimental disease (day 7; þ14%, P < 0.01), and, later on, these same parameters appeared to be very reliable markers of the severity in experimental illness (Table 7) (48,62,63). Interestingly, the whole blood copper amounts (calculated on the basis of body weight, according to the Altman and Dittmer formula) were found comparably higher throughout the experiment, being insignificantly different among them in the asymptomatic, slightly and seriously affected rats (48,70). Thus, all the evidence summarized above seems to suggest that the increase of copper, quantified in the acutely and chronically inflamed rat paw tissue, is not merely a consequence of the increased plasma copper levels, but, rather, the expression of a true and selective metal accumulation in this area. This event can, in turn, be seen as a signal revealing an increased local need for copper, which could have been induced by the reaction that the body promoted to counteract the inflammatory noxa. Finally, taking into account all the organism’s compartments in which acute or chronic inflammation-induced significant changes of the total copper amount were measured [i.e., the whole blood, liver, kidney, spleen, inflamed pleural exudates, and inflame paw (37,38,48,60,62,63)], we recalculated the comprehensive variations of the rat body copper content brought about by the inflammatory processes examined. The data obtained (Fig. 2) clearly show that: (i) both acute and chronic inflammatory processes induced an overall increase amount of the metallo-element in the organism; (ii) although the total whole blood copper content (which we know to be raised to comparable extents as response to any sort of inflammation) and the total content of kidney copper (that, as previously reported, was reduced in the AA of the rat) were taken into account in the calculation, the net accumulation of body copper reported in Figure 2 was found directly proportional to the severity of the experimental pathology studied. Importantly, the above-mentioned accumulation appeared to occur without depleting other compartments of the metal, such as the bone and the muscle, which are known to hold high amounts of copper (49,50,61). Finally, considering that the healthy adult rat contains about 2 mg/g of body weight of copper (71), the total copper content of the control animals that we used for our experiments ranged between 250 and 420 mg. Thus, the statistically and biologically significant inflammationinduced increases of the metal reported in Figure 2 ranged between 5% and 7% in the acutely inflamed rats, and may even approximate 25% in the case of the severely affected arthritic animals. In conclusion, as commented for the inflamed tissue, the above observation seems to suggest that the inflammatory challenge induced also a whole-body increased demand for copper, to be used in the onset and development of the physiological regulatory mechanisms devoted to keep the inflammatory process under a proper control.


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Accumulation of body copper (Total µg) a,b,c

56

42

28

a,b

a a

14

0

Acute inflamm.

Chronic inflamm. Asymptom.

Chronic inflamm. Mild dis.

Chronic inflamm. Severe dis.

Figure 2 Comprehensive copper accumulation in some rat body compartments relevant to the inflammatory process. Accumulation (expressed as the difference of total micrograms between inflamed and noninflamed animals) of total copper measured in the body compartments in which significant inflammation-induced variations of copper status were measured. The bars represent the net body increments of the metal promoted by the different experimental acute and chronic inflammatory processes studied in various rat models. Statistics (Student’s t test): a, P < 0.001 versus age-matched non-inflamed controls; b, P < 0.001 versus age-matched asymptomatic arthritic rats; c, P < 0.001 versus age-matched mild-disease arthritic rats. Source: From Refs. 37, 38, 48, 62, and 63.

HUMAN SUBJECTS: STUDIES ON ‘‘ENDOGENOUS’’ COPPER METABOLISM IN ACUTE AND CHRONIC INFLAMMATIONS, WITH A PARTICULAR REFERENCE TO RHEUMATOID ARTHRITIS The inflammation-induced changes of ‘‘endogenous’’ copper metabolism were also frequently studied in humans, focusing especially, though not exclusively, on rheumatoid arthritis (RA) (Table 9, and references therein). Due to obvious ethical reasons, the experimental approach to this problem was restricted to the body compartments in which an examination by nonor mini-invasive techniques was possible, i.e., in the plasma, red blood cells, urine, and, in few instances, the inflamed areas. The foremost aims of these

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Table 9 Examples of the Changes of Copper Status Induced by Some Acute (A) and Chronic Inflammatory Diseases in Man (Cp ¼ Ceruloplasmin) Disease Pneumonia (A) Periodontal disease (A) Tonsillitis (A) Sandfly fever virus (A) RA RA RA RA Ankylosing spondylitis RA RA RA RA RA (juvenile) RA RA RA Psoriatic arthritis RA RA (juvenile) RA

Serum Serum Blood 24 hours Inflamed total Cu Cp cells urine area References I I I I I I U U I I I I I I I I I I I I I

— I — — U — — — — — I I I I — — — — I — I

U — — — — — — — — — — U — — — — I U U — I

— — — D — — — — — I — — — — — — U — U — U

— I — — — I U — — — I — — — I — — — — — —

72 2 73 74 3 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 59

Abbreviations: I, significantly increased; U, unchanged; D, decreased; RA, human rheumatoid arthritis.

researchers were: (i) to verify the hypothesis that a marginal copper deficiency may be a contributory factor in the etiology of rheumatoid arthritis; (ii) to validate the assumption that the development of a rheumatoid process could, in time, induce a depletion of the body copper stores; and (iii) to evaluate whether or not the copper status in serum could be taken as a reliable index of the severity of the chronic pathology studied (90–93). In particular, an experimental support to either or both the above (i) and (ii) theoretical propositions would offer a rational justification for the use of the ‘‘exogenous’’ copper administration as an important metal re-equilibrating treatment, able to favor the amelioration, if not the resolution, of the rheumatoid condition. In humans, the existence of infectious, immune (or autoimmune), and, in general, any acute or chronic disease with a relevant inflammatory component (cancer included), is typically characterized by a significant increase of total serum (or plasma) copper and ceruloplasmin concentrations, these two parameters being highly and significantly correlated (59,80–83,94). As


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previously noted while discussing the animal models of inflammation, in the case of rheumatoid arthritis, in spite of the very high correlation existing between serum copper and ceruloplasmin levels, some authors found that the increase of the serum metal was essentially determined by a selective rise of the non-Cp-bound fraction of this parameter (3). This claim was proposed on the basis of the data obtained indirectly measuring the non-Cp-bound copper, i.e., calculating this value by subtracting from the total serum copper concentration the amount of the metal supposed to be carried by the ceruloplasmin itself. However, applying the same methodology, other authors confirmed that the serum metal increases were basically due to the rise of circulating ceruloplasmin (59,82). As it was already reported in this paper, a further and, perhaps, conclusive support for the latter evidence was obtained directly quantifying the actual amounts of non-Cp-bound copper in the serum of rheumatoid patients, in whom only minor variations of this circulating fraction of the metal were observed (55). Finally, considering as a whole the data summarized in Table 9, it appears that only very few studies reported the total serum copper or ceruloplasmin concentrations as unchanged, and none noted a decrease in the level of the above two parameters (3,76,77). Total serum copper and ceruloplasmin do not change significantly in relation to the dietary supply of this metal, unless a biologically significant copper-deficient alimentary regimen is adopted, or relevant amounts of intestinal copper-absorption inhibitors, such as zinc, iron, calcium, ascorbic acid, phitate, etc., are present in the food (56,71,95). Conversely, the ceruloplasmin concentration in the serum, and thus the total serum Cu, is strongly influenced by a great number of physiological factors, (e.g., hormonal levels) and pathological conditions (such as stress, inflammation, etc.) (17,56). On the other hand, the erythrocytes are considered to be a blood compartment in which copper is less susceptible to the hormonal as well as pathological stimuli, unless the pathology considered is directly concerning the erythrocyte itself (71,95). For this reason, the red blood cell copper level is taken to be a relatively more reliable index of the actual overall body copper status, at least as far as moderate deficiencies have to be identified (see below for further details) (71,95). In general, the erythrocyte copper concentration was found unchanged in this compartment of blood in RA patients (Table 9), with the exception of two studies carried out by the same research team that reported a significant increase of this parameter (59,86). However, this last claim turned out to be a misleading result since, as previously recalled in this paper, the apparent increase of the erythrocyte copper level (measured as mg/mL of metal in the packed cells) was actually due to the RA-induced significant decrease in the individual volume of red blood cell, and the overall amount of copper contained in this total fraction of blood [calculated according to the formula of Altman and Dittmer (70)] resulted not to be significantly different from that of the age-matched healthy controls (59).

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Another compartment that may be considered a somewhat good index of a possible copper deficiency status is the 24-hour urine, albeit, as for the total serum copper, the level of urinary copper is sensitive to biologically significant body metal depletion, but not predictive of a marginal deficiency condition (71,95). This index was found to be decreased only in one case of acute inflammation [i.e., in the sandfly fever virus infection, carried out on healthy volunteers (74)]. In the rheumatoid arthritis patients, an early report of increased copper urinary excretion was published, whereas more recent works found this parameter unchanged, thus convincingly suggesting that RA does not promote a significant increase of body copper losses via this excretory pathway (59,79,86,88). To our knowledge, the copper status in an inflamed human solid tissue was studied only in patients suffering from acute periodontal disease, in which a 15-fold increase of the metal (measured as Cp levels) was found in the inflamed as compared to the normal periodontal tissue explants (2). A few more data exist on the copper content in the synovial effusions withdrawn from the actively inflamed joints of RA patients, compared with the fluids taken from the human osteoarthritic or traumatic knees used as control values (75,76,80,84). In one single case, the copper concentration of the rheumatoid synovial fluid was found to be insignificantly different from that measured in the osteoarthritic subjects, whereas in the other instances a dramatic increase of the metallo-element levels was measured in this particular compartment (75,76,80,84). We would like to stress that this evidence, together with that obtained evaluating the status of copper in human inflamed blood, closely recalls the behavior of the metal metabolism previously described when studying the development of acute and chronic inflammations in the laboratory animal. The data summarized in the above section of the paper, together with other published information, may help in the attempt to propose some reasonable answers to the questions with which this survey on the human’s ‘‘endogenous’’ copper metabolism in inflammation (especially RA) has been introduced. The hypothesis that a marginal copper deficiency could contribute to the development of the rheumatoid arthritis was detailed by Rainsford and Sorenson (90–92). These authors reported that there was evidence suggesting that the dietary intake of copper in some Western countries might be well below that required for natural physiological functions. Moreover, it was pointed out that high levels of environmental pollutants, toxic metals such as lead and especially cadmium, might act as antagonists of copper absorption or affect the normal development of its biological pathways (91). However, although at least in the United States, the average intakes of copper may even be below the FDA’s recommended dietary allowances, there are still conflicting opinions about the practical need for concern over health in relation to copper status in humans (96). Moreover, this issue cannot be directly solved using the techniques available today for


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defining the existence of a marginal copper deficiency. In fact, the serum copper and Cp concentrations may well reveal a moderate or severe metal deficiency but are not at all responsive to marginal deficiencies (97,98). The same lack in the capacity to reveal a marginal copper-deficiency condition is true for both the 24-hour-urine metal excretion and the total copper red blood cell level (98). Conversely, the evaluation of a body copper marginal deficiency could, perhaps, be achieved by studying erythrocyte SOD 1 activity, although an early work reported that this parameter might increase in conditions in which an oxidative stress of any origin has been caused (97,99,100). More promising seems to be the measure of the cytochrome-c oxidase activity in the platelets. Actually, studies with rats show that this enzymatic marker is a sensitive sign of copper stores (101,102); the cytochrome-c oxidase activity in platelets highly correlates with copper concentration in the liver (r ¼ 0.99, P < 0.0001), an established indicator of copper status in animals (97). In conclusion, the hypothesis that a marginal copper deficiency status could favor the onset of the human rheumatoid arthritis cannot currently be either confirmed or denied. On the contrary, according to data available today, it seems reasonable to suggest that adequately nourished RA subjects do not develop a significant body copper depletion over time (59,85). In particular, studying the copper content of plasma, blood cells, and 24-hour urine in rheumatoid arthritis patients divided into four groups according to their disease duration, i.e., 0–1, 1–5, 5–10, and over 10 years, we have clearly shown that all the above-mentioned parameters were not significantly influenced by the duration of the disease itself (59). It seems worthwhile to stress that, at least for the urinary copper excretion and copper erythrocyte levels, a progressive decrease of these indices would have to be observed if the disease were responsible for a time-dependent biologically significant depletion of body copper stores (71,95). Consequently, it appears reasonable to exclude the need for a dietary copper replenishing therapy in response to the hypothesized RA-induced copper deficiency in humans suffering from rheumatoid arthritis. Finally, focusing on the possibility that the total serum (or plasma) copper could be taken as a predictive clinical marker in the rheumatoid patient, this speculation has been belied by recent work (59,85). For instance, plasma copper was found to correlate significantly with some other plasma indices of RA, such as the iron concentration (r ¼ 0.20, P < 0.050), total proteins (r ¼ 0.33, P < 0.010), a2-globulins (r ¼ 0.46, P < 0.010), c-globulins (r ¼ 0.22, P < 0.050), and ceruloplasmin (r ¼ 0.77, P < 0.001), but no significant correlations were found versus the well-established major clinical markers of the disease, i.e., the number of swollen joints, the grip strength, the functional class, the anatomical stage, and the physician’s assessment index (59). Interestingly enough, the above data confirm, adding more sophisticated details, the evidence already reported in this review when discussing the acute and chronic animal models of inflammation, which led us to propose that the increase of total serum (or plasma) copper is most

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likely an ‘‘all or nothing’’ phenomenon, unable to discriminate between mild and severe inflammatory conditions. EFFECTS OF ‘‘EXOGENOUS’’ COPPER ADMINISTRATION ON THE INFLAMMATORY PROCESS The data summarized so far may be briefly outlined as follows: Dietary copper deficiency acts as a proinflammatory stimulus, at least in animal models of acute inflammation. In rats on a normal Cu-containing diet, the induction of acute and chronic inflammatory processes clearly produces a condition in which more copper is required by some compartments of the organism. In view of the fact that no major redistribution of ‘‘endogenous’’ copper occurs, it seems reasonable to suggest that this increased demand for the metal is mainly fulfilled by enhanced intestinal absorption and/or decreased intestinal excretion of copper (37,57). Interestingly, it has been shown that turpentine edema increases the amount of dietary copper required to maintain hepatic SOD 1 levels equal to those of nonstressed rats (103). Moreover, albeit limited to blood, urine, acutely inflamed periodontal tissue, and rheumatoid synovial fluids, the data reported in humans would appear to give further support to the above hypothesis (57). Due to reasons that are not yet understood, however, the natural antiinflammatory effect promoted by increased intestinal absorption and/or decreased intestinal excretion of the metallo-element appears to be susceptible to significant improvement by the therapeutic administration of copper compounds (7). Thus, the use of ‘‘exogenous’’ copper preparations as remedies to counteract the inflammatory pathologies appears to consistently find its rationale in the above-summarized remarks. In turn, the study of anti-inflammatory potential of administered copper would help to better understand the overall network of relationships existing between this metal, both ‘‘endogenous’’ and ‘‘exogenous,’’ and the onset, development, and remission of the inflammatory process. Problems Related to Routes of Copper Administration The final therapeutic goal regarding the use of copper preparations as anti-inflammatory agents is to identify effective and safe drugs to keep human rheumatoid arthritis, an important deabilitating disease for which a fully satisfactory treatment has not yet been found, under adequate control, if not to cure it (104). To reach this goal, the physician should not only consider an effective and safe copper compound, but also be able to administer this potential drug (which could be possibly used for long periods of time) by a nontraumatic route like oral or topical.


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Many reviews have been published summarizing the anti-inflammatory properties of over 140 different copper(II) complexes with a wide variety of ligands (92,105–108). Examination of the literature reveals that for the large majority of these copper-containing molecules the anti-inflammatory and/or antiarthritic activity was evaluated in animal models, following sc or intraperitoneal (ip) administration. In fact, sc and ip treatments are preferred to demonstrate and reproduce in vivo biological activity of copper. Copper(II) preparations given by any parenteral route tend to disappear rapidly from plasma, being mainly accumulated in the liver and, to some extent, in the kidneys (109). However, when these substances are administered orally, they may not enter the organism unchanged, and their copper content may not become bioavailable, unless very special Cu(II) ligands or particular vehicles and adequately high amounts of complexes are used. Most copper compounds, when exposed to the acidic pH in the stomach, actually undergo nearly complete dissociation, followed by formation and absorption of new, in situ formed Cu(II) complexes (110,111). Copper is then transferred from the portal circulation to the liver, which regulates its subsequent metabolism by:

Storing the metal as Cu-thionein in the hepatic tissue, thus creating a physiological reservoir of the metal to be used according to actual physiological needs (56). Completing the synthesis of the copper-dependent proteins, a process in which a membrane copper P-type ATPase, the Wilsondisease protein (WND) (112), seems to be involved in delivering Cu(I) ions into the Golgi’s vesicles. In particular, the WND protein acts co-operatively with the ClC-4 chlorine channel (113) to assemble the olo-ceruloplasmin in this subcellular apparatus before the multifunctional protein is secreted into the general circulation. Finally, excreting the excess copper via the bile, once again utilizing the activity of the WND protein assisted by that of the Cu-chaperon Murr 1 (114).

As previously stressed the Cp synthesis is not induced, during conditions of adequate dietary copper intake, by increasing the amount of the metal present in the diet, nor by supplementing the organism with copper given by any parenteral route (17,56,62,109). On the other hand, the homeostatic mechanisms regulating the Cp production and secretion appear to react exclusively to endogenous stimulations (17,56). Therefore, if the orally administered copper complex is broken down by gastric juice, the extra copper that enters the liver will, most likely, undergo hepatic first-pass clearance, becoming not bioavailable for any therapeutic purpose. Theoretically, topical administration of copper is the most convenient method of treating rheumatoid arthritis, because it may avoid or minimize the risks of systemic toxicity, and it may direct the drug straight to the

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affected area, i.e., the inflamed joints. The main problem with such treatment is to identify both ligands and formulations that allow to carry therapeutically significant amounts of the active principle through the skin. This problem is made worse by the poor lipophilic nature of most Cu(II) compounds. Thus, the goal to get any important clinical success using topically applied copper preparations in the treatment of RA patients has not yet been accomplished. Last but not least, the iv route of administration has to be considered, although in recent times it only has been used in a few cases. Nevertheless, two of those deserve mention. In the first case, a number of copper-containing proteins was tested for their anti-inflammatory potential, and found to be active in an acute animal model of inflammation. In the second, and certainly in a more significant one, the antirheumatic action of a copper(II) complex with salicylic acid has been clinically evaluated in man. This latter study appears to bear even nowadays great interest, both theoretical and practical. We would like to open our survey on the effects of ‘‘exogenous’’ copper on the development and control of inflammation, reporting on iv administration of these metal-containing molecules. Intravenous Administration of Copper Compounds Laroche and coworkers (20) isolated a number of copper-dependent enzymes from different animal and vegetable sources, and iv-tested them for their acute anti-inflammatory activity in the laboratory animal, using yeastinduced paw edema model in the mouse (Table 10). As reported in the table, the copper proteins ceruloplasmin (isolated from beef erythrocytes) and laccase [isolated from a fungus belonging to the genus Polyporus (70)], which are both biochemically classified as ‘‘bluecopper oxidases,’’ appear to be the more active among the tested molecules. It is relevant to note that, as stressed by the authors to validate their results, Table 10 Examples of Anti-inflammatory Activity of Some Cu-Dependent Enzymes Administered iv in the Mouse, Challenged with a Subplantar Injection of a Yeast Suspension Copper-dependent enzyme Cu-serum albumin Ceruloplasmin SOD 1 Diamine oxidase Cytochrome oxidase Ascorbate oxidase Laccase

Source

Edema inhibition (%)

Human serum Beef serum Beef erythrocytes Pig kidney Pseudomonas aeruginosa Cucumis sativus Polyporus versicolor

None 73 44 35 53 37 72

Abbreviations: SOD 1, cytoplasmatic Cu,Zn superoxide dismutase; iv, intravenous. Source: From Ref. 20.


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the anti-inflammatory activity was measured three hours after the challenge to which the iv treatment followed immediately. This makes it possible to use heterologous proteins without inducing any immune reaction in the experimental animal (20). As mentioned regarding intravenous use of copper complexes in humans, a 20-year-long clinical study demands attention. Hangarter (115) reported that in the treatment of arthritic diseases the therapeutic results with iv copper alone were comparable to those of chrysotherapy, although copper treatment did not give rise to the considerable side effects associated with gold-salts treatment. However, Hangater’s major efforts were dedicated to clinical evaluation of a copper complex marketed as PermalonTM. Chemically, the antirheumatic drug Permalon is a complex of one or two cupric ions coordinated by two or four salicylate molecules, i.e., Cu(II)(salicylate)2 or Cu(II)2-(salicylate)4 (105,116). In the period between 1950 and 1971, Hangarter (115,116) studied the activity of Permalon in 1147 patients suffering from different rheumatic-degenerative diseases such as the arthrosis deformans, sciatica, erythema nodosum, lumbar spine syndrome, cervical spine–shoulder syndrome, acute rheumatic fever, and, particularly, rheumatoid arthritis. Focusing on the RA patients, the treatments with slow iv infusions of cupric salicylate followed the protocol reported in Table 11, where the clinical results obtained are also summarized. Commenting on the above results the author stated that, according to own experience, the antirheumatic effects obtained with intravenous cupric salicylate were considerably superior to those achieved with the antirheumatic agents commonly used at that time, such as nonsteroidal anti-inflammatory drugs (NSAIDs), antimalarial agents, gold salts, as well as cortisone preparations (116). Interestingly, during the open discussion that followed the presentation of that paper at an international symposium held in Little Rock (Arkansas) in 1981, it emerged that the RA patients who responded to the therapy with remission of the disease maintained Table 11 Protocol of Permalon Treatment Within 620 Rheumatoid Arthritis Patients, and Results Obtained by the Therapy—A 20-Year Study Treatment

6–10 infusions at intervals of 2–4 days

Composition of each infusion Salicylate: maximum dose for a full cycle Copper: maximum dose for a full cycle Therapeutic effect reported: Remissions Significant improvements No effects

Salicylate 6–8 g; copper 7.5–10.0 mg 80 g, i.e., average of 2.5 g/die 100 mg, i.e., average of 3.1 mg/die

Source: From Ref. 116.

403 cases (65%) 143 cases (23%) 74 cases (12%)

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their completely symptom-free status for a subsequent average period of three years (116). The therapy was occasionally accompanied by a transient nausea and tinnitus, both of which are very well-known minor side effects due to the administration of high salicylate doses (117). However, no other adverse reactions were observed during the treatments as well as the subsequent follow-up. In particular: (i) no disturbances were reported at the level of the gastrointestinal tract; (ii) the blood sugar, electrolyte levels, and the classical serum electrophoresis markers were maintained within the normal ranges; and finally, (iii) no evidence of cardiac, circulatory, renal, hepatic, respiratory, or central nervous system toxicities were found (115,116). Unfortunately, the above-reported results were obtained following the criteria of an open trial (i.e., in the absence of a double-blind comparison versus both sodium salicylate- and placebo-treated control groups); consequently, they cannot be taken as a valid report on the basis of clinical protocols of evaluation accepted today. In 1971, Prof. Hangarter retired; the production of Permalon was discontinued by the manufacturer for economic reasons (116), and since then no other research team decided to submit cupric salicylate to a currently officially approved clinical trial. Subcutaneous and Intraperitoneal Administration of Copper Compounds Table 12 shows a few representative examples of copper compounds, such as simple copper salts, copper-containing proteins, and copper complexes with widely different inorganic and organic ligands, which have been shown to possess significant anti-inflammatory and/or antiarthritic properties following sc or ip injections in the experimental animals. Notably, a number of these copper complexes are formed using NSAIDs as complexing agents. Although today well established at least in the animal models, the anti-inflammatory activity of copper compounds subcutaneously or intraperitoneally injected, but also given orally, has been the subject of a dispute originated by the actual irritating potential that copper ions could exert at the site of exposure (110,129). In the classical models of experimental acute inflammation, the compounds to be tested are administered one hour before the inflammatory challenge, and their activity is evaluated between three and seven hours after the challenge itself (68,69). Bonta and Noordhoek (130) showed that a preventive ip injection with a well-known promoter of acute inflammation, i.e., a carrageenan suspension, was able to strongly inhibit the normal course of the subsequent inflammatory reaction induced by subplantar injection of a kaolin preparation. The authors proposed that the challenge with an irritant (or potentially irritant) substance done at one site of the body could trigger the organism’s physiological antiinflammatory response, thus enabling the living system to reduce the degree of the inflammation later on induced by the same, or any other irritant,


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Table 12 Representative Examples of Copper Compounds Active as In Vivo Anti-inflammatory and/or Antiarthritic Agents, Following sc or ip Administration in the Animal (Rodents) Models of Inflammation Compound Cu(II)Cl2 Cu(I)2O Cu(II)2-(acetate)4 Cu(OH)2CuCO3 [basic cupric carbonate (malachite)] Cu(I)-thiomalate Cu(II)-ascorbate Cu(II)-(anthranilate)2 Cu(II)2-(3,5-DIPS)4 Cu(II)-diaqutetrakis(p-cresotate)-(H2O)2 Cu(II)-diaqutetrakis(o-cresotate)-(H2O) Cu(II)-histidine Cu(II)-tryptophan Cu(II)-cysteine Cu,Zn-SOD (from bovine liver) Cu(I)-thionein Cu(II)2-(salicylate)4a Cu(II)2-(aspirinate)4a Cu(II)n-(niflumate)2n-(H2O)na Cu(I)-D-penicillamine-(H2O)1.5a Cu(II)n-(fenamole)n-(acetate)2na Cu(II)-piroxicama

Administration route

Reference

sc sc sc sc

118 119 7 120

sc sc sc sc ip ip ip ip ip sc ip sc sc sc sc sc ip

121 7 7 122 123 124 125 125 125 126 127 122 122 122 122 122 128

a The anti-inflammatory activity of the copper complex is significantly greater than that of the parent ligand used in the experiment. Abbreviations: sc, subcutaneous; ip, intraperitoneal.

injected at a remote site of the same organism; this phenomenon was labeled ‘‘counter-irritancy’’ (130). Thus, the parenteral and oral irritation usually ascribed to copper suggested that the anti-inflammatory activity seen with the copper compounds could be explained on the basis of their ability to promote a ‘‘counter-irritant’’ reaction (129,131). However, this hypothesis was consistently disputed by other published data, according to which the ‘‘counter-irritancy’’ of copper compounds was only negligibly responsible for their anti-inflammatory activity (7,132,133). Recently, we carried out an experiment in the attempt to better clear up this issue (the paper has been submitted for publication). Noninflamed female rats were subcutaneously injected with aseptic isotonic solutions of Cu(II)-(acetate)2 containing either 3.0 [i.e., a fully therapeutic dose (7,105)] or 0.3 mg/kg of copper. The animals were sacrificed three, seven, or 24 hours after treatment. Skin specimens of the injected area were removed to perform a full macroscopic

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and microscopic histological examination, and the blood was collected to determine its plasma total copper concentration. The results obtained unequivocally pointed out that, at all considered times, the histology did not showed any significant sign of a local inflammatory response. Moreover, the plasma total copper remained unchanged throughout the experiment, compared with that measured in the saline-treated controls. In particular, this significant marker of inflammation did not increase 24 hours after the sc injection of either copper acetate solutions. The latter evidence seems to convincingly suggest that the treatments did not induce a systemic antiinflammatory reaction capable of interfering with the own anti-inflammatory potential of copper, further sustaining that the pharmacological action of the metal is real, and that it does not depend on any biologically relevant ‘‘counter-irritancy’’ phenomenon. As mentioned in the introduction, Sorenson (7), in his classical paper, not only showed that many sc given copper-containing molecules are very active anti-inflammatory and/or antiarthritic agents, but also proposed a theory that was destined to open an active debate. Sorenson’s (92,107) idea was that the coordination compounds formed in vivo between the surplus of copper present in the plasma of inflamed animals or humans and the clinically used nonsteroidal anti-inflammatory agents could represent the active metabolites of these drugs, and moreover, Sorenson suggested that the superoxide radical disproportionation (i.e., SOD 1 mimetic activity) accounted for the anti-inflammatory action of these copper(II) complexes. Sorenson’s hypothesis was, however, challenged by coordination chemists who, mainly, if not exclusively, using a computer simulation approach, claimed that the complexes between copper(II) and salicylate, acetylsalicylate, as well as many other NSAID-type counter-anions, could not exist under plasma conditions since the ligands are incapable of effectively competing for the metal (108,134–136). Nevertheless, the original observations made over the years that copper chelates of anti-inflammatory drugs have greater potency as anti-inflammatory agents can still be demonstrated with a considerable number of compounds (137). In particular, in a variety of laboratory animal models, parenterally administered copper aspirinate showed to posses relevant anti-inflammatory activity beyond that provided by an equimolar amount of aspirin (137). Referring to aspirin, it seems appropriate to recall that oil–water partition coefficient studies have shown that copper aspirinate, likely in the form Cu(II)2(aspirinate)4, is 10 times more lipophylic than aspirin at comparable pH values of about 4.0 (108,138). Obviously, this implies that copper aspirinate, once entered into the organism, is facilitated in diffusing through the lipophilic cellular barriers and, thus, more bioavailable than aspirin. Another interesting observation was the result of the studies carried out with a compound closely related to the salicylate, i.e., the 3,5-di-isopropylsalicylate (3,5-DIPS), which, although has no anti-inflammatory activity as such, was shown to possess potent


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anti-inflammatory activity when complexed with Cu(II) ions (105). It was found that in plasma Cu(II)(3,5-DIPS)2/Cu(II)2(3,5-DIPS)4 forms stable complexes with human serum albumin (139,140). This, in turn, led researchers to suppose that other, either known or unknown endogenous copper ligands, could allow the existence of some Cu(II)-NSAID or non-NSAID complexes in vivo, by coordinating them in sort of a multimolecular adduct able to survive as such in plasma or in interstitial fluids. Finally, to further sustain the soundness of the Sorenson’s hypothesis, two other in vivo studies deserve to be mentioned. In the first, Korolkiewicz and coworkers (141) showed that copper aspirinate is significantly more active than an equimolar mixture of copper and aspirin, when given orally to inflamed rats. This observation seems to indicate that, in spite of any theoretical prevision obtained from computer simulation models, some copper aspirinate complex should survive intact at the acid pH of the stomach, and be absorbed by the animals treated. In the second study, Milanino and coworkers (142) demonstrated that a standard oral dose of indomethacin (i.e., 2 mg/kg) was remarkably active in reducing the carrageenan-induced rat paw swelling (average inhibition: 49%). However, the drug lost about 60% of its potency when administered to inflamed copper-deficient rats. Oral Administration of Copper Compounds As noted before, the oral route of administration of copper compounds appears to be inconvenient, in general it being improbable that these molecules would survive as intact Cu-complexes in gastric juice. Nevertheless, there are examples that show that this problem could be overcome (Table 13). Table 13 Some Representative Examples of Copper Compounds Active as In Vivo Anti-inflammatory and/or Antiarthritic Agents, Following Oral Administration in the Animal (Rodents) Models of Inflammation Compound Cu(I)2O Cu(II)-(acetate)2 Cu(II)-(diaminoethane)2-Cl2O Cu(II)-D-alanine Cu(II)2-(3,5-diisopropylsalicylate)4 Cu(II)-bis(2-benzimidazolyl)thioethers Cu(II)2-(aspirinate)4b Cu(II)2-(aspirinate)4b Cu(II)2-(benoxaprofen)2b Cu(II)-carbonate a

Vehicle

Reference

Mulgofena Isotonic solution Mulgofena Mulgofena Propylene glycol Gum Arabic Starch mucilage Sunflower oil Sunflower oil Diet

131 143 144 144 92 145 141 146 146 62

Polyoxyethylated vegetable oil. The anti-inflammatory activity of the copper complex is significantly greater than that of the parent ligand used in the experiment. b

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One possibility is to use as ligand a structure able to form stable complexes with Cu(II) ions, thus obtaining a significant degree of resistance to acid attack in the stomach. As a matter of fact, that is the case for the unsubstituted bis(2-benzimidazolyl)thioether (NSN), which is able to bind Cu(II), forming (both in the solid state and in solution) an adduct in which the metal is penta-coordinated by the two benzimidazole N donors and the thioether moiety, all belonging to the ligand, as well as by one water molecule and one perchlorate group (145,147). Significantly, the Cu(II)-NSN is stable enough in vitro to be intact at over 90% of its original amount, at a pH ranging between 4.5 and 4.0 (145). Moreover, about 10% of the complex is still present as such, well above the in vitro addition of the stoichiometric quantities of HCl (i.e., two equivalents) required for the complete protonation of the ligand (pH values near 3.0) (145,147). Thus, as can be reasonably expected to be seen in the above evidence and the intrinsic anti-inflammatory potential of copper, the Cu(II)-NSN complex was shown to be orally active in both the acute (carrageenan paw edema; maximum inhibition 57%, P < 0.001) and chronic (adjuvant-induced arthritis; maximum inhibition 46%, P < 0.01) models of rat inflammation, whereas NSN alone was virtually devoid of any anti-inflammatory activity (145). Moreover, in the acute model the pharmacological response of Cu(II)-NSN showed to be clearly dose-dependent, thus validating the biological meaning of the anti-inflammatory (and antiarthritic) effect observed (145). Unfortunately, it was not possible to carry out further studies on this interesting molecule. Consequently, it remains unknown whether in vivo it acts as intact Cu(II)-NSN or it expresses its pharmacological activity upon transformation, by the organism’s metabolism, into a chemically different active molecule. Moreover, there is no information about its possible mechanism(s) of action as an anti-inflammatory agent, in vivo or in vitro. Conversely, another potentially strong copper-complexing molecule, i.e., the D-penicillamine (a drug still commonly used for the treatment of Wilson’s disease) (9), has been given orally, as Cu(II)-D-penicillamine complex to rats affected by the kaolininduced paw edema, but it remained inactive also at the very high dose of 300 mg/kg (144). An alternative way to overcome the gastric barrier action is to use vehicles able to protect the integrity of the orally administered copper complex, thus assuring both its absorption and bioavailability. One such a vehicle appears to be sunflower oil. In fact, suspending the Cu(II)2(aspirinate)4 complex in sunflower oil, Rainsford (146) was able to show that 150 mg/kg of orally administered complex caused a 38% inhibition (P < 0.05) of the rat paw swelling (carrageenan-induced inflammation), whereas the same dose of aspirin alone was totally inactive. Similar results were also obtained studying the copper complex with another NSAID, i.e., the benoxaprofen (146). Thus, 100 mg/kg of Cu(II)2(benoxaprofen)4 exhibited a 53% inhibition of the edema, whereas 100 mg/kg of the ligand alone were fully inactive.


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Interestingly, both NSAID-copper complexes not only showed a significantly greater potency compared with the parent drugs, but they were also virtually devoid of the gastric ulcerogenic activity that characterized the benoxaprofen (upon repeated daily oral dosing), and in particular aspirin (146). Evidence that Cu-NSAIDs complexes have a much lower gastric toxicity than the parent compounds has been first reported and then repeatedly confirmed by Sorenson (92,105,107,122). Another vehicle that could favor the absorption of intact copper complexes following their oral administration is a polyoxyethylated vegetable oil named mulgofen. For instance, some copper complexes, such as the Cu(II)-(diaminoethane)2 and Cu(II)-D-alanine, resulted to be active inhibitors of the kaolin-induced rat paw edema model when suspend in mulgofen and given orally at 100 mg/kg (144). However, using the same vehicle, oral aspirin and copper aspirinate were both found to be inactive at the dose of 100 mg/kg (131). Nevertheless, regardless of the vehicle used, it seems reasonable to point out that a significant number of studies, which have been reviewed recently (92,148), showed that a remarkable number of copper complexes, and those formed using many NSAIDs as ligands, in particular, are active anti-inflammatory and/or antiarthritic agents not only after iv, sc, and ip dosing but also following oral administration, provided that sufficiently high amounts of the compounds are given. Since it was not possible to show a proinflammatory effect of dietary-induced copper deficiency on the adjuvant-arthritic rat (26,27,33), the behavior of the metal in this experimental chronic pathology was studied by carrying out an ‘‘opposite’’ experiment, i.e., examining the development of the AA in rats fed a copper-supplemented diet. Using this simple ‘‘dietary trick,’’ it was possible to examine the effects of the oral copper administration on the inflammatory process, excluding, at the same time, any possible biological actions of the ‘‘exogenous’’ ligands used to form the conventional copper complexes commonly studied as anti-inflammatory/antiarthritic drugs. According to a preliminary observation, a putative anti-inflammatory diet containing 200 ppm of copper (added as carbonate; control diet, Cu ¼5 ppm) was actually able to inhibit the development of the adjuvant arthritis (28%, P < 0.01) in the copper-supplemented rats after one month of ‘‘prophylactic’’ feeding (149). Thus, the approach of studying the pharmacotoxicology of the 200-ppm copper-supplemented animals was continued, evaluating the development of the AA in rats preliminarily treated with experimental diets containing 50, 100, or 200 ppm of copper (i.e., respectively, 10, 20, and 40 times the standard copper requirements) (62,63,150). The results obtained from the toxicological analyses on noninflamed rats after 30 and 58 days of 200 ppm copper-supplemented feeding are reported in Figure 3 (relative to the changes of copper status in the plasma, liver, and kidneys) and in Table 14. The data plotted in Figure 3 show that the 200 ppm Cu-supplemented diet was able to progressively and significantly

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Figure 3 Status of copper in plasma, liver, and kidney of noninflamed female rats following 30 and 58 days of feeding with either a normal (Cu ¼ 5 ppm) or supplemented (Cu ¼ 200 ppm) copper diet. On the right side of each column referring to the dietary ‘‘copper-supplemented’’ rats (Cu ¼ 200 ppm), the percent increases of copper versus the time-matched control diet (Cu ¼ 5 ppm) are reported in absolute values. Statistics (Student’s t test): P < 0.01. Source: From Refs. 62, 63, and 150.

increase the total copper content in the liver and in the kidneys above the control values, following 30 and 58 days of treatment. Conversely, as expected and repeatedly stated above, such pronounced copper supplementation did not at all influence the metal status in the plasma of the animals. It is worth stressing that, although the same trend described above was observed in the 50 and 100 ppm Cu-supplemented rats, the increases in hepatic- and renal-copper measured were remarkably lower compared to those obtained with the 200 ppm copper-treated animals, and throughout the experiment were not significantly different from the control values (61). Table 14 shows that the 200 ppm copper-containing diet notably increased the total amount of copper present in the hind paws also, albeit this effect was remarkably more evident at day 30 than at day 58. However, possibly more interesting are the following evidences: Copper supplementation did not modify the status of the metal in the cell fraction of blood, as well as in the brain of the treated rats. Massive dietary copper supplementation did not change the zinc status in any of the compartments examined. This observation is very relevant since, on the one hand, the excess of alimentary


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Table 14 The Toxicology of the Non-inflamed Copper-Supplemented Rats Changes versus the normally fed rats (Cu ¼ 5 ppm) Parameter

30 days

58 days

Hind paw copper Blood cells and brain copper Plasma, blood cells, liver, kidney, brain, and hind paws zinc Hematocrit White cells (count, differential count) Red cells (count, mean erythrocyte volume) Platelets (count, mean platelet volume) Serum aspartate aminotransferase Serum alanine aminotransferase Serum alkaline phosphatase Serum creatinine Serum urea Hemoglobin Sodium, potassium, chlorine, calcium, and CO2 Blood proteins (total and fractionated, albumin, a/c ratio) Macroscopic and microscopic examination of liver, kidneys, adrenals, hind paws, eyes, thymus, lymph nodes (cervical, mesenteric), salivary glands, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, spleen, pancreas, trachea, lungs, heart, aorta, skin, mammary glands, urinary bladder, prostate, ovary, thyroid, parathyroids, brain, pituitary, spinal cord, sciatic nerve, skeletal muscle, femur, and sternum

þ36%a None None

þ17%a None None

None None None None None 16%b None None None 2%b None None

þ3%b None None None None None 33%b None None None None None

None

None

Note: A summary of the parameters (other than those reported in Fig. 3, i.e., the plasma, liver, and kidney copper values) evaluated after 30 and 58 days of feeding with the ‘‘anti-inflammatory’’ 200 ppm copper-containing diet. a P < 0.01. b P < 0.05 (Students’ t test). Source: From Refs. 62 and 150.

copper might induce an inhibition of the intestinal zinc absorption, and, on the other, zinc has been shown to be an ‘‘endogenous’’ as well as an ‘‘exogenous’’ anti-inflammatory agent (56,151). The parameters related to the main toxicological markers of the blood cell, blood proteins, and hematochemical conditions, revealed that they were not affected by the treatment.

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The major indices of the hepatic and renal functions were found to be within normal ranges. This evidence bears particular significance, since both liver and kidney (together with the brain and, to some extent, the erythrocytes and bones), are well-known target organs for copper toxicity (9,152). Finally, the macro- and microscopic examination showed that all tissues listed in Table 14 have fully normal anatomical characteristics. As far as the antiarthritic activity is concerned, it should be pointed out that the tail injection of the complete adjuvant was done after 30 days of preliminary feeding with either the control (Cu ¼ 5 ppm) or any of the three copper-supplemented diets. The development of the chronic pathology was then followed for 28 days, during which the dietary regimen of each of the four groups of animals was kept unchanged (62,63,149). The results shown in Figure 4 indicate that the 50 and the 100 ppm copper-containing diets were not able to reduce the arthritic score at any considered time point (i.e., 14, 21, and 28 days after the inoculum). Conversely, the 200 ppm copper-supplemented diet, although ineffective at day 14 and 21, showed a noteworthy and biologically significant inhibition of the arthritic score

35

% inhibition of arthritic score

* 28 Cu = 200 ppm 21

14

7

Cu = 100 ppm

0

Cu = 50 ppm 14

21

28

Days after complete-adjuvant tail injection Figure 4 Effects of three different copper-supplemented diets on the development of adjuvant-induced arthritis in the rat. The plotted data represent the percent inhibition of adjuvant-arthritis scores measured in arthritic rats fed with diets containing 10, 20, or 40 times the amount of dietary copper (Cu ¼ 5 ppm) present in the standard diet given to the adjuvant-arthritic control p < 0.001. Source: From Ref. 62.


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measured at the end of the experiment (day 28). The pharmacological importance of the above results was confirmed when evaluating the total plasma copper concentration in the arthritic rats fed the control diet versus those kept on the 200 ppm copper-added one during the symptomatic phase of the experimental disease (day 14 and day 28). Figure 5 shows that, different from the AA-controls, the total plasma copper concentration in the 28-daysarthritic copper-supplemented animals was dramatically reduced, which, in turn, suggests that an amelioration of the overall clinical status of affected rats may have occurred. The above speculation is further sustained when

150 AA day 14

% copper increase

120 90 60 30 0 plasma

hind paws

150 AA day 28 % copper increase

120 90 60 30

* 0 plasma AA rats on copper normal diet

hind paws AA rats on copper 40 times supplemented diet

Figure 5 Percent increases of total copper content in the plasma and hind paws of adjuvant-arthritic rats, fed with either a standard (Cu ¼ 5 ppm) or copper-supplemented (Cu ¼ 200 ppm) diet. Each column represents the percent increase in total copper induced by chronic disease in affected rats, compared with their time- and diet-matched controls, at 14 (upper panel) or 28 (lower panel) days after challenge. Statistics (Student’s t test): all the values are statistically significant versus the respective control groups; however, emphasis has been put on the comparison between total plasma copper of arthritic copper-supplemented rats at day 14 and 28, P < 0.0001. Source: From Ref. 63.

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taking into account other significant markers of the seriousness of the experimental disease (Table 15). For a correct understanding of the data reported in Table 15, it should be recalled that the paw weight and the circulating white blood-cell and blood-platelet numbers significantly increase in the AA animals; conversely, a remarkable decrease of total kidney-copper amount and total plasma-zinc concentration are brought about by the pathology, being inversely correlated to its gravity (5,48,57). It thus becomes evident that the tendency towards normalization shown by all the above-mentioned disease-induced metabolic and physical changes (the arthritic score and the total plasma copper concentration included) seems to testify in favor of the efficacy of the 200 ppm copper supplementation in counteracting the development of rat adjuvant arthritis. Finally, differently from plasma copper, the total amount of the metal contained in the hind paws of the arthritic animals (expressed as percent increases), does not seem to show biologically significant changes comparing the data of day 14 with those of day 28, in both copper-normal and -supplemented rats (Fig. 5). The maintenance of high level of copper in the inflamed area might be simply due to a slow clearance rate of the metal from this body compartment. However, considering that 28 days after the complete-adjuvant injection the experimental pathology remains unresolved, it could be more likely that copper is kept within the affected site in order to favor the local processes of tissue repair. This hypothesis is supported by the evidence that the physiological equilibrium of connective tissue depends upon a correct functioning of the copperdependent enzyme lysyl oxidase (153–156). Moreover, a tripeptide-copper complex, i.e., the glycyl-L-histidyl-L-lysine-Cu(II) (GHK-Cu), which is naturally formed within the damaged tissue, appears to be also intimately involved in tissue regeneration and remodelling process (155,157–160). Table 15 Percent Differences of Some Markers of Adjuvant-Arthritis Severity in Normally Fed and 200 ppm Cu-Supplemented AA Rats Parameter Mean weight of hinds paws (g) Kidney copper (total mg) Total plasma zinc (mg/mL) White blood cells (103/mm3) Blood platelets (103/mm3)

AA in the 5 ppm copper-fed rats (%)

AA in the 200 ppm copper-fed rats (%)

þ58 53 47 þ41 þ46

þ30a 34a 16a þ12a þ13a

Note: The values were measured at the end of the experiments (day 28). Total number of rats: 30–40 per group. a P < 0.001 (Student’s t test). Source: From Refs. 62 and 63.


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Percutaneous Administration of Copper Compounds Reports related to the concerns and merits of copper preparations topically applied as anti-inflammatory/antiarthritic agents are thoroughly discussed in other chapters of this book. Nevertheless, the issue of the treatment of systemic inflammatory pathologies with percutaneous copper merits concise mention also in this review. The efficacy of the cutaneous treatment with copper-containing preparations in the therapy of wounds and some skin diseases has been empirically known for more than 3500 years ago [papyri of Ebers and Smith, circa 1550–1450 B.C. (6)]. Nowadays, the fact that tissue repair requires the local presence of copper-dependent molecules is an experimentally well-established evidence (155). The copper-dependent enzyme lysyl oxidase belongs to these molecules, which catalyses the formation of the cross-linking compounds that bind together the peptide chain of collagen and elastin, and in so doing impart both support and elastic properties to the tissues (153). Furthermore, when the extracellular-matrix macromolecules are degraded by different pathological stimuli, the protein cleavage leads to a virtuous feedback mechanism, yielding a number of peptides, some of which act as potent signals for the repair and remodelling of the damaged tissue (161). In particular, one of these peptides, the GHK-Cu, appears to be extremely active in promoting the healing of excision wounds and the skin damages caused by scald-burns (both experimentally induced), probably by means of a multifaceted way of action (155,158). Notably, the Cu(II)-tripeptide mentioned above is also fully active following topical treatments (159). At least theoretically, it appears to be easy to obtain relevant therapeutic results giving selected copper-containing preparations by cutaneous route, in the case of open wounds and, perhaps, skin pathologies. However, it is much more difficult to obtain similar effects using the same route of administration on intact skin, as when one has to deal with chronic systemic pathologies, such as adjuvant-arthritis of the rat and the human rheumatoid arthritis. As reported above, in both chronic diseases the untreated organism tends to accumulate copper within the inflamed tissues and inflammatory exudates (48,75,80,84). According to the most currently accepted interpretation, this particular event, together with the increases of the total copper measured in some other body compartments, is likely devoted to contrasting and eventually defeating the chronic pathology (151). This hypothesis appears to be also supported by the observation that the concentrations of copper in the paws of the 28-days-arthritic rats is increased by about 50% to 60% in both copper-normal and copper-supplemented animals, compared to their respective time-matched controls (62,63). However, only in the copper-supplemented arthritic rat, in which the absolute amounts of the metal contained in the hinds paws is about 1.5 times greater than that measured in the arthritic controls, a significant therapeutic effect was observed at the end of the experiment (62,63). Thus, achieving an increased copper concentration

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within the inflamed site seems to be an important therapeutic goal using any potential anti-inflammatory and/or antiarthritic copper-containing molecule. The attempts at using percutaneous administration of copper in the treatment of inflammatory conditions began with the studies of Walker and coworkers (162,163), who claimed that wearing the copper bracelet could have some beneficial effects on rheumatoid arthritis patients, and also showed that the metallic copper might be actually dissolved by some sweat components and permeate the intact cat skin. However, the research on the anti-inflammatory potential of topically applied copper preparations was quickly reoriented toward the use of nonphysiological cupriphores, as soon as it became evident that nonlipophilic NSAIDs can form much more lipophilic molecules by complexing with copper, and at the same time, they proved to be more potent anti-inflammatory/antiarthritic agents compared to the parent compounds (7,107,108,138). A number of studies have been carried out using salicylic acid as cupriphore, although according to the authors, at least one other NSAID, phenylbutazone, has also been, successfully tested as a Cu(II) complex following percutaneous administration in inflamed rats and horses (164,165). Focusing on Cu(II) salicylate, an ethanolic preparation of this complex, registered as Alcusal1, has been the subject of active research, done both in the laboratory animals and humans. For instance, it was reported that topically applied Alcusal caused: (i) a significant relief of swelling and joint stiffness in patients with rheumatoid arthritis; (ii) suppression of established polyarthritis in rats; (iii) prevention of the inflammatory response to the injection into the rat hind paw of carrageenan, histamine, or hydroxyl apatite; (iv) minimal toxicity of the applied compound in treated rats (166,167). Similar anti-inflammatory results were obtained in laboratory animals, with Dermcusal1, a Cu(II) salicylate complex in dimethyl sulfoxide/glycerol, and the choice of this vehicle was claimed to favor the cutaneous absorption of the copper salicylate in comparison with its parent, ethanol-based preparation (Alcusal) (168). Further studies were carried out dissolving 64Cu(II)-labeled salicylate in ethanol/ dimethyl sulfoxide/glycerol (66). This preparation was topically applied to rats affected by two substantially different models of inflammation, i.e., the sponge granuloma (of about six days duration and of proliferative character), and the carrageenan-induced foot edema (which lasts for a much shorter time, and is mostly edemic in nature) (69). This kind of copper salicylate preparations being already known for its anti-inflammatory activity (167–169), the authors focused attention on the body labeled copper distribution in the two models of inflammation. The data reported showed an increase of radioactivity in the kidneys, liver, spleen, adrenals, thymus, and serum from animals with long-lasting granulomatous inflammation. On the other hand, no biologically significant percent changes of 64Cu-relative specific activity were observed in the tissues of rats with carrageenaninduced paw edema (66). Thus, especially in the case of the rats affected


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by sponge granuloma, it appeared that copper was actually absorbed from the skin and then distributed systemically. Although promising on the basis of the studies preformed with experimental animals, the anti-inflammatory/ antiarthritic potential of the above copper salicylate preparations topically applied actually failed to be confirmed in humans. Their beneficial clinical activity in osteoarthritic patients and in subjects affected by sports injuries has been recently reviewed by Beveridge (164). The author based his claim quoting only two unpublished studies with the Alcusal gel, which were, moreover, carried out on a very limited number of patients (i.e., 20 and 19, respectively). Conversely, a recent randomized, double-blind, placebo-controlled trial done with topically applied copper salicylate gel on 93 patients with osteoarthritis of the hip and knee (170) was unsuccessful in showing significant differences of the copper salicylate treatment compared with controls that received placebo. Furthermore, significantly more patients in the copper salicylate group reported adverse reactions, particularly skin rashes (17.0% vs. 1.7% of the placebo group), which caused the exclusion of these subjects from the trial (170). Nonetheless, the contradictory data so far reported on the percutaneous efficacy of copper in the therapy of human inflammatory conditions should, by no means, discourage the pursuit of studies aimed at finding a copper preparation able to efficiently penetrate the cutaneous barrier, and to show a significant anti-inflammatory and/or antiarthritic activity in humans. A recent study, in particular, continues in this effort. In fact, Hostynek et al. (171) examined, by sequential tape stripping, the diffusion of metallic pulverized copper through the human stratum corneum of intact skin in vivo. Copper content was determined (inductively coupled plasmamass spectroscopy) in 20 sequential strips taken from treated and control subjects, either under occlusive (air exclusion) or semiocclusive (access of air) copper applications, for periods of up to 72 hours. Focusing on 72-hour exposure, and there on strips from no. 15 to 20 (that reach the glistening epidermal layer, i.e., the vital layers of epidermis in intimate contact with the derma postcapillary vascular bed), the following evidence was obtained: (i) occlusive copper application let copper permeate the epidermis to a limited extent only; (ii) conversely, under semiocclusive conditions, the presence of both oxygen and some unidentified molecules contained in skin exudates oxidizes Cu(0), and the in situ formed complexes entered the skin accumulating within the stratum corneum in biologically significant amounts; and (iii) in particular, at 72 hours, an average of about 0.6 mg/cm2 of copper was measured in each of layers from 15 to 20. Thus, assuming application of copper on a surface of circa 100 cm2 (which is a reasonable area to be topically treated in the case of an inflamed knee joint), 72 hours after a single treatment the total amount of metal that will accumulate in the inner layer of epidermis (strip no. 20) will be equivalent to 60 mg of copper. Actual bioavailability of such a relevant quantity of the metallo-element is

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unknown; however the evidence stated is a first, coming from direct measurements showing potential, biologically significant concentrations of ionic copper reaching selected anatomical locations in the body following percutaneous treatment. Although in all likelihood most of the metal that permeated the superficial skin layers under such experimental conditions will be lost due to the effect of epidermal desquamation, since it is complexed with molecules that are per se subject to the natural process of excretion, the above results underscore the possibility of successfully using the topical route of administration for copper. In our opinion, the main issues related to the possibility of effectively exploiting the topical route of administration for copper compounds may be briefly outlined as follows: The metal should be complexed by a ligand able to form a stable and ‘‘shielded’’ lipophilic chelate, thus in vivo allowing its penetration into the skin, and protecting the ‘‘exogenous’’ Cu(II) ions from the competition with the numerous potentially complexing molecules present in the tissue, at least during the diffusion of the complexes through the skin. This kind of approach could, at the same time, reduce the risk that ‘‘free’’ copper ions released from the complex may come into direct contact with the tissues, causing occurrence of adverse dermatological reactions (170,172). The ligand also has to be able to coordinate the metal by bonds strong enough to allow the distribution of the complex in the organism, without being so stable as to end up with a molecular structure actually containing copper that is a non-bioavailable. In fact, too stable chelates, such as those in vivo formed with tetrathiomolybdate (173,174), are not capable of exerting any pharmacological action except that of seriously depriving the treated organism of copper. Both at the local and systemic levels, the ligand has to be nontoxic. To this end, the natural amino acids and a number of di- or tripeptides, but also simpler structures such as Cu(II) acetate, appear to be the favorites, disregarding whether or not they are endowed with their own anti-inflammatory potential. Even though the choice of the ligand would have completely fulfilled the above-mentioned suggestions, it appears possible that many copper complexes could turn out to be insufficiently lipophilic in order to permeate intact skin and supply the target tissues with pharmacologically adequate amounts of the metal. Consequently, the use of proper vehicles in which to dissolve or suspend the complex is most likely an important part of the overall task. Aside from the excipients typically used (i.e., ethanol, dimethyl sulfoxide, etc.), a very attractive and new approach could be that of embedding the copper complex in liposomal vesicles (175). As a matter of fact,


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recent papers have shown the high efficiency of such carriers for the percutaneous administration of a variety of drugs, such as asthma medications, kanamycin, and indomethacin (176–178). COPPER ANTI-INFLAMMATORY ACTIVITY: HYPOTHESES EXPLAINING THE POSSIBLE MECHANISMS OF ACTION The essentiality of copper for humans was first recognized early in the past century, when Hart and coworkers (179) showed copper to be critical for erythropoiesis in the rat. Since then, it became apparent that, during their evolution, all the aerobic living systems have recruited specialized copper sites to provide an adequate electron transfer reactivity to the proteins destined to cope with oxygen (180,181). The facts that in vivo this transition metal so easily shifts from the Cu(II) to the Cu(I) oxidation state and vice versa as well as its ability to form stable complexes with electron-donor biomolecules are the basic reasons for the utilization of copper in the living oxygen-dependent organisms (182). Thus, copper is a crucial constituent of many redox enzymes as well as nonenzymatic biologically active molecules, the importance of which in maintaining physiological homeostasis of biochemical, cellular, and tissue functions is a well-established evidence. Table 16 lists some examples of copper-molecules, separated into two groups, depending on their prevailing enzymatic or nonenzymatic activity. Inflammation is a physiological reaction, aimed at defending the organism against exogenous as well as endogenous attack, and copper is deeply involved in this process. However, inflammation develops through an extremely complex and strictly interdependent network of single responses, in which, step by step, different cell types (such as mast cells, polymorphonuclear leukocytes, monocytes, endothelial cells, macrophages, lymphocytes, fibroblasts, etc.), and different mediators (such as, histamine, serotonin, arachidonic acid derivatives, nitric oxide, cytokines, oxygen free radicals, chemokines, complement factors, etc.) come progressively into play (200). Copper was shown to be intimately involved in this overall inflammation network, influencing the behavior of many inflammatory cells, as well as the metabolism and/or activity of numerous inflammatory mediators (200). However, providing a comprehensive treatise on this issue would go beyond the actual purposes of this review; consequently, comments are brief, outlining some representative examples of the possible mechanisms of action on which the anti-inflammatory activity of endogenous and/or exogenous copper may be based. Histamine Release and Activity It is generally accepted that histamine is the first chemical mediator to be released after the inflammatory stimulus, and it is also well established

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Table 16 Examples of Copper-Dependent Molecules Involved in Maintaining Physiological Homeostasis and Ensuring Adequate Copper Management Copper molecules Enzymatic proteins Cytochrome c oxidase

Cu,Zn superoxide dismutase (SOD 1) Ceruloplasmin

Lysyl oxidase Monoamine and diamine oxidases Dopamine b-monooxygenase Monophenol monooxygenase (tyrosinase) Cell surface monoamine oxidase Peptylglycine a- amidating enzyme Nonenzymatic proteins Metallothioneins (MTs) Transcription factors (Mac 1, Amt 1, Ace 1, etc.) Copper chaperones (Atox1, hCCS, hCox17, etc.), and membrane-copper pumps (P-type ATPases) A-domains of the clotting factors V and VIII

In vivo main functions

References

Terminal enzyme of the mitochondrial respiratory chain Dismutation of superoxide anions Oxidation of Fe(II) in plasma; distribution of copper to extra-hepatic tissues; antioxidant activity Cross-linking of collagen and elastin in tissue regeneration Oxidative deamination of histamine, serotonin, etc. Catecholamine biosynthesis

17, 183

Melanin biosynthesis

192

Regulation of glucose uptake and cell adhesion Bioactivation of neuroactive peptides

193

Copper storage and detoxification; stress proteins Regulation of gene transcription for SOD 1, catalase, MTs, etc. Copper handling within different pro- and eukaryotic cell types; maintenance of copper homeostasis Participation in blood coagulation process

195

17, 184 17, 185–187

153, 188 184, 189 190, 191

184, 194

182 196–198

182, 199

that its main role in inflammation is to increase the vascular capillary bed permeability (201–203). According to some early research, a binuclear, hydroxyl-bridged Cu(II) complex is the active form of histamine (105). On the other hand, copper, as cofactor of the diamino oxidases, is essential in the process of histamine degradation (184,189). Furthermore, the metal seems to regulate the release of this bioactive molecule from the mast cells,


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after their stimulation by a wide variety of proinflammatory noxa (92). Nevertheless, other biological pathways, e.g., the triggering of the complement cascade, and bioactive molecules, e.g., the arachidonic acid products and nitric oxide, are know to play a very significant role in regulating the inflammationinduced increase of vascular permeability (31,67,203–205). Note that, in all the above-mentioned processes, some copper-dependent molecules may be involved directly or indirectly. Therefore, the ambivalent role of copper in the processes of histamine release, activity, and catabolism, has a yet unclear relevance in justifying the mechanism of the anti-inflammatory action of this transition metal, either endogenous or exogenous. The Arachidonic Acid Cascade The products of the cyclo-oxygenase pathways, i.e., prostacyclins, prostaglandins, and tromboxanes, are, since the late 1970s, well-known mediators of many different biological reactions (31). As far as inflammation is concerned, the prostaglandins among these molecules are likely to play an important role, and the inhibition of cyclo-oxygenase (COX-1 and COX-2) activities actually represents one of the foremost mechanisms of action of the large majority of NSAIDs (206,207). As previously noted, copper(II), used as simple salts such as CuSO4 and Cu(NO3)2, seems to be able to interact in vitro with the activity of these enzymes, altering the equilibrium in the production of the prostaglandins E2 and F2a, thus promoting an antiinflammatory effect (28,29). These early observations were later confirmed using rabbit kidney medulla slices, and rat peritoneal macrophages (208,209). Nonetheless, as described studying the severe copper-deficient rat, it was observed that metal deprivation did not have significant effects on either the lung COX-1 and/or COX-2 activity(ies), or on the reactivity of the copper-deprived gastrointestinal tissues to the exogenous administration of prostaglandin F2a and E2 (26). This, in turn, seems to suggest that endogenous copper might not play a direct biologically significant role in the arachidonic acid cascade. Moreover, the administration of some copper(II) complexes with non-anti-inflammatory ligands, such as the bis-pyridine, bis(2,4-dimethylpyridine), and bis(2,4,6-trimethylpyridine), showed to have a remarkable anti-inflammatory activity in vivo, but no effect on the prostaglandin biosynthetic pathways (210). Conversely, Cu(II)2(aspirinate)4 and Cu(II)2(indomethacinate)4 were reported to decrease the activity of cycloxygenases to a greater extent compared to the parent ligands (211), thereby strongly limiting the production of the arachidonic acid different endproducts, prostaglandins included (212). Thus, in view of the conflicting results described above, and considering also that the biosynthesis of the various arachidonic acid cascade products is qualitatively as well as quantitatively different in the different body tissues (207), whether or not the endogenous Cu(II) may exert a significant part of its physiological anti-inflammatory

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effects also by regulating the arachidonic acid pathways, is an issue that still remains to be better clarified. Connective Tissue Metabolism Inflammation can be controlled by stimulating the repair processes that ultimately replace damaged tissue with new cells and extracellular matrix; when these processes are impaired, the inflammatory reaction may tend to perpetuate and worsen, eventually degenerating into chronic disease (153). The roles of the copper-dependent enzyme lysyl oxidase in the biochemical pathways of reconstruction of collagen and elastin, as well as the participation of the GHK-Cu in the process of tissue repair and remodelling, were already mentioned briefly (153,158). In particular, the above copper(II) tripeptide complex has been the subject of numerous investigations, which highlight many interesting features of this molecule: (i) GHK-Cu is normally present in human plasma, probably being in a dynamic equilibrium with its ‘‘de-coppered’’ form GHK; (ii) both GHK and GHK-Cu are generated, in situ, by the proteolysis of inflammatory and extracelluar matrix proteins (due to the action of lytic enzymes released by activated phagocytes) during episodes of tissue damage; (iii) GHK-Cu has been found to be a potent chemoattractant selective for macrophages, monocytes, and mast cells, but has no activity in favoring the migration of other inflammatory cell types, such as neutrophils; (iv) GHK-Cu has a high superoxide dismutase activity, which contributes to the detoxification of the superoxide anions produced, particularly in the initial phases of inflammatory response; and (v) in the inflammation-damaged area both GHK and, especially, GHK-Cu induce the removal of tissue debris, capillary growth, differentiation as well as viability and axon outgrowth of neuronal cells, and, finally, the production of fibroblast-mRNA for the synthesis of matrix protein, such as collagen, elastin, proteoglycans, glycosaminoglycans, and decorin (158,213) (see also the website: http://www.skinbiology.com/copperpeptideregeneration.html). Thus, the evidence reported above seems not only to stress the remarkable biological importance of the endogenous copper in regulating the tissue repair process, but may also stimulate research aimed at finding out other copper peptides suitable to bind copper and able to act in vivo as new antiinflammatory and, especially, antiarthritic drugs. Regulation of Nitric Oxide Synthase Activity In vivo the enzyme nitric oxide synthase is devoted to the production of the highly bioactive nitric oxide molecule (N¼O). The physiological roles of nitric oxide are also expressed in decreasing the vascular tone, decreasing the platelet adhesion and aggregation, and increasing the cytotoxic activity of macrophages and neutrophils (205,214). Moreover, it has recently been shown that, in vitro, most of the noxious effects of interleukin-1 (IL-1) on


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the cartilage metabolism (i.e., decrease synthesis of extracellular matrix component, abnormal cell renewal, enhanced sensitivity of chondrocytes to oxidative stress, etc.) are probably mediated by the action of superoxide on nitric oxide that, in turn, acts on IL-1-stimulated chondrocytes (215). The authors speculated that a combined therapy with NO synthase inhibitors and antioxidant, e.g., Cu(II)2(3,5-DIPS)4 that seems to posses both activities, may be promising for full cartilage protection (215). The inflammatory reaction is one of the conditions in which an upregulation of nitric oxide synthase has been reported (205,216). It is noteworthy to remark that the anti-inflammatory agent Cu(II)2(3,5-DIPS)4 has been shown to have a significant downregulatory activity on the nitric oxide synthase in vitro, which would be a mechanism well compatible with the pharmacological actions of this complex (217,218). Moreover, in vitro copper selectively inhibits the catalytic activity of the constitutive nitric oxide synthase I in C6 glioma cells (219). Whether or not a regulatory role of endogenous copper on the activity of the nitric oxide synthases could also occur in vivo, is, at present, unclear. However, it has recently been observed that the copper-dependent monoamine oxidases seem to modulate the expression of the macrophage-inducible isoform II of this enzyme (220), which leaves this intriguing possibility open. Leukocyte Activity and Migration The activity of polymorphonuclear (neutrophils) and mononuclear leukocytes (simply referred to as PMNLs) plays a pivotal role in the development and control of both acute and chronic inflammations. Among other functions, the intense phagocytic activity of these cells is specifically oriented towards eliminating deleterious materials from injured tissue, such as bacteria and other microorganisms, neoplastic cells, cell debris, antigen–antibody complexes, nonbiotic foreign particles, etc. (221). Immediately following the noxa the vascular permeability increases locally, chemotactic/activating factors are released by the endothelial vascular and inflammatory cells (that are also resident in situ), as well as by the invading organisms and/or their degradation products, and, consequently, impressively high amounts of PMNLs are recruited at the inflamed site from blood (222). Following activation by the different chemoattractants, the circulating leukocytes are induced to penetrate into the injured tissue, where they are further activated by a variety of locally present molecules, and, consequently, prompted to carry out their typical defense functions, among which the microcosm of biological pathways expressed by the so-called ‘‘respiratory burst’’ plays a primary role. Actually, both endogenous and exogenous copper are certainly involved in many different steps of the above chain of events, as well as in the overall inflammatory process (Table 17). During the PMNLs phagocytosis that occurs in the inflamed area, these cells dramatically increase (10–15-fold) their oxygen consumption within a few seconds after contact with the stimulating substance (respiratory

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Table 17 Some Examples of Copper-Containing Molecules Involved in the Scavenging of Oxygen-Derived Free Radicals in Chemotaxis, Migration and Oxidative Burst of Polymorphonuclear (Neutrophils) Leukocytes

Compound SOD 1 Cu(I)-thionein Ceruloplasmin Cu(II)-GHK

Cu(II)SO4; Cu(II)-H(1-His-Gly)2OH Cu(II)-rutin Cu(II)-bis-pyridine; Cu(II)bis(2,4-dimethylpyridine); Cu(II)-bis(2,4, 6-trimethylpyridine) Cu(II)2(3,5-DIPS)4-albumin Cu(II)-salicylatea; Cu(II)-acetylsalicylatea Cu(II) (salsalate)2a Cu(II)2(3,5-DIPS)4; Cu(II)2(aspirinate)4a; Cu(II)2(indomethacinate)4a Cu(II)-piroxicama Cu(II)2(niflumate)4a Cu(II)2(dimethylsulfoxide)2(mu-niflumate)4a Cu(II)-carbonate in the diet

Activity on oxygen radicals scavenging, and other PMNLs reactions

References

Scavenging of superoxide Scavenging of superoxide and hydroxyl radicals Scavenging of oxygen radicals Scavenging of superoxide, chemotactic activity, and stimulation of repair processes Scavenging of superoxide

223 127, 224

Scavenging of oxygen radicals Inhibition of superoxide production

227 210

Scavenging of superoxide; inhibition of superoxide production Scavenging of superoxide

139, 140, 228

Scavenging of superoxide Inhibition of chemotaxis, migration, and superoxide production Scavenging of superoxide; inhibition of migration Inhibition of chemotaxis; superoxide production Inhibition of superoxide production Inhibition of ex vivo adhesion

225 158, 213

226

229 230 231, 232

128 233 140 63

a Activity of the copper complex is significantly greater than that of the parent ligand used in the experiment. Abbreviations: PMNLs, polymorphonuclear (neutrophil) and mononuclear leukocytes, SOD 1, cytoplasmic Cu, Zn superoxide dismutase Cu(II)-GHK, glycyl-L-histidyl-L-lysine-Cu(II); DIPS, diisopropylsalicyclic acid.

burst) (234). Inside the PMNLs phagosomes, the enzyme NAPH oxidase utilizes oxygen to give rise to large amounts of superoxide anions, which, in turn, leads to the formation of much more reactive oxyl radicals, such


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as singlet oxygen, hydroxyl radical, and hydroperoxyl radical, as well as hydrogen peroxide (155,234). This sustained production of noxious oxygen products (that are collectively called ‘‘reactive oxygen species,’’ ROS) has the obvious scope of opposing the disease-inducing actions of the ingested particles (either biotic or nonbiotic) during the physiological process of inflammation (235). However, the oxygen radicals, which the PMNLs’ respiratory burst has generated, could be cytotoxic not only for the foreign inflammatory material but also for the PMNLs themselves, as well as for the adjacent tissue cells and matrix or interstitial- and exudate-fluid biomolecules (235,236). It has been proposed that the chemically nonspecific character of the ROS-induced reactions affects the cell membranes and the soluble or matrix biological structures, damaging them and also leading, at least theoretically, to the extemporary formation of endogenous antigens that could, in turn, favor the evolution of the acute inflammatory reaction toward its chronic phase (237). An early study showed that the attack of superoxide anions on the phagocytic cell membrane might initiate the arachidonic acid cascade (238). Furthermore, it was observed that the enzymatic oxidation of arachidonic acid, which ends with the formation of its active metabolites, generates free radicals, the hydroxyl radical in particular, as side products (239). In addition, the enzyme nitric oxide synthase may function to produce superoxide anions (205,240). Thus, a very relevant goal to be achieved for keeping inflammation under proper control appears to be the inactivation of the fairly large excess of ROS that may be released within the inflamed tissue. The enzyme that in vivo is responsible for scavenging superoxide anions is the SOD 1 molecule. This enzyme, however, not only is specific for superoxide but is also largely contained only inside the intact cells; as a consequence, it cannot counteract the other oxygen radicals, and its action outside the cell is probably a secondary one. Nevertheless, other natural copper-containing molecules, such as ceruloplasmin, Cu(I)-thionein, and Cu(II)-GHK, which may be present in the plasma-exudate and/or in situ produced during inflammation, could efficiently replace SOD 1 in scavenging superoxide as well as, at least indirectly, the other ROS (Table 17) (155). Interestingly, when inflammation is localized in the skeletal muscle, the endogenous carnosine and homocarnosine, which may easily bind copper ions in situ forming stable complexes, are able to act as oxygen radical scavengers. This hypothesis was proposed on the basis of the evidence that both Cu(II)-carnosine and Cu(II)-homocarnosine can dismutate the superoxide anions released from the neutrophils activated by in vitro contact with phorbol miristate acetate (PMA) (241). It may be relevant to stress that all the above-summarized evidence could well explain the reason for the observed copper accumulation within the inflamed tissues of both acutely and chronically inflamed laboratory animals and humans, previously described in this review. Also, many small molecular weight copper complexes exogenously administered have been shown to disproportionate

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superoxide anions and/or inactivate other oxygen radicals (Table 17) (218,242). Among these molecules are included structures such as some oligopeptides (not necessarily of endogenous origin), macrocyclic tetraanhydroaminobenzaldehyde, ethylenediaminetetra-acetate, as well as the NSAID piroxicam, which have all been shown to be unable to display any scavenging activity when tested noncomplexed with copper; the complexes between Cu(II) and all the above listed molecules are, in contrast, very active superoxide (or ROS) scavengers (128,227,243–245). Conversely, many other NSAIDs do show an SOD-mimetic activity of their own; this activity, however, is significantly and, sometimes, dramatically enhanced by their complexation with copper(II) ions (Table 17). Another biochemical trait that a relevant number of NSAIDs share with a group of pyridine derivatives, and which may be related to their mechanism of action, is the ability to inhibit the PMNLs respiratory burst, reducing the oxygen uptake of these cells and decreasing their superoxide and other ROS production; once again, this effect is significantly increased when the same substances are evaluated as Cu(II) complexes (Table 17) (218). Notably, a study by Nilsson (246) has shown that the inhibition of the respiratory burst by two copper compounds, i.e., the Cu(II)2(3,5-DIPS)4 and the Cu(II)SO4, may be primarily due to the modulation of the protein kinase C activity. According to Sorenson (107,218), this observation may be extended to other copper complexes of NSAIDs, such as Cu(II)2(aspirinate)4, Cu(II)2(indomethacinate)4, etc.; moreover, interference with the protein kinase C activity pathways of action also accounts for the inhibition of chemotaxis and/or migration shown by the above (and possibly other) Cu(II) complexes (Table 17). The crucial process of migration of circulating leukocytes into the inflamed site, which is restricted to the postcapillary venules of the affected area, is a complex network of cellular adhesion molecule interactions that involves also many inflammatory chemoattractants and mediators (either of ‘‘self’’ or ‘‘non-self’’ origin) such as histamine, cytokines (IL-1, IL-6, tumor necrosis factor a, etc.), chemokines (IL-8, eotaxin, fractalkine, RANTES proteins, etc.), leukotriene B4 and possibly other arachidonic acid derivatives, Cu(II)-GHK, platelet activating factor, complement factor C 5a, nitric oxide, ROS and oxidized low-density lipoproteins, bacterial lipopolysaccharides, bacterial formilated peptides, etc. (213,247,248). All these substances govern, in a time- and stimulation-mediated expression and activity, the different endothelial and inflammatory cells responses, which are aimed at creating the proper conditions for the recruitment and activation at the inflammatory site of the different cells types actually involved in the physiological onset, development, and control of inflammation. Summarily, the PMNLs migration can be divided into three distinct phases: The first one, which immediately follows the insult by the phlogistic agent, is the tethering of the flowing leukocytes to the


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endothelial vascular cells. This event is caused by the exposure on the outer surface of PMNL plasma membrane of the adhesion molecule L-selectin that strongly, but provisionally, binds to a yet unidentified receptor located on the endothelial cell surface (249). After tethering, the leukocyte begins to ‘‘roll’’ along the blood endothelial vessel, to eventually firmly adhere to the endothelium itself before migrating into the inflamed site. The process of rolling appears to be basically mediated by the expression of the endothelial surface molecules P- and E-selectins that bind to selectin-specific leukocyte-surface ligands, the best known of which is the P-selectin glycoprotein ligand-1. Arrival of P- and E-selectins on the scene has the effect of initiating and continuing the leukocyte rolling, then gradually slowing down, thus preparing the last phase of the entire migration process, i.e., the firm adhesion to the endothelial cell of the PMNLs and their subsequent extravasation (249). The processes of firm endothelial leukocyte adhesion and leukocyte transmigration towards the inner tissues of the inflamed site are also extremely complex and not yet fully understood. They imply the exposition on the leukocyte plasma membrane of a number of b-integrin adhesion molecules, in particular the b2-integrins LAF-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), as well as, possibly, of the L-selectin again (250,251). The structures mentioned above bind to some counterpart endothelial-expressed adhesion molecules such as the family of intercellular adhesion molecules (ICAMs), of which the ICAM-1 is the most relevant member, the vascular adhesion molecule-1 (VCAM-1), as well as the vascular adhesion protein-1 (VAP-1) (250–252).

It has been proposed that the expression and activation of VAP-1 could be the initial step in the pathway of firm leukocyte adhesion and transendothelial migration, at least in conditions of a blood flow velocity comparable with those that allow the binding of other adhesion molecules (251). In fact, in the absence or blockade of this protein in vitro, the normal interactions between the leukocyte adhesion molecules, such as the LAF-1, Mac-1, etc., with their endothelial counter-receptors, such as the ICAM-1, VCAM-1, etc., are remarkably prevented (50% inhibition), and the leukocyte transmigration significantly reduced (251,253). Recently, the above observations have been confirmed in vivo, showing that the deficiency or the blocking of VAP-1 remarkably inhibits the PMNLs from migrating into the inflamed sites, thus preventing the customary development of both acute and chronic inflammations in the mouse (254,255). A very relevant feature of the VAP-1 is that this endothelial protein is, at the same time, an adhesion molecule and a copper-dependent enzyme, i.e., a semicarbazidesensitive amine oxidase, also known under the acronym AOC3 (193,252,253).

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The above biological activities of VAP-1 are exerted by two distinct extracellular domains of the protein, and are strictly interdependent since the specific inhibition of the amine oxidase activity also abolishes the ability of VAP-1 to bind to its, yet unidentified, leukocyte counter-receptor (193,252). Apart from the copper dependency of VAP-1 physiological role, it already was reported that the biological action of some inflammatory chemoattractants and mediators, such as histamine, arachidonic acid derivatives, GHK-Cu, nitric oxide, and ROS [that also cause the generation of the oxidized low-density lipoproteins (247)], was modulated by either endogenous and/or exogenous copper. Moreover, nontoxic amounts of copper sulfate or chloride have been shown to induce the expression of ICAM-1 on the surface of chondrocytes in situ in cartilage explant cultures; however, these copper-induced ICAM-1 molecules failed to promote the adhesion to IL-1activated peripheral blood monocytes (256). Another interesting evidence came from studies on the in vivo lysozyme secretion and ex vivo adhesion and superoxide anion production, by neutrophils isolated from rats fed a standard or a 200 ppm coppersupplemented diet, either healthy or affected by complete-adjuvant-induced arthritis (63). Notably, the lysozyme concentration was measured directly in the blood of the experimental animals, whereas the neutrophil adhesion and superoxide generation were evaluated in an ex vivo assay that utilized fetal bovine serum-coated plates, and exogenously added PMA as stimulant (257). The results showed that: (i) the dietary supplementation with copper did not change the blood lysozyme concentration, as well as the adhesion and superoxide production by the neutrophils isolated from the noninflamed rats; (ii) conversely, all the above parameters significantly increased in the adjuvant-arthritic animals of both dietary-treated groups; and (iii) nevertheless, albeit lysozyme secretion and superoxide generation were found comparable to those measured in the nonsupplemented arthritic rats, the ability of the ‘‘inflamed’’ copper-supplemented neutrophils to adhere to the assay plates was markedly reduced (47%; P < 0.001) (Fig. 6). This last observation seems to suggest that exogenous copper, orally administered by means of a copper carbonate-integrated diet, could specifically and significantly impair the expression and/or functioning of the neutrophils’ adhesion molecules, which, in turn, may partly account for the antiarthritic activity of the 200 ppm coppersupplemented diet reported in the same experiment (63). Immune System Development and Reactivity Research mostly coming from trace element depletion studies clearly established the importance of an adequate copper (as well as iron, zinc, and selenium) dietary intake for protection of animals and humans in the process of host resistance to the invading pathogens (Table 18). In fact, a large body of concordant data support the following general


213

40

AA not suppl.

Neutrophil adhesion (%)

30

20

*

*

AA Cu suppl.

Healthy Cu suppl. 10 Healthy not suppl.

0

Diet 30 d Diet 44 d AA inoculum AA 14 d

Diet 58 d AA 28 d

Figure 6 Effects of normal (Cu ¼ 5.0 ppm) or supplemented (Cu ¼ 200 ppm) copper diet on the ex vivo adhesion of neutrophils isolated from either healthy and adjuvantarthritic rats. Percent of ex vivo adhesion of blood neutrophils isolated after 30 days (AA inoculum), 44 days (AA 14 days), and 58 days (AA 28 days) of feeding, from rats kept on normal- (Cu ¼ 5.0 ppm) and copper-supplemented (Cu ¼ 200 ppm) diets. Statistics (Student’s t test): P < 0.01, comparison between Cu supplemented, AA 14 and 28 days, versus not supplemented, AA 14 and 28 days. Note that the adhesion percentages of neutrophils isolated from both Cu-normal and Cu-supplemented arthritic rats are significantly different (P < 0.01) from those measured in the respective control groups (i.e., normal- and Cu-supplemented healthy animals). Source: From Ref. 63.

conclusions (258): (i) an insufficient copper supply is associated with the impairment of numerous activities of cells involved in both innate and acquired immune reactions; (ii) the observed alterations may be due to decreased activities of individual cells, reduction in the total number of active cells, or to a combination of the above two effects; (iii) the extent of the damage to the immune system could be sufficient to increase the host susceptibility to the exogenous (or endogenous) aggressions; and (iv) all negative effects of copper deficiency on immune reactivity can be reversed by restoring the normal copper status in the examined subjects. The evidence reported in Table 18 shows that an analytically measurable copper deficiency status impairs the overall inflammatory reactions, decreasing not only the phagocytic-cell ability to efficiently destroy the invading hosts but also seriously affecting the physiological activity of the whole battery of immune-competent organs (such as the thymus, bone

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Table 18 Some Representative Examples of Dietary Copper-Deficiency on Mammalian Immunity Functions Effects of copper deficiency

References

Decreased phagocytic and cytotoxic activity of rodent-competent cells Reduced superoxide anion production and candidacidal activity of animals neutrophils Increased susceptibility to transplanted leukemia cells in the mouse Decreased resistance to the attack of different pathogens in the mouse Impaired ex vivo activity of rat T cells to different stimulants Alterations of protein and lipid composition of murine plasma membrane Reduced superoxide anion production and candidacidal activity of peritoneal macrophages Decreased thymus weight and antibody titer in rodents, characterized by a male versus female increased vulnerability Decreased rat splenic T-cell reactivity to mitogens, and decreased number of helper and cytotoxic T-cell subset Decreased killing capacity of steer PMNLs Decreased proliferative capacity of rat splenocytes, restored by interleukin-2 or copper addition Decreased proliferative ability of monocytes and increased number of B-cells in the peripheral blood, in humans Decreased production of interleukin-2 by human T-lymphocytes Impaired interleukin-2 gene expression in a human T-lymphocyte line (Jurkat cells)

32 259 260 261 33 262 263 264 265 266 267 268 269 270

Abbreviation: PMNLs, polymorphonuclear (neutrophil) and mononuclear leukocytes.

marrow, and spleen) and cells (such as the B- and T-lymphocyte cell lines). Notably, the above effects of copper depletion were significantly more pronounced in male versus female rats (264). Also, marginal conditions of copper deficiency (obtained in male rats fed with a 2.7 ppm coppercontaining diet) that do not involve evident changes of copper levels and ceruloplasmin activity in the serum nor significantly modify the organ (liver in particular) copper status are capable of markedly inhibiting the proliferative response of the splenic mononuclear cells to challenge by mitogens (271). In addition, the bioactivity of T-lymphocyte-produced IL-2 (which plays a central role in the regulation of the acquired immune responses) was dramatically reduced in those experimental conditions (270,271). More recently, it has been shown that, at least in the Jurkat cells, the decrease synthesis of IL-2 appears to be caused by the copper deficiency-induced inhibition of the expression of the NA-AT transcription factor (258,272). Much


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less data are available on the effects that copper supplementation may have on normal immune reactions. For example, the subcutaneous injection of Cu(II)2(3,5-DIPS)4 significantly stimulated the production of the different immune cell lines by the lymphoid tissues and the spleen, thus favoring an early recovery of injured mice after their exposure to 8 Gy whole-body irradiation (273). Furthermore, a significant inhibition of delayed-type hypersensitivity reactions was reported in mice fed with copper-supplemented diets, and this effect was also shown to be dependent on the amount of the metallo-element present in the diet as well as on the duration of the supplemented-feeding treatment (274). However, the leukocytes, such as neutrophils and monocytes, as well as the injured endothelia, actively participate in triggering the immune response, also by means of the production and coordinated action of numerous inflammatory mediators, such as ROS, nitric oxide, arachidonic acid derivatives, interleukins, chemokines, adhesion molecules, etc. (213,247,248,256). As we already stressed, albeit the production of superoxide and other ROS by tissue phagocytes has an essential role in counteracting the noxious actions of the invading pathogens, the persistence of an excess of these active oxygen products may be itself potentially dangerous for the organism. Thus, it has been previously proposed that an efficient control of the oxygen radicals generation could be an important goal in ensuring a proper control of the overall inflammatory reaction development. However, recent data may appear to challenge the above hypothesis. In fact, it has been reported that transgenic mice overexpressing SOD 1 showed a significant increase in delayed-type hypersensitivity reactions; conversely, the selective blockade of SOD 1 activity by means of disulfiram resulted in a significant decrease in the development of both delayed-type hypersensitivity reactions and adjuvant-induced arthritis in the rat (275). In other words, the above results would suggest that an overexpression of ROS could inhibit the development of the immune-mediated chronic phase of the inflammatory process, a final result comparable to that previously described as an effect of severe copper deficiency in the laboratory animals (26,27,33). Even so, a great number of copper complexes, both of endogenous as well as exogenous origin, are endowed with free-radical scavenging potential; furthermore, they are also able to inhibit the phagocyte respiratory burst, thus lowering the amount of ROS generated at the inflamed site (Table 17). These copper complexes have been unequivocally demonstrated to have significant anti-inflammatory and antiarthritic roles in vivo (Tables 10–13). Moreover, as previously detailed, the dietary deficiency of copper not only promotes an enhanced acute inflammatory reaction but also causes a dramatic decrease in the SOD 1 activity in the total blood cells (22,23). In addition, the development of acute- and chronic-inflammatory reactions is also critically influenced by the processes of chemotaxis, endothelial adhesion, and migration of the circulating inflammatory cells, the immunocompetent ones included (276).

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Interestingly, a number of the copper complexes listed in Table 17 also have antichemotactic and/or antimigratory effects. Moreover, it has also been mentioned that the in vitro addition of copper induces the expression of nonfunctional ICAM-1 molecules on the chondrocyte surface, and the dietary-administration of high amounts of copper significantly impairs the ex vivo neutrophils’ ability to adhere to the assay plates, as well as remarkably inhibits the AA arthritis development in the treated rats (63,256). On the other hand, it has also been shown that the neutrophils isolated from copper-deficient mice expressed ‘‘ex vivo’’ a 50% reduced level of the adhesion molecule CD11b (i.e., Mac-1) on their surface (277). In conclusion, the experimentally observed activity of many copper complexes, which significantly ameliorate the conditions of organisms affected by chronic pathologies characterized by relevant inflammatory and immunological components, may depend upon a delicate balance in which the chemical nature of the copper complex, its actual bioavailability, the site and, especially, the preferred biological process of intervention, as well as the phase of development of the disease itself are all concurrently involved. CONCLUSIONS The evidence summarized seems to reasonably lead to the following general conclusions: The nutritional deficiency of copper significantly impairs the organism’s ability to develop a normal acute inflammatory reaction to counteract either biotic or nonbiotic attacks. Notably, this effect is strictly and simultaneously dependent on the amount of copper contained in the diet, on the length of treatment, as well as on the gender of the animals studied, males being remarkably more sensitive than females to dietary copper depletion. Both acute and, especially, chronic experimentally induced inflammations cause a net increase of the total copper in some body compartments, such as blood, liver, and the inflamed area. This accumulation of copper seems to be directly correlated with the severity of the pathology considered; furthermore, with the exception of the kidneys, it does not appear to involve any comprehensive and biologically significant redistribution of the metallo-element between the main body compartments. As a consequence, it is conceivable that the inflammatory process induces an overall enhanced requirement for copper, which may be accomplished increasing the metallo-element absorption/retention and/or decreasing its hepatic excretion, by the affected organism. It may not be secondary to underscore that a dramatic accumulation of copper has been reported also in the inflamed synovial fluid collected from rheumatoid


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arthritic patients, as well as of ceruloplasmin in the human inflamed periodontal tissue; these observations could imply that the need for more copper to better cope with the inflammatory pathologies may also characterize inflammations in humans. In spite of the previously described inflammation-induced increase in body copper levels, administration of extra amounts of copper, either by prophylactic feeding of animals with a metalsupplemented diet or by therapeutically treating them with exogenous copper salts or complexes, has clearly shown a significant anti-inflammatory and anti-arthritic potential in vivo.

In general, copper may be considered an anti-inflammatory trace metal per se, as evidenced by the active participation of the endogenous metallo-element in the modulation of inflammatory reaction, by the protective effect of the dietary-supplemented copper on the development of the rat adjuvant-induced arthritis, as well as by the activity of therapeutically administered copper, observed regardless of the counter-anion used to carry it into the body. Nevertheless, the actual effects (i.e., the real potency) of exogenous copper as an anti-inflammatory and anti-arthritic drug have been shown to be significantly dependent on the route of administration chosen, as well as on the ligand used to form the complex. Focusing on the ligands, molecules that are endowed with their own anti-inflammatory activity, i.e., a significant majority of the clinically used NSAIDs, appear to potentiate their effects when administered complexed with Cu(II) ions. However, albeit a number of data seem to suggest that the observed anti-inflammatory activity is mainly due to the in vivo action of the intact complexes, the possibility that some Cu(II)-NSAIDs may also transfer their copper to one (or more) endogenous ligand(s) cannot be entirely ruled out. As a consequence, the final results would be, at least partly, obtained by the distinct effects of the NSAID given, and that of the complex(es) in vivo formed between the copper ions carried into the body by the drug and some endogenous ligand(s) of this essential metallo-element. An accurate survey of the data, which could explain the mechanisms of copper action in modulating the phlogosis’ development and remission, does not help much in solving the above problem, neither can it answer the question whether or not the endogenous and the exogenous metallo-element may somehow overlap in their anti-inflammatory actions. Actually, the whole scenery is obscured by the fact that the copper-dependent defense mechanisms (as well as those of NSAIDs) may simultaneously address different inflammatory pathways; their targets would also change according to the type of inflammation developed, as well as to its specific phase of progress. For instance, it has already been reported that copper has an unclear and, perhaps, secondary role in regulating the arachidonic acid cascade in vivo, whereas most of the NSAIDs can strongly inhibit this chain of reactions. Consequently,

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administering a Cu(II)-NSAID complex, the observed influence on prostaglandins, prostacyclins, and tromboxanes production is, possibly, mainly due to the effect of the ligand alone. On the other hand, endogenous copper in the form of SOD 1, ceruloplasmin, Cu(I)-thionein, Cu(II)-GHK, and perhaps other in vivo existing copper complexes, has remarkable antioxidant properties. The same has been reported for some NSAIDs, and their complexation with copper has been described to potentiate these ROSscavenging effects. It is also well documented that many NSAIDs inhibit the respiratory burst and, consequently, the oxygen free radicals generation by activated phagocytes; once again their copper complexes appear to be more active, but a possible autonomous role for endogenous copper in this sequence of PMNLs reactions has not yet been recognized with certainty. The physiological processes of tissue repair and remodelling are known to be governed by copper-dependent enzymes in vivo (e.g., the lysyl oxidases), as well as by copper-oligopeptides complexes formed in situ [e.g., Cu(II)GHK]; in this case it may be possible that the role of the endogenous copper is a predominant one, and the administration of exogenous copper might have the prevailing function of supplying the damaged tissue with the metallo-element. Finally, it is well established that the pathway of leukocyte migration towards target tissues represents a key step in the development and control of acute and chronic inflammation, and it also eventually involves the activation of the immune response. Some clinically used NSAIDs have an inhibitory effect on the inflammatory cells’ chemotaxis and migration, and their Cu(II) complexes clearly show a significantly enhanced activity. Recently, endogenous copper has been discovered to play a very remarkable role in the process of leukocyte rolling, adhesion, and extravasation, being an essential component of the multifunctional vascular adhesion protein-1, the activity of which appears to be crucial for normal leukocyte migration. Moreover, addition of simple Cu(II) salts to the assay medium in vitro promotes the synovial chondrocyte layers to express nonfunctional ICAM-1 molecules on their surface. Furthermore, neutrophils isolated from arthritic rats fed with a 200 ppm copper-supplemented diet have a significantly decreased ability to adhere to the test plates ex vivo, whereas the ‘‘parent’’ inflamed cells coming from animals fed a diet containing standard amounts of copper carry out this process normally. Thus, the examples summarized above clearly suggest that further research is needed to better understand which one, if any, of the copper-dependent processes examined could have a predominant relevance in explaining the natural anti-inflammatory activity of endogenous copper. Moreover, similar caution is also advisable in speculating on the possible biological pathways in which the endogenous and the exogenous copper activities may synergistically overlap. Finally, a last issue remains to be mentioned briefly, i.e., the potential toxicity of administered copper, a concern that, unfortunately, is still raised


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by many clinicians not accustomed with the action of copper compounds in the therapy of rheumatoid arthritis and other degenerative-inflammatory human diseases. However, existing data appear to clearly indicate that this concern is not justified. In fact, copper may have noxious effects only following the chronic oral (or parenteral) exposure to very large amounts of the metallo-element; in particular: (i) by chronic oral ingestion of foodstuff (e.g., water) if it supplies the organism with over 5 mg/kg of copper per day; and (ii) in the case of prolonged hemodialysis with apparatus that cause the introduction into the circulation of the metallo-element coming from copper-containing semipermeable membranes or copper tubing (278,279). Thus, a real risk of copper toxicity may exist only by administering amounts of the metallo-element certainly far greater than those that may be actually used for therapeutic purposes. In this context, it seems very relevant to recall that rheumatoid arthritis patients treated with a full cycle of PermalonTM did not show the appearance of significant adverse reactions, in particular none that could be due to iv administered copper (116). This evidence is noteworthy in that, although the antiarthritic activity of the above drug cannot be acknowledged in light of modern clinical evaluation protocols, the absence of significant adverse reactions reported is reliable, being largely obtained by means of objective measurements. In conclusion, the research oriented towards the study of the roles of copper in inflammation, and the utilization of copper compounds as anti-inflammatory and antiarthritic remedies, is by no means obsolete. In particular, the chance of targeting the copper preparations directly to the inflamed sites deserves notable attention. In fact, the percutaneous route of administration (using liposomes as copper carriers through the skin barrier) can both minimize the possible, although remote, toxicological hazards, as well as, especially, favor the accumulation of the metallo-element where its therapeutic characteristics may be better expressed.

ACKNOWLEDGMENTS Review of the manuscript by Mrs. E. M. Hostynek-Welch and Dr. J. J. Hostynek is gratefully acknowledged. ABBREVIATIONS ppm CPE CP AA SOD 1 RA sc

part per million (mg/kg) carrageenan-induced paw edema carrageenan-induced pleurisy adjuvant-induced arthritis cytoplasmatic Cu,Zn superoxide dismutase human rheumatoid arthritis subcutaneous (Continued)

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ip iv Cp NSAID DIPS NSN GHK-Cu PMNLs ROS PMA IL ICAM-1 VCAM-1 VAP-1

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intraperitoneal intravenous ceruloplasmin nonsteroidal anti-inflammatory drug di-isopropylsalicylic acid unsubstituted bis(2-benzimidazolyl)thioether glycyl-L-histidyl-L-lysine-Cu(II) polymorphonuclear (neutrophil) and mononuclear leukocytes reactive oxygen species phorbol miristate acetate interleukin intercellular adhesion molecule-1 vascular adhesion molecule-1 vascular adhesion protein-1

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10 Copper Jewelry and Arthritis Brenda J. Harrison Department of Earth and Ocean Sciences, Copper Research Information Flow Project, University of British Columbia, Vancouver, British Columbia, Canada

Pseudoscience differs from erroneous science. Science thrives on errors, cutting them away one by one. False conclusions are drawn all the time, but they are drawn tentatively. Hypotheses are framed so they are capable of being disproved. A succession of alternative hypotheses is confronted by experiment and observation. Science gropes and staggers toward improved understanding. Proprietary feelings are of course offended when a scientific hypothesis is disproved, but such disproofs are recognized as central to the scientific enterprise. —Carl Sagan (1)

INTRODUCTION This paper provides an overview, from the point of view of a nonspecialist, of the ‘‘copper bracelet’’ issue, based on an exploration of the available scientific literature, popular press, promotional material made available on the Internet, discussions of the issue of complementary and alternative health care methods, and books for the lay reader. The focus will be on copper bracelets, since they are currently the ‘‘in vogue’’ appliances. The discussion is applicable to other means of wearing metallic copper on the human body, 237

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from copper rings, to copper disks worn under watchbands, or copper pennies worn inside socks! ‘‘Arthritis’’ is the general term for a suite of over 100 distinct diseases and conditions. The most common forms are rheumatoid (involving persistent inflammation of the synovial membranes of joints) and osteoarthritis (a degenerative process involving the cartilage and bone of joints). Arthritis affects nearly 43 million Americans (one in six); although arthritis can strike at any age, aging of the ‘‘baby boom’’ generation will bring an increase in the number of sufferers in the coming decades (2). Most forms of arthritis are incurable, but effective interventions (including use of analgesics, steroidal and nonsteroidal anti-inflammatory drugs—NSAIDs), are available. They are underused (3). ‘‘The wearing of copper bracelets is a folk remedy for arthritis, but there are little data to support the efficacy of this remedy’’ (4). The role of copper in the treatment of arthritis was reviewed by Milanino et al. (5) to ‘‘stimulate a thoughtful and unbiased reconsideration of the old idea of using copper as a therapeutic agent in the treatment of those chronic inflammatory diseases—in particular rheumatoid arthritis.’’ Serum copper is elevated in rheumatoid arthritis although there is evidence that serum copper does not correlate with disease severity (6–8). From animal studies it has been established that the rise in serum copper is accompanied by an increase in liver copper levels (9–12). The modern use of copper complexes to treat arthritis dates from the 1940s, and the theory that copper complexes of nonsteroidal anti-inflammatory drugs are more active and less toxic than the parent compounds is supported by a substantial body of literature (13,14). The role of copper in arthritis is complex; the prevailing paradigm includes the following elements: 1. Copper is an essential trace element; copper deficiency may have an adverse effect on inflammation and connective tissue disease such as arthritis. 2. Serum copper is elevated in arthritis. 3. The plasma protein ceruloplasmin, which binds copper, is an ‘‘acute phase reactant’’; the observed elevation of serum copper is often attributed to an increase in this protein as a result of the disease. 4. D-penicillamine, used to treat arthritis, binds free copper ions. 5. Copper is an essential cofactor for the enzyme Cu, Zn-superoxide dismutase; this enzyme neutralizes the destructive free radicals that may play a role in tissue damage in the disease and it possesses anti-inflammatory activity. 6. The enzyme lysyl oxidase requires copper as a cofactor and is required for connective tissue formation.

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The established role of copper in both superoxide dismutase and ceruloplasmin offers a framework in which copper could be expected to play a role in arthritis treatment; however, ‘‘unfortunately, other than for goldsulfhydryl compounds, there exists a chronic deficiency of solid scientific information about any role of trace metallic elements, whether they are administered parenterally, orally, or topically as therapeutic agents in the management of rheumatoid arthritis (RA) (15). In an intriguing study using an animal model, a mixture of copper, gold, and silver exhibited significant antirheumatic function not shown by the individual elements (16). THE COPPER BRACELET ‘‘MYTH’’ AND HYPOTHESIS Copper bracelets, armbands, rings, etc., are a folk remedy promoted and sold for the relief of the symptoms of arthritis. They may be purchased from pharmacies, health food stores, catalogues, ‘‘new-age stores,’’ and online suppliers through the Internet. A Google search of the Internet for (þ ‘‘copper bracelets’’ þarthritis) conducted on January 17, 2005 uncovered approximately 12,300 Web pages. A Google Scholar search the same day uncovered only 32 pages using the same search terms. Google Scholar allows specific searching of scholarly literature (such as peer-reviewed papers, theses, books, and technical reports). Most of the pages turned up by the standard Google search are promotional sites, featuring enthusiastic testimonials. A variety of health-related claims are made on these sites—many claim therapeutic bracelets date from antiquity and are ‘‘powerful’’ and ‘‘natural.’’ Along with the testimonials, some sites also provide descriptions of the ‘‘theory’’ behind how the bracelets work. Claims for the Antiquity of the Remedy Many websites describe the use of copper bracelets for arthritis as having a very long history, although they are generally vague about how long that history might be. Many talk of ‘‘ancient writings’’ and ‘‘ancient times’’; or refer to Roman, Greek, or Egyptian times; or use by ancient Aztecs, Persians, or Hindus. Others refer to a period of thousands of years, centuries, even ‘‘over 200 years.’’ Some refer to lost knowledge (especially knowledge lost or abandoned by modern medicine), while others suggest that bracelets have been in constant use for a very long time. Claims for the Power of the Remedy Many websites tell of folklore and ‘‘old wives’ tales’’ that support the idea that copper bracelets can ease the suffering of arthritis patients. They use such terms as ‘‘healing power,’’ ‘‘medicinal value,’’ ‘‘ease the pain,’’ ‘‘ease discomfort,’’ ‘‘relief of pain,’’ or ‘‘increase mobility’’—all of which suggest

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that the bracelets can alleviate or heal arthritis. Many cite the use of copper bracelets by athletes, especially professional golfers, as evidence of their effectiveness. Claims for the ‘‘Naturalness’’ of the Remedy The ‘‘naturalness’’ of copper bracelets is a prominent feature on promotional websites. Key phrases include ‘‘natural,’’ ‘‘natural home remedy,’’ ‘‘natural remedy,’’ ‘‘natural healing,’’ and ‘‘natural relief.’’ Claims About the Underlying Science Behind the Remedy and Theories About How It Works Some web pages claim that copper bracelets work by increasing blood or oxygen flow, or increasing circulation, and thus speeding healing or relieving pain. Others claim that copper interacts with ‘‘positive fields’’ or energy flow in the body, or in some way balances electrical potentials. Many claim that arthritis patients are deficient in copper and that the bracelets supply the essential element and so promote health and healing; bracelets are promoted as a worry-free, time-released source of supplemental copper that may remedy or prevent inflammation. Another group of sites claims that the copper absorbs toxins by some mechanism or that copper will combat superoxide radical damage. Consideration of the Reputed Antiquity of the Remedy There is a pervasive belief that the use of copper jewelry as a remedy for arthritis stretches across many cultures and back into history for centuries or even millennia. The basis for this belief is not clear, although it is likely that people have worn copper jewelry and amulets since the earliest working of copper. Arthritis as a condition predates civilization and even the origin of the human species. Evidence of arthritic deformation can be seen in dinosaur skeletons and cave bears of the Pleistocene epoch were also afflicted (17). Neanderthal and Neolithic skeletons, early Egyptian skeletons dating back to 4000 BC—all show evidence that arthritis was widespread. The use of copper compounds as therapeutics by early cultures is well documented and probably dates to the beginning of recorded history and earlier. Most applications relate to copper’s antibiotic properties (18). The ancient Egyptian medical text, the Smith Papyrus dating from 1600 to 1300 BC, recounts the use of copper to treat infected wounds and to sterilize drinking water. Other ancient texts from Egypt, Greece, Rome, the Aztec empire, ancient India, Persia, and China make it clear that early healers used a number of copper compounds to treat a wide variety of conditions ranging from infections to ulcers, inflammation, hemorrhoids, neuralgia, leprosy, eye ailments, venereal disease, intestinal worms, lung diseases, and in the


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promotion of wound healing (19). Dresher (20) gives a good, updated overview of historical and modern uses of copper in medicine. The Pharmaceutical Journal in 1974 carried a small column called ‘‘Copper on the up and up,’’ which gives an entertaining (and prophetic) history of the use of copper through the ages and the prospects for its medical development (21). This brief account includes the statement, ‘‘How long ago people started to wear copper or bronze ornaments for their supposed magical or remedial effects is unknown.’’ The discovery that the skeleton of a Durotrigian defender of Maiden Castle in Dorset who was killed in 43 AD had been buried with a spiral bronze ring on one of the toes led the author to speculate that it is ‘‘ . . . not too fanciful to suppose that such rings were employed to ward off the gout or the rheumatism . . . ’’ This story was repeated by Dr. John Sorenson (22) in a paper on the therapeutic uses of copper; however, Sorenson (23), in the next sentence classified copper bracelets per se as ‘‘modern copper-containing folklore remedies for the treatment of arthritis.’’ In an e-mail message to the author on November 1, 2004, Dr. Niall Sharples (24), Senior Lecturer in Archaeology at Cardiff University and author of an English Heritage book about the history of Maiden Castle, was able to confirm part of the skeleton story. There is ‘‘ . . . indeed a copper alloy probably bronze ring around the toe of a skeleton from Maiden Castle. . . . These rings are fairly common in the Iron Age of Britain and they are clearly jewellry worn on both fingers and toes. Burials however, are less common and there are very few burials with rings if any . . . Whether they also functioned in a medicinal fashion I could not say.’’ The Maiden Castle book provides a drawing on page 120 of the skeleton; a tankard had also been buried with the tanker. Spiral bronze rings were common trade goods of the period and may have been worn as status symbols (24). Native North Americans have a copper bracelet myth of their own, in which copper bracelets are worn not for healing, but as a source of mystical ‘‘power’’ (25). Theophrastus Phillippus Aureolus Bombastus von Hohenheim (1493– 1541), better known as the celebrated Renaissance physician, alchemist, and philosopher Paracelsus, crafted ‘‘constellation rings’’ in metals that corresponded to the known planets including copper to bring the protective influence of Venus and so ‘‘was one of the first recorded copper bracelet wearers, not to ward off arthritis however, but rather to assure for himself the benevolence of Venus’’ (19). As described by Kelly Patricia O’Meara in a Washington Times article on POWs/MIAs (February 7, 2000), copper bracelets became immensely popular in 1970 when three American college students began making them to draw attention to missing-in-action servicemen. The original bracelets were modeled on a metal bracelet that Robert Dornan (later a Congressman) obtained from hill tribesmen in Vietnam. Called MIA bracelets, each had the name of a serviceman missing in Vietnam inscribed on the outside.

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They sold at the time for $3 each, became very popular, and eventually were worn by 5 million Americans. They are still worn today. In a 1982 essay, Dr. Gerald Weissmann, NYU School of Medicine, speculated that the origin of copper adornments as a remedy for arthritis might have lain with the astrology followers of the 1960s, since the bracelet wearers Weissmann (26) interviewed talked about ‘‘ . . . the healing power of the metal and the conjunction of the stars . . . clear connection between the planets in their orbit and their metal talisman . . . clear faith that gravitational and electrical forces generated by heavenly bodies were transmitted to their joints by way of the copper bracelet.’’ It would seem then that the copper bracelet (for arthritis) myth might have an origin more recent than ‘‘ancient times,’’ and more rooted in superstition and magic than in any deep, mysterious scientific knowledge. Many of the ancient texts and new age websites allude to the mystical connection between elemental copper and the planet Venus. Consideration of the Reputed Power of the Remedy For the majority of arthritic conditions (gout is a notable exception), there is no known cause or cure. There are, however, effective drugs that can slow the progress of the disease and prevent irreversible joint damage. A number of ‘‘self-care’’ measures (including relaxation, diet, and exercise) are also helpful and are routinely recommended by doctors. Copper bracelets are not among the measures generally regarded as having any effect. Claims have also been made for a number of dietary supplements, some of which include copper; however, as of December 1996 the U.S. Food and Drug Administration (FDA) had not authorized any health claims for any food or dietary supplement and arthritis (27). This situation remains unchanged in 2005 according to information on the FDA website. Consideration of the Naturalness of the Remedy Copper is a naturally occurring element; a copper bracelet is, of course, a fabricated object. ‘‘Natural’’ by itself does not confer safety (elemental arsenic is ‘‘natural,’’ but deadly in a sufficient dose). There is a hypothetical risk of suffering an accidental injury while wearing a copper bracelet—for example, an instruction on the Q-Ray Ionized Bracelet1 website warns against wearing the bracelets while using an electric blanket ‘‘because the blanket could heat up the metal bracelet and cause it to burn your skin.’’ Other than suffering from such an accident, the only imaginable risk is developing a skin reaction to the copper. Hostynek and Maibach (28) recently conducted an extensive review of available case reports and concluded, ‘‘ . . . true allergic reactions to copper appear rare, particularly those induced by skin contact . . . ’’ The authors could find very few studies reporting


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reactions (allergic contact dermatitis and eczema) among copper jewelry wearers, and it is likely that some of those were actually caused by sensitivity to nickel, not copper. Consideration of the Proposed Underlying Mechanism of the Remedy In common with materials promoting other unproven remedies (treatments that have not been shown in repeated scientific studies to work and to be safe), claims on the bracelet web pages range from the plausible to the absurd; a survey of the pseudoscience on these pages would make an interesting paper on its own. Many betray an underlying misunderstanding of basic science. ‘‘Negative and positive’’ charges and fields echo the pseudoscientific claims behind magnets and magnet therapy, also in vogue (29). There seems to be indecision on whether bracelets work by making something flow into or out of the body. Some sites do cite the scientific literature, although many do it in a highly selective manner, reprinting a sentence or two to support their claims. Among the scientific ‘‘facts’’ that are correctly claimed are the essentiality of copper, its role in superoxide dismutase and antioxidant role, formation of chelates with substances (amino acids) in sweat, and the possible role of copper deficiency in the disease. A few sites attempt a balanced treatment of the current state of scientific evidence and correctly cite supporting publications but they are the exception. THE COPPER BRACELET TRIAL Scientific Evidence for the Use of Bracelets for Arthritis Therapy Considered The International Copper Research Association in 1975 published a critical review of copper in medicine. The authors advocated the use of copper chelates as anti-inflammatory agents for arthritis treatment. Interestingly, while copper bracelets were covered briefly, their use was suggested as a means of exploiting copper’s bactericidal and fungicidal properties: Finally, it should be kept in mind that people who have a high frequency of minor skin afflictions of a fungal or a bacterial type, rather than using copper salts in liniment or salves, may wish to use the much more convenient metallic form of copper: say in the form of a copper barrette, a copper chain, or a copper bracelet worn close to the skin (21). Such a metallic copper device will slowly release copper ions, most probably in the form of amino acid complexes, which will permeate both the surrounding skin and eventually also the body. A maximum figure of the amount of copper released most likely will not exceed that released from the Copper-T device, about 45 mg per day. Such a slow, daily release of ionic copper will

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act as a bactericide and fungicide on the skin environment of the metallic copper. At the same time, it cannot cause any harmful general effects to the individual, since the amount of copper released will only constitute a small percent (

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