Reviews in Food and Nutrition Toxicity, Volume 4 (Reviews in Food and Nutrition)

REVIEWS IN FOOD AND NUTRITION TOXICITY Volume 4 REVIEWS IN FOOD AND NUTRITION TOXICITY Edited by Victor R. Preedy and...

Author: Victor R. Preedy | Ronald R. Watson

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REVIEWS IN FOOD AND NUTRITION TOXICITY Volume 4

REVIEWS IN FOOD AND NUTRITION TOXICITY Edited by Victor R. Preedy and Ronald R. Watson

Volume 1 Volume 2 Volume 3 Volume 4

REVIEWS IN FOOD AND NUTRITION TOXICITY Volume 4

Edited by Victor R. Preedy and Ronald R. Watson

Boca Raton London New York Singapore

A CRC title, part of the Taylor & Francis imprint, a member of the Taylor & Francis Group, the academic division of T&F Informa plc.

Published in 2005 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW Boca Raton, FL 33487-2742

© 2005 by Taylor & Francis Group CRC Press is an imprint of Taylor & Francis Group 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: 0-8493-3519-1 (Hardcover) International Standard Book Number-13: 978-0-8493-3519-8 (Hardcover) Library of Congress Card Number 2004047814 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.

Library of Congress Cataloging-in-Publication Data Reviews in food and nutrition toxicity / edited by Victor R. Preedy and Ronald Watson. p. cm. Includes bibliographical references and index. ISBN 0-8493-3519-1 (alk. paper) 1. Nutrition policy. I. Preedy, Victor R. II. Watson, Ronald R. (Ronald Ross). III. Title. TX359.A56 2004 363.8'561–dc22

2004047814

Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com Taylor & Francis Group is the Academic Division of T&F Informa plc.

and the CRC Press Web site at http://www.crcpress.com

Preface In this fourth volume of reviews, we present state-of-the-art chapters pertaining to the potential and actual harm that arises as a consequence of consuming food substances and other components in the diet. We emphasize the terms potential and actual, as very often there are threshold boundaries that need to be crossed before an innocuous substance becomes toxic and/or induces a cascade of cellular and pathological changes. However, as evidenced from the present chapters, there is some debate as to what these thresholds are, and this illustrates the fundamental need for constant scientific dialogue. The chapters in Reviews in Food and Nutrition Toxicity fulfill these scientific, academic, and intellectual needs. The first part of the present volume can be considered thematic in that five chapters cover chemical elements: heavy metals (mercury, lead, cadmium, and arsenic), selenium, arsenic, sulfur, and fluoride. The chapters on arsenic, sulfur, and fluoride are general and may be considered as overviews, whereas the two chapters on heavy metals and arsenic specifically pertain to their occurrence in breast milk and fish, respectively. There follows three chapters on bacterial and fungal components. These include contamination of ready-to-eat foods, T-2 mycotoxin, and aflatoxin B1. Finally, there are two very comprehensive reviews on cycad consumption and dietary lectins. As with previous volumes in Reviews in Food and Nutrition Toxicity, we believe that the present coverage will stimulate broad-based interests as well as specific applicability to other food or nutritional substances. It is difficult to highlight a single chapter for meritous mention, as they are all equally well focused and scientific stimulating. The present chapters are written by nationally and internationally recognized experts and essentially complement the previous three volumes to give wide coverage of food nutrition and toxicity in a holistic manner.

Editors Victor R. Preedy, Ph.D., D.Sc., F.R.C.Path., is a professor in the Department of Nutrition and Dietetics, King’s College, London. He directs studies regarding protein turnover, cardiology, nutrition, and, in particular, the biochemical aspects of alcoholism. Dr. Preedy graduated in 1974 from the University of Aston with a combined honors degree in biology and physiology with pharmacology. He received his Ph.D. in 1981 in the field of nutrition and metabolism, specializing in protein turnover. In 1992, he received membership in the Royal College of Pathologists based on his published works, and in 1993 a D.Sc. degree for his outstanding contribution to the study of protein metabolism. At the time, he was one of the university’s youngest recipients of this distinguished award. Dr. Preedy was elected a fellow of the Royal College of Pathologists in 2000. He has published more than 475 articles, which include more than 150 peer-reviewed manuscripts based on original research, and 70 reviews. His current major research interests include the role of alcohol in enteral nutrition and the molecular mechanisms responsible for alcoholic muscle damage. Ronald R. Watson, Ph.D., attended the University of Idaho but graduated from Brigham Young University in Provo, Utah, with a degree in chemistry in 1966. He earned his Ph.D. in biochemistry from Michigan State University in 1971. His postdoctoral schooling in nutrition and microbiology was completed at the Harvard School of Public Health, where he gained 2 years of postdoctoral research experience in immunology. From 1973 to 1974, Dr. Watson was assistant professor of immunology and performed research at the University of Mississippi Medical Center in Jackson. He was assistant professor of microbiology and immunology at the Indiana University Medical School from 1974 to 1978 and associate professor at Purdue University in the Department of Food and Nutrition from 1978 to 1982. In 1982, Dr. Watson joined the faculty at the University of Arizona Health Sciences Center in the Department of Family and Community Medicine of the School of Medicine. He is currently professor of health promotion sciences in the Mel and Enid Zuckerman Arizona College of Public Health. Dr. Watson is a member of several national and international nutrition, immunology, cancer, and alcoholism research societies. He is presently funded by the National Heart Blood and Lung Institute to study nutrition and heart disease in mice with AIDS. Dr. Watson has edited more than 35 books on nutrition and 53 scientific books and has contributed to more than 500 research and review articles.

Contributors Gabriella Augusti-Tocco Department of Cellular and Developmental Biology “La Sapienza” University Rome, Italy

Willy Baeyens Brussels Research Unit of Environmental, Geochemical and Life Sciences Department of Analytical and Environmental Chemistry Vrije Universiteit Brussel Brussels, Belgium

Tapan K. Basu Department of Agricultural, Food and Nutritional Science The University of Alberta Edmonton, Alberta, Canada

Marjan De Gieter Brussels Research Unit of Environmental, Geochemical and Life Sciences Department of Analytical and Environmental Chemistry Vrije Universiteit Brussel Brussels, Belgium Tony J. Fang Department of Food Science National Chung Hsing University Taiwan, Republic of China Hanne Frøkiær Biocentrum-DTU Biochemistry and Nutrition Technical University of Denmark Lyngby, Denmark Claudia Gundacker Center for Public Health Medical University of Vienna Vienna, Austria

Emanuele Cacci Department of Cellular and Developmental Biology “La Sapienza” University Rome, Italy

Erin L. Hawkes Graduate Program in Neuroscience University of British Columbia Vancouver, British Columbia, Canada

Thomas F.X. Collins Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration Laurel, Maryland

Ziad W. Jaradat Department of Biotechnology and Genetic Engineering Jordan University of Science and Technology Irbid, Jordan

Tanja Maria Rosenkilde Kjær Biocentrum-DTU Biochemistry and Nutrition Technical University of Denmark Lyngby, Denmark Lioudmila A. Komarnisky Department of Agricultural, Food and Nutritional Science The University of Alberta Edmonton, Alberta, Canada

Christopher A. Shaw Graduate Program in Neuroscience Departments of Ophthalmology, Physiology, and Experimental Medicine University of British Columbia Vancouver, British Columbia, Canada Robert L. Sprando Center for Food Safety and Applied Nutrition U.S. Food and Drug Administration Laurel, Maryland

Ruggero Ricordy Institute of Molecular Biology and Pathology CNR Rome, Italy

Ujang Tinggi Centre for Public Health Sciences Queensland Health Scientific Services Brisbane, Australia

Jeff D. Schulz Graduate Program in Neuroscience University of British Columbia Vancouver, British Columbia, Canada

Bettina Zödl Center for Physiology and Pathophysiology Medical University of Vienna Vienna, Austria

Table of Contents Chapter 1 Heavy Metals in Breast Milk: Implications for Toxicity .........................................1 Claudia Gundacker and Bettina Zödl Chapter 2 Selenium Toxicity and Its Adverse Health Effects .................................................29 Ujang Tinggi Chapter 3 Arsenic in Fish: Implications for Human Toxicity.................................................57 M. De Gieter and W. Baeyens Chapter 4 Biological and Toxicological Considerations of Dietary Sulfur ............................85 Lioudmila A. Komarnisky and Tapan K. Basu Chapter 5 Fluoride – Toxic and Pathologic Aspects: Review of Current Literature on Some Aspects of Fluoride Toxicity..................................................................105 Thomas F.X. Collins and Robert L. Sprando Chapter 6 Bacterial Contamination of Ready-to-Eat Foods: Concern for Human Toxicity .....................................................................................................143 Tony J. Fang Chapter 7 T-2 Mycotoxin in the Diet and Its Effects on Tissues..........................................173 Ziad W. Jaradat Chapter 8 Aflatoxin B1 and Cell Cycle Perturbation.............................................................213 Ruggero Ricordy, Emanuele Cacci, and Gabriella Augusti-Tocco Chapter 9 Cycad Consumption and Neurological Disease....................................................233 Jeff D. Schulz, Erin L. Hawkes, and Christopher A. Shaw

Chapter 10 Dietary Lectins and the Immune Response ..........................................................271 Tanja Maria Rosenkilde Kjær and Hanne Frøkiær Index ......................................................................................................................297

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Heavy Metals in Breast Milk: Implications for Toxicity Claudia Gundacker and Bettina Zödl

CONTENTS Abstract ......................................................................................................................2 Abbreviations .............................................................................................................2 Introduction................................................................................................................2 Sources of Heavy Metals and Transfer in the Environment ........................3 Exposure Routes and Biological Half-Life...................................................4 Heavy Metals in Breast Milk ....................................................................................5 Exogenous and Endogenous Sources............................................................5 The Process of Milk Production ...................................................................7 Transfer of Heavy Metals into Milk .............................................................8 Mercury ................................................................................................9 Lead ......................................................................................................9 Cadmium ............................................................................................13 Arsenic ...............................................................................................14 Metal Concentrations in Breast Milk..........................................................14 Factors Influencing Milk Metal Contents ...................................................14 Mercury ..............................................................................................15 Lead ....................................................................................................15 Cadmium ............................................................................................15 Arsenic ...............................................................................................16 Toxicological Implications ..........................................................................16 Mercury ..............................................................................................17 Lead ....................................................................................................19 Cadmium ............................................................................................20 Arsenic ...............................................................................................20 Exposure Guidelines....................................................................................20 Conclusions..............................................................................................................21 References................................................................................................................22

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2

Reviews in Food and Nutrition Toxicity

Abstract

Breast milk is unique as a matrix for biomonitoring, providing information about the metal body burden of women as well as the exposure of infants. The heavy metals mercury, lead, cadmium, and arsenic are widespread and persistent agents with significant dose-related toxicological implications at high exposure levels. However, the interrelationships under conditions of chronic exposure are not fully known. Metal in breast milk originates from exogenous sources, i.e., uptake via contaminated air, food, and drinking water, and endogenous release along with essential trace elements, which is characteristic for the reproductional period. Metal transfer into breast milk depends on the chemical form and the distribution of the metal in maternal blood fractions. Methylmercury is strongly bound to erythrocytes. A small quantity of methylmercury passes into breast milk and is easily absorbed by the suckling infant. Inorganic mercury is readily transferred into breast milk, but is not well absorbed by infants. Lead transfer is associated with casein. Human milk has a very low casein content; therefore, the excretion rate of lead is low. Because cadmium binds to metallothioneins, the mammary gland, like the placenta, is considered to serve as a barrier for cadmium and to protect the infant. Inorganic arsenic is not excreted in breast milk to any significant extent. The suckling infant may be exposed to toxic influences in a period of highest susceptibility. Metal toxicity is dependent on the chemical form involved, which determines the bioavailability, absorption rate, and retention time. The brain is regarded as the most important target organ of toxic impairment even at low doses. There is some epidemiological evidence that prenatal metal exposure (in particular, methylmercury exposure) correlates with neurodevelopmental deficits. Yet, it remains unclear whether and to what extent postnatal metal exposure through breastfeeding impairs the infant’s health. The toxicokinetics of arsenic among neonates and infants has been scarcely reported. As environmental and maternal conditions lead to significant differences in milk metal levels, all measures must be taken to avoid additional metal exposure of infants via breastfeeding.

Abbreviations

As: arsenic; Ca: calcium; Cd: cadmium; Hg: mercury; K: potassium; Mg: magnesium; Na: sodium; Pb: lead; Po4: phosphate; Zn: zinc

INTRODUCTION The American Academy of Pediatrics (AAP) firmly adheres to the position that breastfeeding ensures the best possible health as well as the best developmental and psychosocial outcomes for the infant. It is recommended that breastfeeding continue for at least 12 months, and thereafter for as long as mutually desired (AAP, 1997). There is no doubt that exclusive breastfeeding is ideal nutrition; yet it has to be considered that breast milk may contain pollutants, which implies the need to evaluate breast milk contents. Analyses of breast milk metal concentrations provide data about the metal burden in the woman’s body on the one hand, and metal exposure of neonates and infants via breastfeeding on the other. Therefore, breast milk is “unique as a matrix for biomonitoring, and analyses of breast milk for

Heavy Metals in Breast Milk: Implications for Toxicity

3

environmental chemicals as well as for nutrients are of wide scientific interest” (Needham and Wang, 2002). Among diverse environmental pollutants, heavy metals belong to the most harmful xenobiotics, as they are widespread and persistent agents with significant doserelated toxicological implications. The persistence of metals, i.e., that they are not degradable, is one of their most problematic features and a major factor in the ecotoxicological relevance of heavy metals. The toxicology of metals is related to approximately 80 elements, including those heavy metals that, per definition, exceed a density of 5 g/cm3. Heavy metals of relevance in this context are mercury, lead, and cadmium. Arsenic is usually regarded as a hazardous heavy metal although it is actually a semimetal. Humans are routinely exposed to environmental metal concentrations and accumulate metals accordingly, which results in a variety of health impacts. Heavy metals are known, or at least suspected, to possess an immunotoxic, mutagenic, carcinogenic, embryotoxic, and teratogenic potential. Their dose–effect relationships, however, are not fully known, especially under chronic exposure. Long-term, low-level metal exposure results in elevated metal burdens for the body. Such burdens are considered nontoxic as long as they are below health-based exposure guidelines; nonetheless they may impair human health. Women of reproductive age are subject to a process known as body clearance, which may be defined as the loss of essential and nonessential elements during pregnancy and lactation due to the high nutrient demand at this stage. Lactating women (and subsequently their offspring) are affected by heavy metal exposure not only via exogenous sources, i.e., environmental exposure, but also through endogenous metal release. Hence the infant may be exposed to toxic influences in a period of highest susceptibility due to rapid growth, immaturity of kidneys and liver, and the unique vulnerability of the myelinizing central nervous system (CNS) to neurotoxic exposure. Furthermore, in cases of maternal element deficiency, the risk of toxic effects for both the infant and mother may be higher (Vahter et al., 2002); yet very few data are available on the interrelationships of essential and nonessential trace elements in breast milk. Data on mercury, lead, cadmium, and arsenic transfer into and concentrations in breast milk are described in the following, as are the factors apparently responsible for increasing milk metal levels. Very few studies have been carried out on the distinct effects of metal exposure via breastfeeding, illustrating the difficulties in this concern: effects of postnatal exposure do not clearly separate from those of prenatal exposure.

SOURCES

OF

HEAVY METALS

AND

TRANSFER

IN THE

ENVIRONMENT

Heavy metals spread through natural and anthropogenic sources. Heavy metals are natural constituents of Earth’s crust, emitted by volcanic activity, forest fires, and rock weathering. Anthropogenic sources of heavy metals include various processing and manufacturing industries, mining, foundries and smelters, piping, waste disposal, and diffuse sources such as combustion of fossil fuels and by-products, constituents of products, and corrosion. Human activities throughout the last century have

4


dramatically altered the biochemical and geochemical cycles of some heavy metals. Stumm and Keller (1984) presumed that, on a global scale, the anthropogenic emissions significantly exceed the natural emissions. Once released into the environment, metals move between the atmosphere, land, and water. Physical properties determine whether an element is predominantly transported by the atmosphere or the lithosphere, which is particle-bound aquatic transport for the latter. Relatively volatile heavy metals and those that become attached to airborne particles can be widely dispersed on a very large scale. The biosphere absorbs and accumulates various quantities of metals at certain trophic levels, depending on the environmental metal concentration, metal bioavailability, the feeding behavior, and the physiological state of organisms. In addition, some organic metal forms tend to accumulate along the food chain, e.g., methylated mercury. As a consequence, metallic elements are found in all living organisms and have potential toxicological implications for humans if the latter frequently consume species known to be accumulators of heavy metals, such as predatory fish, sea mammals, crustaceans, or shellfish.

EXPOSURE ROUTES

AND

BIOLOGICAL HALF-LIFE

The main exposure routes for humans are (1) inhalation of metal aerosols and metal vapor, (2) metal uptake through food and drinking water, (3) dermal metal absorption, and in case of the fetus, (4) uptake via the placenta. After a metal has been taken into the lung or into the gastrointestinal tract, it will be deposited on the walls of the airways or will be taken up in the mucosa of the gastrointestinal tract, and a certain fraction of the deposited amount will be transferred to the systemic circulation and distributed among tissue compartments throughout the body (Camner et al., 1986). Several chemical and physical characteristics of metals in exposure media, such as air, water, and food, are important for absorption, excretion, and retention of metals by humans, and determine the specific biological half-life of metals. Mercury is readily absorbed (especially methylmercury in the gastrointestinal tract) and distributed throughout the body. Biological half-life varies from a few days to months; the organs with the longest retention times are the brain and kidneys (Figure 1.1). Vahter et al. (2000) presumed that the half-life of methylmercury is longer in fetal blood than in maternal blood, about 2 months in the latter. About 10% of ingested lead is absorbed in the gastrointestinal tract. Infants and children may absorb as much as 50% of dietary lead. The main target organ of lead is the skeleton. The half-life varies among different tissue types. Lead retention in soft tissues is about 3 weeks, but in bone it may range from a few years to a few decades (Figure 1.2). Raghunath et al. (1997) reported retention times of 20 and 9 days for blood-lead and blood-cadmium in 6- to 10-year-old children, respectively. Gulson et al. (1999) described a longer lead half-life for infants than for mothers: 91 vs. 59 days. Cadmium predominantly accumulates in the kidney. On account of its low excretion rate, cadmium has a very long half-life of 10 to 30 years in the muscle, kidney, and liver (Figure 1.3). Organic and inorganic arsenic have been shown to be readily absorbed via the gastrointestinal tract, and also by inhalation (Figure 1.4).


Invasion:

Hg:

Anorganic

•Anorganic •Organic

•Ingestion •Inhalation •Dermal

Organic

5

Absorption: Anorganic •1–7% •80–95%

Organic


Distribution via blood and lymph (mostly bound to plasma proteins)

Excretion: •Urine (60% of total elimination) •Feces (methyl-Hg) •Sweat •Saliva •Exhalation •Breast milk

Body Depots: •CNS (organic Hg) •Liver •Pancreas •Kidney

Target Organs: •Kidney •CNS (organic Hg-Minamata disease)

T1/2: •70–90 d •1–18 a in CNS (metallic Hg)

FIGURE 1.1 Mercury distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.) Note: a = annus; d = days.

Absorbed arsenic is widely distributed in the body; the highest levels are found in the hair, nails, and skin. The major part of arsenic in humans is eliminated within 10 days.

HEAVY METALS IN BREAST MILK EXOGENOUS

AND

ENDOGENOUS SOURCES

Metals circulating in the maternal bloodstream originate from endogenous (metals released from storage organs and tissues) as well as exogenous sources (metal uptake via inhaled air, food, and drinking water). It is assumed that the chemical similarity of nonessential and essential elements, for example, calcium and lead, leads to the incorporation of nonessential elements via the same routes into the same storage organs available for essential element uptake, e.g., the skeleton for calcium and lead. This is true also for the extraction process taking place in phases of high nutrient demand, which is characteristic of the reproduction period. During this time, metals are eliminated along with essential trace elements from the target organs. Mobilization of lead from bone is likely to occur during periods of altered mineral metabolism. Because calciotropic factors determine the uptake and storage of lead in this compartment, changes in calcium-related regulatory factors are likely to affect lead compartmentation (Silbergeld, 1991).

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Pb:

Invasion:

•Anorganic •Organic

Absorption

Anorganic

Anorganic •5–10% (children up to 50%) •50–80%

•Ingestion •Inhalation

Organic •Ingestion •Inhalation •Dermal

Organic •>90% •>90% •>90%

Distribution via blood (90% bound to erythrocytes)

Body Depots: •Bone [Pb3(PO4)2] (90–95% of body burden)

Target Organs:

Excretion: •Urine (75– 80% of absorbed Pb) •Feces (90% of oral uptake) •Breast milk •Sweat

•Kidney •CNS •Smooth muscle •Peripheral nervous system •Red bone mark

T1/2: •20–30 a in bone •20 d in soft tissues

FIGURE 1.2 Lead distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.)

Cd:

Invasion:

Absorption:

•Ingestion •Inhalation

•1–7% •25–50%

Distribution via blood (95% bound to erythrocytes -most likely complexed to metallothioneins)

Excretion: •Urine •Feces •Breast milk •Placenta

Body Depots: •Kidney (50–75% of body burden) •Liver (metallothioneins) •Pancreas •Thyroid gland

Target Organs:

T1/2: >10 a

•Kidney •Lung (cancer) •Bone (Itai Itai disease) •Gonads

FIGURE 1.3 Cadmium distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.)


As:

Invasion:

Absorption:


•95% •30–60% •1–40%

Distribution via blood (95–99% in erythrocytes bound to globin)

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Body Depots: •Hair, nails (keratin) •Erythrocytes •Thyroid gland •Liver, kidney •Epididymis

Excretion:

Target Organs:

•Urine •Feces •Sweat •Exhalation

•Skin (hyperpigmentary, cancer) •Lung (cancer) •Heart muscle •Liver •Kidney •

T1/2: 5d (except hair, nails, bone)

FIGURE 1.4 Arsenic distribution and half-life in the human body. (Modified from Oehlmann and Markert, 1997.)

Both endogenous and exogenous metal concentrations are responsible for the actual metal content in milk. Which of these sources is the more important, especially for lead, is under discussion (see, e.g., Gulson et al., 2003). Oskarsson et al. (1998) presume that the clearance of chemicals during lactation is the major factor. In fact, between 45 and 70% of blood-lead in adult females arises from long-term lead stores in the tissue (Gulson et al., 1995) and the mobilization of lead from the skeleton during the postnatal period is greater than that during pregnancy; this might be attributed to an inadequate calcium intake. Breast milk lead levels were related to a 5.6% bone loss and to significant bone turnover in a study conducted by Sowers et al. (2002).

THE PROCESS

OF

MILK PRODUCTION

Lactation, i.e., the production of milk by the mammary gland, is a highly complex procedure. It can occur only after a series of developmental processes have taken place in the breast of the pregnant woman. Interaction between the mother and the child is an essential aspect of the process. Unless the child starts suckling or the breast is emptied artificially, the secretion of milk will stop within a few days (Philip and WHO Working Group, 1988). The production of milk varies according to the infant’s demands, age, and ability to suckle. The normal output of mature milk ranges from 600 to 1000 ml per day, but may vary between 300 and 1200 ml per day. The process of milk production and ejection is triggered by the hormones progesterone, estrogen, prolactin, and oxytocin. Lactogenesis begins about 40 h after the birth. The foremilk colostrum is produced within 3 to 5 days after delivery; it is low in volume and fat content (2.9%). Over the next 2 to 6 weeks, the transitional milk matures and increases in fat content to about 4%; the major class of milk lipids

8


are the triglycerides. Breast milk is composed of several other components including carbohydrates, proteins, and minerals, especially calcium (Needham and Wang, 2002). Milk is synthesized in the mammary alveolar gland, prior to which the components of milk and their precursors have to pass through a membrane that separates the blood flowing in capillaries from the alveolar epithelial cells of the breast. Alveolar secretory cells are involved in four processes that occur simultaneously: exocytosis; fat synthesis and excretion; secretion of ions, water, and proteins; and the transfer of milk components across the cell. Oskarsson et al. (1998) presumed that the exocrine pathway is quantitatively the most important. The major milk proteins casein and lactose, along with calcium and phosphate, form the so-called micelles, which are transported via Golgi vesicles to the apical membranes and then released into the milk alveoli. Small molecules such as sodium, potassium, chloride, and glucose can pass across the apical membrane.

TRANSFER

OF

HEAVY METALS

INTO

MILK

The transport of xenobiotics into milk is supposed to follow the same pathways as milk components and to proceed according to the principles of cellular metal uptake (Table 1.1). In general, there is a low transfer of toxic metals through milk when maternal exposure levels are low. However, knowledge concerning the lactational transport of metals and the potential effects of metals on milk secretion and composition is scarce (Oskarsson et al., 1998).

TABLE 1.1 Cellular Uptake of Heavy Metals Passive Transport

Active Transport

Diffusion Energy (ATP)-dependent via carrier-proteins Channel-mediated diffusion (e.g., Na+, K+, Ca2+) 2+ Carrier-mediated diffusion (e.g., Ca ) Simple diffusion Filtration Depends on: Similarity to essential metals (e.g., Ca-Pb, Zn-Cd) (chemical mimicry) Size and molecular weight Chemical bonding Lipophilic character Grade of ionization Transport through epithelial cells can take place: Transcellular (active or passive) Paracellular (passive through tight junctions of epithelial cells) Source: Data from Merian, 1984; Oehlmann and Markert, 1997.


9

Experimental data have shown that each metal is distributed in a characteristic way between the milk fractions. Lead is almost exclusively found in the casein fraction, while cadmium and methylmercury are found in fat, and inorganic mercury is largely found in whey fractions (Oskarsson et al., 1998). In human milk, mercury is mainly bound to caseins (Mata et al., 1997). Thus, mercury possesses a greater ability to interact with milk proteins than with low-weight molecules. In contrast, it appears that cadmium and lead are equally distributed among milk components with high and low molecular weights (Coni et al., 2000). Mercury Findings of Sundberg et al. (1999b) showed serum albumin is a major mercurybinding protein in whey and plasma fractions of mice for both methylmercury and inorganic mercury. The authors suggested passive transfer from plasma into milk using albumin as a passive carrier. Following the administration of lead and mercury to lactating and nonlactating mice, metal elimination from plasma was significantly greater in lactating mice, while about 30, 8, and 4% of the administered dose of lead, inorganic mercury, and methylmercury, respectively, was excreted in milk (Oskarsson et al., 1998; Sundberg et al., 1998). The transfer of mercury from plasma to milk was found to be higher in dams exposed to inorganic mercury than to methylmercury. In contrast, the uptake of mercury from milk was higher in the sucklings of dams exposed to methylmercury than to inorganic mercury (Oskarsson et al., 1995). Almost all methylmercury delivered via milk was absorbed and the suckling pups had a very low elimination rate until lactational day 17. Sundberg et al. (1999a) concluded that, on account of differences in kinetics, lactational exposure to methylmercury is a greater hazard for the breastfed infant than is inorganic mercury. However, both inorganic and organic mercury can be excreted in breast milk and the demethylation that takes place in vivo is thought to play an important role in the lactational transfer (Abadin et al., 1997). In contrast to lipophilic chemicals such as the persistent organic pollutants, metals do not bind to fat and usually do not accumulate at higher concentrations in breast milk than in blood. Oskarsson et al. (1995) established that milk mercury levels are approximately 30% of the levels in blood. On account of the placental transfer of mercury, it may be concluded that prenatal mercury exposure is generally more important than lactational exposure. In contrast, Drexler and Schaller (1998) and Ramirez et al. (2000) found lower maternal blood-mercury levels compared to milk mercury concentrations (Table 1.2). Lead It has been suggested by Palminger Hallen et al. (1996) that lead is transported into milk via the same pathway as calcium because of its high affinity to casein. In lactating mice, lead was found to be associated with casein micelles inside the alveolar cells and the milk lumen, indicating that lead is excreted into milk, bound to casein, via the Golgi secretory system. Oskarsson et al. (1995) reported that tissue

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TABLE 1.2 Mercury, Lead, Cadmium, and Arsenic Concentrations in Breast Milk Country

μg/l Hg

SD/Range

Austria

7.70

11

Austria

0.85 95 48 – >95

Lobsters Prawns/shrimp Crabs

Crustaceans 4.7–26 5.5–20.8 3.5–8.6

77 – >95 55 – >95 79 – >95

Bivalves 1 Bivalves 2 Gastropods Cephalopods

Mollusks 0.7–2.8 1.0–2.3 3.1–116.5 49

Echinoderms Coelenterates Sponges

12.4 7.5 3.2–6.8

44–88 12–50 58 – >95 72 – >95 60 15 13–15

TABLE 3.4 Experimental LD50 Values of Arsenic Species Arsenic Species As(III) MMA DMA TeMA TMAO AC AB

LD50 (g/kg) 0.0345 1.8 1.2 0.89 10.6 >6.5 >10.0

Source: Shiomi, 1994. With permission.

Table 3.5. This table provides evidence for the relatively small amounts of toxic arsenicals in seafood. In fish, the concentrations of inorganic and other toxic As compounds generally stay below 5% of the total As content.

Arsenic in Fish: Implications for Human Toxicity

69

TABLE 3.5 Toxic Arsenic in Fish, Mollusks, and Crustaceans Biota Species

Dogfish Thornback ray European conger Atlantic cod Saithe Pouting Whiting Ling Angler European seabass Dab European plaice Lemon sole Common sole Sand sole Witch sole Megrim Brill Turbot Herring Haddock Mullet Blue mussel Giant cupped oyster Great scallop Sea scallop Whelk

Common shrimp Delta prawn Fiddler crab Edible crab a

mg As kg–1 ww

As Species

Fish 0.046–0.60a 0.057–0.42a 0.028a 0.038–0.11a 0.039–0.057a 0.072–0.1a 0.051–0.094a 0.038–0.099a 0.036–0.20a 0.044a 0.034–0.46a 0.033–0.44a 0.02c 0.024–0.48a 0.49–0.57a 0.062–0.38a 0.13–0.40a 0.056–0.23a 0.031–0.087a 0.16a 0.03c 0.02c 0.01–0.03d

Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Inorganic Toxic Toxic Toxic Toxic Toxic Toxic Toxic Inorganic Inorganic Inorganic

0.55 0.92 1.2 1.5 1.6 2.3 1.4 2.8 0.94 4.0 1.96 1.4 0.1 0.68 1.4 1.52 0.92 1.25 2.6 0.89 3.6 0.8 2.8

Mollusks 0.07b 2.01b 0.04–0.09d 0.036 –0.72a 0.6–0.7 dwe 0.065–0.43a 0.06–0.18c

DMA TMAO Inorganic Toxic Me4As+ Toxic Inorganic

1.6 47 2.4 9.1 7.5 0.42 1.3

Crustaceans 0.17a 0.03d 0.02–0.04d 0.04–0.22d 0.18–0.40a

Toxic Inorganic Inorganic Inorganic Toxic

3.3 0.74 2.2 8.0 0.77

% of Total As

Toxic As as the sum of As(V), As(III), MMA, and DMA (De Gieter et al., 2002). Kaise et al., 1988. cBrooke and Evans, 1981. dSuñer et al., 1999. eLai et al., 1999. b

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HUMAN EXPOSURE TO FISH ARSENIC FATE

OF INGESTED

ORGANIC ARSENIC

As early as 1919, Bang stated that the organic As contained in fish and other marine food was readily excreted by humans. It appeared to be excreted in the feces, but only to a very limited extent. Experiments showed that, in animals fed homogenized shrimp, fecal excretion was only 5 to 25% of the ingested As (Coulson et al., 1935). Excretion in human feces turned out to be even less pronounced (Charbonneau et al., 1980). One reason for this low fecel excretion can be proposed: rapid and almost complete absorption of As in the gastrointestinal tract. Subsequent to absorption in the gastrointestinal tract, As is transported by the blood to other tissue in the body. Siewicki (1981) conducted a study of rats fed witch flounder, with low (4.7 mg As kg–1), medium (15.8 mg As kg–1), or high (28.8 mg As kg–1) As concentrations. The rats fed medium and higher As diets exhibited higher retention of As in the liver and spleen than did the control group rats fed the low As diet. Retention of As in the erythrocytes was not observed in the rats fed fish; on the contrary, it was observed in rats fed 22.1 mg DMA kg–1. Vahter et al. (1983) administered 73As-labeled AB to mice, rats, and rabbits and demonstrated that this major arsenical in marine products is rapidly cleared from plasma and other tissue. Longest retention was observed in the cartilage, testes, and epididymis of the animals, and in rabbits, also in muscle. AB itself was the only arsenical that was detected in extracts of the tissue and 98% of the ingested AB dose was excreted unchanged in the urine. A parallel experiment with 73As-labeled AC concluded that the clearance of AC from plasma and tissue was somewhat slower than that of AB. Tissues with the longest retention were again the reproductive organs, prostate, epididymis, and testes, and the myocardium, liver, adrenal cortex, pancreas, dental pulp, and pituitary gland, but in contrast to AB, the major compound excreted in the urine after administration of AC was AB, indicating that AC was oxidized to AB (Marafante et al., 1984). In humans, the National Academy of Science (1977) mentions an estimated total human body content of As of between 3 and 4 mg. This As is widely distributed in the body, in liver, kidney, lung, spleen, skin, hair, and nails. The uterus, bone, muscle, and neural tissue have also been shown to accumulate As. Following the ingestion of seafood, however, there are no records of tissue distribution in humans. Despite this, it has been observed that the organoarsenicals in fish and shellfish are readily excreted in the urine in an unchanged form, thus indicating that the kidneys play a key role in the fate of As taken up from diet. Chapman (1926) ascertained that ingestion of 33 μg As by eating lobster led to the excretion of 74% of this dose in the urine in 48 h. From a similar experiment, Coulson et al. (1935) concluded that almost 100% of the ingested As is eliminated within 1 week. Freeman et al. (1979) observed urinary excretions by six volunteers fed flounder, varying from 64 to 90% of the ingested As after 9 days. The excreted As was moreover in the same chemical form as in the fish. A similar observation was made by Luten et al. (1982): after the consumption of plaice, 69 to 85% of AB was excreted in the urine. Consequently,


71

consumption of seafood may result in substantial increases in As absorption by the human body, but also in increased elimination by the kidneys. The fate of the simpler organic As forms MMA and DMA seems to be similar to that of AB. Buchet et al. (1981) reported a study in which these arsenicals were ingested by volunteers; 75% of the ingested DMA was excreted unchanged in the urine within 4 days after exposure. From initial MMA, 78% of the dose was recovered in the same form, but 10% had been converted to DMA, suggesting some kind of methylation mechanism. Yamauchi and Yamamura (1984) observed an unchanged excretion of 90% of the trimethylarsinic acid from prawns in the urine within 60 h.

FATE

OF INGESTED INORGANIC

ARSENIC

Because the greater toxicological risks of As result from the presence of inorganic As in sources of exposure, the fate of ingested inorganic As has been studied more elaborately than that of organic As. The ingestion of water-soluble inorganic As was observed to proceed similarly to that of organoarsenicals; ingestion also results in high excretions in urine and only minor amounts in the feces. Arsenate and arsenite, as well, are rapidly absorbed by the gastrointestinal tract, and their presence in blood decreases shortly after exposure. The latter could not be confirmed in rats, as their erythrocytes seem to retain As, leading to much slower excretion in the urine (Vahter, 1994). In dogs, mice, rabbits, monkeys, and humans, however, as much as 90% of an administered dose is cleared from the blood with a half-time of 1 to 2 h. The half-times of the second and third phases have been estimated at 30 and 200 h, respectively (Vahter and Norin, 1980). Opposed to the pathway of organoarsenicals that causes AB, AC, DMA, and MMA to be largely excreted unchanged, inorganic As appears to be biotransformed and metabolized in vivo. Analyses of human urine from individuals exposed to inorganic As show the production of substantial amounts of DMA and MMA; 24 h after ingestion of 500 μg of inorganic As, 8, 5.3, and 9.3% of the dose is eliminated in the urine, respectively, as inorganic As, MMA, and DMA. Another 24 h later, an additional 2.3, 2.3, and 8.5% of the respective compounds is excreted (Buchet et al., 1980). Following exposure to inorganic As, elevated As concentrations in the liver, kidneys, lungs, and intestinal mucosa of rabbits and mice were noted. Vahter (1981) proposed that at low doses mammals are capable of methylating the inorganic As absorbed from the gastrointestinal tract. The mechanism for this methylation is believed to be the same mechanism as that suggested by Challenger (1945) for the methylation of As in microorganisms (Figure 3.4). It occurs via alternating reduction of pentavalent As to trivalent As and methylation. Subsequent to the reduction of As(V), As(III) is possibly bound to dithiol (Thompson, 1993); methylgroups are transferred from methyldonor S-adenosyl-methionine (SAM) to this As(III); and the resulting derivative is either transformed into MMA or used as a substrate for a second methylation to a dimethylated derivative, which later transforms into DMA. This reaction is believed to be mediated by the methyltransferases arsenite

72


OH As

HO

O

HO

CH3

As

As

HO

CH3

OH

OH

OH

As(V)

As(III)

MMA(V)

CH3 As

H3 C

O

OH

red.

CH3 CH3

red.

H3C

As O

trimethylarsine

TMAO

red.

HO

CH3

As

CH3

OH

MMA(III)

CH3

CH3

CH3

As

CH3 CH3

red. O

OH

DMA(III)

As

CH3

OH

DMA(V)

FIGURE 3.4 Methylation pathway as suggested by Challenger (1945).

methyltransferase and MMA methyltransferase. Although they have not been fully characterized yet, they have been purified from liver of rabbits, hamsters, and rhesus monkeys (Zakharyan et al., 1995). The liver is thus believed to be the prime organ for As methylation. Arsenic methylating activity has also been detected in several other tissues of mice — in the testes, kidney, liver, and lung — but to a much lesser extent (Healy et al., 1998). Although microorganisms generally produce trimethylated derivatives, the end point of the methylation in mammals is DMA. It is this DMA that is readily excreted in the urine. Note that, since the liver is believed to be the main site of methylation, ingestion leads to a higher degree of methylation than does inhalation or parental administration (Vahter, 1981). Because inorganic As(III) is more toxic than As(V), the initial step in the reaction, the reduction of As(V) to As(III), might be interpreted as a kind of bioactivation of the toxicity of As. However, it has been shown that As(III) is taken up much more readily by the hepatocytes, where the methylation occurs, than is As(V) (Lerman et al., 1983). Additionally, both end products of the methylation, MMA and DMA, are less reactive with tissue constituents and are more easily excreted in the urine. Therefore, this methylation reaction is generally believed to be a detoxification mechanism for As. Some considerations do, however, oppose this assumed beneficial effect: •

The rate of methylation decreases with increasing dose concentration (Vahter, 1981). This can either be explained by an overload of the methylation capacity or by inhibition of the reaction at high concentrations of As(III). High As(III) concentrations were indeed observed to inhibit the second step of the methylation and, thus, the production of DMA. Increased inorganic As concentrations therefore allow and induce the buildup of significant amounts of inorganic As in the body tissue. Next to organs, high concentrations of As were measured in keratin-rich tissues such as skin, hair, and nails. These tissues contain several sulfhydryl groups and allow efficient binding of trivalent As. Sectional analysis of


73

arsenatereductase As(V)

As(III)

2GSH

GS-SG

SAM arsenite-methyltransferase SAH

MMA(V)reductase MMA(III)

MMA(V)

SAM MMA-methyltransferase

SAM = S-adenosylmethionine SAH = S-adenosylhomocysteine GSH = reduced glutathione GS-SG = oxidized glutathione

SAH

DMA(V)

DMA(III)

FIGURE 3.5 In vivo methylation in mammals.

•

•

hair and nails indicated that the time of exposure to As can be demonstrated from peak concentrations, when the As entered the hair and nail roots via the bloodstream (Curry and Pounds, 1977; Pounds et al., 1979). The distribution of As in human organs following fatal acute intoxication by arsenic trioxide provided data on increased As levels in liver and kidney. These organs contained 7 to 350 times more As than blood (Benramdane et al., 1999). As mentioned above, the initial step in the biotransformation requires the reduction of As(V) to As(III) to allow uptake of As in the hepatocytes to occur more readily. Glutathione (GSH) and possibly other thiols serve as reducing agents. The first step in the methylation reaction is thus facilitated by the presence of reduced glutathione (Figure 3.5) and this step is at the same time believed to be the limiting step of the methylation. Significant differences in excretion pattern were observed in cases of liver insufficiencies. This difference is probably attributable to a reduced GSH content of the liver. Buchet and Lauwerys (1987) indeed reported reduced uptake of inorganic As by the hepatocytes of animals with low hepatic GSH. In addition to this observation, the authors also remarked that an increased amount of MMA was excreted. GSH is thus also believed to mediate the dimethylation step of the reaction. The existence of by-products from the reduction and methylation of inorganic As has recently been discovered. Suzuki et al. (2002) demonstrated the release of trivalent As containing MMA and DMA from the site of As methylation into body fluids. These intermediates, in particular

74


MMA(III), are today known to be even more reactive and toxic to rat and human cells than both As(III) and As(V) (Styblo et al., 2000). Petrick et al. (2000) observed an MMA(III) toxicity to Chang human hepatocytes, 26 times greater than that of inorganic arsenite. Lin et al. (1999) estimated MMA(III) to be over 100 times more potent as an inhibitor of thioredoxin reductase than As(III). Also the potent cytotoxicity and genotoxicity of DMA(III) has been reported (Kenyon and Hughes, 2001). The formation of MMA(III) and DMA(III) can therefore be considered to represent toxification of inorganic As, rather than detoxification. Exactly to what extent this trivalent MMA and DMA contribute to the total toxicity following exposure to inorganic As remains to be elucidated, but their discovery leads to a changed understanding of the purpose of the methylation. Understanding the metabolism of As is thus a critical factor in the risk assessment of chronic As exposure.

TOXICITY TO HUMANS CONCENTRATIONS

VS.

LEGAL LIMITS

Some authors suggest that at low concentrations As might be an essential element for organisms (Uthus, 1994). These studies imply that As plays a physiological role in the methionine metabolism, which is contrary to the toxicity of As that has been sufficiently documented over the centuries. Arsenite’s toxicity is believed to arise from reaction with sulfhydryl groups, among others. The compound would thus inhibit sulfhydryl enzymes, necessary for cellular metabolism. Arsenate may replace phosphate in the ATP/ADP mechanism and thus inhibit oxidative phosphorylation. Arsenic compounds are also described as antagonistic to the essential trace elements iodine and selenium (Levander, 1977). Toxicity arising from ingestion of inorganic As is believed to manifest itself in systemic effects involving the skin, the cardiovascular system, and the neurological system. Additionally, the IARC (1980) concluded that there is enough evidence to associate the exposure to inorganic As with skin, lung, and bladder cancer and classified As as a so-called group 1 carcinogen to humans. DMA is equally shown to induce organ-specific lesions in the lungs of mice, rats, and humans (Kenyon and Hughes, 2001). The same authors also mention dose-dependent increases in urinary bladder tumors upon lifetime exposure to DMA from diet or drinking water. DMA is believed to act either as a cancer promoter or as a complete carcinogen in different animals. Despite this extensive knowledge of certain As compounds’ toxicities, it seems that little attention has been paid to the probability of exposure to the element through fish consumption. Since recent discoveries of major calamities involving As in drinking water in Bangladesh and West Bengal, several reports and committees have focused on setting or adjusting maximum permissible concentrations for drinking water, but studies on the bioavailability of inorganic As in various foodstuffs are not given high priority. This results in a set of ambiguous formal As norms for fish and shellfish; existing legal limits vary from 0.1 mg kg–1 in Venezuela to 10 mg kg–1 in Hong Kong. Also,


75

in some countries, norms for As are related to total concentrations, while in other countries they express only the allowable inorganic As fraction. These discrepancies far from simplify judgments on the potential human risk related to seafood consumption and, thus, the meaning of these norms is not at all straightforward. The Joint FAO/WHO Expert Committee (1983) set a limit of 0.1 mg kg–1 wet weight (ww) for inorganic As in fish and seafood. In 1967, 50 μg kg–1 bodyweight (bw) was enforced as the tolerable daily intake (TDI) of As, but this norm dates from before epidemiological studies indicated that inorganic As might be carcinogenic to humans. Therefore, the committee arrived at an updated provisional TDI, specific for inorganic As, of 2 μg inorganic As kg–1 bw, thus a provisional tolerable weekly intake (PTWI) of 0.015 mg inorganic As kg–1 bw. Friberg (1988) stated that extensive consumption of seafood, in portions of 150 g day–1, might lead to ingested amounts of inorganic As that are high enough to affect the risk of cancer. Extrapolation of a 7-day-a-week consumption of 150 g day–1 of marine products, containing a realistic concentration of 10 mg kg–1 total As of which 5% inorganic, by a person of 60 kg, results in a weekly intake of 0.0087 mg kg–1 bw. When considering the FOA/WHO Expert Committee’s PTWI for inorganic As of 0.015 mg kg–1 bw, the toxic dose is thus not met. Conversely, this PTWI can produce either permitted amounts of seafood consumption or permitted levels of contamination. To arrive at the level of concern described as PTWI, the same person of 60 kg would daily have to ingest 128.6 μg inorganic As. Considering 150-g portions of fish, the inorganic As content of the fish would have to be 0.85 mg kg–1 ww. This concentration level is a lot higher than generally encountered in seafood.

ADDITIONAL CAUTIONS Nevertheless, PTWIs should be regarded with caution. Additional care should be taken when combining high levels of seafood consumption with other exposures to As. Several exposures to drinking water containing high levels of As have been recorded over the past decades in various countries. In Chile, Taiwan, West Bengal, India, Mexico, Argentina, China, and Bangladesh, thousands of people were exposed to high amounts of inorganic As of natural origin in drinking water (Mandal and Suzuki, 2002). This water often appeared to contain As in concentrations much higher than all existing drinking water limits. For example, in Bangladesh, 52 of 64 districts had well water As concentrations far above the Bangladesh drinking water standard of 50 μg l–1, and in 17 of the districts the maximum As level in groundwater exceeded 1 mg l–1. Another matter of concern is that there is inadequate information on the impact of storage, transport, and processing of seafood on As accumulation in the fish tissue. Until now, only limited consideration has been given to this issue: •

A comparative study of fresh and frozen fish pointed out that As concentrations in frozen species are higher than in fresh species (Juma et al., 2002).

76


•

•

•

Vélez et al. (1995) showed that, converse to AB being the major arsenical in fresh seafood, this might not be the case in manufactured products. They found an AB content in frozen and canned products of 47 and 30% of the total As, respectively, vs. 80% in the fresh product. The DMA content was observed to be higher in preserved products than in fresh ones; 4% of the total As content in the processed vs. 1% in the fresh seafood was in the form of DMA. In a later study of canned seafood, the same authors demonstrated the possible transfer of AB and DMA from the tissue to the accompanying liquid (Vélez et al., 1997). These observations led them to believe that AB is likely to be solubilized in the intercellular liquids of the fish and released into the brine, and second, that AB might be degraded during processing. Mürer et al. (1992) showed that AB can be converted efficiently to inorganic As(V) upon ultraviolet radiation and speculated that this conversion might occur during storage or preparation of seafood. Reinke et al. (1975) discussed the rapid reduction of arsenate to arsenite after fish death, and also Hanaoka et al. (1993) observed the degradation of accumulated AB to inorganic As in shark’s muscle, after the death of the animal.

Thus, studies on concentrations, and especially speciation of As compounds, in processed seafood are absolutely necessary. The effects of transport, storage, and preparation on modifications in As speciation in seafood should be determined. This study may be used to recommend regulations for conservation and cooking in order to avoid or reduce the presence of toxic As species.

DOSE–RESPONSE ASSESSMENTS Doses responsible for effects after exposure to inorganic As have until now been deduced from incidents of mass poisoning. Fatal doses of ingested As(III) oxide have been reported in the range of 1 to 2.5 mg kg–1 bw. Additionally, it appears that ingestion of 3 mg As(V) daily over a period of few weeks may give rise to severe poisoning in infants and symptoms of toxicity in adults (WHO, 1981). A report of the NRC (1999) also cited noncancer effects from chronic ingestion of inorganic As, at doses of 10 μg kg–1 day–1 and higher. This report concludes that epidemiological studies are needed to characterize the dose–response relationship for Asassociated cancer and noncancer effects. Most laboratory animals, however, appear to be substantially less susceptible to As than humans. It has been reported that chronic oral exposure to 0.05 to 0.1 mg inorganic As kg–1 day–1 causes neurological and hematological toxicity in humans but not in monkeys, dogs, and rats (Byron et al., 1967). Also with regard to carcinogenicity, all models to test the effects of arsenicals in humans failed to mimic the actual human mechanism satisfactorily. Thus, quantitative dose-dependent data for animals should not be considered a reliable source for application to humans. Thus, one of the recommendations is to establish suitable animal models for As toxicity assessments.


77

CONCLUSIONS Although extensive research has provided information either on the form of As in seafood or on its fate in the human body after consumption of this seafood, several questions that are of concern for the accurate assessment of potential dangers of exposure to As via the consumption of seafood remain: •

•

•

•

•

The exact origin and metabolical pathway of As in higher marine organisms remain hypothesized. Also the origin of several concentration differences, both interspecies and intraspecies, remains speculative to date. Further investigation of the type and concentration of As compounds in marine products are highly desirable. Several reports demonstrate the large differences in toxicity among the different arsenicals in marine animals. There is thus comprehensive knowledge that the majority of As in seafood is nontoxic. Nevertheless, many formal limits do not seem to take these notations into account and remain largely ambiguous. Formulations of maximum permissible concentrations for health regulations in seafood should recognize the chemical nature of As in seafood. Additionally, they should be lowered for populations exposed to other sources of As. The recent discovery of highly toxic trivalent by-products of the methylation reaction of inorganic As in mammals has changed the current assumptions of methylation as a detoxification mechanism for inorganic As. Understanding the metabolism of As in humans may thus be considered a critical factor in the assessment of the risk associated with chronic exposure to As. There is inadequate information on seafood processing effects on the concentrations and especially on the speciation of As in this seafood. The effects of transport, storage, and preparation on potential modifications in As content should be investigated. This study may be used to recommend regulations for conservation and cooking to avoid or reduce the presence of toxic As species. Reliable animal models and their transpositions to humans should be developed to assess the toxicity of As to humans.

REFERENCES Andreae, M.O. and Klumpp, D. (1979) Biosynthesis and release of organoarsenic compounds by marine algae, Environmental Science and Technology, 13: 738–741. Ballin, U., Kruse, R., and Rüssel, H.-A. (1994) Determination of total arsenic and speciation of arsenobetaine in marine fish by means of reaction — headspace gas chromatography utilizing flame-ionization detection and element specific spectrometric detection, Fresenius Journal of Analytical Chemistry, 350: 54–61. Bang, I. (1919) in H. Lund, Ed., Report by the Swedish Arsenic Commission, Vol. 11, Ohlssons Boktryckeri, 1–56.

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Benramdane, L., Accominotti, M., Fanton, L., Malicier, D., and Vallon, J.J. (1999) Arsenic speciation in human organs following fatal arsenic trioxide poisoning — a case report, Clinical Chemistry, 45: 301–306. Bohn, A. (1975) Arsenic in marine organisms from West Greenland, Marine Pollution Bulletin, 6: 87–89. Brooke, P.J. and Evans, W.H. (1981) Determination of total inorganic arsenic in fish, shellfish and fish products, Analyst, 106: 514–520. Brown, C.J. and Eaton, R.A. (1999) Leaching of copper-chrome-arsenic (CCA) wood preservative in sea water, Material und Organismen, 33: 213–233. Buchet, J.P. and Lauwerys, R. (1987) Study of factors influencing the in vivo methylation of inorganic arsenic in rats, Toxicology and Applied Pharmacology, 91: 65–74. Buchet, J.P., Lauwerys, R., and Roels, H. (1980) Comparison of several methods for the determination of arsenic compounds in water and in urine, International Archives of Occupational and Environmental Health, 46: 11–29. Buchet, J.P., Lauwerys, R., and Roels, H. (1981) Comparison of the urinary-excretion of arsenic metabolites after a single oral dose of sodium arsenite, monomethylarsonate, or dimethylarsinate in man, International Archives of Occupational and Environmental Health, 48: 71–79. Byron, W.R., Bierbower, G.W., Brouwer, J.B., and Hanse, W.H. (1967) Pathological changes in rats and dogs from two year feeding of sodium arsenate or sodium arsenate, Toxicology and Applied Pharmacology, 10: 132–147. Challenger, F. (1945) Biological methylation, Chemical Reviews, 36: 315–361. Chapman, A.C. (1926) On the presence of compounds of arsenic in marine crustaceans and shell fish, Analyst, 51: 548–563. Charbonneau, S.M., Tam, G.K.H., Bryce, F., and Sandi, E. (1980) in Proceedings of the 19th Annual Meeting of the Society of Toxicology, Washington, D.C., 9–13 March 1980, Abstr. 362, p. A121. Chilvers, D.C. and Peterson, P.J. (1987) Global cycling of arsenic, in T.C. Hutchinson and K.M. Meema, Eds., Lead, Mercury, Cadmium and Arsenic in the Environment, Scope 31, New York: John Wiley, 279–301. Coulson, E.J., Remington, R.E., and Lynch, K.M. (1935) Metabolism in the rat of the naturally occurring arsenic of shrimp as compared with arsenic trioxide, Journal of Nutrition, 10: 255–270. Cullen, W.R. and Dodd, M. (1989). Arsenic speciation in clams of British Columbia, Applied Organometallic Chemistry, 3: 79–88. Cullen, W.R., Hettipathirana, D., and Reglinski, J. (1989) The effect of arsenicals on cell suspension cultures of the Madagascar periwinkle (Catharanthus roseus), Applied Organometallic Chemistry, 3: 515–521. Curry, A.S. and Pounds, C.A. (1977) Arsenic in hair, Journal of the Forensic Science Society, 17: 37–44. De Gieter, M., Leermakers, M., Van Ryssen, R., Noyen, J., Goeyens, L., and Baeyens, W. (2002) Toxic and total arsenic levels in North Sea fish, Archives of Environmental Contamination and Toxicology, 43: 406–417. Edmonds, J.S. and Francesconi, K.A. (1977) Methylated arsenic from marine fauna, Nature, 265: 436. Edmonds, J.S. and Francesconi, K.A. (1981a) The origin and chemical form of arsenic in the school whiting, Marine Pollution Bulletin, 12: 92–96. Edmonds, J.S. and Francesconi, K.A. (1981b) Isolation and identification of arsenobetaine from the American lobster Homarus americanus, Chemosphere, 10: 1041–1044.


79

Edmonds, J.S. and Francesconi, K.A. (1981c) Arseno-sugars from brown kelp (Ecklonia radiata) as intermediates in cycling of arsenic in a marine ecosystem, Nature, 289: 602–604. Edmonds, J.S. and Francesconi, K.A. (1983) Arsenic-containing ribofuranosides: isolation from brown kelp Ecklonia radiata and NMR spectra, Journal of the Chemical Society–Perkin Transactions I, 10: 2375–2382. Edmonds, J.S. and Francesconi, K.A. (1987) Trimethylarsine oxide in estuary catfish (Cnidoglanis macrocephalus) and school whiting (Sillago bassensis) after oral administration of sodium arsenate; and as a natural component of estuary catfish, Science of the Total Environment, 64: 317–323. Edmonds, J.S., Francesconi, K.A., Healy, P.C., and White, A.H. (1982) Isolation and crystal structure of an arsenic-containing sugar sulphate from the kidney of the giant clam, Tridacna maxima, X-ray crystal structure of (2S)-3[5-deoxy-5 (dimethylarsinoyl)-βD-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulphate, Journal of the Chemical Society–Perkin Transactions I, 12: 2989–2993. Fowler, S.W. and Ünlü, M.Y. (1978) Factors affecting bioaccumulation and elimination of arsenic in the shrimp Lysmata seticaudata, Chemosphere, 7: 711–720. Francesconi, K.A., Edmonds, J.S., and Stick, R.V. (1989) Accumulation of arsenic in yelloweyed mullet (Aldrichetta-Forsteri) following oral-administration of organoarsenic compounds and arsenate, Science of the Total Environment, 79: 59–67. Francesconi, K.A. and Edmonds, J.S. (1993), in A.D. Ansell, R.N. Gibson, and M. Barnes, Eds., Oceanography and Marine Biology: An Annual Review, Vol. 31, UCL Press, London, 11. Francesconi, K.A., Hunter, D.A., Bachmann, B., Raber, G., and Goessler, W. (1999) Uptake and transformation of arsenosugars in the shrimp Crangon crangon, Applied Organometallic Chemistry, 13: 669–679. Freeman, H.C., Uhthe, J.F., Fleming, R.B., Oduse, P.H., Ackman, R.G., Landry, G., and Musial, C. (1979) Clearance of arsenic ingested by man from arsenic contaminated fish, Bulletin of Environmental Contamination and Toxicology, 22: 224–229. Friberg, L. (1988) The GESAMP evaluation of potentially harmful substances in fish and other seafood with special reference to carcinogenic substances, Aquatic Toxicology, 11: 379–393. Hamilton, E.I. and Minski, M.J. (1973) Abundance of the chemical elements in mans diet and possible relations with environmental factors, Science of the Total Environment, 1: 375–394. Hanaoka, K., Kogure, T., Miura, Y., Tagawa, S., and Kaise, T. (1993) Post-mortem formation of inorganic arsenic from arsenobetaine in a shark under natural conditions, Chemosphere, 27: 2163–2167. Hanaoka, K., Goessler, W., Yoshida, K., Fujitaka, Y., Kaise, T., and Irgolic, K.J. (1999) Arsenocholine- and dimethylated arsenic-containing lipids in starspotted shark Mustelus manazo, Applied Organometallic Chemistry, 13: 765–770. Healy, S.M., Casarez, E.A., Ayala-Fierro, F., and Aposhian, H.V. (1998) Enzymatic methylation of arsenic compounds V. Arsenite methyltransferase activity in tissues of mice, Toxicology and Applied Pharmacology, 148: 65–70. IARC (1980) IARC Monographs, Arsenic and Its Compounds, Vol. 23, Lyons: International Agency for Research on Cancer, 39–141. Irgolic, K.J., Woolson, E.A., Stockton, R.A., Newman, R.D., Bottino, N.R., Zingaro, R.A., Kearney, P.C., Pyles, R.A., Maeda, S., McShane, W.J., and Cox, E.R. (1977) Characterisation of arsenic compounds formed by Daphnia Magna and Tetraselmis chuii from inorganic arsenate, Environmental Health Perspectives, 19: 61–66.

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Joint FAO/WHO Expert Committee on Food Additives (1983) 27th Report, Technical Report Series, No 696, Geneva: World Health Organization. Jongen, W.M.F., Cardinaals, J.M., Bos, P.M.J., and Hagel, P. (1985) Genotoxicity testing of arsenobetaine, the predominant form of arsenic in marine fishery products, Food and Chemical Toxicology, 23: 669–673. Juma, H., Battah, A., Salim, M., and Tiwari, P. (2002) Arsenic and cadmium levels in imported fresh and frozen fish in Jordan, Bulletin of Environmental Contamination and Toxicology, 68: 132–137. Kaise, T., Yamauchi, H., Hirayama, T., and Fukui, S. (1988) Determination of inorganic arsenic and organic arsenic compounds in marine organisms by hydride generation/cold trap/gas chromatography-mass spectrometry, Applied Organometallic Chemistry, 2: 339–347. Kaise, T., Oya-Ohta, Y., Ochi, T., Okubo, T., Hanaoka, K., Irgolic, K.J., and Sakurai, T. (1996) Toxicological study of organic arsenic compound in marine algae using mammalian cell culture technique, Journal of the Food and Hygienic Society of Japan, 37(3): 135–141. Kenyon, E.M. and Hughes, M.F. (2001) A concise review of toxicity and carcinogenicity of dimethylarsinic acid, Toxicology, 160: 227–236. Klumpp, D.W. (1980) Characteristics of arsenic accumulation by seaweeds Fucus spiralis and Ascophyllum nodosum, Marine Biology, 58: 257–264. Lai, V.W.-M., Cullen, W.R., and Ray, S. (1999) Arsenic speciation in scallops, Marine Chemistry, 66: 81–89. Langston, W.J. (1980) Arsenic in U.K. estuarine sediments and its availability to benthic organisms, Journal of the Marine Biological Association of the United Kingdom, 60: 869–881. Lerman, S.A., Clarkson, T.W., and Gerson, R.J. (1983) Arsenic uptake and metabolism by liver cells is dependent on arsenic oxidation state, Chemico-Biological Interactions, 45: 401–406. Levander, O.A. (1977) Metabolic interrelationships between arsenic and selenium, Environmental Health Perspectives, 19: 159–164. Lin, S., Cullen, W.R., and Thomas, D.J. (1999) Methylarsenicals and arsinothiols are potent inhibitors of mouse liver thioredoxin reductase, Chemical Research in Toxicology, 12: 924–930. Lunde, G. (1968) Analysis of arsenic in marine oils by neutron activity. Evidence of arsenoorganic compounds, Journal of the American Oil Chemists Society, 45: 331–332. Lunde, G. (1969) Water soluble arseno-organic compounds in marine fishes, Nature, 224: 185–187. Lunde, G. (1973) The synthesis of fat and water soluble arseno organic compounds in marine and limnetic algae, Acta Chemica Scandinavica, 27: 1586–1594. Lunde, G. (1975) A comparison of organoarsenic compounds from different marine organisms, Journal of the Science of Food and Agriculture, 26: 1257–1262. Luten, J.B., Riekwel-Booy, G., and Rauchbaar, A. (1982) Occurrence of arsenic in plaice, nature of organo-arsenic compound present and its excretion by man, Environmental Health Perspectives, 45: 165–170. Luten, J., Riekwel-Booy, G., Van Der Greef, J., and ten Noever de Brauw, M. (1983) Identification of arsenobetaine in sole, lemon sole, flounder, dab, crab and shrimp by field desorption and fast atom bombardment mass spectrometry, Chemosphere, 12: 131–141. Maeda, S., Ohki, A., Tokuda, T., and Ohmine, M. (1990) Transformations of arsenic compounds in a freshwater food chain, Applied Organometallic Chemistry, 4: 251–254.


81

Maher, W.A. (1985) Distribution of arsenic in marine animals: relationship to diet, Comparative Biochemistry and Physiology, 82C: 433–434. Mandal, B.K. and Suzuki, K.T. (2002) Arsenic around the world: a review, Talanta, 58: 201–235. Marafante, E., Vahter, M., and Dencker, L. (1984) Metabolism of arsenocholine in mice, rats and rabbits, Science of the Total Environment, 34: 223–240. Mariner, P.E., Holzmer, F.J., Jackson, E., Meinardus, H.W., and Wolf, F.G. (1996) Effects of high pH on arsenic mobility in a shallow sandy aquifer and on aquifer permeability along the adjacent shoreline, Commencement Bay superfund site, Tacoma, Washington, Environmental Science and Technologies, 30: 1645–1651. Mason, R.P., Laporte, J.-M., and Andres, S. (2000) Factors controlling the bioaccumulation of mercury, methylmercury, arsenic, selenium, and cadmium by freshwater invertebrates and fish, Archives of Environmental Contamination and Toxicology, 38: 283–297. Michel, P. (1993) L’Arsenic en Milieu Marin: Biogeochimie et Ecotoxicologie, Repères Océan Ifremer, 4, 41–42. Ministry of Agriculture, Fisheries and Food. (1982) Survey of arsenic in food, the eight report of the steering group of food surveillance, the working party on the monitoring of foodstuffs for heavy metals, Food surveillance paper 8, London: HMSO. Morita, M. and Shibata, Y. (1987) Speciation of arsenic compounds in marine life by high performance liquid chromatography combined with inductively coupled argon plasma atomic emission spectroscopy, Analytical Sciences, 3: 575–577. Mürer, A.J.L., Abildtrup, A., Poulsen, O.M., and Chritensen, J.M. (1992) Effect of seafood consumption on the urinary levels of total hydride-generating arsenic compounds — instability of arsenobetaine and arsenocholine, Analyst, 117: 677–680. National Academy of Science. (1997) Medical and Biological Effects of Environmental Pollutants — Arsenic, Washington, D.C.: National Academy of Science. Norin, H., Christakopoulos, A., Sandström, M., and Ryhage, R. (1985) Mass fragmentographic estimation of trimethylarsine oxide in aquatic organisms, Chemosphere, 14: 313–323. NRC (1999) Arsenic in Drinking Water, Washington, D.C.: National Research Council, National Academy Press. Penrose, W.R., Black, R., and Hayward, M.J. (1975) Limited arsenic dispersion in sea water, sediments and biota near a continuous source, Journal of Fisheries Research Board of Canada, 32: 1275–1281. Petrick, J.S., Ayala-Fierro, F., Cullen, W.R., Carter, D.E., and Aposhian, H.V (2000) Monomethylarsenous acid (MMAIII) is more toxic than arsenite in Chang human hepatocytes, Toxicology and Applied Pharmacology, 163: 203–207. Pounds, C.A., Pearson, E.F., and Thurner, T.D. (1979) Arsenic in fingernails, Journal of the Forensic Science Society, 19: 165–173. Reinke, J.U., Freeman, H.C., and Johnston, J.R. (1975) The determination of arsenite and arsenate ions in fish and shellfish by selective extraction and polarography, Environmental Letters, 8: 371–380. Saha, J.C., Dikshit, K.A., and Bandyopadhyay, M. (1999) A review of arsenic poisoning and its effects on human health, Critical Reviews in Environmental Science and Technology, 29: 281–313. Sakurai, T., Kaise, T., and Matsubara, C. (1996) Immunotoxicity of organic arsenic compounds in marine animals, Applied Organometallic Chemistry, 10: 727–732. Sanders, J.G. (1979) Effects of arsenic speciation and phosphate concentration on arsenic inhibition of Skeleonema costatum (Bacillariophyceae), Journal of Phycology, 15: 424–428.

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Sanders, J.G. and Cibick, S.J. (1985) Adaptive behavior of euryhaline phytoplankton communities to arsenic stress, Marine Ecology Progress Series, 22: 199–205. Shibata, Y., Morita, M., and Edmonds, J.S. (1987) Purification and identification of arseniccontaining ribofuranosides from the edible brown seaweed, Laminaria japonica (Makonbu), Agricultural and Biological Chemistry, 51: 391–398. Shinagawa, A., Shiomi, K., Yamanaka, Y., and Kikuchi, T. (1983) Selective determination of inorganic arsenic (III), (V) and organic arsenic in marine organisms, Bulletin of the Japanese Society of Scientific Fisheries, 49: 75–78. Shiomi, K. (1994) Arsenic in marine organisms: chemical forms and toxicological aspects, in J.O. Niagru, Ed., Arsenic in the Environment. Part II: Human Health and Ecosystem Effects, Vol. 27, New York: John Wiley & Sons, chap. 12. Shiomi, K., Shinagawa, A., Igarashi, T., Hirota, K., Yamanaka, H., and Kikuchi, T. (1984) Contents and chemical forms of arsenic in shellfishes in connection with their feeding habits, Bulletin of the Japanese Society of Scientific Fisheries, 50: 293–297. Siewicki, T.C. (1981) Tissue retention of arsenic in rats fed witch flounder or cacodylic acid, Journal of Nutrition, 111: 602–609. Smith, D.C., Leduc, R., and Tremblay, L. (1975) Pesticide-residues in total diet in Canada. 4. 1972 and 1973, Pesticide Science, 6: 75–82. Styblo, M., Del Razo, L.M., Vega, L., Gerrmolec, D., LeCluyse, E.L., Hamilton, G.A., Reed, W., Wang, C., Cullen, W.R., and Thomas, D.J. (2000) Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells, Archives of Toxicology, 74: 289–299. Suñer, M.A., Devesa, V., Muñoz, O., López, F., Montoro, R., Arias, A.M., and Blasco, J. (1999) Total and inorganic arsenic in the fauna of the Guadalquivir estuary: environmental and human health implications, Science of the Total Environment, 242: 261–270. Suzuki, K.T., Mandal, B.K., and Ogra, Y. (2002) Speciation of arsenic in body fluids, Talanta, 58: 111–119. Taylor, D.D. and Bright, T.J. (1973) The distribution of heavy metals in reef-dwelling groupers in the Gulf of Mexico and Bahama Islands, Report of the Department of Oceanography, Texas A&M University, College Station. Thompson, D.J. (1993) A chemical hypothesis for arsenic methylation in mammals, ChemicoBiological Interactions, 88: 89–114. U.S. FDA (1993) Guidance Document for Arsenic in Shellfish, Washington, D.C.: U.S. Food and Drug Administration, 27. Uthus, E.O. (1994) Arsenic essentiality and factors affecting its importance, in W.R. Chappell, C.O. Abernathy, and C.R. Cothern, Eds., Arsenic Exposure and Health, Science and Technology Letters, London: Northwood, 199–208. Vahter, M. (1981) Biotransformation of trivalent and pentavalent inorganic arsenic in mice and rats, Environmental Research, 25: 286–293. Vahter, M. (1994) Species differences in the metabolism of arsenic compounds, Applied Organometallic Chemistry, 8: 175–182. Vahter, M. and Norin, H. (1980) Metabolism of As-74-labelled trivalent and pentavalent inorganic arsenic in mice, Environmental Research, 21: 446–457. Vahter, M., Marafante, E., and Dencker, L. (1983) Metabolism of arsenobetaine in mice, rats and rabbits, Science of the Total Environment, 30: 197–211. Vélez, D., Ybáñez, N., and Montoro, R. (1995) Percentages of total arsenic represented by arsenobetaine levels of manufactured seafood products, Journal of Agricultural and Food Chemistry, 43: 1289–1294.


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Vélez, D., Ybáñez, N., and Montoro, R. (1997) Migration of arsenobetaine from canned seafood to brines, Journal of Agricultural and Food Chemistry, 45: 449–453. Vos, G., Hovens, J., and Hagel, P. (1986) Chromium, nickel, copper, zinc, arsenic, selenium, cadmium, mercury and lead in Dutch fishery products, 1977–1984, Science of the Total Environment, 52: 25–40. Weis, J.S. and Lores, E. (1993) Uptake of metals from chromated copper arsenate (CCA) treated lumber by epibiota, Marine Pollution Bulletin, 26: 428–431. WHO (1981) Arsenic, Environmental Health Criteria 18, Geneva: International Programme on Chemical Safety, World Health Organization. Yamauchi, H. and Yamamura, Y. (1984) Metabolism and excretion of orally ingested trimethylarsenic in man, Bulletin of Environmental Contamination and Toxicology, 32: 682–687. Zakharyan, R.A., Wu, Y., Bogdan, G.M., and Aposhian, H.V. (1995) Enzymatic methylation of arsenic compounds. I: Assay, partial purification, and properties of arsenite methyltransferase and monomethylarsonic acid methyltransferase of rabbit liver, Chemical Research in Toxicology, 8: 1029–1038.

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Biological and Toxicological Considerations of Dietary Sulfur Lioudmila A. Komarnisky and Tapan K. Basu

CONTENTS Abstract ....................................................................................................................85 Introduction..............................................................................................................86 Properties of Elemental Sulfur ................................................................................86 Occurrence of Sulfur in Nature...............................................................................88 Global Cycle ............................................................................................................89 Biological Role of Sulfur ........................................................................................91 Sulfur Metabolism ...................................................................................................92 Sulfur Deficiencies ..................................................................................................93 Sulfur Toxicity .........................................................................................................94 Toxicity Due to SO2 ....................................................................................94 Routes of SO2 Entry into Living Organisms.....................................95 Maximum and Threshold Limit Values of SO2 .................................95 SO2-Sensitive Subjects.................................................................................95 Diseases and Sulfur .....................................................................................96 Pathogenesis of SO2-Linked Toxicity .........................................................96 Clearance of SO2 from the Organism .........................................................98 Toxicity of Secondary Origin (Sulfur Metabolism)....................................98 Conclusion .............................................................................................................100 References..............................................................................................................100

Abstract

Essential amino acids, including methionine (a sulfur-containing amino acid), must be supplied through the diet, as humans and animals, except for ruminant animals, are unable to synthesize them. Sulfur is present in body tissues as part of the amino acids: methionine, cysteine, and taurine. The relative reductionoxidation state of the cell depends primarily on the precise balance between

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concentrations of reactive oxygen species (ROS) and the cysteine-dependent (thiol) antioxidant buffers, glutathione (GSH) and thioredoxin. These antioxidants have high affinity for ROS, thus protecting other intracellular molecules from oxidative damage. Dietary deficiency of sulfur is relatively uncommon, whereas its toxicity is increasingly recognized as a serious concern in our environment. Human activities, such as burning fossil fuels, refining petroleum, smelting sulfur compounds of metallic minerals into free metals, and other industrial processes, have a large impact on the atmospheric sulfur balance. The atmospheric sulfur is lost into water reservoirs, where some of the sulfur enters marine communities and soil as it moves through the food chain. The metabolic product, sulfur dioxide (SO2), is thus considered one of the major air pollutants worldwide. Sulfur toxicity is recognized to be a consequence of metabolic derangement of methionine. Its metabolic product, homocysteine, is thought to be an independent risk factor for arterial diseases.

INTRODUCTION Sulfur is a major inorganic element, essential to the entire biological kingdom because of its incorporation into amino acids, proteins, enzymes, vitamins, and other biomolecules. Unlike humans and monogastric animals, plants have the ability to synthesize sulfur-containing amino acids (e.g., methionine and cysteine) utilizing inorganic sulfur, and thereby become important sources for the element. The maintenance of sufficient levels of sulfur in the biosphere, however, involves the biogeochemical sulfur cycle (Figure 4.1). The primary components in this cycle are the lithosphere, the atmosphere, and the hydrosphere, which provide sulfur for the habitat of the biosphere. In living species, sulfur compounds undergo metabolic processes; sulfur metabolites are excreted to re-enter the biogeochemical cycle (Miller, 1998). Although sulfur is a dietary essential, its excess leading to toxicity is more of a concern than its dietary deficiency. Sulfur-containing food additives, for example, may increase indigenous sulfur in human and animal diets and may cause allergic reactions in sulfur-sensitive individuals. This chapter describes dietary sulfur as an important element to all living cells and delineates its potential toxicological characteristics.

PROPERTIES OF ELEMENTAL SULFUR Sulfur is uniquely classified within the group of macroelements, for it belongs to the group 6A of nonmetals in the periodic table of the chemical elements. Unlike other macrominerals, sulfur exists predominantly in organic molecules; there are only very small amounts of free metabolic sulfite (SO3=) and sulfate (SO4=). It is one of the most abundant elements in Earth’s crust and is cycled and metabolized through the lithosphere, hydrosphere, atmosphere, and biosphere in the biogeochemical cycle (Kellogg et al., 1972). Table 4.1 illustrates the major chemical and physical properties and oxidation states of sulfur. Sulfate (oxidation state +6) is the most stable and abundant form of sulfur available for use by living organisms in the

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ATMOSPHERE (hydrogen sulfide + oxidation = sulfur dioxide + hydration = sulfurous and sulfuric acids)

hydrogen sulfide

Precipitation (sulfurous and sulfuric acids)

dimethyl sulfide

LITHOSPHERE (sulfur compounds from volcanoes, mines, industries)

HYDROSPHERE (sulfur compounds from oceans, hot springs)

sulfates, sulfites

sulfates, sulfites decay, waste

decay, waste

BIOSPHERE Plants (reduced sulfur)

Animals

Humans

FIGURE 4.1 Simplified diagram of the global sulfur cycle. (Adapted from Komarnisky et al., 2003.)

biosphere. Table 4.2 lists the inorganic sulfur compounds available for biosynthesis of sulfur amino acids (SAAs) and other biological sulfur compounds. SAAs and other organic compounds found in living cells contain sulfur in the lowest oxidation state, –2 [–S–, –S–S–, or –SH (thiol) forms], found in sulfides. To be used for the synthesis of amino acids, sulfates or other sulfur compounds in the oxidation state greater than –2 must be reduced to sulfides. Essential amino acids, including methionine (a sulfur-containing amino acid), must be supplied through diet, as humans and animals other than ruminants are unable to synthesize them. Ruminants can obtain SAAs from the microbial protein synthesized in the rumen and have various populations of sulfur-reducing bacteria that can utilize plant amino acids and inorganic sulfur for the synthesis of their own proteins. Ruminants, therefore, can obtain proteins and amino acids from the microbial cells that have been subjected to the process of acid digestion and proteolytic hydrolysis in the postruminal part of the digestive tract (Komarnisky et al., 2003).

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TABLE 4.1 The Most Common Inorganic Sulfur Compounds Compound

Chemical Formula

Sulfur dioxide

SO2

Sulfur trioxide Sulfuric acid

Process Involved

Reaction State

End Product

Oxidation

Intestinal microbial fermentation

SO3 H2SO4

S + O2 or H2S + O2 SO2 + O2 SO3 + H2O

Oxidation Hydration

Sulfurous acid

H2SO3

SO2 + H2O

Hydration

Bisulfate ions

HSO4−

H2SO4 + H2O

Intestinal microbial fermentation Contact with moist mucous membranes, skin Contact with moist mucous membranes, skin Buffering system of body fluids

Sulfate ions

SO4=

Hydrogen sulfite ions Sulfite ions

HSO3−

Hydrogen sulfide

SO3= H2S

Ionization of sulfuric acid HSO4− + H2O Ionization of sulfuric acid H2SO3 + H2O Ionization of sulfurous acid HSO3− + H2O Ionization of sulfurous acid SO4= + 2e−, Reduction SO3= + 6e−

Body metabolic processes Buffering system of body fluids Body metabolic processes Intestinal microbial fermentation

Sources: Adapted from Komarnisky et al., 2003.

OCCURRENCE OF SULFUR IN NATURE In humans and animals, three forms of sulfur exist, predominantly as organic compounds. These include thiomethyl of methionine residues in protein, sulfhydryl disulfides of protein (cysteine–cystine residues), and compounds, such as ester- or amide-bound sulfates (e.g., glycosaminoglycansins, steroids, and xenobiotic metabolites). In its native state, elemental sulfur is present in metal ores or sulfide minerals. In fossil fuels, sulfur is found in a variety of complex organic compounds and as hydrogen sulfide (H2S). This sulfur is released into atmosphere as ubiquitous air pollutant sulfur dioxide (SO2). In plants, humans, and animals, sulfur occurs in various biological structures. This sulfur is released into environment as SO4=, SO3=, or H2S either in waste or after the decay of dead plants and animal and human carcasses. Table 4.3 summarizes the occurrence of sulfur in nature and outlines sources of dietary sulfur available for humans and animals. For example, in onions there are three main sulfoxides including methyl, propyl and 1-propyl, and 1propenyl, which give rise to the onion’s tear-producing effect. Onion flavor results from these organosulfur compounds arising from the enzymatic decomposition of the flavor precursors (Randle, 1997). Intact cells of the onion have no odor, but when cells are disrupted, the enzyme, allinase, hydrolyzes S-alk(en)yl sulfoxides to produce pyruvate, ammonia, and volatile sulfur compounds associated with flavor and odor.


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TABLE 4.2 Occurrence of Sulfur in Nature Sources

Volcanic deposits Subterranean deposits Hot springs, geysers Fossil fuels

Food Vitamins Amino acids Preservatives

Organic compounds Microorganisms

Forms Available Natural Gypsum, pumice stone Sulfur ore (So), metallic sulfides, nonmetallic sulfates Sulfurous water Coal, petroleum, natural gas Dietary Onion, cabbage, cauliflower, broccoli, oil of garlic, mustard, eggs Thiamin, biotin Methionine, keto-methionine, cysteine, cystine, homocysteine, cystathionine, taurine, cysteic acid Sulfites and sulfiting agents: sulfur dioxide, sodium, bisulfite, potassium bisulfite, sodium metabisulfite, potassium metabisulfite, sodium sulfite Biological Proteins, lipoic acid, coenzyme A, glutathione, chondroitin sulfate, heparin, fibrinogen, ergothionine, estrogens, ferredoxin Aerobic heterotrophic, Desulfovibrio, Desulfotomaculum, chemoautotrophic, photoautotrophic

Fertilizers

Industrial Phosphates and ammonium sulfate

Combustion of fossil fuels

SO2, H2S

Anthropogenic Sources: Adapted from Komarnisky et al., 2003.

GLOBAL CYCLE Large amounts of Earth’s sulfur are found as a part of metal ores and minerals. These forms of sulfur are available for plants and microorganisms to synthesize SAAs and other forms of sulfur compounds. Humans obtain SAAs by consuming plants or animal meats. In all living organisms, sulfur is found in organic (thiol) form as a constituent of some proteins, vitamins, and amino acids (Table 4.3). When organisms die and decompose, some of the sulfur is taken up by the microorganisms and some is released again into the soil as sulfate. Large volumes of sulfur gases (H2S and SO2) are emitted into the atmosphere via volcanic eruptions and the processing of fossil fuels (Hobbs et al., 1981). In addition, dimethyl sulfide (DMS), a metabolic waste product of marine phytoplankton (Hay and Kubanek, 2002), is released into the atmosphere from oceans. It has been estimated that the total natural flux of gaseous sulfur to the atmosphere is 65 to 125 Tg (1 Tg = 1012 g)

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TABLE 4.3 Chemical and Physical Properties of Sulfur Characteristics General

Appearance

Physical properties

Allotropic modifications

Structure of solid sulfur Chemical properties

Properties Symbol Atomic number Atomic weight Group Valence Color Smell Texture of solid sulfur Density at 20°C Boiling point Solubility: In water In carbon disulfide Flammability Rhombic Monoclinic Amorphous S8 rings Minimum oxidation number Maximum oxidation number Reactivity Oxidation state: +6 +5 +4 +4 +3 +2 0 –2

Description S 16 32.064 Nonmetallic oxygen element group 2, 4, 6 Light yellow Odorless, tasteless Brittle 2.06 g/cm3 444.6°C Insoluble Soluble Flammable Rhombic or octahedral crystals Needle-shaped crystals Noncrystalline (liquid) Ring molecules are composed of eight sulfur atoms –2 6 Highly reactive Sulfate (SO42–) Dithionate (S2O62–) Sulfite (SO32–) Disulfite (S2O52–) Dithionite (S2O42–) Thiosulfate (S2O32–) Elemental sulfur (So) Sulfide (S2–)

Source: Adapted from Komarnisky et al., 2003.

(Rodhe, 1989). Sulfur returns back to Earth as sulfates and sulfites as a result of encountering sulfur gases with humid atmosphere. Thus, sulfur circulates through the atmosphere, lithosphere, hydrosphere, and biosphere in a continuous global cycle. A simplified diagram of the sulfur cycle is presented in Figure 4.1. The activities of microorganisms are essential in the global cycling of sulfur. Sulfonates and sulfate esters are widespread in nature and make up over 95% of the sulfur content of most aerobic soils. Many microorganisms can use sulfonates and sulfate esters as a source of sulfur for growth, even when they are unable to metabolize the carbon skeleton of the compounds (Kertesz, 2000). From terrestrial ecosystems,


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sulfur is continually lost into the water reservoirs, where some of the sulfur cycles through marine communities as it moves through food chains.

BIOLOGICAL ROLE OF SULFUR Sulfur is present in body tissues as part of the amino acids methionine, cysteine, and taurine. The sulfur atoms in cysteine are responsible for the major covalent cross-links in protein structures. Disulfide bridges formed between two cysteine molecules are important in stabilizing protein conformation. Hair and fingernails have a high percentage of cysteine to facilitate strength and rigidity of shape. Sheep have wool of a lower quality when the nutritional level of cystine and high-sulfur protein contents is restricted (Campbell et al., 1975). Propionibacterium acne has a requirement not only for amino nitrogen but also for reduced sulfur that is satisfied by the constant availability of this substance in the form of sulfhydryl groups in the sebaceous follicle during keratinization. The relationship between P. acne and its nutritional substrate may give this organism a selective advantage in this ecological niche (Nielsen, 1983). Sulfur also has a structural function as a part of mucopolysaccharides and sulfolipids. It occurs in the iron–sulfur proteins of the coenzyme Q/cytochrome c reductase complex of the respiratory chain. Sulfur atoms are also important in ironcontaining flavoenzymes, such as succinate dehydrogenase and NADH dehydrogenase. Intracellular reduction-oxidation status is increasingly recognized as a primary regulator of cellular growth and development. The relative reduction-oxidation state of the cell depends primarily on the precise balance between concentrations of reactive oxygen species (ROS) and the cysteine-dependent (thiol) antioxidant buffers, glutathione (GSH) and thioredoxin. These antioxidants have high affinity for ROS, thus protecting other intracellular molecules from oxidative damage (Deplancke and Gaskins, 2002). Sulfur, as a part of the sulfhydryl groups, forms thioester linkages that are necessary for the activation of molecules such as acetate. Interconversions between disulfide (GSSG) and sulfhydryl groups in oxidationreduction reactions occur as a result of reduction and oxidation of the sulfurcontaining compound GSH: Glutathione reductase GSSG + NADPH + H+ → 2GSH + NADP+ Glutathione peroxidase 2GSH + H2O2 → GSSG + 2H2O Other forms of endogenous sulfur compounds such as aminoethylcysteine ketimine dimer (Pecci et al., 2000) and hypotaurine (a sulfinate found in various biological tissues) are also able to protect against the ROS-induced damage (Pecci et al., 1999).

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Lipid peroxidation is the primary mechanism by which food deteriorates during storage in the presence of oxygen. Free radicals including peroxil, alkoxyl, and hydroxyl have been implicated in the mechanism of lipid peroxidation. Natural sulfur-containing compounds such as cysteine, glutathione, and lipoic acid, as well as synthetic compounds including N-acetylcysteine and a-mercaptopropionylglycine, protect against oxidative stress in biological systems through the scavenging and reduction of various oxidants (Eiserich and Shibamoto, 1994). Cyanide (CN–), a potent respiratory poison, is released by the hydrolysis of cyanogenic glycosides. The metabolic process that detoxifies cyanide is catalyzed by the ubiquitous enzyme rhodanese. The latter transfers sulfur from various sulfur compounds, but mostly from thiosulfate (SSO3=), producing the nonpoisonous compound thiocyanate (SCN–), which is excreted from the organism (Josephy, 1997). CN– + SSO3= → SCN– + SO3= Some individuals have a diminished capacity to detoxify CN– to SCN– as a consequence of either a genetic predisposition, which occurs in patients with Leber’s optic atrophy, or a diet low in sulfur-containing amino acids (Calabrese, 1979). Taurine (2-aminoethanesulfonic acid), a sulfur-containing amino acid, is the most abundant intracellular amino acid in humans, and is implicated in numerous biological and physiological functions. In healthy individuals, the diet is the usual source of taurine, although in the presence of vitamin B6 it is also synthesized from methionine and cysteine. With the exception of cow’s milk, taurine is widely distributed in foods of animal origin but not plant sources (Kendler, 1989). Taurine has antioxidant and anti-inflammatory properties. It is involved in bile acid conjugation, cholestasis prevention, and antiarrhythmic, inotropic, and chronotropic effects, as well in modulation of calcium flux and neuronal excitability, osmoregulation, detoxification, and membrane stabilization (Lourenco and Camilo, 2002). Taurine is an essential amino acid for preterm neonates and is assured by breast milk. It is also suggested that patients requiring long-term parenteral nutrition, those with chronic hepatic, heart, or renal failure, including premature and newborn infants, are at risk for taurine deficiency and may benefit from supplementation (Lourenco and Camilo, 2002). Taurine may be also essential for patients in the postinjury state (Paauw and Davis, 1990).

SULFUR METABOLISM Methionine is the only dietary essential sulfur amino acid. From methionine are synthesized all other important sulfur compounds, including cysteine, cystine, glutathione, acetylcoenzyme A, thiamin, biotin, lipoic acid, and taurine. During sulfur metabolism, sulfur is oxidized from its organic form, sulfides (S=), to sulfites (SO3=) and sulfates (SO4=). Most of the ingested sulfur is excreted in the urine after oxidation to sulfate as free ion, SO4=. Production of sulfate proceeds through cysteine sulfonic acid to sulfinyl pyruvate, SO2, and finally SO4=. Sulfite oxidase (a molybdenumdependent enzyme) is the last step in this process. Inorganic sulfate is an end product


93

ATP + SO 4= sulfurylase Adenosine-5´-phosphosulfate (APS) ATP-adenosine phosphosulfate kinase Adenosine-3´-phosphate-5´-phosphosulfate (PAPS) sulfotransferase

+R

R-SO3H (sulfate ester)

FIGURE 4.2 Role of SO4= in the biosynthesis of sulfate esters. R = phenols, steroids, indoles, hexosamine.

of sulfur amino acid metabolism, but it is also the cosubstrate for the biosynthesis of a wide array of complex sulfoesters (Figure 4.2).

SULFUR DEFICIENCIES Dietary sulfur deficiency is relatively rare, and hence there exists no recommended dietary intake for this element. Unlike in monogastric animals the rumen microbes in ruminants can use inorganic sulfur to synthesize sulfur-containing amino acids. Sulfur deficiency may therefore lead to methionine deficiency in polygastric animals. The sulfur requirements of these animals grazing on sorghum may thus be increased because of the need for sulfur in the detoxification of the cyanogenic glycosides found in most sorghum forages (Subcommittee on Beef Cattle Nutrition et al., 1996). It was also shown that calves fed the dietary urea-supplemented diets with added 0.15 or 0.30% of sulfur had higher weight gains and a trend toward improved efficiency compared with those with no added sulfur. Plasma total amino acid concentrations were also increased by the addition of sulfur to urea-supplemented diets compared to the basal urea diet (Hill et al., 1985). Although, in humans, primary deficiency of sulfur is relatively uncommon, there are some situations where a deficiency of secondary origin may exist. Newborn infants may thus be at risk of amino acid deficiency and toxicity, due to lack of small intestinal metabolism and metabolic immaturity. Impaired small intestinal metabolism (or lack of first-phase metabolism) alters the whole-body requirement for methionine, threonine, and arginine (Brunton et al., 2000). Considerable fractions of sulfhydryls in blood are present in erythrocytes (RBCs), which among others participate in intraorgan amino acid transport. The metabolism of SAAs and sulfhydryls is usually altered in patients with end-stage renal disease (ESRD). It was found that nutritional status influences plasma, but not RBC, concentrations of sulfhydryls in patients with ESRD. The study also shows that GSH concentrations in RBC and whole blood are related to hematocrit and not to nutritional parameters,

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indicating that anemia status rather than nutritional status determines RBC and whole-blood GSH levels in patients with ESRD (Suliman et al., 2002a). Dietary cyanide exposure from consumption of insufficiently processed bitter cassava roots may be a factor aggravating growth retardation (Banea-Mayambu et al., 2000) and paralytic disease konzo (Banea-Mayambu et al., 1997) in the Bandundu region, Democratic Republic of Congo (former Zaire). Cassava roots contain naturally occurring cyanogens that are associated with the seasonal outbreaks of these ailments. The signs of diseases mainly appeared in dry seasons when the diet lacked supplementary foods with sulfur-containing amino acids that promote cyanide detoxification (Tylleskar et al., 1991). Urinary linamarin, the cyanogenic glucoside and source of cyanide in cassava, was implicated in the development of konzo. This suggests that a specific neurotoxic effect of linamarin, rather than the associated general cyanide exposure resulting from glucoside breakdown in the gut, may be the cause of this disease (Banea-Mayambu et al., 1997).

SULFUR TOXICITY Awareness that SO2 is an environmental pollutant arose in the middle of 20th century (Amdur, 1974). The magnitude of SO2 toxicity may be even greater when present in combination with other air pollutants. Air pollution in the environment consists of a complex mixture of compounds, and various atmospheric conditions can alter the toxicity of air pollutants. In addition, the metabolism or detoxification of a single chemical in the body may be altered by the mixture of chemicals as well as by the pre-existing health state of the affected organism itself (Oehme et al., 1996). It is generally believed that the toxicity of SO2 in ambient air is significantly influenced by the coincident presence of particulates (Mehlman, 1983). For example, proteincontaining dust and SO2 may promote toxic allergic reactions (Sosedova and Benemanskii, 2000). It was shown that the surface of inorganic particles containing effluent gases, produced during combustion of fossil fuels, may interact with SO2 to form an irritant aerosol. The submicron fraction of this inorganic material may penetrate deep into the lung and cause serious health effects (Peoples et al., 1988). The studies on mice suggest that fine carbon particles can be an effective vector for the delivery of toxic amounts of SO4= to the periphery of the lung (Jakab et al., 1996). Sulfur toxicity may also be a consequence of deranged metabolism of sulfurcontaining amino acids, especially methionine.

TOXICITY DUE

TO

SO2

Sulfites and sulfating agents are sulfur-based preservatives that occur naturally or are used in the food-processing industry. They are used to prevent or reduce discoloration of light-colored fruits and vegetables, prevent black spots on shrimp and lobster, inhibit the growth of microorganisms in fermented foods such as wine, condition dough, and maintain the stability and potency of certain medications. Sulfites can also be used to bleach food starches, to prevent rust and scale in boiler water that is used to steam food, and even in the production of cellophane for food packaging. SO2, sodium bisulfite, potassium bisulfite, sodium metabisulfite,


95

potassium metabisulfite, and sodium sulfite are the forms of these preservatives. Sulfites exert the antimicrobial properties through release of free SO2, which inhibits propagation of yeasts, molds, and bacteria by killing the microorganisms entirely or by blocking their ability to reproduce. Despite their broad use in the food industry, sulfites may produce deleterious health effects in sulfite-sensitive individuals, although clinical responses can vary. Sulfites can cause chest tightness, nausea, hives, or even anaphylactic shock. However, difficulty in breathing is the most common symptom reported by sulfite-sensitive people (Field et al., 1994). Sulfites, bisulfites, and metabisulfites are all dry chemical forms of the SO2 gas that may cause irritation in the lungs and a severe asthma attack for those who suffer from asthma. A person can develop sulfite sensitivity at any point in life; as yet, the mechanism for sulfur sensitivity is not fully understood. Routes of SO2 Entry into Living Organisms Mammals and birds are exposed to air pollutants by inhalation through the nose and mouth, as well via cutaneous or ocular routes (Oehme et al., 1996). People can be exposed to environmental pollutants through food (e.g., ingesting residues of pesticides used in agriculture), in the workplace (e.g., inhalation of chemicals used in industrial processes), and directly from the environment (e.g., breathing polluted air in cities). Maximum and Threshold Limit Values of SO2 In North America, the threshold limit values (TLV) of SO2 in atmospheric air have been established for humans: 2 ppm for a normal 8-h workday or 40-h workweek. It is assumed that workers may be repeatedly exposed to this amount of SO2 without adverse effects. The maximum concentration that should not be exceeded at any time during a 15-min exposure period is 5 ppm SO2 (Katzung, 1998). However, epidemiological studies have shown a direct relationship between moderate concentrations of air pollution and airway disease. Thus, during the summer of 1999 the respiratory symptoms of chronic cough and phlegm, wheeze, and shortness of breath observed in 3709 Chinese adults were associated with the median indoor concentrations of SO2 in Beijing: 14 μg/m3; Anqing City: 25 μg/m3; and rural Anqing: 20 μg/m3 (Venners et al., 2001).

SO2-SENSITIVE SUBJECTS Individuals with asthma are most sensitive to inhaled SO2, which is a common air pollutant found in the workplace (Riedel et al., 1992). Despite that there are considerable interindividual variations in response to SO2 in patients with asthma (Winterton et al., 2001), bronchoconstriction seems to be the most common sensitivity response (Lester, 1995). The bronchoconstrictive effect in asthmatic subjects may be produced by inhalation of air amended with 0.75 ppm or even lower concentrations of SO2 (Wiebicke et al., 1990). SO2-induced bronchoconstriction is mediated by parasympathetic pathways (Sheppard et al., 1980). The mechanism of this type of bronchoconstriction involves release of leukotrienes (Balmes et al., 1987; Gong, Jr.

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et al., 2001). Mast cells lying in the surface mucosa of the lung are thought to be less stable in asthmatic subjects and may also be implicated in the mechanism of response to SO2 (Rocchiccioli and Riley, 1989). It has been suggested that the genetic biomarker associated with the wild-type allele of the tumor necrosis factor-alpha (TNF-α) promoter polymorphism may be employed in identification of sensitivity to inhaled SO2 in subjects with asthma (Winterton et al., 2001). Human lungs develop throughout childhood until age 20. Children have higher metabolic rates than adults and therefore require more air to breathe to inhale more oxygen. Their lungs may thus be more affected by damage due to air pollution. Epidemiological studies have shown acute effects of ambient air pollution on the occurrence of respiratory symptoms in children having breathing disorders. In the Netherlands children susceptible to SO2 had bronchial hyperresponsiveness and relatively high serum concentrations of total IgE (Boezen et al., 1999).

DISEASES

AND

SULFUR

It was concluded from a variety of animal experiments that long-term exposure to SO2 alone did not cause cancer (Mehlman, 1983), and no clear evidence exists that SO2 or bisulfite causes mutagenicity in mammals (Pool-Zobel et al., 1990). However, it was found that workers exposed to SO2 at a sulfuric acid factory in Taiyuan City (northern China) had a higher number of lymphocytes with chromosomal aberrations compared to controls. Thus, these observations show that SO2 is a possible clastogenic and genotoxic agent (Meng and Zhang, 1990, 2002). Air pollution as a trigger for exacerbations of chronic obstructive pulmonary disease (COPD) has been recognized for more than 50 years, leading to the development of air quality standards in many countries, which have substantially decreased the levels of air pollutants derived from the burning of fossil fuels, such as black smoke and SO2 (MacNee and Donaldson, 2000). Subjects with diabetes mellitus are also at risk for SO2-induced toxicity. Exposure of chemically induced diabetic rats to 10 ppm of SO2 potentiated visual evoked potential (VEP) changes and lipid peroxidation caused by the increased release of free radicals (Agar et al., 2000). It has been suggested that the hypercalciuria induced by a high-meat diet is mainly caused by the high content of SAAs and may be reversed by the ingestion of potassium-rich foodstuffs (Kaneko et al., 1990).

PATHOGENESIS

OF

SO2-LINKED TOXICITY

SO2, a highly water-soluble gas, dissolves in water to form bisulfite (HSO3–), sulfite (SO3), and hydrogen (H+) ions. These reactions are described by the following equilibrium relationships: SO2 + H2O β ↔ HSO3– + H+ β ↔ SO3 = 2H+ The hydrolysis of SO2 occurs very rapidly in aqueous environments. As a consequence, within fluid-filled structures such as cells, tissues, and blood vessels, any


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effects of SO2 exposure must be due to the effects of bisulfite and/or sulfite anions. However, during inhalation of SO2 gas, SO2 itself will be present at the air–liquid interface, so the initial effects of SO2 in cells at the luminal surface of the airways could be due to the direct chemical effects of SO2 gas rather than to the effects of bisulfite or sulfite (Amdur, 1989). The latter two major hydrolysis products are present in roughly equal concentrations at physiologic pH as determined by the pKa of their equilibrium reaction (~7.2). However, at the pH level reported at the luminal surface of the airways (6.6), the ratio of bisulfite to sulfite is approximately 5:1. This may be unfortunate, because bisulfite is generally more chemically reactive than sulfite. Bisulfite is a nucleophile that reacts with many biomolecules through substitution at electrophilic sites (Neta and Huie, 1985). One of these reactions leads to the disruption of disulfide bonds and the production of thiosulfates (RSH) through the following reaction (Petering and Shih, 1975). R–S–S–R + HSO3– ↔ RSSO3– + RSH Because disulfide bonds are widely found in tissue proteins, it is possible that bisulfite formed at the airway surface during SO2 inhalation initiates bronchoconstriction by such an effect on surface proteins. In addition, sulfite ions (SO3=) in reaction with superoxides (O2•–) form very reactive bisulfite radicals (SO3•) and hydrogen peroxide (H2O2). SO3• participates in radical chain processes such as lipid peroxidation (Hippeli and Eltsner, 1995). The chemical relationship between SO2, SO3•, and SO3= has led to speculation that the bronchoconstriction that follows oral ingestion of sulfite-containing foods and beverages in some patients with asthma is mechanistically related to SO2-induced bronchoconstriction (Stevenson and Simon, 1984). The SO3• ion is converted to sulfate by sulfite oxidase, an enzyme found in the lung, the liver, and a variety of other tissues (Petering and Shih, 1975). Sulfite can also be converted to sulfate by nonenzymatic autooxidation, which occurs in the presence of oxygen. Although this reaction is usually slow, it can be catalyzed by trace metal ions. This reaction is also a source of free radicals that may contribute to the tissue toxicity of SO2 (Neta and Huie, 1985). The distribution, metabolism, and toxicity of sulfite in the respiratory tract and other tissues have been studied on sulfite oxidase-deficient rats. The animals were exposed to 10 and 30 ppm of SO2. The endogenously generated sulfite and Ssulfonate compounds (a class of SO2 metabolites) were accumulated in the respiratory tract tissues and in the plasma of these rats. In addition, their testes were severely atrophied and as a result were devoid of spermatogenic cells. In contrast, in normal, sulfite oxidase-competent rats exposed to the same concentrations of SO2, sulfite and S-sulfonate compounds were restricted to the airways (Gunnison et al., 1987). In addition, studies on five mongrel dogs exposed to 22 ppm and four dogs to 50 ppm of 35SO2 for 30 to 60 min suggested that the blood plasma contained more 35S, which was associated with α-globulin proteins (half of 35S), than erythrocytes, which contained intracellular sulfur. Most of the urinary radioactive sulfur was excreted in the form of inorganic sulfate (Yokoyama et al., 1971).

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CLEARANCE

OF

SO2

FROM THE

ORGANISM

Mucociliary clearance of the respiratory tract is an important defense mechanism against inhaled pathogens and depends on ciliary and mucous factors. Airway mucus, a complex airway secretion, functions as a renewable and transportable barrier against inhaled particulates and toxic agents. Inhalation of a number of air pollutants, including SO2 and cigarette smoke, may increase mucus secretion and alter mucus rheology (Samet and Cheng, 1994). Cilia, which line both the upper and lower airways, are covered by a thin layer of mucus. The rapid, coordinated beat of cilia propel particles trapped in the mucus layer to the pharynx. Although no evidence for direct nervous control of ciliary function has been demonstrated, it was suggested that adrenergic agonists might enhance it. Cilial defects may be either congenital (primary) or acquired (secondary) as a result of infection, toxins, or drugs (Verdugo et al., 1980). Degradation of the pulmonary surfactant dynamic interfacial properties due to inhalation of SO2-polluted air may result in a slowdown of the pulmonary clearance rate and an increase in the lung burden (Podgorski et al., 2001). For highly soluble gases such as SO2, the upper airways have been shown to be a very effective scrubber with much of the gas removed during a single pass. For example, it has been shown that, in nose-breathing rabbits inhaling 400 ppm SO2, less than 10 ppm reached the trachea. It was also noted that SO2 inhalation did not affect lung function in normal subjects, but induced bronchoconstriction in subjects with asthma. It was suggested that nasal breathing, which is often impaired in people with asthma, reduces the pulmonary effects of SO2 because this water-soluble gas is absorbed by the nasal mucosa (Peden, 1997). Sulfite oxidase, a mitochondrial molybdoenzyme in mammals, is essential for detoxication of the sulfite arising from metabolism of sulfur-containing amino acids, from ingestion of bisulfite preservatives, and from inhalation of SO2 (Coughlan, 1983). Endogenous sulfite is generated as a consequence of the body’s normal processing of sulfur-containing amino acids. Sulfites occur as a result of fermentation, and they also occur naturally in a number of foods and beverages. Despite that they have been safely used as food additives for a long time, sulfiting food preservatives have been found to be allergy-causative agents. Sulfite sensitivity occurs most often in adults with asthma and in preschool children (Lester, 1995).

TOXICITY

OF

SECONDARY ORIGIN (SULFUR METABOLISM)

Homocysteine (Hcy) is a sulfur-containing amino acid produced by the breakdown of methionine (Figure 4.3). Plasma Hcy levels can be elevated due to a variety of genetic and nutritional factors. Poor nutrition from diets low in folate and vitamins B12 and B6 can lead to hyperhomocysteinemia. Hyperhomocysteinemia is regarded as a public health problem of increasing importance likely to contribute to vascular disorders and premature mortality. Mildly elevated levels of Hcy have been implicated in a number of disease processes such as atherosclerotic vascular disease and adverse obstetrical outcomes. High levels of plasma Hcy are also associated with abnormal collagen cross-linking. Hyperhomocysteinemia in pregnancy is associated with preterm, premature rupture of membranes, an important public health concern,


Methionine CH2THF

Folate Cycle

SAM

DMG

THF

B12MS BHMT

S

Methionine Cycle

Homocysteine CBS

B6

DNA RNA Protein

Methyltransferase

Betaine

CH3THF

99

SCH3 SAH

Transsulfuration Pathway

Cystathionine B6

Cysteine

SO4=

FIGURE 4.3 Metabolic fate of homocysteine. THF: tetrahydrofolate; DMG: dimethylglycine; SAM: S-adenosylmethionine; CBS: cystathionine ß-synthase; BHMT: betaine homocysteine hydroxymethyltransferase. (Modified from Basu and Fisher, 2002.)

due to the effects of homocysteine on connective tissue integrity (Ferguson et al., 2001). Folate, cobalamin, pyridoxine, and riboflavin dietary deficiencies are currently regarded as causative factors of hyperhomocysteinemia, which may arise from the shrinking of endogenous nitrogen pools as a result of decreased protein intake or stress-induced increased losses. Raised total Hcy may result from the attempt of the malnourished or stressed body to preserve methionine homeostasis (Ingenbleek et al., 2002). Malnutrition, hypoalbuminemia, and diabetes mellitus in patients with chronic renal failure influence SAA levels, mainly plasma total Hcy, which should be considered when evaluating hyperhomocysteinemia as a cardiovascular risk factor (Suliman et al., 2002b). Homocystinuria, another inborn disease related to sulfur metabolism, is implicated as the result of enzyme cystathionine synthase deficiency (Poole et al., 1975). Intestinal gas is thought to be the cause of abdominal discomfort in infants. Gas release by infant feces is strongly influenced by an infant’s diet. It was found that the highly toxic sulfur gases hydrogen sulfide (H2S) and methanethiol (CH3SH) are high in soy-formula-fed infants and low in breastfed infants (Jiang et al., 2001). Mercaptides (sodium hydrogen sulfide and sodium methanethiol) and mercaptofatty acid (sodium mercaptoacetate) are reducing agents that help to maintain anaerobic conditions in the colonic lumen. Metabolic effects of sodium hydrogen sulfide on butyrate oxidation along the length of the colon closely resemble metabolic abnormalities observed in active ulcerative colitis. The increased production of sulfide in ulcerative colitis suggests that the action of mercaptides may be involved in the genesis of ulcerative colitis (Roediger et al., 1993). Problems associated with excess in dietary sulfur intake in ruminants are being increasingly recognized. Excessive levels of sulfur-containing compounds in domestic ruminant animals’ rations and clinical problems associated with low to moderate levels of exposure to dietary sulfur may be more common than previously thought

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(Olkowski, 1997). Subsequent excessive ruminal sulfide production is an important factor in the pathogenesis of polioencephalomalacia (PEM), without concurrent thiamine deficiency. Most cases of PEM were developed between 15 and 30 days after introduction to a high-sulfur diet. In cases where water is an important source of dietary sulfur, risk of PEM may increase during hot weather (McAllister et al., 1997).

CONCLUSION Sulfur is one of the most abundant chemical elements on Earth and is an important nutritive constituent in all biological systems. In nature, sulfur is commonly found in its most stable form as sulfate. The latter must be reduced to sulfite by microorganisms in order to be metabolized by animals and humans for the synthesis of SAA. As a constituent of certain amino acids, sulfur performs a number of functions in enzyme reactions and protein synthesis. Mammals acquire organic (thiol) forms of sulfur from their diets. Sulfur and sulfur compounds exhibit protective and antioxidative properties during a number of metabolic disorders in living cells. Anthropogenic emissions of sulfur have a large impact on the balance of this element in the environment, which in turn may influence health of plants, animals, and humans.

REFERENCES Agar, A., Kucukatay, V., Yargicoglu, P., Aktekin, B., Kipmen-Korgun, S., Gumuslu, D., and Apaydin, C. (2000) The effect of sulfur dioxide inhalation on visual evoked potentials, antioxidant status, and lipid peroxidation in alloxan-induced diabetic rats, Archives of Environmental Contamination and Toxicology, 39: 257–264. Amdur, M.O. (1974) 1974 Cummings Memorial Lecture. The long road from Donora, American Industrial Hygiene Association Journal, 35: 589–597. Amdur, M.O. (1989) Health effects of air pollutants: sulfuric acid, the old and the new, Environmental Health Perspectives, 81: 109–113. Balmes, J.R., Fine, J.M., and Sheppard, D. (1987) Symptomatic bronchoconstriction after short-term inhalation of sulfur dioxide, American Review of Respiratory Disease, 136: 1117–1121. Banea-Mayambu, J.P., Tylleskar, T., Gitebo, N., Matadi, N., Gebre-Medhin, M., and Rosling, H. (1997) Geographical and seasonal association between linamarin and cyanide exposure from cassava and the upper motor neurone disease konzo in former Zaire, Tropical Medicine & International Health, 2: 1143–1151. Banea-Mayambu, J.P., Tylleskar, T., Tylleskar, K., Gebre-Medhin, M., and Rosling, H. (2000) Dietary cyanide from insufficiently processed cassava and growth retardation in children in the Democratic Republic of Congo (formerly Zaire), Annals of Tropical Paediatrics, 20: 34–40. Basu, T.K. and Fisher, A. (2002) Niacin-induced hyperhomocysteinemia, an independent risk factor for arterial occlusive disease, in P. Mitro, D. Pella, R. Rybár, and G. Valoik, ˇ Eds., Cardiovascular Diseases 2002, Kosice, Slovakia: Monduzzi Editore S.p.A.


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Boezen, H.M., van der Zee, S.C., Postma, D.S., Vonk, J.M., Gerritsen, J., Hoek, G., Brunekreef, B., Rijcken, B., and Schouten, J.P. (1999) Effects of ambient air pollution on upper and lower respiratory symptoms and peak expiratory flow in children, Lancet, 353: 874–878. Brunton, J.A., Ball, R.O., and Pencharz, P.B. (2000) Current total parenteral nutrition solutions for the neonate are inadequate, Current Opinion in Clinical Nutrition and Metabolic Care, 3: 299–304. Calabrese, E.J. (1979) Possible adverse side effects from treatment with laetrile, Medical Hypotheses, 5: 1045–1049. Campbell, M.E., Whiteley, K.J., and Gillespie, J.M. (1975) Influence of nutrition on the crimping rate of wool and the type and proportion of constituent proteins, Australian Journal of Biological Sciences, 28: 389–397. Coughlan, M.P. (1983) The role of molybdenum in human biology, Journal of Inherited Metabolic Disease, Suppl. 1, 6: 70–77. Deplancke, B. and Gaskins, H.R. (2002) Redox control of the transsulfuration and glutathione biosynthesis pathways, Current Opinion in Clinical Nutrition and Metabolic Care, 5: 85–92. Eiserich, J.P. and Shibamoto, T. (1994) Sulfur-containing heterocyclic compounds with antioxidative activity formed in Maillard reaction model systems, in C.J. Mussinan and M.E. Keelan, Eds., Sulfur Compounds in Foods, Washington, D.C.: American Chemical Society, 247–251. Ferguson, S.E., Smith, G.N., and Walker, M.C. (2001) Maternal plasma homocysteine levels in women with preterm premature rupture of membranes, Medical Hypotheses, 56: 85–90. Field, P.I., McClean, M., Simmul, R., and Berend, N. (1994) Comparison of sulfur dioxide and metabisulfite airway reactivity in subjects with asthma, Thorax, 49: 250–256. Gong, H., Jr., Linn, W.S., Terrell, S.L., Anderson, K.R., and Clark, K.W. (2001) Antiinflammatory and lung function effects of montelukast in asthmatic volunteers exposed to sulfur dioxide, Chest, 119: 402–408. Gunnison, A.F., Sellakumar, A., Currie, D., and Snyder, E.A. (1987) Distribution, metabolism and toxicity of inhaled sulfur dioxide and endogenously generated sulfite in the respiratory tract of normal and sulfite oxidase-deficient rats, Journal of Toxicology and Environmental Health, 21: 141–162. Hay, M.E. and Kubanek, J. (2002) Community and ecosystem level consequences of chemical cues in the plankton, Journal of Chemical Ecology, 28: 2001–2016. Hill, G.M., Boling, J.A., and Bradley, N.W. (1985) Plasma amino acid profiles and growth of the bovine fed different sources of nitrogen and levels of sulfur, International Journal for Vitamin and Nutrition Research, 55: 439–446. Hippeli, S. and Eltsner, E.F. (1995) Biological and biochemical effects of air pollutants: synergistic effects of sulfite, Biochemical Society Symposia, 61: 153–159. Hobbs, P.V., Radke, L.F., Eltgroth, M.W., and Hegg, D.A. (1981) Airborne studies of the emissions from the volcanic eruptions of Mount St. Helens, Science, 211: 816–818. Ingenbleek, Y., Hardillier, E., and Jung, L. (2002) Subclinical protein malnutrition is a determinant of hyperhomocysteinemia, Nutrition, 18: 40–46. Jakab, G.J., Clarke, R.W., Hemenway, D.R., Longphre, M.V., Kleeberger, S.R., and Frank, R. (1996) Inhalation of acid coated carbon black particles impairs alveolar macrophage phagocytosis, Toxicology Letters, 88: 243–248. Jiang, T., Suarez, F.L., Levitt, M.D., Nelson, S.E., and Ziegler, E.E. (2001) Gas production by feces of infants, Journal of Pediatric Gastroenterology and Nutrition, 32: 534–541.

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Josephy, P.D. (1997) Sidebar: sulfate, sulfite, thiosulfate, and cyanide toxicity, in P.D. Josephy, Ed., Molecular Toxicology, Oxford: Oxford University Press, 130–131. Kaneko, K., Masaki, U., Aikyo, M., Yabuki, K., Haga, A., Matoba, C., Sasaki, H., and Koike, G. (1990) Urinary calcium and calcium balance in young women affected by high protein diet of soy protein isolate and adding sulfur-containing amino acids and/or potassium, Journal of Nutritional Science and Vitaminology (Tokyo), 36: 105–116. Katzung, B.G. (1998), Basic & Clinical Pharmacology, 7th ed., Stamford, CT: Appleton & Lange. Kellogg, W.W., Cadle, R.D., Allen, E.R., Lazrus, A.L., and Martell, E.A. (1972) The sulfur cycle, Science, 175: 587–596. Kendler, B.S. (1989) Taurine: an overview of its role in preventive medicine, Preventive Medicine, 18: 79–100. Kertesz, M.A. (2000) Riding the sulfur cycle — metabolism of sulfonates and sulfate esters in Gram-negative bacteria, FEMS Microbiology Reviews, 24: 135–175. Komarnisky, L.A., Christopherson, R.J., and Basu, T.K. (2003) Sulfur: its clinical and toxicologic aspects, Nutrition, 19: 54–61. Lester, M.R. (1995) Sulfite sensitivity: significance in human health, Journal of the American College of Nutrition, 14: 229–232. Lourenco, R. and Camilo, M.E. (2002) Taurine: a conditionally essential amino acid in humans? An overview in health and disease, Nutricion Hospitalaria, 17: 262–270. MacNee, W. and Donaldson, K. (2000) Exacerbations of COPD: environmental mechanisms, Chest, Suppl. 2, 117: 390S–397S. McAllister, M.M., Gould, D.H., Raisbeck, M.F., Cummings, B.A., and Loneragan, G.H. (1997) Evaluation of ruminal sulfide concentrations and seasonal outbreaks of polioencephalomalacia in beef cattle in a feedlot, Journal of the American Veterinary Medical Association, 211: 1275–1279. Mehlman, M.A. (1983) Current toxicological information as the basis for sulfur oxide standards, Environmental Health Perspectives, 52: 261–266. Meng, Z.Q. and Zhang, L.Z. (1990) Chromosomal aberrations and sister-chromatid exchanges in lymphocytes of workers exposed to sulfur dioxide, Mutation Research, 241: 15–20. Meng, Z. and Zhang, B. (2002) Induction effects of sulfur dioxide inhalation on chromosomal aberrations in mouse bone marrow cells, Mutagenesis, 17: 215–217. Miller, G.T., Jr. (1998) Living in the Environment. Principles, Connections, and Solutions, 10th ed., Belmont, CA: Wadsworth. Neta, P. and Huie, R.E. (1985) Free-radical chemistry of sulfite, Environmental Health Perspectives, 64: 209–217. Nielsen, P.A. (1983) Role of reduced sulfur compounds in nutrition of Propionibacterium acnes, Journal of Clinical Microbiology, 17: 276–279. Oehme, F.W., Coppock, R.W., Mostrom, M.S., and Khan, A.A. (1996) A review of the toxicology of air pollutants: toxicology of chemical mixtures, Veterinary and Human Toxicology, 38: 371–377. Olkowski, A.A. (1997) Neurotoxicity and secondary metabolic problems associated with low to moderate levels of exposure to excess dietary sulfur in ruminants: a review, Veterinary and Human Toxicology, 39: 355–360. Paauw, J.D. and Davis, A.T. (1990) Taurine concentrations in serum of critically injured patients and age- and sex-matched healthy control subjects, American Journal of Clinical Nutrition, 52: 657–660. Pecci, L., Costa, M., Montefoschi, G., Antonucci, A., and Cavallini, D. (1999) Oxidation of hypotaurine to taurine with photochemically generated singlet oxygen: the effect of azide, Biochemical and Biophysical Research Communications, 254: 661–665.


103

Pecci, L., Antonucci, A., Pinnen, F., and Cavallini, D. (2000) Identification of an oxidation product of aminoethylcysteine ketimine dimer, Amino Acids, 18: 61–67. Peden, D.B. (1997) Mechanisms of pollution-induced airway disease: in vivo studies, Allergy, Suppl. 52: 37–44. Peoples, S.M., McCarthy, J.F., Chen, L.C., Eppelsheimer, D., and Amdur, M.O. (1988) Copper oxide aerosol: generation and characterization, American Industrial Hygiene Association Journal, 49: 271–276. Petering, D.H. and Shih, N.T. (1975) Biochemistry of bisulfite-sulfur dioxide, Environmental Research, 9: 55–65. Podgorski, A., Sosnowski, T.R., and Gradon, L. (2001) Deactivation of the pulmonary surfactant dynamics by toxic aerosols and gases, Journal of Aerosol Medicine, 14: 455–466. Pool-Zobel, B.L., Schmezer, P., Zeller, W.J., and Klein, R.G. (1990) In vitro and ex vivo effects of the air pollutants SO2 and NOx on benzo(a)pyrene activating enzymes of the rat liver, Experimental Pathology, 39: 207–212. Poole, J.R., Mudd, S.H., Conerly, E.B., and Edwards, W.A. (1975) Homocystinuria due to cystathionine synthase deficiency. Studies of nitrogen balance and sulfur excretion, Journal of Clinical Investigation, 55: 1033–1048. Randle, W.M. (1997) Onion flavor chemistry and factors influencing flavor intensity, in S.J. Risch and C.T. Ho, Eds., Spices: Flavor Chemistry and Antioxidant Properties, 6th ed., Washington, D.C.: American Chemical Society, 41–44. Riedel, F., Naujukat, S., Ruschoff, J., Petzoldt, S., and Rieger, C.H. (1992) SO2-induced enhancement of inhalative allergic sensitization: inhibition by anti-inflammatory treatment, International Archives of Allergy and Immunology, 98: 386–391. Rocchiccioli, K.M. and Riley, P.A. (1989) Clinical pharmacology of nedocromil sodium, Drugs, 37: 123–126. Rodhe, H. (1989) Acidification in a global perspective, Ambio, 18: 155–160. Roediger, W.E., Duncan, A., Kapaniris, O., and Millard, S. (1993) Reducing sulfur compounds of the colon impair colonocyte nutrition: implications for ulcerative colitis, Gastroenterology, 104: 802–809. Samet, J.M. and Cheng, P.W. (1994) The role of airway mucus in pulmonary toxicology, Environmental Health Perspectives, Suppl., 102: 89–103. Sheppard, D., Wong, W.S., Uehara, C.F., Nadel, J.A., and Boushey, H.A. (1980) Lower threshold and greater bronchomotor responsiveness of asthmatic subjects to sulfur dioxide, American Review of Respiratory Disease, 122: 873–878. Sosedova, L.M. and Benemanskii, V.V. (2000) The combined action of protein-containing dust and sulfur dioxide (experimental studies) [in Russian], Medicina Truda i Promyshlennaya Ekologia, 8: 21–24. Stevenson, D.D. and Simon, R.A. (1984) Sulfites and asthma, Journal of Allergy and Clinical Immunology, 74: 469–472. Subcommittee on Beef Cattle Nutrition, Committee on Animal Nutrition, Board on Agriculture and National Research Council, Eds. (1996) Nutrient Requirements of Beef Cattle, 7th ed., Washington, D.C.: National Academy Press. Suliman, M.E., Barany, P., Divino Filho, J.C., Qureshi, A.R., Stenvinkel, P., Heimburger, O., Anderstam, B., Lindholm, B., and Bergstrom, J. (2002a) Influence of nutritional status on plasma and erythrocyte sulfur amino acids, sulphhydryls, and inorganic sulphate in end-stage renal disease, Nephrology, Dialysis, Transplantation, 17: 1050–1056.

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Suliman, M.E., Stenvinkel, P., Heimburger, O., Barany, P., Lindholm, B., and Bergstrom, J. (2002b) Plasma sulfur amino acids in relation to cardiovascular disease, nutritional status, and diabetes mellitus in patients with chronic renal failure at start of dialysis therapy, American Journal of Kidney Diseases, 40: 480–488. Tylleskar, T., Banea, M., Bikangi, N., Fresco, L., Persson, L.A., and Rosling, H. (1991) Epidemiological evidence from Zaire for a dietary etiology of konzo, an upper motor neuron disease, Bulletin of the World Health Organization, 69: 581–589. Venners, S.A., Wang, B., Ni, J., Jin, Y., Yang, J., Fang, Z., and Xu, X. (2001) Indoor air pollution and respiratory health in urban and rural China, International Journal of Occupational Medicine and Environmental Health, 7: 173–181. Verdugo, P., Johnson, N.T., and Tam, P.Y. (1980) beta-Adrenergic stimulation of respiratory ciliary activity, Journal of Applied Physiology, 48: 868–871. Wiebicke, W., Jorres, R., and Magnussen, H. (1990) Comparison of the effects of inhaled corticosteroids on the airway response to histamine, methacholine, hyperventilation, and sulfur dioxide in subjects with asthma, Journal of Allergy and Clinical Immunology, 86: 915–923. Winterton, D.L., Kaufman, J., Keener, C.V., Quigley, S., Farin, F.M., Williams, P.V., and Koenig, J.Q. (2001) Genetic polymorphisms as biomarkers of sensitivity to inhaled sulfur dioxide in subjects with asthma, Annals of Allergy, Asthma and Immunology, 86: 232–238. Yokoyama, E., Yoder, R.E., and Frank, N.R. (1971) Distribution of 35S in the blood and its excretion in urine in dogs exposed to 35SO2, Archives of Environmental Health, 22: 389–395.

5

Fluoride — Toxic and Pathologic Aspects: Review of Current Literature on Some Aspects of Fluoride Toxicity Thomas F.X. Collins and Robert L. Sprando

CONTENTS Abstract ..................................................................................................................106 Abbreviations .........................................................................................................106 Introduction and Background ................................................................................106 Exposure ................................................................................................................107 Fluoride Compounds Used to Fluoridate Water ...................................................112 Acute Toxicity........................................................................................................114 Subchronic Toxicity...............................................................................................115 Dental Effects ............................................................................................115 Skeletal Effects ..........................................................................................117 Renal Effects..............................................................................................118 Pulmonary Effects .....................................................................................118 Hormonal Effects.......................................................................................118 Genetic Effects...........................................................................................119 Neural Effects ............................................................................................119 Reproductive Toxicity Aspects ..................................................................119 Correlation with Decreased Fertility in Humans ............................119 Correlation with Down’s Syndrome in Humans .............................120 Correlation with Male Reproduction and Male Offspring..............120 Correlation with Female Reproduction ...........................................129

105

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Developmental Toxicity Aspects ...............................................................132 Human Studies .................................................................................132 Animal Studies.................................................................................132 References..............................................................................................................134

Abstract

Humans are exposed to fluorides, which are ubiquitous compounds, primarily through water, food, dental products, and air. The use of fluoridated water in the preparation of foods and beverages at commercial establishments and at home, coupled with fluoride in dental products, has led to increased consumption of fluorides. Fluorides can produce wide-ranging effects on many tissues, organs, and systems in the body. Fluorides are toxic at high concentrations, but at low concentrations they are added to drinking water to prevent the formation of dental caries. In mammals, fluorides have a high affinity for teeth and bones. Consumption of excess fluoride during the time of tooth enamel formation in children can cause dental fluorosis, marked by discolored or “mottled” teeth. Skeletal fluorosis, marked by symptoms ranging from slight pain to crippling deformities, is an additive disease, for which daily consumption of high levels of fluoride for many years is usually required. In addition to teeth and bones, fluoride can also affect kidneys, lungs, and the nervous system, and it can disturb hormones and possibly change genetics. Treatment of male animals with high levels has indicated that fluoride can affect testicular production in mice and rabbits, but not always in rats. Treatment of female rats and rabbits with sodium fluoride during several generations failed to produce reproductive effects, but a high concentration of fluoride decreased bone ossification in rats.

Abbreviations

ATPase: adenosine triphosphatase; CDC: Centers for Disease Control; EPA: Environmental Protection Agency; F1 generation: first generation; FDA: Food and Drug Administration; FSH: follicular stimulating hormone; HSD: hydroxysteroid dehydrogenase; l: liter; LH: luteinizing hormone; mg/kg: milligrams per kilogram bodyweight; ml: milliliter; μg: microgram; NRC: National Research Council; NTP: National Toxicology Program; P generation: parental generation; PHS: Public Health Service; ppm: parts per million (= one milligram per liter); WHO: World Health Organization

INTRODUCTION AND BACKGROUND Much has been said and will continue to be said, and much ink has been used to praise or denounce fluoride. Proponents constantly laud its effectiveness in reducing dental caries, and opponents are equally steadfast in citing its toxic effects. For most trace elements in food or water such as chromium or zinc, the effect depends on the dose. Fluoride, however, is unusual among trace elements because the same range of exposure can produce beneficial or harmful effects, depending on the developmental stage of the individual and nutritional factors.

Fluoride — Toxic and Pathologic Aspects

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Fluoride is the ionic form of the halogen fluorine, which is the most electronegative and reactive of all elements. Fluoride salts are readily formed. In nature, fluoride occurs chiefly in fluorspar (calcium fluoride) and cryolite (sodium aluminum fluoride), but it is widely distributed in other minerals. Volcanoes are a major source of hydrogen fluoride, a compound that dissolves readily in water to form hydrofluoric acid. Coal combustion, aluminum production plants, and phosphate fertilizer plants are the main anthropogenic sources of hydrogen fluoride (PHS, 2001). In general, exposure to hydrogen fluoride in the air is low, although persons living near industrial sources of the gas or workers in fluoride-processing industries may be exposed to higher levels of the gas in the air. Also, vegetables and fruits grown near these sources could contain fluoride from fluoride-containing dust settling on the plants (PHS, 2001). During ancient and medieval times, caries and periodontal disease occurred without the benefit of efficient treatments. With the ready availability of cheap sugar in Europe and North America, incidence of caries reached such high levels that a substantial number of young people lost their teeth because of them. In the early 1900s, particularly in the southwestern U.S., it was observed that people with mottled (i.e., discolored) teeth had fewer cavities than people without mottled teeth. Naturally occurring fluoride in the drinking water was identified as responsible for this hardening of tooth enamel. Prevention of caries on a public-health scale began in 1945 with the fluoridation of water in Grand Rapids, Michigan. Starting in the 1950s, fluoride was added to fluoride-deficient drinking water in many communities to bring the total level of fluoride to 1 mg/l (1 ppm). This level was considered the optimal level to reduce caries and to minimize the risk of dental fluorosis. In developed industrial countries where the incidence of caries has decreased substantially since then, emphasis in dentistry has shifted to cosmetic dentistry (Marthaler, 2002). Most recently, the variety and availability of teeth-whitening products appear to be expanding almost exponentially. The 1984 World Health Organization Guidelines (WHO, 1984) suggested that in areas with a warm climate, the optimal fluoride concentration in drinking water should remain below 1 ppm, and in cooler climates it could go as high as 1.2 ppm (Table 5.1). This difference is based on the fact that persons perspire more in hot weather and drink more water to compensate for the liquid lost. The upper limit of concentration was set at 1.5 ppm, a level considered a threshold between the benefit of resistance to tooth decay and the risk of dental fluorosis. The nutritional status also influences the rate at which fluoride is absorbed by the body. For example, calcium in the diet binds with fluoride and thus decreases the body’s retention of fluoride.

EXPOSURE For humans, the common sources of fluoride are water, food, dental products, and air. Fluoride concentrations and exposure vary among these sources. The fluoride concentration in fresh surface water is low, ranging from 0.01 to 0.03 mg/l (WHO, 1984). The fluoride concentration in groundwater fluctuates from less than 0.1 to more than 25 mg/l (WHO, 1984). Fluoride enters groundwater by natural processes,

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TABLE 5.1 Recommended Water Fluoride Concentrations Average Maximum Daily Air Temp.

Recommended Control Limits (ppm)

°F

°C

Lower-Upper

Optimum

50.0–53.7 53.8–58.3 58.4–63.8 63.9–70.6 70.7–79.2 79.3–90.5

10.0–12.0 12.1–14.6 14.7–17.7 17.8–21.4 21.5–26.2 26.3–32.5

0.9–1.7 0.8–1.5 0.8–1.3 0.7–1.2 0.7–1.0 0.6–0.8

1.2 1.1 1.0 0.9 0.8 0.7

Source: Data from NRC (1993).

and the concentration depends on the amount of leaching of rock. Waters from some areas of the Southwest have higher concentrations of fluoride than most areas in the East. On the basis of Environmental Protection Agency (EPA, 1985) estimates, more than 86% of people who are served by public water systems are exposed to fluoride levels of 1.0 mg/l or less. Approximately 0.4% are exposed to drinking water that is greater than 2.0 mg/l, particularly from groundwater sources. A person’s daily intake of fluoride from drinking water is a function of the person’s age, size, and the fluoride concentration of the water. In general, the fluoride intake from drinking water increases proportionally as the fluoride content of water increases. See Table 5.2 for adequate intake (AI) and upper-level (UL) intake. AI is based on fluoride

TABLE 5.2 Adequate Intake (AI) and Upper Limit (UL) of Fluoride Intake (mg/day) Age

AIa

ULb

Reference Weight kg (lb)

0–6 months 7–12 months 1–3 years 4–8 years 9–13 years, males 9–13 years, females 14–18 years, males 14–18 years, females 19 years and over, males 19 years and over, females

0.01 0.5 0.7 1.0 2.0 2.0 3.0 3.0 4.0 3.0

0.7 0.9 1.3 2.2 10.0 10.0 10.0 10.0 10.0 10.0

7 (16) 9 (20) 13 (29) 22 (48) 40 (88) 40 (88) 64 (142) 57 (125) 76 (166) 61 (133)

a

Fluoride intake of 0.05 mg/kg/day from all sources (for ages over 6 months). Fluoride intake of 0.10 mg/kg/day from all sources.

b

Source: Data from Food and Nutrition Board (1999).


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consumption of 0.05 mg/kg/day from all sources, and UL is based on fluoride consumption of 0.10 mg/kg/day from all sources (Food and Nutrition Board, 1999). The use of fluoridated water in the preparation of foods and beverages at commercial establishments or at home has led to increased consumption of fluoride. Increased fluoride content is seen in carbonated beverages prepared with fluoridated municipal water. The fluoride in foods and beverages contributes to the total fluoride daily intake. At the time the optimal level in drinking water was set, drinking water was the only source of fluoride. Now, the addition of fluoride in food during food preparation is causing the ingestion of fluoride in doses that could be detrimental. The concentration of fluoride available in foods tends to be below 0.05 mg/100 g (Taves, 1983), except in fluoridated drinking water, some beverages, and foods made with or cooked in fluoridated water (Food and Nutrition Board, 1999). When foods and beverages most commonly consumed by adolescents were analyzed for fluoride, no significant differences were seen between an optimally and a negligibly fluoridated community (Jackson et al., 2002). However, a significant difference was found between the two communities in the fluoride content of fountain beverages and in cooked or reconstituted foods prepared using local water. In some of the coal-burning areas of rural China, fluoride can be adsorbed by corn dried over unvented ovens burning high-fluorine coal (Zheng and Huang, 1989). Where fluoride in indoor air was adsorbed by food, the daily mean intake of fluoride from corn, wheat, chiles, potatoes, and vegetables was 37.6, 0.32, 5.77, 3.54, and 0.69 mg per person, respectively (Ando et al., 1998). In these areas of rural China, fluoride exposure was estimated to be 97% via food and 2% via inhalation (Ando et al., 1998). More than 10 million people in the Guizhou Province of China and surrounding areas suffer from dental and skeletal fluorosis (Ando et al., 1998; Finkelman et al., 1999). When 238 commercially available infant foods were examined (Heilman et al., 1997), fluoride concentrations ranged from 0.01 to 8.38 ppm, with the highest concentration found in infant foods containing chicken (Table 5.3). Foods made with mechanically deboned chicken may contribute significantly to total fluoride intake because the mechanical separation process removes attached meat from bone along

TABLE 5.3 Fluoride Concentrations in Infant Foods Type

N

Range (ppm)

Mean Concentration (ppm)

Fruits and desserts Vegetables Mixed foods Meats Chicken

88 48 42 19 6

0.01–0.49 0.01–0.42 0.01–0.63 0.01–8.38 1.05–8.38

0.10 0.12 0.21 1.46 4.49

Ref. Heilman Heilman Heilman Heilman Heilman

et et et et et

al., al., al., al., al.,

1997 1997 1997 1997 1997

Note: Ranges and mean concentrations of fluoride in infant foods, based on 203 samples.

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with a small fraction of finely powdered bone (Fein and Cerklewski, 2001). The bone fraction is likely to contain elements such as calcium and fluoride. Mechanically separated chicken, such as chicken sticks and luncheon meats that are likely to be consumed by young children, was tested for fluoride content, and elevated levels of fluoride were found, but the levels varied with the brands tested. Two brands of pureed chicken contained 2.82 and 5.58 μg fluoride/g, chicken sticks contained 3.61 μg fluoride/g, two brands of luncheon meat contained 1.60 and 2.35 μg fluoride/g, and two brands of Vienna sausage contained 1.45 and 2.18 μg fluoride/g. These levels are above the desirable intake levels for a child (Fein and Cerklewski, 2001). Foods made with mechanically separated turkey, however, contained much less fluoride than their chicken counterparts. It was proposed that turkey bones are more difficult to crush and powder during the mechanical separation process than are chicken bones (Grunder and MacNeil, 1973). Several studies have been done on the fluoride content of beverages such as juices, part-juice drinks, carbonated soft drinks, tea, and wine. When 43 fruit juices were examined, fluoride concentration was 0.15 to 6.80 ppm (Stannard et al., 1991). When 532 juices and juice drinks were analyzed for fluoride concentration, the concentration ranged from 0.02 to 2.80 ppm (Kiritsy et al., 1996). Fluoride levels in 332 soft drinks ranged from 0.02 to 1.28 ppm (Heilman et al., 1999). Some of the fluoride concentrations are summarized in Table 5.4. Within each type, the beverages are arranged in ascending order according to mean fluoride concentration. All the beverages had wide ranges of fluoride concentration, and in many cases the concentration was over 1 ppm. Studies by Kiritsy et al. (1996) and Heilman et al. (1999) were done as part of the Iowa Fluoride Study, and Stannard’s study (1991) was done to evaluate fluoride concentration in beverages obtained in the Boston area. When the same beverages were tested from different areas of the country, the concentrations were sometimes similar (e.g., apple juice) and other times quite varied (e.g., prune juice). This finding was not surprising because of the known production and distribution routes of juices and other beverages throughout the country. For some juices (e.g., apple juice), the fruit is grown and processed locally and shipped to locations throughout the country. For other juices, the juices or juice concentrates are purchased from other national and international locations, processed and/or reconstituted, and sent to locations throughout the country (Kiritsy et al., 1996). Any reconstitution alters the fluoride concentration based on the fluoride content of the water used for reconstitution. Individual companies also may have several sites of production. The fluoride content of the finished product then depends on the level of fluoride in the water. If the companies use water from several production sites with different levels of fluoride, the fluoride content may differ from one location to the next. The lowest fluoride values were found in juices that needed the least amount of water. Grape juice and other juices containing grape juice contained concentrations of fluoride that were higher than expected. By extracting the juice only from the insides of the grapes, a great reduction in the fluoride content occurred, indicating that the fluoride was on the skin (Stannard et al., 1991). By international agreement, the fluoride content of wine should not exceed 1 ppm (Burns and Gump, 1993). A study of California wines showed that the fluoride


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TABLE 5.4 Fluoride Concentrations in Beverages Type

N

Range (ppm)

Mean (ppm)

Ref.

Fruit nectar Pineapple Prune Orange Apple Apple Grapefruit Mixed fruit Red grape Pear Cranberry Mixed fruit Prune White grape Red grape White grape

7 12 2 88 8 83 34 119 37 8 45 11 10 10 5 3

Juices 0.05–0.27 0.15 0.03–0.88 0.16 0.18–0.26 0.22 0.02–1.85 0.37 0.16–1.30 0.47 0.03–2.64 0.54 0.05–1.30 0.55 0.03–2.02 0.57 0.05–2.45 0.74 0.16–1.64 0.75 0.04–1.73 0.81 0.22–2.60 0.96 0.05–2.13 1.18 0.15–2.80 1.45 1.25–2.60 1.81 1.95–6.80 3.92

Pepsi Cola Coca-Cola Royal Crown Cola Dr. Pepper/Seven-Up

104 79 2 47

Carbonated Soft Drinks 0.02–1.22 0.60 Heilman 0.02–1.10 0.70 Heilman 0.95–0.99 0.97 Heilman 0.70–1.28 1.02 Heilman

Rosé Red White

Lemonade Fruit drinks Tea

Kiritsy et al., 1996 Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996 Kiritsy et al., 1996 Stannard et al., 1991 Stannard et al., 1991

et et et et

al., al., al., al.,

1999 1999 1999 1999

2 9 8

California Wines 0.90–1.28 0.96 0.23–2.80 1.05 0.41–1.50 1.09

Burgstahler and Robinson, 1997 Burgstahler and Robinson, 1997 Burgstahler and Robinson, 1997

17 8 5

Other Beverages 0.03–0.84 0.25 0.15–1.13 0.54 0.95–2.33 1.41

Kiritsy et al., 1996 Stannard et al., 1991 Kiritsy et al., 1996

content of wines from five brands of California grapes ranged from 0.83 to 5.20 ppm (Table 5.4) (Burgstahler and Robinson, 1997). The elevated fluoride levels may be due to the use of cryolite as a pesticide in the vineyards. Where tea drinking is a daily occurrence, tea can contribute to the total fluoride intake. The tea tree can selectively absorb fluoride from soil and accumulate it in the leaves, and the concentration of fluoride is related to the age of the tree (Gupta, 1991; Xu, 1987). In Tibet and some parts of western China, brick tea is considered a necessity. The fluoride concentration of brick tea, which is made from old stems and leaves of the tea tree, is 200 to 300 times higher in fluoride than ordinary green

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or black tea, which is made from the tender leaves and buds (Cao et al., 1996). During the 1980s, brick tea–type fluorosis was found in groups of people living in remote west and north border districts of China (Cao et al., 2003). In Tibet and in the Sichuan Province of China, Tibetans with a long history of brick tea consumption were studied for total fluoride intake, dental fluorosis, and skeletal fluorosis (Cao et al., 1996). The fluoride intake of Tibetan children and adults was 5.49 and 10.43 mg/person/day, respectively. Of this intake, over 94% was from brick tea and zanba (roasted highland barley flour processed with brick tea water). Among the Tibetans older than 16 years of age, nearly one third had skeletal fluorosis. Among those older than 50 years of age, more than 50% had skeletal fluorosis. An epidemiological study in Tibet in 2001 showed that natural fluoride in water was very low, but foods processed with brick tea water (zanba and buttered tea) had fluoride contents of 4.52 and 3.21 mg/kg, respectively (Cao et al., 2003). The adult daily fluoride intake reached 12 mg, of which 99% originated from the brick tea–containing foods (Cao et al., 2003). Osteosclerosis-type skeletal fluorosis (overall increased bone matrix density) affected 74% of the persons studied, and ossification and tendon attachment calcification affected 63% (Cao et al., 2003). More than 90% of the toothpaste sold in the U.S. contains fluoride at concentrations of 1000 to 1500 ppm (Beltran and Szpunar, 1988; Whitford, 1987). Based on the amount of toothpaste used for each brushing, the number of brushings, and the amount swallowed, children can ingest more than 2 mg fluoride/day (Barnhart et al., 1974; Baxter, 1980; Brunn and Thylstrup, 1988; Dowell, 1981; Hargreaves et al., 1972). Fluoride in the air originates both from natural sources and from human activities (WHO, 1984). The natural sources of fluoride include dusts from soil and droplets of seawater dispersed by wind. The urban sources of fluoride in the air are generated by industries (Smith and Hodge, 1979). Intake of fluoride by air is considered negligible in the U.S. However, in some rural areas of China, the most important energy source is coal with high-fluoride content. Coal-burning stoves are used with or without chimneys for heating, cooking, and drying food for storage, and high concentrations of fluoride have been detected in indoor air (Ji, 1993). Fluoride in indoor air is directly inhaled by the residents, and it is easily absorbed in stored food. The combination of inhalation and ingestion can provide high fluoride intake. A correlation between high concentrations of airborne fluoride and a high prevalence of fluorosis has been observed in some rural areas of China (Ando et al., 1998).

FLUORIDE COMPOUNDS USED TO FLUORIDATE WATER In the United States, the regulation of fluoride in drinking water is the responsibility of the EPA. Under the Safe Drinking Water Act of 1974 (Public Law 93-523), the EPA sets primary and secondary maximum contaminant levels for natural levels of fluoride in drinking water. The primary maximum contaminant level of 4 mg/l is the level that drinking water is not allowed to exceed and the secondary maximum


113

TABLE 5.5 Water Fluoridation Agents in the U.S. Agent

Chemical Formula

Community Size

Population Served (in millions)

No. of Utilities

Sodium fluoride Sodium fluorosilicate Fluorosilicic acid

NaF Na2SiF6 H2SiF6

1–10,000 10,000–25,000 25,000+

11.7 36.1 80.0

2491 1635 5876

Note: Of the 1992 population of 258.5 million, 144.2 million (55.8%) drank water treated with fluoridation agents. The figures in this table account for 88.6% of the population served by water fluoridation agents. The remaining 11.4% (16.5 million) are served by systems using unspecified chemical agents. Source: 1992 Census by the Centers for Disease Control (CDC, 1993).

contaminant level of 2 mg/l is the level that the EPA recommends that drinking water not exceed. At the time of the Fluoridation Census in1992 (CDC, 1993), three major fluoride chemicals were used for water fluoridation: sodium fluoride, sodium fluorosilicate (also known as sodium silicofluoride), and fluorosilicic acid (also known as hydrofluosilicic acid). Fluoridation agents are summarized in Table 5.5. Sodium fluoride is a by-product of the aluminum industry and the silicates are by-products of the phosphate fertilizer industry. Sodium fluoride, a white odorless salt with a solubility that remains constant at all water temperatures, is the additive of choice for small communities, i.e., with populations 25,000. The methods and equipment used in fluoridation are described by Reeves (1996). At the time of the 1992 census, 62.1% of the U.S. population was drinking fluoridated water, and of this water 6.3% was naturally fluoridated (CDC, 1993). Thus, 55.8 million persons were drinking water treated with fluoridating agents. In the U.K., fluorosilicic acid and sodium fluorosilicate are the chemicals most commonly used. In Central and South America, sodium fluoride, sodium fluorosilicate, fluorosilicic acid, and calcium fluoride are used. Calcium fluoride can be used in tropical climates because the temperature of the drinking water is warm enough to dissolve the chemical. Fluoride is the active part of treated water. Sodium fluoride in water dissociates readily into free fluoride and sodium ions. It has been assumed that silicofluoride complexes in water would behave similarly to produce free fluoride ions and other fluoride compounds such as aluminum fluoride, and that the treated water, when consumed, would have no silicofluoride residues. There are, however, still unresolved

114


problems concerning the fate of fluorosilicates added to drinking water (Urbansky, 2002). There are also possible problems in that the silicofluorides obtained from phosphate industry scrubbers could add a variety of impurities such as arsenic and lead, albeit at low levels.

ACUTE TOXICITY High concentrations of fluoride are toxic. Acute oral exposure to sodium fluoride can result in significant dysfunction, such as nausea, vomiting, abdominal pains, diarrhea, and death (Table 5.6). A fatal ingestion of sodium fluoride was reported as early as 1899 (Sharkey and Simpson, 1933). Hodge and Smith (1965) estimated the lethal dose for a 70-kg man was 5 to 10 g sodium fluoride, or 32 to 64 mg fluoride/kg bodyweight. One 3-year-old boy who swallowed 200 mg sodium fluoride for a dose of 16 mg/kg bodyweight died 7 hours after ingestion (Eichler et al., 1982). One 27-month-old child died 5 days after ingesting about 100 mg sodium fluoride (Whitford, 1990). When given orally, the lethal dose of fluoride in animals was 20 to 100 mg/kg bodyweight (Davis, 1961). The LD50 of sodium fluoride in Sprague-Dawley male rats was 101 mg/kg (Skare et al., 1986), and the LD50 values for female rats ranged from 52 to 31 mg/kg, depending on bodyweight (De Lopez et al., 1976). The LD50 value of sodium fluoride observed for mice was 44.3 mg/kg (Lim et al., 1978).

TABLE 5.6 Potential Adverse Effects of Excess Fluoride Organ or Organ System

Potential Adverse Effects Acute Dose

All systems

Teeth Bones Kidneys Lungs Hormones Neural system Male reproductive system Female reproductive system

Death Subacute Dose Dental fluorosis (mottled teeth) Skeletal fluorosis (joint pain, calcification of ligaments, osteoporosis, muscle wasting, neurological defects) Renal failure Congestion and histopathological changes Increased parathyroid hormone secretion, inhibition of thyroid hormones, decreased testosterone concentration Behavioral effects Testicular changes, decreased fertility Skeletal variations in offspring

Note: Potential adverse effects were described in one or more animal species.


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SUBCHRONIC TOXICITY Some of the potential adverse effects of subacute doses of fluoride are summarized in Table 5.6. Most fluoride is ingested orally, and 50 to 80% of the fluoride ingested may be absorbed from the gastrointestinal tract. The amount absorbed depends on the dietary concentrations of calcium and other substances with which fluoride may form insoluble and poorly absorbed compounds. The amount absorbed also depends on the individual features of absorption and excretion. For example, healthy, young, or middle-aged adults may retain approximately 50% of ingested fluoride and excrete the remaining quantity in the urine. Young children, however, may retain as much as 80% due to uptake by developing skeletons and teeth (Food and Nutrition Board, 1999). The uptake rate of fluoride is faster in the bones of children than in adults; hence fluoride is cleared faster from the bloodstream in children than in adults (PHS, 2001). Body fluid and tissue fluid concentrations are proportional to the long-term level of intake, and they are not homeostatically regulated (Food and Nutrition Board, 1999; Guy, 1979). Because the fluoride concentration is proportional to intake, it is the total amount of fluoride ingested on a daily basis, regardless of the source, that provides the difference between a beneficial, caries preventive effect and an undesirable dental, skeletal, or other effect. Once fluoride is absorbed into the body, it passes into the blood for distribution and partial excretion. In plasma, it may exist as a nonionic form or an ionic form (Guy, 1979). The ionic or free form is the most important form for toxicity. It has been shown to circulate unbound in plasma (Ekstrand et al., 1977; Taves, 1968), to complex with calcified tissues, to be distributed to the soft tissues, or to be excreted. Most of the ionic fluoride retained in the body enters the calcified tissues (bones and teeth), either by substitution for the hydroxyl or the bicarbonate ion in hydroxyapatite in bone or enamel to form fluoroapatite, or as an ionic exchange within the crystalline surface (McCann and Bullock, 1957). Approximately half of the fluoride absorbed each day is deposited in the calcified tissues, and the result is that more than 99% of the fluoride in the body is found in calcified tissue (Whitford,1983). Fluorosis refers to the toxic condition that results from exposure to excessive amounts of fluorine or fluorides. Because of the affinity of fluoride for calcified tissue, most of the toxic effects of fluoride are manifested primarily in teeth or bones, and the conditions are referred to as dental fluorosis or skeletal fluorosis. Dental fluorosis is usually considered an adverse cosmetic effect, and skeletal fluorosis is considered an adverse functional effect. Endemic or chronic skeletal fluorosis, seen in some parts of the world where high fluoride levels are found in drinking water, is characterized by bone, joint, and muscle pain, progressive ankylosis of various joints and crippling deformities (Gupta et al., 1993b). Such severe cases are rare in the U.S. (Bowen, 2002; NRC, 1993).

DENTAL EFFECTS Exposure to excessive levels of fluoride during the period of tooth development (birth to approximately 8 years of age) can lead to dental fluorosis. This condition is

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characterized by a mottling of tooth enamel that ranges from barely discernible flecks on enamel to obviously pitted and brown-stained enamel (NRC, 1993). The staining, characteristic of more severe forms of fluorosis, develops after tooth eruption, but is seen only when porous enamel has formed before eruption (Fejerskov et al., 1990). The tooth enamel maturation process consists of increased mineralization within the developing tooth and a concurrent loss of early-secreted matrix proteins. Excess fluoride available to the enamel during maturation disrupts mineralization and results in retention of excessive enamel proteins (NRC, 1993). Animal studies have indicated that the early-maturation stage is the period during which enamel is most sensitive to fluoride effects (Den Besten, 1986; Richards, 1990; Richards et al., 1986). In humans, severe fluorosis follows the breakdown of the enamel surface layers shortly after eruption and results in mineral uptake in the exposed hypomineralized subsurface lesions (Fejerskov et al., 1991; Thylstrup, 1983). Fluorosis may be severe in permanent teeth, but it is rarely reported in primary teeth except in areas of the world where high amounts of fluoride are ingested (Larsen et al., 1987; McInnes et al., 1982; Mann et al., 1990; Nair and Manji, 1982; Olsson, 1979; Thylstrup, 1978). The low degree of fluorosis in primary teeth was once believed to be due to the placental barrier preventing the passage of fluoride from maternal to fetal blood. However, additional evidence demonstrated that the placenta acts only as a partial barrier (Gedalia and Shapira, 1989). Fetal blood concentrations of fluoride are usually lower than maternal levels. Most fluoride in the outer enamel layer of teeth is deposited during the enamel maturation period before eruption. The maturation period lasts a short time in primary teeth (1 to 2 years), but permanent teeth take 4 to 5 years to mature. The combination of shorter maturation period for primary teeth and the lower blood fluoride concentrations during prenatal development probably is the reason for the low incidence of fluorosis in primary teeth (NRC, 1993). Fluoride intake by children aged 2 to 5 years is especially important because the front teeth are at the early-maturation stage, and during this period they are particularly susceptible to fluoride-induced changes. Dental fluorosis in permanent teeth appears to be a dose–response condition (Dean, 1942; Eklund et al., 1987; Fejerskov et al., 1990; Gedalia and Shapira, 1989; Larsen et al., 1987). Animal studies have shown that fluoride can disturb enamel maturation by several pathways, by spikes of fluoride caused by daily injections, by long-term administration of low doses of fluoride, or by a single high dose of fluoride. These studies have been summarized by the National Research Council (NRC, 1993). The role of fluoride as an anticaries agent was examined when an inverse relationship was noted in many areas of the country between the level of fluoride in the drinking water and the incidence of dental caries. A large amount of literature is available on this relationship (Dean, 1938; Newbrun, 1989; Whitford, 1983). Since the 1950s, the prevalence and severity of caries have been considerably reduced (NRC, 1993), and dentists can now concentrate some of their efforts on cosmetic dentistry such as teeth whitening procedures. Fluoride can act on dental enamel, dentin, and cementum while it is forming or after it erupts, through saliva or blood or by direct contact with fluoridated water or dental products. Fluoride replaces hydroxyl ions in the crystal lattice structure of enamel, forming fluoroapatite (Ten Cate, 1990; Whitford, 1983). This change in


117

enamel is considered to enhance the resistance of the teeth to caries. The capacity of teeth to absorb fluoride into the enamel’s structure diminishes as the enamel matures (Weatherell et al., 1977). This quality makes it different from bone, in which there can be constant alteration. The concentration of fluoride in sound, mature tooth enamel averages 1700 ppm in people living in areas with drinking water concentrations of 0.1 ppm or less, and 2200 to 3200 ppm in areas with drinking water concentrations of approximately 1 ppm. At these fluoride levels, fluoride increases the enamel’s resistance to dissolution and decay (NRC, 1993). In areas with drinking water concentrations of 5 to 7 mg/l, enamel fluoride concentrations have been observed at 4800 ppm (Aasenden, 1974). People living in areas of high fluoride concentrations usually exhibit severe dental fluorosis, and their tooth enamel can become brittle enough to fracture. Teeth in this condition often require treatment to restore function (NRC, 1993).

SKELETAL EFFECTS Fluoride has a high affinity for bone, but the bond formed is a reversible one (Whitford, 1990). Fluoride in bone can be mobilized rapidly by interstitial ionic exchange or slowly as a result of the constant process of bone alteration. Bone alteration is more active in the young where bone is more hydrated and has a greater surface area than older bone. The greater surface area provides greater area for fluoride exchange. Fluoride deposition in bone has been found to be inversely proportional to age (Whitford, 1990). As fluoride deposition in bone decreases with age, fluoride levels increase in plasma (Parkins and Greenlimb, 1974). In bone, fluoride replaces the hydroxyl ion in hydroxyapatite and forms fluoroapatite, a compound with different physical and chemical properties. Continuous ingestion of high levels of fluoride can lead to skeletal fluorosis, a condition whose effects can range from increased bone density to crippling skeletal fluorosis, characterized by complete rigidity of the spine. Most cases have been reported from developing countries, particularly India, where drinking water sources in 15 states in India and 15 districts of Rajasthan contain over 1.5 ppm fluoride (Purohit et al., 1999). High levels of fluoride are necessary to cause fluorosis, but nutritional status and individual variation must also be considered contributors to this condition. Smith and Hodge (1979) described the preclinical and clinical stages of skeletal fluorosis. The preclinical stage, characterized by a slight increase in bone mass, is asymptomatic. Stage 1 of skeletal fluorosis is characterized by occasional stiffness or pain in the joints and some osteosclerosis of the pelvis and vertebral column. At Stage 2 and 3, the clinical signs are chronic joint pain, calcification of ligaments, osteosclerosis, possibly osteoporosis of long bones, muscle wasting, and neurological defects in severe cases. Crippling skeletal fluorosis might occur in persons who have ingested 10 to 20 mg fluoride/day for at least 10 years. From 1963 to 1993, only five cases of skeletal fluorosis were reported in the U.S. (NRC, 1993). As a compound with high affinity for bone, sodium fluoride has been investigated as a preventative or therapy for osteoporosis in postmenopausal women. The studies have been reviewed by NRC (1993) and the Food and Nutrition Board (1999), which found that fluoride therapy did not demonstrate a significant reduction in fractures.

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RENAL EFFECTS Most of the removal of fluoride that occurs from the body (approximately 50% of daily intake) is done by renal excretion (Whitford, 1990). The kidney cells are therefore a possible target of fluoride toxicity because they can be exposed to high concentrations of fluoride. Studies of renal effects have shown that structural renal changes occurred in rats exposed to fluoride and that there was a relationship between renal effects and pH of urine (Daston et al., 1985; Hodge and Smith, 1977; Greenberg, 1986; Manocha et al., 1975; Taylor et al., 1961; Whitford et al., 1976). In humans, the efficiency of the renal cells in removing fluoride from blood has been shown to be greater than for other halogens. Clearance values of 0.5 to 2.0 ml/min were observed for chloride and bromide (Whitford, 1990). Clearance values of 12.4 to 89.1 ml/min have been reported for fluoride (Jarnberg et al., 1983; Whitford, 1990). Two teen-age patients who suffered renal failure also suffered from fluorosis of teeth and bones (Juncos and Donadio, 1972). Renal failure and indications of chronic fluoride intoxication were reported in a person who ingested 2 to 4 l/day of mineral water containing 8.5 mg fluoride/l for 20 years (Lantz et al., 1987). Despite this observation, several epidemiological investigations have shown no human kidney disease from long-term exposure to fluoride at concentrations up to 8 mg/l (EPA, 1985). In another study of two communities, the renal status of persons who drank 8 mg/l and the status of persons who drank 0.4 mg/l was similar (Leone et al., 1954).

PULMONARY EFFECTS Excess fluoride in drinking water that causes dental mottling and skeletal fluorosis may also be responsible for lung damage. Rabbits fed 10 or 20 mg sodium fluoride/kg/day for 6 months showed gross lung changes (pale areas on the surface and dark brown congested areas in cross sections) and histopathological changes (alveolar hemorrhage, congestion, edema, etc.) (Purohit et al., 1999). The fluoride content of lung tissue homogenate was more than ten times greater in treated animals than in control animals, and the content was dose related. Lung damage was tested because patients of skeletal fluorosis in India may have been wrongly treated for tuberculosis.

HORMONAL EFFECTS In a study of the effect of high fluoride ingestion on serum parathyroid hormone, 200 children consumed water containing 2.4, 4.6, 5.6, or 13.5 mg fluoride/l (Gupta et al., 2001). High fluoride ingestion increased the level of parathyroid hormone secretion. The authors suggested that, due to the role of the parathyroid hormone in maintaining serum calcium levels, fluoride might play a role in toxic manifestations of fluorosis. Fluorides have also been implicated in the inhibition of thyroid hormones and in goiter formation (Jooste et al., 1999; Zhao et al., 1998). Decreased testosterone concentration was observed in patients with skeletal fluorosis, and in males without skeletal fluorosis who drank water with high fluoride concentration (Susheela and Jethanandani, 1996). The authors suggested that fluoride


119

toxicity may cause adverse effects on the reproductive system of males living in endemic fluorosis areas. Testosterone levels in males in areas nonendemic for fluorosis were normal.

GENETIC EFFECTS The genetic toxicity of fluoride has been tested extensively in microbes, cultured mammalian cells, and animals. These test results have been reviewed by NRC (1993) and Zeiger et al. (1993), and they concluded that there is evidence that fluoride exposure can lead to chromosomal aberrations in in vitro test systems but that aberrations in in vivo systems are unresolved. The genetic toxicity of fluoride in human blood lymphocyte cells was determined after long-term exposure to various concentrations of fluoride in drinking water (Li et al., 1995). Subjects who ingested low-fluoride water had higher frequencies of sister chromatid exchanges than did subjects who ingested higher levels of fluoride. Skare et al. (1986) demonstrated that oral administration of up to 84 mg/kg of sodium fluoride to adult male rats did not induce DNA strand breaks in testicular cells when measured by alkaline elution. They also observed that although plasma fluoride levels were as high as 12 ppm, testicular fluoride levels were only 10 to 12% of the plasma levels and fluoride did not accumulate in the testis after five daily treatments. The authors concluded that sodium fluoride is unlikely to pose a hazard with respect to heritable genetic effects.

NEURAL EFFECTS When the effects of fluoride on the developing rat brain were tested, sex- and dosespecific behavioral changes were observed (Mullenix et al., 1995). Males were most sensitive to exposure at prenatal days 17 to 19, and females were most sensitive to exposure as weanlings and adults. The severity of the effect on behavior increased directly with plasma fluoride levels and fluoride concentrations in specific brain regions. The plasma fluoride levels were similar to those reported in humans exposed to high levels of fluoride. The molecular mechanism underlying brain dysfunction from chronic fluorosis was studied in rats that received either 30 or 100 ppm fluoride in their drinking water for 7 months (Long et al., 2002). At 100 ppm, but not at 30 ppm, the level of the nicotinic acetylcholine receptors (NAChR) α-4 subunit protein in the brains of the rats was significantly lowered. The expression of the α-7 subunit protein was significantly decreased by both 100 and 30 ppm. These decreases in the number of receptors may be an important factor in the mechanism of brain dysfunction in chronic fluorosis.

REPRODUCTIVE TOXICITY ASPECTS Correlation with Decreased Fertility in Humans Despite that fluoride has been added to municipal water supplies to diminish the occurrence of dental caries for more than half a century, there are limited data on

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the potential of fluoride to induce reproductive effects in humans and in animals. A statistically significant association was reported between decreasing total fertility and increasing fluoride levels in water (Freni, 1994). In the meta-analysis, birth data for more than 525,000 women (10 to 49 years old) living in areas with high fluoride drinking water levels (up to 3 ppm or higher) were compared with birth data for approximately 985,000 women living in adjacent areas with low fluoride drinking water levels. The results have not been duplicated by other investigators. Correlation with Down’s Syndrome in Humans The possible association between fluoride concentration in drinking water and the incidence of Down’s syndrome was proposed by Rapaport (1957, 1963). These studies have been used to support the claim that fluoridation of drinking water leads to increased incidence of Down’s syndrome. However, no support for this claim was found in other studies (Berry, 1958; Erickson, 1980; Erickson et al., 1976; Needleman et al., 1974). When the studies of correlation between fluoridation and Down’s syndrome were reviewed, Erickson (1980) found no correlation. A systematic review of the association of Down’s syndrome and water fluoride level was also done by Whiting et al. (2001). Based on a comprehensive literature search, they identified six studies worldwide for review. These were the same studies as the ones identified above. Of the six studies, four studies showed no association, and two were significantly associated. Based on their review, Whiting et al. (2001) concluded that the association between water fluoride level and Down’s syndrome was inconclusive. Correlation with Male Reproduction and Male Offspring Studies in Mice Table 5.7 provides a summary of some of the available studies in male mice. Kour and Singh (1980) exposed mice to 0, 10, 500, or 1000 ppm sodium fluoride in their drinking water for 30, 60, or 90 days. Microscopic effects were not observed in the testis of the animals receiving sodium fluoride at a concentration of 10 ppm, but necrotic seminiferous tubules were observed in animals from the 500 and 1000 ppm treatment groups. Chinoy and Sequeira (1989a) examined the effect of sodium fluoride exposure on the histology and histocytochemistry of the reproductive organs of male mice. Three groups received sodium fluoride, by gavage, at a dose of 10 mg/kg bodyweight/mouse/day and one group received sodium fluoride at a dose of 20 mg/kg/mouse for 30 days. The treatment of 10 mg/kg/mouse/day was withdrawn from two groups for 30 and 60 days, respectively, and the animals were used for recovery studies. Testicular effects characterized by a disorganization and denudation of cells of the germinal cells of the seminiferous epithelium were observed with some tubules showing a lack of sperm in the lumen in both treatment groups. Effects were also observed in the cauda epididymis, caput epididymis, and vas deferens of both treatment groups. The Leydig cells, the seminal vesicles, and the prostate were not affected by the treatment. Removal of treatment resulted in a complete recovery of these organs.

Kour and Singh

Chinoy and Sequeira

Chinoy and Sequeira

Shashi

1989a

1989b

1990

Authors

1980

Year

Duration

Subcutaneous injection

Feed

Gavage

0, 10, 500, 1000 ppm

Dose

100 days

5, 10, 20, 50 mg/kg/day

Two groups treated 30 days and 10, 20 euthanized; two groups treated 30 mg/kg/day days, then treatment withdrawn for 30 or 60 days

Two groups treated 30 days and 10, 20 euthanized; two groups treated 30 mg/kg/day days, then treatment withdrawn for 30 days

Drinking water 30, 60, 90 days

Route of Exposure

Rabbits

Albino mice

Swiss mice

Albino mice

Species

(continued)

Spermatogenic arrest and seminiferous necrosis; abnormal spermatocyte maturation and differentiation

Decreased: bodyweight, testicular succinic dehydrogenase, epididymides sialic acid, ATPase Increased: prostate and seminal vesicle weight, seminal vesicle fructose levels, prostate acid phosphatase, and total protein After treatment withdrawal, levels returned to normal

Testicular effects: denudation of germinal epithelium; Leydig cell, no effect Epididymal effects: caput epididymis — epithelial cell nuclear pyknosis and absence of luminal sperm Cauda epididymis: nuclear pyknosis, denudation of vas deferens, nuclear pyknosis, clumped stereocilia, and cellular debris, absence of sperm and increased lamina propria Treatment withdrawal: recovery of histoarchitecture

Lack of germ cell maturation and differentiation at 500 and 1000 ppm; necrotic seminiferous tubules after 90 days

Results

TABLE 5.7 Summary of Fluoride Effects on Male Reproduction Parameters in Mice, Rats, and Rabbits (from 1980 to the present)

Fluoride — Toxic and Pathologic Aspects 121

Authors

Chinoy et al.

Chinoy et al.

Susheela and Kumar

Chinoy and Sequeira

Shashi and Kaur

Year

1991b

1991a

1991

1992

1992

Treated 30 days followed by withdrawal for 30 days; animals given ascorbic acid, calcium, or calcium and acorbic acid

Duration

Subcutaneous injection

Orally

Orally (?)

3.5 months

Two groups treated 30 days, then treatment withdrawn and 30–60 days; mating during withdrawal phase

18, 29 months

Direct injection Single injection in vas deferens

Feed

Route of Exposure

Swiss strain mice

Rabbits

Holtzman strain rats

Rabbits

Species

0, 5, 10, 20, 50 Rabbits mg/kg/day

10, 20 mg/kg/day

10 mg/kg/day

50 μg/50 μl

20, 40 mg/kg/day

Dose

Depletion of testicular structural, nuclear, and total proteins in all test groups; reduction of testicular DNA

Decreased sperm motility, count, and fertility Withdrawal of treatment: significant recovery in sperm count, motility, and fertility

29 months: seminiferous tubules: spermatogenic arrest, spermotagenic cells disrupted, tubules devoid of spermatozoa Epididymal effects at 18 and 29 months

Spermatogenic arrest, absence of spermatozoa in seminiferous tubule lumen, decreased cauda epididymal sperm count Cauda epididymis and vas deferens: sperm deflagellated or with tail abnormalities

Reduced fertility due to decreased sperm motility and sperm counts; abnormal sperm morphology; recovery pronounced in ascorbic acid treatment group; recovery most pronounced in animals supplemented with calcium and ascorbic acid

Results

TABLE 5.7 (CONTINUED) Summary of Fluoride Effects on Male Reproduction Parameters in Mice, Rats, and Rabbits (from 1980 to the present)

122 Reviews in Food and Nutrition Toxicity

Kumar and Susheela

Kumar and Susheela

Sprando et al.

Sprando et al.

1994

1995

1996

1997, 1998

Single injection

20, 23 months

18 months

Drinking water Exposure: in utero through 14 weeks postlactation

Intratesticular injection

Orally

Orally

50 days treatment; 50 days treatment + withdrawal for 70 days; 50 days treatment + withdrawal for 70 days + supplements: ascorbic acid, calcium, or ascorbic acid + calcium

Narayana and Chinoy

1994

Gavage

Krasowska and Drinking water 6, 16 weeks Wlostowski

1992

0, 25, 100, 175, 150 ppm

0, 25. 100, 175, 250 ppm

10 mg/kg/day

10 mg/kg/day

10 mg/kg/day

SpragueDawley rats

SpragueDawley rats

Rabbits

Rabbits

Albino rats

100, 200 ppm Wistar rats

No spermatogenic or endocrine effects

No spermatogenic effects

(continued)

Loss of stereocilia, decreased height of pseudostratified columnar epithelium and increased diameter of caput and cauda epididymis Fragmented sperm in cauda epididymis

Acrosomal, nuclear, and flagellar abnormalities

Sperm acrosomal hyaluronidase and acrosin reduced, sperm acrosomal damage and deflagellation Cauda epididymal sperm count decreased, fluoride withdrawal, incomplete recovery Supplementation with ascorbic acid, calcium, or a combination of both: significant recovery from fluorideinduced effects

Testicular fluoride levels not increased, testicular zinc levels decreased, testicular effects resembled those of zinc deficiency


29 days

NaF = sodium fluoride; HSD = hydroxysteroid dehydrogenase.

Gavage

Ghosh et al.

Duration

2002

Route of Exposure

Elbetieha et al. Drinking water 4, 10 weeks, then mated to untreated females

Authors

2000

Year

20 mg/kg/day

100, 200, 300 ppm

Dose

Wistar rats

Swiss mice

Species

Decreased testicular, prostate, and seminal vesicle weights with decreased 3- and 17β-HSD activities Decreased epididymal sperm count, dilated seminiferous tubules

Fertility affected by 10 weeks but not by 4 weeks of treatment Reduction in number of implantation sites and viable fetuses in females mated to NaF-treated males (200 ppm NaF) Increased seminal vesicle and preputial gland weights in mice exposed to 200 and 300 ppm NaF for 4 weeks but not 10 weeks

Results

TABLE 5.7 (CONTINUED) Summary of Fluoride Effects on Male Reproduction Parameters in Mice, Rats, and Rabbits (from 1980 to the present)

124 Reviews in Food and Nutrition Toxicity


125

To determine if sodium fluoride induced biochemical changes in selected reproductive organs of albino male mice, Chinoy and Sequeira (1989b) fed sodium fluoride to groups of mice at doses of 10 or 20 mg/kg bodyweight/day for 30 days. Treatment was then withdrawn from selected groups for 1 and 2 months. At the end of the treatment period, testicular, epididymal (caput and cauda), vas deferens, seminal vesicle, and prostate weights were obtained. Significant changes in testicular cholesterol and serum testosterone concentrations were not observed. Testicular succinic dehydrogenase, epididymal sialic acid, and ATPase levels were decreased. Vas deferens glycogen, seminal vesicle fructose, prostate gland acid phosphatase, and total protein were increased. Chinoy and Sequeira (1992) fed male albino mice sodium fluoride at doses of 10 or 20 mg/kg bodyweight for 30 days. The treatment of 10 mg/kg/mouse/day was withdrawn from two groups for 1 and 2 months, and the animals were used for recovery studies. Normally cycling females were mated with treated males on the 31st day after treatment and, in the groups from which sodium fluoride was withdrawn, at the end of 1 and 2 months, respectively. Sperm motility and cauda epididymal sperm counts were decreased significantly in both treatment groups after 30 days of treatment and subsequently recovered after withdrawal from sodium fluoride treatment for 2 months. Additionally, fertility was almost absent in the treated animals but increased significantly after withdrawal of sodium fluoride treatment. Effects were observed on sperm head morphology, including effects on the acrosomal, post-acrosomal, and midpiece regions. Elbetieha et al. (2000) exposed 60-day-old male Swiss mice to sodium fluoride at concentrations of 100, 200, or 300 ppm in their drinking water for 4 or 10 weeks. Fertility was assessed by breeding the sodium fluoride–treated male mice to untreated females after the exposure period. Fertility was reduced in a dose-related manner in the 100, 200, and 300 ppm dose groups after 10 weeks of exposure but not after 4 weeks of exposure. The number of pregnancies resulting from mating treated males to nontreated females was 50, 45, and 36% for the 100, 200, and 300 ppm dose groups, respectively. Seminal vesicle and preputial weights were increased in mice exposed to 200 or 300 ppm sodium fluoride for 4 weeks, but not for 10 weeks. The authors concluded that long-term exposure to sodium fluoride adversely affected fertility in male mice. Studies in Rats Table 5.7 provides a summary of some of the available studies in male rats. Chinoy et al. (1991a) injected a single microdose (50 μg/50 μl) of sodium fluoride into the vasa differentia of the adult male albino rat resulting in an arrest of spermatogenesis and absence of spermatozoa in the lumina of the seminiferous tubules and a reduction in cauda epididymal sperm numbers, which consequently led to an impairment of fertility in experimental animals. Krasowska and Wlostowski (1992) exposed male Wistar rats to fluoride at concentrations of 100 or 200 ppm fluoride in their drinking water for 6 or 16 weeks. The results of this study indicated that although testicular fluoride levels increased, the increase was not associated with dose or time of exposure. The data also suggested that fluoride exposure to 100 or 200 ppm decreased significantly the

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concentrations of zinc in the testis, particularly in the 16-week treatment groups. Testicular iron and copper concentrations were not adversely affected by fluoride treatment. After 16 weeks of exposure, approximately 50% of the rats treated with 100 and 200 ppm of fluoride exhibited testicular histopathological effects in the germinal epithelium, reminiscent of zinc-deficient rats. The authors concluded the observed testicular effects were induced by a deprivation of testicular zinc resulting from high fluoride intake. Narayana and Chinoy (1994) examined the effects of sodium fluoride ingestion on sperm structure and metabolism. They also examined the effects of sodium fluoride withdrawal and the effects of administering fluoride with ascorbic acid or calcium alone or in combination on sperm structure and metabolism. The results of this study indicated that sperm hyaluronidase and acrosin were reduced after 50 days of treatment. Staining with alcoholic silver nitrate revealed acrosomal damage and deflagellation. These findings could account for the observed reduction in sperm motility. A reduction in cauda epididymal sperm count was attributed to an arrest of spermatogenesis. A recovery of the observed effects was not complete after withdrawal of sodium fluoride for 70 days. The administration of ascorbic acid and calcium alone or in combination appeared to reverse the fluoride-mediated effects. The authors suggested that the effect of sodium fluoride on sperm structure and metabolism in rats was reversible. Sprando et al. (1996) utilized intratesticular injections to characterize the effect of the short-term sodium fluoride exposure on spermatogenesis. One testis from each experimental animal was injected with sodium fluoride (50, 175, or 250 ppm) in vehicle (0.9% physiological saline). One testis from each control animal was injected with vehicle. Testicular effects were not observed at any of the doses utilized in this study and the authors concluded that spermatogenesis was not adversely affected by direct short-term exposure to sodium fluoride even at levels 200 times greater than those under normal conditions. Sprando et al. (1997) examined the potential of sodium fluoride to affect spermatogenesis and endocrine function in parental (P) and first (F1) generation male rats. In this study, male and female experimental rats received sodium fluoride in their drinking water at one of four concentrations (25, 100, 175, or 250 ppm). P generation male and female rats were exposed to sodium fluoride in their drinking water for 10 weeks, then mated within the same treatment groups. F1 generation male rats remained within the same treatment groups as their parents and were exposed to sodium fluoride in their drinking water for 14 weeks after weaning, at which time reproductive tissues were collected. Dose-related effects were not observed within the P and F1 treatment groups for testis weights, prostate/seminal vesicle weights, nonreproductive organ weight, testicular spermatid counts, testicular spermatid counts, luteinizing hormone (LH), follicular-stimulating hormone (FSH), or serum testosterone concentrations. Histological changes were not observed in testicular tissues from either the P or F1 generation males. The authors concluded that prolonged exposure to sodium fluoride in drinking water in the rat at the doses utilized in this study did not adversely affect spermatogenesis or endocrine function in the P and F1 generation male rats.


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In a further study of the rats treated throughout several generations (described above; Sprando et al., 1997), Sprando et al. (1998) obtained quantitative morphometric information on the testis of sodium fluoride–treated F1 generation male rats. No statistically significant changes were observed in the absolute volumes of the blood vessels, boundary layer, Leydig cells, lymphatic space, macrophages, tubular lumen, seminiferous epithelium, seminiferous tubules, interstitial space, and testicular capsule. These findings suggested that the volumetric composition of the various testicular components was not altered by exposure to sodium fluoride. When the number of Sertoli cell nucleoli was enumerated to detect tubular shrinkage resulting from germ cell loss or germ cell degeneration, the number of Sertoli cell nucleoli was not statistically different between control and treated rats. This result suggested that germ cell number in the seminiferous tubules was not significantly affected by sodium fluoride exposure. The mean diameters of the seminiferous tubules from the treatment groups were not significantly different from the control groups; this suggested that sodium fluoride treatment did not adversely affect spermatogenic activity in the sodium fluoride–treated animals. The length of the seminiferous tubule, seminiferous tubule length per unit volume, and the surface area of the seminiferous tubules from the treatment groups were not significantly different from the control groups, again suggesting that sodium fluoride exposure did not affect spermatogenesis in the treated animals. Ghosh et al. (2002) examined the effect of sodium fluoride on testicular steroidogenic and gametogenic activities in relation to testicular oxidative stress. Adult male albino Wistar rats were given sodium fluoride by oral gavage at a concentration of 20 mg/kg/day for 29 days. Decreases in testis, prostate, and seminal vesicle wet weight were observed without a concomitant decrease in bodyweight. Testicular Δ-5,3-β-hydroxysteroid dehydrogenase (HSD), 17-β-HSD, testosterone, and epididymal sperm counts were decreased in the fluoride treated group in comparison to the control. Dilated seminiferous tubules were also observed. Fluoride treatment was associated with oxidative stress as evidenced by an increase in conjugated dienes in the testis, epididymis, and cauda epididymal sperm. The authors concluded that fluoride, at doses encountered in contaminated areas, may exert toxic effects on the male reproductive system and these effects are associated with oxidative stress. Studies in Rabbits Table 5.7 provides a summary of some of the available studies in male rabbits. Shashi (1990) evaluated the relationship between infertility and the histological structure of the testes in albino rabbits following the subcutaneous administration of sodium fluoride at 5, 10, 20, or 50 mg/kg/day for 100 days. Effects were observed on spermatocyte maturation and differentiation in the experimental animals. Spermatogenesis ceased and necrotic seminiferous tubules were observed in the higher dosage groups. Chinoy et al. (1991b) assessed the effects of fluoride on metabolism and function of cauda epididymal sperm in rabbits. Male rabbits were fed either 20 or 40 mg/kg bodyweight sodium fluoride for 30 days, then cauda epididymal sperm was collected and assessed. Alteration in the specific activity of ATPase, acid phosphatase, succinate dehydrogenase, and protein was observed. Sodium and potassium levels were

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reduced. The reduced fertility observed in the treated animals was attributed to the reduction in sperm motility and counts and changes in morphology. After 30 days of treatment, selected groups of animals were withdrawn from fluoride treatment for 30 days. During the withdrawal phase, groups of animals were given ascorbic acid, calcium, or ascorbic acid and calcium. Recovery was more pronounced in the ascorbic acid–treated group than the calcium-treated group; however, if both ascorbic acid and calcium were administered together they interacted synergistically and recovery was most pronounced. Susheela and Kumar (1991) administered sodium fluoride orally at a concentration of 10 mg sodium fluoride/kg bodyweight to male rabbits for 18 or 29 months at which time the structure of the testis, epididymis, and vas deferens was studied using light and scanning electron microscopy. A disruption of spermatogenesis characterized by degenerating germ cells and seminiferous tubules devoid of germ cells and/or spermatozoa was observed in animals treated for 29 months. Histological effects were also observed in the ductuli efferentes and vas deferens in animals treated for 18 or 29 months. These effects were characterized by a loss of cilia on the epithelial cells lining the lumen of the ductuli efferentes and of stereocilia on the epithelial cells lining the lumen of the vas deferens. A spermatogenic arrest was observed only in animals treated for 29 months. Shashi and Kaur (1992) investigated the effect of fluoride toxicosis on testicular protein and DNA biosynthesis by exposing male albino rabbits to 0, 5, 10, 20, or 50 mg sodium fluoride via subcutaneous injections for 3.5 months in order to induce experimental fluorosis. The results of this study suggested that both testicular protein and DNA synthesis decreased as a result of fluorosis, further indicating that fluoride may interfere with RNA metabolism and consequently with the synthesis of specific testicular enzymes. Kumar and Susheela (1994) investigated the ability of sodium fluoride to disrupt spermiogenesis and induce defects in rabbit spermatids and epididymal spermatozoa. Male rabbits were treated with 10 mg sodium fluoride/kg bodyweight daily for 18 months. An ultrastructural examination of testicular spermatids and caput epididymal sperm revealed a wide variety of structural defects in the flagellum, the acrosome, and the nucleus of the spermatids and epididymal spermatozoa of fluoride-treated rabbits. These abnormalities included an absence of outer microtubules, complete absence of axonemes, structural and numeric aberrations of outer dense fibers, breakdown of the fibrous sheath, and structural defects in the mitochondria of the middle piece of the flagellum. Detachment and peeling of the acrosome from the flat surfaces of the nucleus were also observed. Kumar and Susheela (1995) utilized both light and scanning electron microscopy to observe the effect of chronic fluoride toxicity on the structure of the ductus epididymis, testis, and spermatozoa in rabbits. Rabbits were treated with 10 mg sodium fluoride/kg bodyweight/day for 20 or 23 months. Serum fluoride levels were significantly elevated in the sera of rabbits treated for both 20 and 23 months. Multiple effects were observed in the cauda and caput epididymis and testis in rabbits treated with sodium fluoride for 23 months. Epididymal effects included a loss of stereocilia, a significant decrease in the height of the pseudostratified columnar


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epithelium, and a significant increase in the diameter of both the caput and cauda ductus epididymis. Additionally, cauda and caput epididymal weights and the number of secretory granules were also reduced in comparison to the control animals. Testicular effects included decreased epithelial cell height and tubular diameter of the testis. The authors noted fragmentation of spermatozoa in the caput and cauda ductus epididymis of animals treated for 23 months but not in the testis and caput and cauda epididymis of the animals treated for 20 months. The authors concluded that sperm maturation might be adversely affected as a result of the structural changes observed in the caput and cauda ductus epididymis. Correlation with Female Reproduction Effect on Estrous Cycle Del Castillo (1928) reported suppression of the estrous cycle when 0.05 mg sodium fluoride per day was fed to two female rats. Phillips et al. (1933) reported that the estrous cycles of females treated with 430 ppm sodium fluoride in the diet were similar to those of the control animals (cycle of 4.5 to 6.5 days). In a second study in the same report, Phillips et al. (1933) reported that high levels of fluoride (greater than 25 mg/kg/day) suppressed estrus, but that the suppression was attributable to inanition, which occurred at high levels of fluoride. Maternal–Fetal Transfer of Fluoride Fluoride crosses the placenta and is found in fetal and placental tissue. Placental transfer has been documented in mice (Ericsson and Hammarstrom, 1964), rats (Theuer et al., 1971), rabbits (Nedeljkovic and Matovic, 1991), and in pregnant women (Caldera et al., 1988; Feltman and Kosel, 1955; Forestier et al., 1990; Gedalia et al., 1961; Malhotra et al., 1993; Shi and Zhang, 1995). In rabbits, fluoride content of bones and teeth of the newborn was significantly increased and dose dependent (Nedeljkovic and Matovic, 1991). When transplacental passage of fluoride was studied in 25 randomly selected neonates in India, fluoride concentration in cord blood was 60% of that in mother’s blood (Gupta et al., 1993a). Several analyses of human milk have shown that the daily fluoride intake of infants, from those drinking colostrum to 3-month-old infants drinking mature milk, ranged from 5 to 10 μg (Esala et al., 1982; Spak et al., 1983). This was the same regardless of the fluoride content of the water consumed. At 0.2 ppm fluoride, the daily fluoride intake was closer to 5 μg (Esala et al., 1982; Spak et al., 1983); at 1.0 or 1.2 ppm, the daily fluoride consumption was 7.3 to 8.5 μg (Esala et al., 1982). A survey of fluoride in human milk showed a strong correlation between fluoride level in milk and presence of fluoride in drinking water (Dabeka et al., 1986). In a study done in remote areas of Thailand, the fluoride content of human milk was 0.017 ppm, and there was no correlation between breast milk fluoride content and fluoride concentration in drinking water (Chuckpaiwong et al., 2000). Human breast milk tested from women living in Mangalore City, India, showed that a minimal amount of fluoride was in breast milk while infant formulae had higher fluoride levels (Rahul et al., 2003).

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Studies in Mice Table 5.8 provides a summary of some of the available studies in female rats and mice. Messer et al. (1973) gave female mice drinking water containing 0, 50, 100, or 200 ppm sodium fluoride and fed them a low-fluoride diet (0.1 to 0.3 ppm). After the first litter, the F1 animals were remated for up to four litters. At 50 ppm, the animals gained weight at the same rates in the F1 and F2 generations and bodyweight of pups was similar to controls. However, there was a progressive decrease in the number of litters produced by the control group and less than 50% of the animals in each generation produced four litters. At 100 ppm, the number of litters was reduced and six of nine litters were stillborn or eaten at birth. Third and fourth litter females were mated to produce a second generation. At 200 ppm fluoride, all had died by 20 weeks of age. Because the weight gain and health signs appeared normal in the control and 50 ppm groups, the investigators determined that the diet was nutritionally adequate except for fluoride, and thus impaired reproduction in the control group was due to fluoride deficiency. When the study by Messer et al. (1973) was repeated by Tao and Suttie (1976), they also noted impaired reproduction, but their results suggested that the diet was marginally deficient in iron and that fluoride improved iron utilization. Studies in Rats Schulz and Lamb (1925) fed high levels of sodium fluoride (equivalent to 500, 1000, 1500, or 2500 ppm) to rats for 9 months before mating. The two females given 500 ppm sodium fluoride each successfully reared a third generation of offspring. Four litters were reared (of six litters) from the animals given 1000 ppm, but the offspring grew at a slower rate than the control animals, and there were fewer third-generation offspring reared than in the control animals. The rats given 1500 ppm sodium fluoride produced four litters, of which only two litters lived, and the offspring grew slowly. In the group of seven rats given 2500 ppm, all died after 8 to 14 weeks. In the same report, Schulz and Lamb (1925) reported that they tested a second series of rats with 10 to 2500 ppm sodium fluoride, and that the animals exhibited no toxic effects until they were given at least 1000 ppm. They also reported that an unfavorable effect on reproduction began at 250 ppm. Unfortunately, the authors provided no information on the treatment or number of animals treated in the second series of studies. Lamb et al. (1933) and Phillips et al. (1933) fed 0 (basal diet) or 0.043% (430 ppm) sodium fluoride to rats, and the animals’ reproduction, growth, and estrous cycles were monitored for five generations. Based on lighter offspring in the treated animals at the time of weaning, Lamb et al. (1933) suggested that either the vigor of the young or the quality or quantity of the milk was affected by fluoride in the diet. Lamb et al. (1933) observed that in the third generation, the treated females produced only one litter each and then failed to produce subsequent litters; but they stated that they could not attribute this interruption to sodium fluoride, because it could have resulted from a chronic lung infection in these animals. Ream et al. (1983) tested the effects of fluoride on bone morphology. They dosed female rats with 0 (distilled water) or 150 ppm sodium fluoride in drinking water for 10 weeks prior to mating and during three successive pregnancy and lactation

Messer et al.

Tao and Suttie

Ream et al.

1973

1976

1983

NaF = sodium fluoride.

2001a Collins et al.

Lamb et al., Phillips et al.

1933

Route of Exposure

Drinking water

Throughout three generations

Three litters

Drinking water (with Same as Messer et al. addition of iron and copper to feed) Drinking water

0, 500, 1000, 1500, 2500 ppm NaF

Dose

Mice

Rats

Rats

Species

Rats

0, 25, 100, 175, Rats 250 ppm

150 ppm NaF

Same as Messer Mice et al.

0, 50, 100, 200 ppm NaF

Throughout five generations 0, 430 ppm

9 months prior to mating, and throughout the study

Duration

Drinking water (with Two generations with four low-fluoride diet) litters in first generation

Feed

Schulz and Lamb Feed

Authors

1925

Year

Results

25–250 ppm: no reproductive or developmental effects

No effects on reproduction Femurs from third litter pups were tested: no visible structural alterations

50–200 ppm: no effects

200 ppm: all dead by 20 weeks of age 100 ppm: growth retardation, reduced number of litters, 6/9 litters stillborn 50 ppm: no effect

Offspring lighter at the time of weaning

2500 ppm: fatal to 7 females after 8–14 weeks 1000 and 1500 ppm: females grew more slowly and produced smaller third-generation litters 500 ppm: no effects on reproduction

TABLE 5.8 Summary of Fluoride Effects on Female Reproductive Parameters in Rats and Mice


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periods. The investigators reported no effects of the compound on fertility or reproduction. The femurs of the 3-week-old third-pregnancy offspring were measured and no pathological changes in the femurs of the offspring were reported. In the early 1990s, the existing reproductive studies (many of which were summarized in the preceding text) were reviewed in several reports and were considered to be inadequate to determine potential reproductive or developmental hazards. These reviews included the report of the National Toxicology Program (NTP, 1990), the review of fluoride benefits and risks (PHS, 1990), and the report by the National Research Council (NRC, 1993). The inadequacies of the studies were attributed to insufficient or sometimes unknown numbers of animals per group and inadequate descriptions of the experimental procedures. None of the studies had been done according to currently accepted international guidelines. In response to the inadequacies demonstrated in the available studies, a multigeneration study was done at the U.S. Food and Drug Administration. Collins et al. (2001a) measured the effects of sodium fluoride ingestion at 0, 25, 100, 175, or 250 ppm in drinking water throughout three generations of rats. Low-fluoride diet was given to the treated animals throughout the study to minimize interference with the fluoride in water. Mating, fertility, survival, and offspring development were not affected.

DEVELOPMENTAL TOXICITY ASPECTS Human Studies Analysis of birth certificates for the period 1973–1975 showed that rates of congenital malformations (except Down’s syndrome) were similar in people ingesting fluoridated or nonfluoridated water (Erickson, 1980). An increased incidence of spina bifida was reported in sections of India where the fluoride content of drinking water is high (4.5 to 8.5 ppm) and this increase was associated with skeletal or dental fluorosis (Gupta et al., 1995). The investigators, however, did not examine the role of nutrients such as folic acid. Animal Studies Table 5.9 provides a summary of some of the available studies in female rats, mice, and rabbits. Pillai et al. (1989) gave 5.2 or 17.3 mg fluoride/kg bodyweight to mice daily on gestation days 6 to 15 (administered orally, as a single dose per day). At euthanasia on day 21, the treated mice showed no sign of pregnancy, and bodyweight and hemoglobin were decreased. The authors suggested that fluoride adversely affected implantation. Sprague-Dawley rats were given sodium fluoride in their drinking water at concentrations of 0, 50, 150, or 300 ppm on gestation days 6 to 15, and New Zealand rabbits were given 0, 100, 200, or 400 ppm sodium fluoride in the drinking water on gestation days 6 to 19 (Heindel et al., 1996). The number of live births, fetal bodyweight, sex ratio, and the incidence of external, visceral, or skeletal anomalies were similar in fluoride-treated and untreated rats and rabbits.

Pillai et al.

Collins et al.

Heindel et al.

Heindel et al.

Collins et al.

1989

1995

1996

1996

2001b

Drinking water

Drinking water

Drinking water

Drinking water

Gavage

Route of Exposure

Throughout 3 generations

Gestation days 6–19




Duration

0, 25, 100, 175, 250 ppm

0, 100, 200, 400 ppm

0, 50, 150, 300 ppm

0, 10, 25, 100, 175, 250 ppm

0, 5.2, 17.3 mg/kg/day

Dose

Rats

Rabbits

Rats

Rats

Mice

Species

250 ppm: decreased ossification of hyoid bone in F2 fetuses 25–175 ppm: no dose-related developmental effects

100–400 ppm: no reproductive or developmental effects

50–300 ppm: no reproductive effects

250 ppm: increased number of fetuses with three or more skeletal variations 10–175 ppm: no fetal effects

At gestation day 21, treated mice showed no sign of pregnancy, and bodyweight and hemoglobin decreased

Results

Note: Summary of studies of fluoride effects on reproductive parameters and on developing fetuses of mice, rats, and rabbits.

Authors

Year

TABLE 5.9 Summary of Developmental Toxicity Studies


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When pregnant rats were given fluoridated drinking water at 0, 10, 25, 100, 175, or 225 ppm throughout gestation, there was no effect on the development of specific bones, although there was an increase in the average number of fetuses per litter with three or more skeletal variations at 250 ppm (Collins et al., 1995). In a developmental toxicity study of pregnant rats that had been treated with sodium fluoride in utero and had continued to be given the same concentrations (0, 25, 100, 175, or 250 ppm) throughout gestation, decreased ossification of the hyoid bone was observed at 250 ppm (Collins et al., 2001b). The number of live births, fetal bodyweight, sex ratio, and the incidence of external and visceral anomalies were similar in fetuses of rats whose parents had also been exposed in utero and throughout their lifetime.

REFERENCES Aasenden, R. (1974) Fluoride concentrations in the surface tooth enamel of young men and women, Archives of Oral Biology, 19: 697–701. Ando, M., Tadano, M., Asanuma, S., Tamura, K., Matsushima, S., Watanabe, T., Kondo, T., Sakurai, S., Ji, R., Liang, C., and Cao, S. (1998) Health effects of indoor fluoride pollution from coal burning in China, Environmental Health Perspectives, 106: 239–244. Barnhart, W.E., Hiller, H.L., Leonard, G.F., and Michaels, S.E. (1974) Dentifrice usage and ingestion among four age groups, Journal of Dental Research, 3: 1317–1322. Baxter, P.M. (1980) Toothpaste ingestion during toothbrushing by school children, British Dental Journal, 148: 125–128. Beltran, E.D. and Szpunar, S.M. (1988) Fluoride in toothpastes for children. Suggestion for change, Pediatric Dentistry, 10: 185–188. Berry, W. (1958) A study of the incidence of mongolism in relation to the fluoride content of water, American Journal of Mental Deficiency, 62: 634–636. Bowen, W.H. (2002) Fluorosis: is it really a problem? Journal of the American Dental Association, 133: 1405–1407. Brunn, C. and Thylstrup, A. (1988) Dentifrice usage among Danish children, Journal of Dental Research, 67: 1114–1117. Burgstahler, A.W. and Robinson, M.A. (1997) Fluoride in California wines and raisins, Fluoride, 30: 142–146. Burns, G.H. and Gump, B.H. (1993) Technological advances in the analysis of wines, in B.H. Gump and D.A. Pruett, Eds., Beer and Wine Production: Analysis, Characterization, and Technological Advances, American Chemical Society Symposium Series 536, Washington, D.C.: American Chemical Society. Caldera, R., Chavinie, J., Fermanian, J., Tortrat, D., and Laurent, A.M. (1988) Maternal–fetal transfer of fluoride in pregnant women, Biology of the Neonate, 54: 263–269. Cao, J., Bai, X., Zhao, Y., Liu, J., Zhou, D., Fang, S., Jia, M., and Wu, J. (1996) The relationship of fluorosis and brick tea drinking in Chinese Tibetans, Environmental Health Perspectives, 104: 1340–1343. Cao, J., Zhao, Y., Liu, J., Xirao, R., Danzeng, S., Daji, D., and Yan, Y. (2003) Brick tea fluoride as a main source of adult fluorosis, Food and Chemical Toxicology, 41: 535–542. CDC (Centers for Disease Control and Prevention). (1993) Fluoridation Census 1992, Atlanta: U.S. Department of Health and Human Services, Public Health Service.


135

Chinoy, N.J. and Sequeira, E. (1989a) Effects of fluoride on histoarchitecture of reproductive organs of the male mouse, Reproductive Toxicology, 3: 261–268. Chinoy, N.J. and Sequeira, E. (1989b) Fluoride induced biochemical changes in reproductive organs of male mice, Fluoride, 22: 78–85. Chinoy, N.J. and Sequeira, E. (1992) Reversible fluoride induced fertility impairment in male mice, Fluoride, 25: 71–76. Chinoy N., Rao, M.V., Narayana, M.V., and Neelakanta, E. (1991a) Microdose vasal injection of sodium fluoride in the rat, Reproductive Toxicology, 5: 505–512. Chinoy, N.J., Sequiera, E., and Narayana, M.V. (1991b) Effects of vitamin C and calcium on the reversibility of fluoride-induced alterations in spermatozoa of rabbits, Fluoride, 24: 29–40. Chuckpaiwong, S., Nakornchai, S., Surarit, R., and Soo-Ampon, S. (2000) Fluoride analysis of human milk in remote areas of Thailand, Southeast Asian Journal of Tropical Medicine and Public Health, 31: 583–586. Collins, T.F.X., Sprando, R.L., Shackelford, M.E., Black, T.N., Ames, M.J., Welsh, J.J., Balmer, M.F., Olejnik, N., and Ruggles, D.I. (1995) Developmental toxicity of sodium fluoride in rats, Food and Chemical Toxicology, 33: 951–960. Collins, T.F.X., Sprando, R.L., Black, T.N., Shackelford, M.E., Bryant, M.A., Olejnik, N., Ames, M.J., Rorie, J.I., and Ruggles, D.I. (2001a) Multigenerational evaluation of sodium fluoride in rats, Food and Chemical Toxicology, 39: 601–613. Collins, T.F.X., Sprando, R.L., Black, T.N., Shackelford, M.E., Olejnik, N., Ames, M.J., Rorie, J.I., and Ruggles, D.I. (2001b) Developmental toxicity of sodium fluoride measured during multiple generations, Food and Chemical Toxicology, 39: 867–876. Dabeka, R.W., Karpinski, K.F., McKenzie, A.D., and Bajdik, C.D. (1986) Survey of lead, cadmium, and fluoride in human milk and correlation of levels with environmental and food factors, Food and Chemical Toxicology, 24: 913–921. Daston, G.P., Rehnberg, B.F., Carver, B., and Kavlock, R.J. (1985) Toxicity of sodium fluoride to the postnatally developing rat kidney, Environmental Research, 37: 461–474. Davis, R.K. (1961) Fluorides: a critical review. V. Fluoride intoxication in laboratory animals, Journal of Occupational Medicine, 3: 593–601. De Lopez, O.H., Smith, F.A., and Hodge, H.C. (1976) Plasma fluoride concentrations in rats acutely poisoned with sodium fluoride, Toxicology and Applied Pharmacology, 37: 75–83. Dean, H.T. (1938) Endemic fluorosis and its relation to dental caries, Public Health Reports, 53: 1443–1452. Dean, H.T. (1942) The investigation of physiological effects by the epidemiological method, in F.R. Moulton, Ed., Fluorine and Dental Health, Publication 19, Washington, D.C.: American Association for the Advancement of Science, 23–31. Del Castillo, E.B. (1928) Action des intoxications par le fluor ou le thallium sur le cycle oestral du rat blanc, Comptes Rendus des Seances, Societe de Biologie, 99: 1405. Den Besten, P.K. (1986) Effects of fluoride on protein secretion and removal during enamel development in the rat, Journal of Dental Research, 65: 1272–1277. Dowell, T.B. (1981) The use of toothpaste in infancy, British Dental Journal, 150: 247–249. Eichler, H.G., Lenz, K., Fuhrmann, M., and Hruby, K. (1982) Accidental ingestion of NaF tablets by children: report of a poison control center and one case, International Journal of Clinical Pharmacology, Therapy, and Toxicology, 20: 334–338. Eklund, S.A., Burt, B.A., Ismail, A.I., and Calderone, J.J. (1987) High-fluoride drinking water, fluorosis, and dental caries in adults, Journal of the American Dental Association, 114: 324–328.

136


Ekstrand, J., Alvan, G., Boreus, L.O., and Norlin, A. (1977) Pharmacokinetics of fluoride in man after single and multiple oral doses, European Journal of Clinical Pharmacology, 12: 311–317. Elbetieha, A., Darmani, H., and Al-Hiyasat, A.S. (2000) Fertility effects of sodium fluoride in male mice, Fluoride, 33: 128–134. EPA (Environmental Protection Agency) (1985) Drinking Water Criteria Document on Fluoride, Contract 68-03-3279, Cincinnati: Office of Drinking Water. Erickson, J.D. (1980) Down syndrome, water fluoridation, and maternal age, Teratology, 21: 177–180. Erickson, J.D., Oakley, G.P., Jr., Flynt, J.W., Jr., and Hay, S. (1976) Water fluoridation and congenital malformations: no association, Journal of the American Dental Association, 93: 981–984. Ericsson, Y. and Hammarstrom, L. (1964) Mouse placental transfer of F-18 in comparison with Ca-45, Acta Odontologica Scandinavica, 22: 523–538. Esala, S., Vuori, E., and Helle, A. (1982) Effect of maternal fluorine intake on breast milk fluorine control, British Journal of Nutrition, 48: 201–204. Fein, N.J. and Cerklewski, F.L. (2001) Fluoride content of foods made with mechanically separated chicken, Journal of Agricultural and Food Chemistry, 49: 4284–4286. Fejerskov, O., Manji, F., and Baelum, V. (1990) The nature and mechanisms of dental fluorosis in man, Journal of Dental Research, 69: 692–700. Fejerskov, O., Yanagisawa, T., Tohda, H., Larsen, M.J., Josephsen, K., and Mosha, H.J. (1991) Posteruptive changes in human dental fluorosis — a historical and ultrastructural study, Proceedings of the Finnish Dental Society, 87: 607–619. Feltman, R. and Kosel, G. (1955) Prenatal ingestion of fluorides and their transfer to the fetus, Science, 122: 560–561. Finkelman, R.B., Belkin, H.E., and Zheng, B. (1999) Health effects of domestic coal use in China, Proceedings of the National Academy of Sciences of the United States of America, 96: 3427–3431. Food and Nutrition Board (1999) Fluoride, in Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride, Washington, D.C.: National Academy of Science Press, 218–313. Forestier, F., Daffos, F., Said, R., Brunet, C.M., and Guillaume, P.N. (1990) Passage transplacentaire du fluor. Etude in utero, Journal de Gynecologie, Obstetrique et Biologie de la Reproduction, 19: 171–175. Freni, S.C. (1994) Exposure to high fluoride concentrations in drinking water is associated with decreased birth rates, Journal of Toxicology and Environmental Health, 42: 109–121. Gedalia, I. and Shapira, L. (1989) Effect of prenatal and postnatal fluoride on the human deciduous dentition. A literature review, Advances in Dental Research, 3: 168–176. Gedalia, I., Brzezinski, A., Bercovici, B., and Lazarov, F. (1961) Placental transfer of fluorine in the human fetus, Proceedings of the Society for Experimental Biology and Medicine, 106: 147–149. Ghosh, D., Das Sarkar, S., Maiti, R., Jana, D., and Das, U.B. (2002) Testicular toxicity in sodium fluoride treated rats: association with oxidative stress, Reproductive Toxicology, 16: 385–390. Greenberg, S.R. (1986) Response of the renal supporting tissues to chronic fluoride exposure as revealed by a special technique, Urologia Internationalis, 41: 91–94. Grunder, L.P. and MacNeil, J.H. (1973) Examination of bone content in mechanically deboned poultry meat by EDTA and atomic absorption spectrophotometric methods, Journal of Food Science, 38: 712–713.


137

Gupta, S. (1991) The fluorine concentrations of foods and toothpastes in India, Fluoride, 3: 113–116. Gupta, S., Seth, A.K., Gupta, A., and Gavane, A.G. (1993a) Transplacental passage of fluorides, Journal of Pediatrics, 123: 139–141. Gupta, S.K., Gambhir, S., Mithal, A., and Das, B.K. (1993b) Skeletal scintigraphic findings in endemic skeletal fluorosis, Nuclear Medicine Communications, 14: 384–390. Gupta, S.K., Gupta, R.C., Seth, A.K., and Chaturvedi, C.S. (1995) Increased incidence of spina bifida occulta in fluorosis prone areas, Acta Paediatrica Japonica, 37: 503–506. Gupta, S.K., Khan, T.I., Gupta, R.C., Gupta, A.B., Gupta, K.C., Jain, P., and Gupta, A. (2001) Compensatory hyperparathyroidism following high fluoride ingestion — a clinicobiochemical correlation, Indian Pediatrics, 38: 139–146. Guy, W.S. (1979) Inorganic and organic fluorine in human blood, in E. Johansen, D.R. Taves, and T.O. Olsen, Eds., Continuing Evaluation of the Use of Fluorides, Boulder, CO: Westview Press, 124–147. Hargreaves, J.A., Thompson, G.W., and Wagg, B.G. (1972) A gravimetric study of the ingestion of toothpaste by children, Caries Research, 6: 237–243. Heilman, J.R., Kiritsy, M.C., Levy, S.M., and Wefel, J.S. (1997) Fluoride concentrations of infant foods, Journal of the American Dental Association, 128: 857–863. Heilman, J.R., Kiritsy, M.C., Levy, S.M., and Wefel, J.S. (1999) Assessing fluoride levels of carbonated soft drinks, Journal of the American Dental Association, 130: 1593–1599. Heindel, J.J., Bates, H.K., Price, C.J., Marr, M.C., Myers, C.B., and Schwetz, B.A. (1996) Developmental toxicity evaluation of sodium fluoride administered to rats and rabbits in drinking water, Fundamental and Applied Toxicology, 30: 162–177. Hodge, H.C. and Smith, F.A. (1965) Biological properties of inorganic fluorides, in J.H. Simons. Ed., Fluorine Chemistry, New York: Academic Press, 1–42. Hodge, H.C. and Smith, F.A. (1977) Occupational fluoride exposure, Journal of Occupational Medicine, 19: 12–39. Jackson, R.D., Brizendine, E.J., Kelly, S.A., Hinesley, R., Stookey, G.K., and Dunipace, A.J. (2002) The fluoride content of foods and beverages from negligibly and optimally fluoridated communities, Community Dentistry and Oral Epidemiology, 30: 382–391. Jarnberg, P.O., Ekstrand, J., and Ehrnebo, M. (1983) Renal excretion of fluoride during water diuresis and induced urinary pH changes in man, Toxicology Letters, 18: 141–146. Ji, R.D. (1993) Research on fluoride level of indoor air in burning coal fluorosis areas, Journal of Hygienic Research, 22: 10–13. Jooste, P.L., Weight, M.J., Kriek, J.A., and Louw, A.J. (1999) Endemic goitre in the absence of iodine deficiency in schoolchildren of the Northern Cape Province of South Africa, European Journal of Clinical Nutrition, 53: 8–12. Juncos, L.I. and Donadio, J.V. (1972) Renal failure and fluorosis, Journal of the American Medical Association, 222: 783–785. Kiritsy, M.C., Levy, S.M., Warren, J.J., Guha-Chowdhury, N., Heilman, J.R., and Marshall, T. (1996) Assessing fluoride concentrations of juices and juice-flavored drinks, Journal of the American Dental Association, 127: 895–902. Kour, K. and Singh, J. (1980) Histological finding of mice testes following fluoride ingestion, Fluoride, 13: 160–162. Krasowska, A. and Wlostowski, T. (1992) The effect of high fluoride intake on tissue trace elements and histology of testicular tubules in the rat, Comparative Biochemistry and Physiology, C, 103: 31–34. Kumar, A. and Susheela, A.K. (1994) Ultrastructural studies of spermiogenesis in rabbit exposed to chronic fluoride toxicity, International Journal of Fertility and Menopausal Studies, 39: 164–171.

138


Kumar, A. and Susheela, A.K. (1995) Effects of chronic fluoride toxicity on the morphology of ductus epididymis and the maturation of spermatozoa of rabbit, International Journal of Experimental Pathology, 76: 1–11. Lamb, A.R., Phillips, P.H., Hart, E.B., and Bohstedt, G. (1933) Studies on fluorine in the nutrition of the rat. I. Its influence upon growth, American Journal of Physiology, 106: 350–355. Lantz, O., Jouvin, M.H., De Vernejoul, M.C., and Druet, P. (1987) Fluoride-induced chronic renal failure, American Journal of Kidney Diseases, 10: 136–139. Larsen, M.J., Kirkegaard, E., and Poulsen, S. (1987) Pattern of dental fluorosis in a European country in relation to the fluoride concentration of drinking water, Journal of Dental Research, 66: 10–12. Leone, N.C., Shimkin, M.B., Arnold, F.A., Stevenson, C.A., Zimmerman, E.R., Geiser, P.B., and Lieberman, J.E. (1954) Medical aspects of excessive fluoride in a water supply, Public Health Reports, 69: 925–936. Li, Y., Liang, C.K., Katz, B.P., Brizendine, E.J., and Stookey, G.K. (1995) Long-term exposure to fluoride in drinking water and sister chromatid exchange frequency in human blood lymphocytes, Journal of Dental Research, 74: 1468–1474. Lim, J.K., Renaldo, G.J., and Chapman, P. (1978) LD-50 of SnF2, NaF, and Na2PO3F in the mouse compared to the rat, Caries Research, 12: 177–179. Long, Y.G., Wang, Y.N., Chen, J., Jiang, S.F., Nordberg, A., and Guan, Z.Z. (2002) Chronic fluoride toxicity decreases the number of nicotinic acetylcholine receptors in rat brain, Neurotoxicology and Teratology, 24: 751–757. Malhotra, A., Tewari, A., Chawla, H.S., Gauba, K., and Dhall, K. (1993) Placental transfer of fluoride in pregnant women consuming optimum fluoride in drinking water, Journal of the Indian Society of Pedodontics and Preventive Dentistry, 11: 1–3. Mann, J., Mahmoud, W., Ernest, M., Sgan-Cohen, H., Shoshan, N., and Gedalia, I. (1990) Fluorosis and dental caries in 6–8-year-old children in a 5 ppm fluoride area, Community Dentistry and Oral Epidemiology, 18: 77–79. Manocha, S.H., Warner, H., and Olkowski, B.L. (1975) Cytochemical responses of kidney, liver, and nervous system to fluoride ions in drinking water, Histochemical Journal, 7: 343–355. Marthaler, T.M. (2002) Dentistry between pathology and cosmetics, Community Dentistry and Oral Epidemiology, 30: 3–15. McCann, H.G. and Bullock, F.A. (1957) The effects of fluoride ingestion on the composition and solubility of mineralized tissues of the rat, Journal of Dental Research, 36: 391–398. McInnes, P.M., Richardson, B.D., and Cleaton-Jones, P.E. (1982) Comparison of dental fluorosis and caries in primary teeth of preschool-children living in arid high and low fluoride villages, Community Dentistry and Oral Epidemiology, 10: 182–186. Messer, H.H., Armstrong, W.D., and Singer, L. (1973) Influence of fluoride intake on reproduction in mice, Journal of Nutrition, 103: 1319–1326. Mullenix, P.J., Denbesten, P.K., Schunior, A., and Kernan, W.J. (1995) Neurotoxicity of sodium fluoride in rats, Neurotoxicology and Teratology, 17: 169–177. Nair, K.R. and Manji, F. (1982) Endemic fluorosis in deciduous dentition: a study of 1276 children in typically high fluoride area (Kiambu) in Kenya, Odonto-Stomatologie Tropicale, 5: 177–184. Narayana, M.V. and Chinoy, N.J. (1994) Reversible effects of sodium fluoride ingestion on spermatozoa of the rat, International Journal of Fertility and Menopausal Studies, 39: 337–346.


139

Nedeljkovic, M. and Matovic, V. (1991) The effect of dose on maternal–fetal transfer of fluoride in rabbits, Arhiv za Higijenu Rada I Toksikologiju, 42: 43–46. Needleman, H.L., Pueschel, S.M., and Rothman, K.J. (1974) Fluoridation and the occurrence of Down’s syndrome, New England Journal of Medicine, 291: 821–823. Newbrun, E. (1989) Effectiveness of water fluoridation, Journal of Public Health Dentistry, 49: 279–289. NRC (National Research Council) (1993) Health Effects of Ingested Fluoride, Washington, D.C.: National Academy Press. NTP (National Toxicology Program) (1990) Technical Report on the Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No. 7681-49-4) in F344/N Rats and B6C3F1 Mice (Drinking Water Studies). NNTP TR 393, NIH Publication 91-2848. Bethesda, MD: U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health. Olsson, B. (1979) Dental findings in high-fluoride areas in Ethiopia, Community Dentistry and Oral Epidemiology, 7: 51–56. Parkins, F.M. and Greenlimb, P.E. (1974) Fluoride secretion in rat bile during periods of intestinal fluoride absorption, Journal of Dental Research, 53: 1296. Phillips, P.H., Lamb, A.R., Hart, E.B., and Bohstedt, G. (1933) Studies on fluorine in the nutrition of the rat. II. Its influence upon reproduction, American Journal of Physiology, 106: 356–364. PHS (Public Health Service) (1990) Review of Fluoride Risks and Benefits, Washington, D.C.: Department of Health and Human Services. PHS (Public Health Service) (2001) Toxicological Profile for Fluorides, Washington, D.C.: Department of Health and Human Services. Pillai, K.S., Mathai, A.T., and Desmukh, P.B. (1989) Effect of fluoride on reproduction in mice, Fluoride, 22: 165–168. Purohit, S.D., Gupta, R.C., Mathur, A.K., Gupta, N., Jeswani, I.D., Choudhary, V.K., and Purohit, S.K. (1999) Experimental pulmonary fluorosis, Indian Journal of Chest Diseases and Allied Sciences, 41: 27–34. Rahul, P., Hegde, A.M., and Munshi, A.K. (2003) Estimation of the fluoride concentrations in human breast milk, cow’s milk, and infant formulae, Journal of Clinical and Pediatric Dentistry, 27: 257–260. Rapaport, I. (1957) Contribution a l’etude du mongolisme, role pathogenique du fluor, Bulletin de l’Academie Nationale de Medecine, 140: 529–531. Rapaport, I. (1963) Oligotrophie mongolienne et caries dentaires, Revue de Stomatologie et de Chirurgie Maxillo-Faciale, 46: 207–218. Ream, L.J., Scott, J.N., and Pendergrass, P.B. (1983) Bone morphology of weanling rats from dams subjected to fluoride, Cell and Tissue Research, 223: 689–691. Reeves, T.G. (1996) Technical aspects of water fluoridation in the United States and an overview of fluoridation engineering world-wide, Community Dental Health, 13: 21–26. Richards, A. (1990) Nature and mechanisms of dental fluorosis in animals, Journal of Dental Research, 69: 701–705. Richards, A., Kragstrup, J., Josephsen, K., and Fejerskov, O. (1986) Dental fluorosis developed in post-secretory enamel, Journal of Dental Research, 65: 1406–1409. Schulz, J.A. and Lamb, A.R. (1925) The effect of fluorine as sodium fluoride on the growth and reproduction of albino rats, Science, 61: 93–94. Sharkey, T.P. and Simpson, W.M. (1933) Accidental sodium fluoride poisoning: report of eight cases, with one fatality, Journal of the American Medical Association, 100: 97–100.

140


Shashi, C.D. (1990) Histological changes in rabbit testes during experimental fluorosis, Folia Morphologica, 38: 63–65. Shashi, C.D. and Kaur, D. (1992) Testicular proteins and DNA in experimental fluorosis, Indian Journal of Pathology and Microbiology, 35: 351–356. Shi, J., Dai, G., and Zhang, Z. (1995) Relationship between bone fluoride content, pathological change in bone of aborted fetuses and maternal fluoride level, Chinese Journal of Preventive Medicine, 29: 103–105. Skare, J.A., Schrotel, K.R., and Nixon, G.A. (1986) Lack of DNA-strand breaks in rat testicular cells after in vivo treatment with sodium fluoride, Mutation Research, 170: 85–92. Smith, F.A. and Hodge, H.C. (1979) Airborne fluorides and man. Part I., Critical Reviews in Environmental Control, 8: 293–371. Spak, C.J., Hardell, L.I., and De Chateau, P. (1983) Fluoride in human milk, Acta Paediatrica Scandinavica, 72: 699–701. Sprando, R.L., Black, T.N., Ames, M.J., Rorie, J.I., and Collins, T.F.X. (1996) Effect of intratesticular injection of sodium fluoride on spermatogenesis, Food and Chemical Toxicology, 34: 377–384. Sprando, R.L., Collins, T.F.X., Black, T.N., Ames, M.J., and Rorie, J.I. (1997) Testing the potential of sodium fluoride to affect spematogenesis in the rat, Food and Chemical Toxicology, 35: 881–890. Sprando, R.L., Collins, T.F.X., Black, T., Olejnik, N., and Rorie, J. (1998) Testing the potential of sodium fluoride to affect spematogenesis: a morphometric study, Food and Chemical Toxicology, 36: 1117–1124. Stannard, J.G., Shim, Y.S., Kritsincli, M., Labropoulou, P., and Tsamtsouris, A. (1991) Fluoride levels and fluoride contamination of fruit juices, Journal of Clinical Pediatric Dentistry, 16: 38–40. Susheela, A.K. and Jethanandani, P. (1996) Circulating testosterone levels in skeletal fluorosis patients, Journal of Toxicology and Clinical Toxicology, 34: 183–189. Susheela, A.K. and Kumar, A. (1991) A study of the effect of high concentrations of fluoride on the reproductive organs of male rabbits, using light and scanning electron microscopy, Journal of Reproduction and Fertility, 92: 353–360. Tao, S. and Suttie, J.W. (1976) Evidence for lack of an effect of dietary fluoride level on reproduction in mice, Journal of Nutrition, 106: 1115–1122. Taves, D.R. (1968) Evidence that there are two forms of fluoride in human serum, Nature, 217: 1050–1051. Taves, D.R. (1983) Dietary intake of fluoride ashed (total fluoride) v. unashed (inorganic fluoride) analysis of individual foods, British Journal of Nutrition, 49: 295–301. Taylor, J.M., Scott, J.K., Maynard, E.A., Smith, F.A., and Hodge, H.C. (1961) Toxic effects of fluoride on the rat kidney. I. Acute injury from single large doses, Toxicology and Applied Pharmacology, 3: 278–289. Ten Cate, J.M. (1990) In vitro studies on the effects of fluoride on de- and remineralization, Journal of Dental Research, 69: 614–619. Theuer, R.C., Mahoney, W., and Sarrett, H.P. (1971) Placental transfer of fluoride and tin in rats given various fluoride and tin salts, Journal of Nutrition, 101: 525–532. Thylstrup, A. (1978) Distribution of dental fluorosis in the primary dentition, Community Dentistry and Oral Epidemiology, 6: 329–337. Thylstrup, A. (1983) Posteruptive development of isolated and confluent pits in fluorosed enamel, Scandinavian Journal of Dental Research, 91: 243–246. Urbansky, E.T. (2002) Fate of fluorosilicate drinking water additives, Chemical Reviews, 102: 2837–2854.


141

Weatherell, J.A., Deutsch, D., Robinson, C., and Hallworth, A.S. (1977) Assimilation of fluoride by enamel throughout the life of the tooth, Caries Research, 11: 85–115. Whitford, G.M. (1983) Fluorides: metabolism, mechanics of action and safety, Dental Hygiene, 57: 16–18, 20–22, 24–29. Whitford, G.M. (1987) Fluoride in dental products: safety considerations, Journal of Dental Research, 66: 1056–1060. Whitford, G.M. (1990) The physiological and toxicological characteristics of fluoride, Journal of Dental Research, 69: 539–549. Whitford, G.M., Pashley, D.H., and Stringer, G.I. (1976) Fluoride renal clearance: a pHdependent event, American Journal of Physiology, 230: 527–532. Whiting, P., McDonagh, M., and Kleijnen, J. (2001) Association of Down’s syndrome and water fluoride level: a systematic review of the evidence, BMC (BioMed Central) Public Health, 1: 6. WHO (World Health Organization). (1984) Fluorine and Fluorides, Environmental Health Criteria 36, International Programme on Chemical Safety (IPCS), Geneva, Switzerland. Xu, C.C. (1987) The study on the ability of selectively accumulating fluorine in tea leaves, Food Science, 5: 10–12 [in Chinese]. Zeiger, E., Shelby, M.D., and Witt, K.L. (1993) Genetic toxicity of fluoride, Environmental and Molecular Mutagenicity, 21: 309–318. Zhao, W., Zhu, H., Yu, Z., Aoki, K., Misumi, K., and Zhang, X. (1998) Long-term effects of various iodine and fluorine doses on the thyroid and fluorosis of mice, Endocrine Regulation, 32: 63–70. Zheng, B. and Huang, R. (1989) Human fluorosis and environmental geochemistry in southwest China, in Developments in Geoscience, Beijing: Chinese Academy of Sciences, 171–176.

6

Bacterial Contamination of Ready-to-Eat Foods: Concern for Human Toxicity Tony J. Fang

CONTENTS Abstract ..................................................................................................................143 Abbreviations .........................................................................................................144 Food-Borne Disease Outbreaks and Ready-to-Eat Foods ....................................144 Microbiological Quality of Ready-to-Eat Food Products.....................................146 Bacillus cereus...........................................................................................147 Escherichia coli and Coliforms .................................................................151 Listeria monocytogenes .............................................................................154 Salmonella spp...........................................................................................155 Staphylococcus aureus...............................................................................158 Risk Assessment and Food-Borne Microorganisms .............................................159 Improvement of Microbiological Quality of RTE Foods through HACCP .........161 Conclusions............................................................................................................163 References..............................................................................................................164

Abstract

The increasing availability of ready-to-use (RTU) and ready-to-eat (RTE) foods reflects consumer demand for convenient foods. In addition to convenience, consumers are also looking for RTE foods that are fresh, healthy, safe, additive free, and nutritious. Microbiological data from food-borne disease outbreaks have indicated that microorganisms play a very important role of the incidence. In Taiwan, Republic of China, the most frequent causes were attributable to Vibrio parahaemolyticus, followed by Staphylococcus aureus and Bacillus cereus. Salmonella spp., Campylobacter, and Escherichia coli O157 were the causative agents for most food-borne illness in Scotland. In the years 1990 to 2000, the U.S. Centers for Disease Control and Prevention (CDC) reported a total of 8797 food-borne outbreaks in the U.S., with Salmonella responsible for 1138 outbreaks (13% of the total

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outbreaks). Detection of pathogens including B. cereus, E. coli, E. coli O157, Listeria monocytogenes, Salmonella spp., and S. aureus on various types of RTE foods, such as 18°C products, are discussed. Risk assessment is the estimation of severity and the likelihood of harm resulting from exposure to a hazard. Four steps, including hazard identification, hazard characterization, exposure characterization, and risk characterization are involved in the risk assessment process. Microbiological risk assessments of pathogens including L. monocytogenes, Salmonella spp., E. coli O157:H7, B. cereus, Staphylococcus aureus, Vibrio spp., Campylobacter, and Clostridium have been published. The relationship between the hazard analysis critical control point (HACCP) system and microbiological quality of RTE foods is discussed. Because microbiological quality can be improved by implementing HACCP, the incidence of food-borne disease could also be reduced by HACCP implementation.

Abbreviations

CCP: critical control point; CDC: U.S. Centers for Disease Control and Prevention; CFU: colony-forming unit; DOH: Department of Health (Taiwan, Republic of China); EAggEC: enteroaggregative Escherichia coli; EHEC: enterohemorrhagic Escherichia coli; EIEC: enteroinvasive Escherichia coli; EPEC: enteropathogenic Escherichia coli; ETEC: enterotoxigenic Escherichia coli; HACCP: hazard analysis and critical control point; GMP: good manufacturing practice; MAP: modified atmosphere packaging; MRA: microbiological risk assessment; MPV: minimally processed vegetables; RTE: ready-to-eat; RTU: ready-to-use SFP: staphylococcal food poisoning

FOOD-BORNE DISEASE OUTBREAKS AND READY-TO-EAT FOODS In the past, people have bought foodstuffs in grocery stores to prepare meals at home. However, a growing number of people are buying ready-to-use (RTU) or ready-to-eat (RTE) foods so that they do not have to spend time cooking. RTU vegetables are fresh-cut, packaged vegetables requiring minimal or no further processing prior to consumption; RTE food products are preprocessed or precooked foods that are ready to eat without further processing before consumption, although some of them are heated before eaten. As the demand for RTE foods increases, a greater variety of RTE foods are becoming available. Many street-vended foods, for example, are RTE foods prepared and sold by vendors on streets and in similar public places (Dawson and Canet, 1991; Ekanem, 1998). They provide a source of readily available, inexpensive, nutritional meals, while providing a source of income for the vendors (Bryan et al., 1992; Ekanem, 1998). Another example of RTE products is RTE vegetables. Many prepared RTE vegetables are packaged in bags, and there is an increasing market for this type of product. In the U.K., sales of salad vegetables between 1995 and 2000 increased from US $1.56 billion to $1.70 billion. Prepared salad sales also rose significantly during the same period, from $471 million to $592 million (Sagoo et al., 2003). Various fresh produce products, such as salad

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vegetables and minimally processed vegetables (MPV), which are fresh raw vegetables sold RTE, have now become available to consumers year-round in areas such as European Union and the U.S. (Aureli, 1991). In Taiwan, Republic of China, there has been a marked increase in the sales of 18°C RTE food products in recent years. The concept of 18°C RTE food products was originally developed in Japan and adopted by the food industry in Taiwan. In the production of such food products, controlling the processing conditions is emphasized. For example, in the case of 18°C box meals, the critical control point (CCP) includes controlling cooking time and temperature. In addition, after the box meals are packaged, they are vacuum-cooled to 18 ± 2°C within 5 min. The rapid cooling method retards the growth of contaminating microorganisms. In general, the shelf life of 18°C RTE food products is about 20 h when they were displayed in stores and kept at 18°C. Rice balls rolled in seaweed, sandwiches, sushi, box meals, and cold noodles are the most common 18°C RTE food products sold in convenience stores in Taiwan (Fang, 2000). Although 18°C RTE food products have become more popular in recent years, the microbial quality of these products needs to be considered because 18°C is a temperature at which most microorganisms grow well. RTE food products provide a source of readily available and nutritious meals for the consumers; however, questions have been raised about the safety and microbiological quality of these food products. Microbiological quality or data from foodborne disease outbreaks can provide us with valuable information. Many countries publish statistical data on food-borne illness annually. In Taiwan, the data have been available from the Department of Health (DOH) since 1981 (Department of Health, 2003). A total of 1873 outbreaks were cumulatively reported to DOH from 1991 through 2002 (Table 6.1). The most frequent causes were Vibrio parahaemolyticus (802 outbreaks), Staphylococcus aureus (169 outbreaks), and Bacillus cereus (126 outbreaks). Chang and Chen (2003) reported that from 1991 through 2000, 274 outbreaks of food-borne illness including 12,845 cases and three deaths were confirmed in central Taiwan. Of the 274 reported outbreaks, 171 (62%) were caused by bacterial pathogens. Bacillus cereus (41%, 113 of 274 outbreaks), S. aureus (18%, 49 of 274 outbreaks), and V. parahaemolyticus (16%, 43 of 274 outbreaks) were the main etiologic agents (Table 6.2). In Scotland, 8, 13, and 22 food-borne outbreaks were reported in the years 1996, 1997, and 1998, respectively (World Health Organization, 2000). Salmonella spp., Campylobacter, and Escherichia coli O157 were the causative agents for most of the food-borne illness in Scotland, accounting for 44, 23, and 19%, respectively (World Health Organization, 2000). Although the U.S. food supply is among the safest in the world, large numbers of food-borne illness outbreaks continue to occur. In 1990, the Centers for Disease Control and Prevention (CDC) documented 533 food-borne outbreaks; however, the number of outbreaks increased to 1417 in the year 2000 (Table 6.3). From 1990 to 2000, the CDC reported a total of 8797 food-borne outbreaks in the U.S., with Salmonella responsible for 1138 outbreaks (13% of the total outbreaks) (Table 6.3). Lindqvist et al. (2000) summarized the food-borne disease incidents in Sweden from 1992 to 1997. A total of 555 incidents, involving 11,076 ill people, were reported. Diseases are a persistent threat to public health worldwide. The three main causative agents involved have bacterial, chemical, or natural origins, with bacterial

146


TABLE 6.1 Food-Borne Disease Outbreaks in Taiwan, 1991–2002 Percentage (%) of Outbreaks Caused by Bacterial Agents Year

No. of Outbreaks

Vibrio parahaemolyticus

Staphylococcus aureus

Bacillus cereus

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

93 88 77 102 123 178 234 180 150 208 178 262

13 (12)a 23 (20) 33 (25) 34 (35) 37 (46) 59 (105) 68 (160) 57 (102) 50 (75) 40 (84) 29 (52) 33 (86)

25 (23)b 21 (18) 31 (24) 13 (13) 10 (12) 4 (7) 6 (14) 2 (3) 4 (6) 11 (22) 5 (9) 7 (18)

14 (13)c 18 (16) 16 (12) 12 (12) 9 (11) 4 (7) 6 (15) 7 (12) 8 (12) 2 (5) 5 (8) 2 (4)

Total

1873

43 (802)

9 (169)

7 (126)

a

Data in parentheses indicate the number of outbreaks caused by Vibrio parahaemolyticus. Data in parentheses indicate the number of outbreaks caused by Staphylococcus aureus. cData in parentheses indicate the number of outbreaks caused by Bacillus cereus. b

Source: Department of Health (2003).

food-borne agents playing a leading role. Epidemiological data of the U.S. CDC between 1990 and 2000 reveal that bacterial pathogens accounted for 23% of total disease outbreaks (Table 6.4). In Taiwan, pathogens account for 58% of total incidents (Table 6.4). From 1981 to 1989, 622 outbreaks of food-borne illness were reported in Taiwan, and pathogenic microorganisms accounted for 80% of confirmed incidents (Chiou et al., 1991). Data collected from 1992 to 1997 in Sweden showed that, in 555 incidents, no disease agent was determined for 66% of the incidents. Bacterial agents were implicated in 25% and viruses in 8% of the incidents (Lindqvist et al., 2000).

MICROBIOLOGICAL QUALITY OF READY-TO-EAT FOOD PRODUCTS RTE foods, including 18°C products, provide a source of readily available and nutritious meals for consumers; however, assuring the safety and microbiological quality of these foods should become the first priority, especially because no heat treatment is given immediately before the foods are consumed. Investigations of the microbiological quality of various RTE or RTU food products, such as 18°C food products (cold noodles, box meals, rice balls rolled in seaweed, cone-shaped handrolled sushi, etc.) (Fang et al., 2002), vegetable salads (Albrecht et al., 1995;


147

TABLE 6.2 Food-Borne Disease Outbreaks in Central Taiwan, 1991–2000 Percentage (%) of Outbreaks Caused by Bacterial Agents Year

No. of Outbreaksa

1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

16 20 20 18 24 36 46 39 25 30

Total

274

Vibrio parahaemolyticus 13 5 5 6 8 17 20 28 16 20

(2)b (1) (1) (1) (2) (6) (9) (11) (4) (6)

16 (43)

Staphylococcus aureus 31 40 40 22 13 6 11 3 24 23

(5)c (8) (8) (4) (3) (2) (5) (1) (6) (7)

18 (49)

Bacillus cereus 25 (4)d 35 (7) 40 (8) 28 (5) 58 (14) 44 (16) 57 (26) 54 (21) 28 (7) 17 (5) 41 (113)

a

In the total of 274 outbreaks, the number of outbreaks caused by bacterial agents were 171. bData in parentheses indicate the number of outbreaks caused by Vibrio parahaemolyticus. cData in parentheses indicate the number of outbreaks caused by Staphylococcus aureus. dData in parentheses indicate the number of outbreaks caused by Bacillus cereus. Source: Chang, J.M. and Chen, T.H. (2003) Journal of Food and Drug Analysis, 11: 53–59. With permission.

Garcia-Gimeno et al., 1996; Kaneko et al., 1999; Odumeru et al., 1997; Sagoo et al., 2003), vacuum-packed vegetarian foods (Fang et al., 1999), bagged salad vegetables (Sagoo et al., 2003), cold and hot meals served by airlines (Hatakka, 1998a, b), cooked rice (Nichols et al., 1999), point-of-sale RTE rice (Nichols et al., 1999), street-vended foods (King et al., 2000; Kubheka et al., 2001; Mosupye and von Holy, 1999), RTE poultry stuffing from retail premises (Richardson and Stevens, 2003), hot-held foods (Chiou et al., 1996), catering dishes (Alberghini et al., 2000; Gillespie et al., 2000), sliced meat and meat products (Gillespie et al., 2000; Levine et al., 2001; Soriano et al., 2000; Tessi et al., 2002), and seafood (Hatha et al., 1998; Heinitz et al., 2000; Valdimarsson et al., 1998), have been reported. Some pathogens associated with RTE food products are discussed below.

BACILLUS

CEREUS

Bacillus cereus is an aerobic, spore-forming rod normally present in soil, dust, and water. It has been associated with food poisoning in Europe since at least 1906 (Jay, 2000a). This pathogen can be found in a number of food products, both fresh and processed. Bacillus cereus can give rise to two distinct forms of food-borne disease:

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TABLE 6.3 Food-Borne Disease Outbreaks in the U.S., 1990–2000 Percentage (%) of Outbreaks Caused by Bacterial Agents Year

No. of Outbreaks

No. of Cases

Salmonella spp.

Staphylococcus aureus

Escherichia coli O157:H7

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000

533 531 411 514 690 645 602 806 1,314 1,334 1,417

19,231 15,052 11,083 14,080 16,995 13,497 15,421 18,802 26,719 25,286 26,043

26 (138)a 23 (123) 20 (80) 19 (95) 14 (98) 15 (94) 13 (80) 11 (91) 9 (124) 9 (113) 8 (112)

2 (13)b 2 (9) 2 (7) 1 (7) 2 (13) 1 (6) 1 (8) 1 (10) 1 (15) 2 (18) 2 (22)

0.4 (2)c 1 (3) 1 (3) 3 (14) 4 (24) 4 (25) 2 (12) 1 (11) 2 (26) 2 (24) 2 (25)

Total

8,797

202,209

13 (1,148)

2 (128)

2 (169)

a

Data in parentheses indicate the number of outbreaks caused by Salmonella spp. Data in parentheses indicate the number of outbreaks caused by Staphylococcus aureus. cData in parentheses indicate the number of outbreaks caused by Escherichia coli O157:H7. b

Source: Centers for Disease Control and Prevention (2003).

emetic and diarrheal syndromes. Cooked rice was first recognized as a cause of foodborne disease outbreak through contamination with B. cereus in 1971 (Mortimer and McCann, 1974). Since then, many outbreaks associated with this pathogen have been reported in Japan, Canada, Finland, the Netherlands, and the U.S. (Beckers, 1976; Khodr et al., 1994; Raevuori et al., 1976; Schmitt et al., 1976; Shinagawa et al., 1979; Terranove and Blake, 1978). Contamination may be introduced from boiled rice, mashed potatoes, and other cooked foods. Moreover, the incidence of B. cereus has been directly related to the temperature of storage and the length of time the food is kept before serving (Jaquette and Beuchat, 1998; Nichols et al., 1999). Bacillus cereus is emerging as an important food-poisoning organism because of its cosmopolitan distribution. A review by Granum and Lund (1997) indicated that B. cereus had become one of the more important causes of food poisoning in the industrialized world. This pathogen has usually been isolated from the samples of raw rice and thus can be considered part of its normal flora (Parry and Gilbert, 1980). The incidence of Bacillus in RTE foods varies widely, from 0 to 100% (Table 6.5). Mosupye and von Holy (1999) investigated the microbiological quality of RTE street-vended food products in Johannesburg, South Africa. In their study, 51 samples were taken for determination of the microbiological quality; B. cereus was detected in 22%. Kaneko et al. (1999) collected 196 samples from two food factories located in the suburbs of Tokyo to examine the bacterial contamination of RTE vegetables


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TABLE 6.4 Percentage of Total Food-Borne Disease Outbreaks Caused by Bacterial Pathogens Reported in the U.S. and Taiwan Percentage (%) of Total Food-Borne Outbreaks Caused by Bacteria Year

U.S.a

Taiwan, ROCb

1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002

37 33 29 31 25 25 23 19 20 17 16 NA NA

NAc 45 56 70 61 61 69 76 63 61 56 44 42

Average

23

58

a

The total number of food-borne illness outbreaks in the U.S. from 1990 to 2000 was 8797. bThe total number of food-borne illness outbreaks in Taiwan from 1991 to 2002 was 1873. cNA: Data not included. Source: Centers for Disease Control and Prevention (2003) and Department of Health (2003).

in the various processing steps including trimming, washing, slicing, soaking, dehydrating, blending, and packaging. High aerobic plate counts were found in most samples even after preparation. Bacillus cereus was detected at rates of 10 and 20% before and after preparation, respectively. Fang et al. (2002) investigated the microbiological quality of 18°C RTE foods. For 18°C RTE sushi, 18°C RTE cone-shaped hand-rolled sushi, 18°C RTE sandwiches, 18°C RTE rice balls rolled in seaweed, and 18°C RTE cold noodles, 18, 40, 54, 56, and 67% of samples were positive for B. cereus, respectively (Table 6.5). In one study, all mashed potatoes and skim milk tested were contaminated with B. cereus (Harmon and Kautter, 1991); similar results were found when cooked vegetables and salad were examined for incidence of B. cereus (Tessi et al., 2002). Bryan et al. (1992) isolated B. cereus from various potato items offered for sale by street vendors in Pakistan. They indicated that the source of contamination was thought to be human handling.

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TABLE 6.5 Bacillus cereus Contamination in Some RTE Products

Product

No. of Samples Analyzed

Percentage (%) of Positive Samples

Range (log CFU g–)

Ref.

4

0 (0)a

NAb

147

0 (0)

NA

12 4162 113

0 (0) 1 (29) 2 (2)

NA 2.0–6.0 7.0

Reviews in Food and Nutrition Toxicity, Volume 4 (Reviews in Food and Nutrition)

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