Hair in Toxicology: An Important Bio-Monitor

Hair in Toxicology An Important Bio-monitor

Issues in Toxicology Series Editors Professor Diana Anderson, University of Bradford, UK Dr Michael D Waters, National Institute of Environmental Health Science, N Carolina, USA Dr Timothy C Marrs, Food Standards Agency, London, UK This Series is devoted to coverage of modern toxicology and assessment of risk and is responding to the resurgence in interest in these areas of scientific investigation. Ideal as a reference and guide to investigations in the biomedical, biochemical and pharmaceutical sciences at the graduate and post graduate level. Titles in the series: Hair in Toxicology: An Important Bio-Monitor Edited by Desmond John Tobin, University of Bradford Visit our website on www.rsc.org/issuesintoxicology For further information please contact: Sales and Customer Services Royal Society of Chemistry Thomas Graham House Science Park, Milton Road Cambridge CB4 0WF Telephone +44 (0)1223 432360, Fax +44 (0)1223 426017, Email [email protected]

Hair in Toxicology

An Important Bio-monitor

Edited by

Desmond John Tobin University of Bradford, UK

ISBN 0-85404-587-2 A catalogue record for this book is available from the British Library # The Royal Society of Chemistry 2005 All rights reserved Apart from fair dealing for the purposes of research for non-commercial purposes or for private study, criticism or review, as permitted under the Copyright, Designs and Patents Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royal Society of Chemistry, or in the case of reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of the licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printed on this page. Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 0WF, UK Registered Charity Number 207890 For further information see our web site at www.rsc.org Typeset by Alden Bookset, Northampton, UK Printed by Athenaeum Press Ltd, Gateshead, Tyne and Wear, UK

Dedication I dedicate this book to my former mentors who were instrumental in providing me with the opportunities and support to lay the foundations of my academic career: the late Aoda´n Breathnach who took me under his wing while he was Emeritus Professor at St. Thomas’s Hospital, London, during my PhD student days; JeanClaude Bystryn, who patiently and generously facilitated my development as an independent scientist while a post-doctoral fellow in his laboratory at NYU Medical Center, New York; and Ralf Paus, who through his unique combination of fun, fairness and frenetic activity, inspired me to attempt cultivation of these same qualities. This book is also dedicated to my parents and family in Ireland, who from the start encouraged, supported and indulged me – Go raibh mı´le maith agaibh go le´ir – foai sca´th a che´ile a mhaireann na daoine! [English: Thank you all very much – people depend on each other in life!]

Preface While the estate agent may chant ‘location, location, location!’, and the stock exchange trader shout ‘‘Buy, sell, buy!’’, the editor of a book devoted to hair has to engage in a little bit of buying, selling and unmasking of the hair follicle as the dream location for unrivalled access to myriad processes underlying much of modern and ancient human life and lifestyle. This book, entitled Hair in Toxicology – An Important Bio-Monitor, is part of a new book series Issues in Toxicology from The Royal Society of Chemistry. It is the first book of this kind to be devoted exclusively to the hair follicle and its shaft as an important tool in modern toxicology. This multi-author book will serve as both a reference and a guide to investigations in the biomedical, biochemical and pharmaceutical sciences. Written by investigators from the fields of chemistry, biochemistry and biology, the authors have first-hand knowledge from their chosen sub-specialities and are active contributors to the peer-reviewed scientific literature. Hair in Toxicology – An Important Bio-monitor is divided into four cognate sections, reflecting the range of interest in the exploitation of this bio-resource to provide valuable information about the world we live in today and have lived in yesterday. The book’s ‘womb to tomb/cradle to grave’ scope begins with a section reviewing our current knowledge of the biology of hair growth: how it cycles, how it generates a fibre, and the value of its component pigment. This section sets the scene by explaining why the hair should serve as a unique bio-resource in toxicology and is followed by a section that considers issues that emerge from the hair’s ability to capture snap-shots of our diverse interactions with the environment, both during and after life, and is contributed by experts in the forensic and environmental sciences. From there we proceed to a section that concentrates on several important toxicological issues to emerge from current strategies for the ‘personal care’ of hair. Finally, the book draws to a close with an assessment of the hair fibre’s contribution to our understanding of human life from archeological and historical investigations. A final perspective on future directions in the use of hair in toxicology closes the book. The increasing sophistication required of forensics, toxicology, and health and personal care product development requires the exploitation of novel non-invasive approaches to source accurate and vital information. The hair fibre and follicle stand alone in fulfilling these criteria. Thus, there is no better time to shout ‘location, location, location!’ for the hair and its follicle within the mammalian system, and even ‘Buy, sell, buy’ for those who make their business from this wondrous miniorgan. But for even the most detached observer, we hope that a perusal of these chapters may instill some sense of the ‘Wow!’ factor that is the hair follicle.

vii

Contents Editor

xix

Contributors

xx Part 1 – Biology of Hair

Chapter 1 The Biogenesis and Growth of Human Hair Desmond J. Tobin 1.1

1.2 1.3

1.4 1.5

The Hair Follicle Mini-organ 1.1.1 Introduction 1.1.2 A Unique Mammalian Epithelial-MesenchymalNeuroectodermal Interactive System 1.1.3 Comparison with Other Keratinised Skin Appendages – The Nail Embryology of the Hair Follicle 1.2.1 Hair Follicle Induction 1.2.2 Hair Follicle Cell Differentiation Regulation of Hair Growth 1.3.1 The Hair Growth Cycle 1.3.1.1 Introduction 1.3.1.2 Catagen 1.3.1.3 Telogen 1.3.1.4 Anagen 1.3.2 Hair Follicle Stem Cells 1.3.3 Systemic and Intrinsic Influences on Hair Growth 1.3.3.1 Introduction 1.3.3.2 Hair Follicle Immunology 1.3.3.3 Hormones and Hair Growth Hair Growth Pattern and Type Hair Growth Rate and Fibre Diameter/Calibre ix

3 3 3 4 4 5 5 7 10 10 10 13 14 14 16 17 17 17 19 21 21

x

Contents

1.6

Racial and Ethnic Characteristics of Hair Growth 1.6.1 Body Site Variation in Hair Growth Patterns 1.7 Aging of the Hair Follicle and Human Scalp 1.8 Common Disorders Affecting Human Hair Growth 1.8.1 Introduction 1.8.2 Male Pattern Alopecia 1.8.3 Female Pattern Alopecia 1.8.4 Alopecia Areata 1.8.5 Telogen Effluvium 1.9 Conclusion 1.10 Acknowledgements 1.11 References

Chapter 2 2.1 2.2

2.3

2.4

2.5

2.6 2.7

The Human Hair Fibre Desmond J. Tobin Psychological Issues Surrounding Hair Anatomy of the Human Scalp Hair Fibre 2.2.1 Hair Fibre Surface – The Cuticle 2.2.2 Hair Fibre – The Cortex 2.2.3 Hair Fibre – The Medulla Biosynthesis of the Hair Fibre 2.3.1 Hair Keratins 2.3.1.1 Keratin-associated Proteins 2.3.1.2 Biochemical Aspects of Hair Keratinisation 2.3.1.3 Nutrition and Hair Fibre Proteins 2.3.1.4 Genetic Variation in Hair Fibre Proteins Physical Properties of the Hair Fibre 2.4.1 Hair fibre Lipids 2.4.2 Mechanical Properties of the Hair Fibre 2.4.2.1 Hair Fibre Curvature 2.4.3 Ethnic Variations in Hair Structure 2.4.4 Aging and Hair Fibre Form Hair Shaft Abnormalities 2.5.1 Features of Damage in Normal Hairs 2.5.2 Hair Shaft Dysplasias 2.5.3 Hair Shaft Abnormalities with Increased Hair Fragility 2.5.4 Hair Shaft Abnormalities without Increased Hair Fragility 2.5.5 Hair Shaft Abnormalities Due to Cosmetic Use Summary References

22 23 23 24 24 26 27 27 29 29 29 29

34 34 35 36 39 39 40 41 42 43 43 43 44 44 44 46 47 48 50 50 50 50 52 52 54 54

Contents

xi

Chapter 3 Pigmentation of Human Hair

57

Desmond J. Tobin 3.1 3.2 3.3

Overview Evolutionary Context for Hair Pigmentation Embryological Development of the Hair Follicle Pigmentary Unit 3.4 Biology of the Mature Hair Follicle Pigmentary Unit 3.4.1 Melanocyte – Keratinocyte Interactions in the Hair Follicle 3.5 Biochemistry of Melanin Biosynthesis 3.5.1 Melanosome Organellogenesis 3.5.1.1 Intra- and Inter-racial Variation in Human Scalp Hair Colour 3.5.2 Biosynthesis of Melanins (Eumelanin and Pheomelanin) 3.5.3 Physico-Chemical Aspects of Hair Melanins 3.6 Regulation of Hair Pigmentation 3.6.1 Hair Growth Cycle Influences 3.6.1.1 Telogen to Anagen Transition 3.6.1.2 Anagen to Catagen Transition 3.6.1.3 Fate of Pigmented Melanocytes During Catagen 3.6.1.4 Pigment Incontinence During Catagen 3.6.2 Hormonal Influences and Pigmentation 3.6.2.1 Positive Regulators of Follicular Melanogenesis 3.6.2.2 Negative Regulators of Follicular Melanogenesis 3.6.3 Nutrition and Hair Pigmentation 3.6.4 Transfer and Degradation of Melanin Granules to Hair Shaft Keratinocytes 3.7 Aging of the Hair Follicle Pigmentary Unit 3.7.1 Molecular Aspects of Melanocyte Aging 3.7.2 Onset and Progression of Hair Graying 3.7.3 Pathogenesis of Loss of Hair Pigmentation 3.7.4 Impact of Pigment Loss on Hair Fibre Structure 3.7.5 Can Canities Serve as a Marker for Disease? 3.7.6 Is Canities Reversible? 3.8 Conclusion 3.9 Acknowledgements 3.10 References

57 57 59 59 61 63 63 64 64 65 66 66 67 68 68 70 70 70 72 72 73 73 74 75 76 78 79 79 80 80 80

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Contents

Part 2 – Application of Hair Biology to Environmental Assessments Chapter 4

Hair in Forensic Toxicology with a Special Focus on Drug-Facilitated Crimes

89

Pascal Kintz and Marion Villain 4.1 4.2 4.3 4.4 4.5 4.6 4.7

4.8 4.9

Chapter 5

Introduction Mechanisms of Drug Incorporation into Hair Specimen Collection Stability of Drugs in Hair Hair Analysis Applications of Hair Analysis Special Focus on Drug-Facilitated Crimes 4.7.1 Case 1 4.7.2 Case 2 4.7.3 Case 3 4.7.4 Case 4 4.7.5 Case 5 4.7.6 Case 6 4.7.7 Case 7 4.7.8 Case 8 4.7.9 Case 9 4.7.10 Case10 Discussion References

Hair and Human Identification

89 90 91 91 91 93 94 95 95 95 96 96 97 97 98 98 99 99 103

104

Bruce A. Benner Jr. and Barbara C. Levin 5.1 5.2

5.3

5.4 5.5

Introduction Background 5.2.1 General Composition and Characteristics of Hair 5.2.2 Growth Phases of Hair 5.2.3 Hair as Forensic Evidence: Interpretation and Legal Considerations Forensic Analysis of Hair for Identification 5.3.1 Microscopy 5.3.2 DNA Sequencing 5.3.2.1 Nuclear DNA 5.3.2.2 Mitochondrial DNA 5.3.3 Chemical Characterisations of Hair – Internal and Surface Components Summary Acknowledgements

104 105 105 106 107 108 108 110 110 112 116 121 121

Contents

xiii

5.6 5.7

Disclaimer References

Chapter 6 Hair and Metal Toxicity

122 122

125

Stefanos N. Kales and David C. Christiani 6.1 6.2

6.3

6.4

6.5

6.6

6.7

6.8 6.9

Overview Mercury 6.2.1 Toxicology 6.2.2 Kinetics and Relation to Hair 6.2.3 Hair vs. Other Biomarkers 6.2.4 Indications for Hair Analysis Arsenic 6.3.1 Toxicology 6.3.2 Kinetics and Relation to Hair 6.3.3 Hair vs. Other Biomarkers 6.3.4 Indications for Hair Analysis Lead 6.4.1 Toxicology 6.4.2 Kinetics and Relation to Hair 6.4.3 Hair vs. Other Biomarkers 6.4.4 Indications for Hair Analysis Cadmium 6.5.1 Toxicology 6.5.2 Kinetics and Relation to Hair 6.5.3 Hair vs. Other Biomarkers 6.5.4 Indications for Hair Analysis Manganese 6.6.1 Toxicology 6.6.2 Kinetics and Relation to Hair 6.6.3 Hair vs. Other Biomarkers 6.6.4 Indications for Hair Analysis Thallium 6.7.1 Toxicology 6.7.2 Kinetics and Relation to Hair 6.7.3 Hair vs. Other Biomarkers 6.7.4 Indications for Hair Analysis Commercial Hair Tests and Their Potential Misuses Important Methodological Issues in Hair Metal Analysis 6.9.1 Sampling 6.9.2 Cosmetic/Hygiene Products 6.9.3 Sample Washing 6.9.4 Sample Preparation 6.9.5 Analytical Methods 6.9.6 Targeted Analyses

125 126 126 127 127 135 135 135 135 136 140 140 140 140 140 141 141 141 141 142 142 142 142 143 143 146 146 146 146 147 147 147 151 151 152 152 152 152 153

xiv

Contents

6.10

Chapter 7

7.1 7.2 7.3 7.4 7.5 7.6 7.7

Chapter 8 8.1 8.2 8.3 8.4

8.5

8.6

6.9.7 Interpretation References

Hair and Exposure to Environmental Pollutants Vladimı´r Bencko Monitoring of Environmental Pollution The Hair Fibre as a Biomarker of Human Exposure to Metals and Inorganic Substances Advantages and Limitations of Hair Fibre Analysis as a Biomarker of Human Exposure to Trace Elements Washing of Hair Samples Discussion Conclusions References

Hair and Nutrient/Diet Assessment Tamsin O’Connell Hair and Diet Diet and Nutritional Investigations Using Hair – a Brief Outline Advantages of Using Hair as a Study Tissue Problems Associated with the Use of Hair as a Study Tissue 8.4.1 Correlation with Diet and Body Pools 8.4.1.1 Isotope Ratios of Light Elements 8.4.1.2 Trace Metal Analysis in Hair 8.4.2 Endogenous Variability in Chemical Signals from Hair 8.4.2.1 Isotope Ratios of the Light Elements Carbon, Nitrogen, Sulfur, Oxygen and Hydrogen 8.4.2.2 Trace Metals 8.4.3 Hair Growth Rates 8.4.4 Hair Contamination Analysis and Data Interpretation 8.5.1 Analytical Methods 8.5.2 Pre-treatment of Hair for Analysis 8.5.3 Data Interpretation Applications 8.6.1 Dietary Intake and Hair Signals 8.6.2 Nutritional Status and Hair Signals 8.6.3 Diseases and Illnesses Associated with Changes in Hair Composition

153 153

159 159 160 166 170 170 171 172

175 175 176 176 177 177 177 178 178 178 180 181 181 182 182 183 183 183 184 185 187

Contents

xv

8.7 8.8

Summary References

188 189

Part 3 – Chemistry and Toxicology of Personal Hair Care Products Chapter 9 Hair Colorant Chemistry Thomas Clausen and Wolfgang Balzer 9.1 9.2 9.3

9.4 9.5 9.6 9.7 9.8

Chapter 10

197

Introduction Hair Dyes: Bleaching 9.2.1 Chemistry of Bleaching 9.2.2 Bleaches Hair Dyeing 9.3.1 Permanent Hair Dyes 9.3.1.1 Dye Precursors 9.3.1.2 Dye Oxidation 9.3.1.3 Primary Intermediates 9.3.1.4 Couplers 9.3.1.5 Other Dye Intermediates 9.3.2 Semipermanent and Temporary Hair Dyes 9.3.2.1 Nitro Dyes 9.3.2.2 Cationic (Basic) Dyes 9.3.2.3 Anionic (Acidic) Dyes 9.3.2.4 Other Dyes 9.3.3 Dyeing with Inorganic Compounds Product Forms Dye-removal Preparations Testing Hair Dyes Toxicology References

197 197 197 199 199 199 199 200 201 202 203 203 203 204 205 205 205 206 207 207 208 208

Hair Dyes and Skin Allergy G. Frank Gerberick and Cindy A. Ryan

212

10.1 Introduction 10.1.1 Biology of Skin Sensitisation and Allergic Contact Dermatitis 10.1.2 Skin Penetration 10.1.3 Protein Reactivity and Skin Metabolism 10.2 Predictive Testing of Hair Dyes for Skin Sensitisation 10.2.1 Animal Models 10.2.2 Predictive Testing of Hair Dyes in Humans 10.3 Human Diagnostic Patch Testing

212 212 213 215 216 216 219 220

xvi

Contents

10.3.1 Dermatology Patient Population 10.3.1.1 Occupational Skin Allergy to Hair Dyes 10.3.2 General Population 10.4 Summary and Future Directions 10.5 References

Chapter 11

Hair Colorant Use and Associated Pathology – Cancer? Tongzhang Zheng, Yawei Zhang, Yong Zhu and Lindsay Morton

11.1 Introduction 11.2 Epidemiological Studies of Hair Dye Use and Human Cancer Risk 11.2.1 Personal Use of Hair Dyes and Human Cancer Risk 11.2.1.1 Prospective Follow-up Studies 11.2.1.2 Case-control Studies 11.2.2 Hair Dye Use and Human Cancer Risk by Gene Type 11.2.3 Occupational Exposure to Hair Dyes and Risk of Human Cancer 11.2.3.1 Bladder Cancer 11.2.3.2 Breast Cancer 11.2.3.3 Ovarian Cancer 11.2.3.4 Lung Cancer 11.2.3.5 Non-Hodgkin’s Lymphoma 11.2.3.6 Hodgkin’s Disease 11.2.3.7 Multiple Myeloma 11.2.3.8 Leukemia 11.2.3.9 Cancers at Other Sites 11.2.3.10 Childhood Cancers 11.3 Biological Plausibility 11.4 Conclusions 11.5 References

Chapter 12

The Chemistry of Hair Care Products: Potential Toxicological Issues for Shampoos, Hair Conditioners, Fixatives, Permanent Waves, Relaxers and Depilatories Janusz Jachowicz

12.1 Introduction 12.2 Safety Evaluations 12.2.1 Animal Safety Tests

220 222 223 224 224

229

229 230 230 230 232 253 254 254 260 260 260 267 267 267 267 276 276 276 280 281

286 286 287 288

Contents

xvii

12.2.1.1 12.2.1.2 12.2.1.3 12.2.1.4 12.2.1.5 12.2.1.6

12.3 12.4

12.5 12.6 12.7 12.8 12.9

Chapter 13

Acute Toxicity Acute Dermal Irritation/Corrosion Acute Eye Irritation Skin Sensitisation 90-Day Inhalation Chronic Toxicity – Repeated Insult Patch Test 12.2.1.7 Mutagenicity and Carcinogenicity 12.2.1.8 Teratogenicity 12.2.1.9 Photo-toxicity and Photo-allergenicity 12.2.2 Non-animal Safety Testing 12.2.2.1 Cell Toxicity Test 12.2.2.2 Primary Eye Irritation 12.2.3 Biodegradability Cosmetic Ingredient Review Review of the Chemistry of the Main Hair Care Product Categories 12.4.1 Hair Shampoos 12.4.2 Hair Conditioners 12.4.3 Hair Waving and Depilating Products 12.4.4 Hair Relaxing Products 12.4.5 Hair Styling Products Preservatives Ingredients with Restricted Use Conclusions Acknowledgements References

Hair Care Products – Regulatory Issues P. Raniero De Stasio

13.1 Introduction 13.2 Hair Product Regulations 13.2.1 European Regulations 13.2.1.1 The EU Cosmetics Directive 13.2.1.2 Specific Considerations on the EU Cosmetic Directive 13.2.2 US Regulations 13.2.3 Japanese Regulations 13.2.4 Some Regulatory Considerations for Other Countries 13.3 Hair Colorants – Special Considerations 13.3.1 Cancer Claims 13.3.2 Allergy 13.4 Conclusions 13.5 References

288 288 288 288 289 289 289 290 290 290 290 291 291 291 291 291 295 298 300 301 302 305 305 307 308

311 311 312 312 312 314 314 315 316 316 316 317 317 318

xviii

Contents

Part 4 – Hair in Archaeology Chapter 14

Hair as a Bioresource in Archaeological Study Andrew S. Wilson

14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 14.10 14.11 14.12 14.13 14.14

Chapter 15

The Value of Hair in Bioarchaeology – An Introduction The Early Use of Hair as a Resource for Physical Anthropology Hair as an Indicator of Past Diet and Population Movement Trace Elements from Archaeological Hair and Their Controversial Use in Dietary Analysis and Disease Recognition Hair as an Indicator of Exposure to Pollutants and as a Record of Micro-environment A Record of Drug Use in Archaeological Hair Samples Hair as a Source of Genetic Information Accelerator Mass Spectrometry Radiocarbon Dating of Hair Hair Grooming Practice and Cosmetic Treatment Hair Stylistic Information and Symbolism Caveats in Using Archaeological Hair – Taphonomy, Contamination and Curation History Summary Acknowledgements References

A Perspective on Future Directions Desmond J. Tobin

Subject Index

321 321 323 324 327 328 330 332 332 333 334 335 335 336 336

345

349

Editor Desmond J. Tobin, PhD, was born in Ireland in 1965. After graduating with a BSc. (Biology, Chemistry and Maths) from the National University of Ireland, Maynooth in 1986, and a graduateship of the Institute of Biology in Immunology from NESCOT, Surrey, England in 1988, he joined the Division of Biochemsitry at St.Thomas’s Hospital Medical School, London where he obtained his PhD degree in 1991. His post-doctoral training was conducted at the Dept. of Dermatology, New York University Medical School, which was followed by a period as Assistant Research Professor in this Department until 1996. Returning to Europe in 1996 for a Lectureship in Biomedical Sciences at the University of Bradford, England, Desmond Tobin is currently Professor of Cell Biology at this Department. Prof.Tobin has authored or co-authored over 100 papers, reviews and chapters.

xix

Contributors Wolfgang Balzer PhD Corporate Vice President R&D Colorants, Wella AG, Berliner Allee 65, D – 64274 Darmstadt, Germany. [email protected]

Prof. David C. Christiani, MD Professor and Director, Occupational Health Program & Professor of Occupational Medicine & Epidemiology, Harvard School of Public Health, Professor of Medicine, Harvard Medical School, Cambridge, MA, USA. [email protected]

Bruce A. Benner PhD Analytical Chemistry Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD20899, USA. [email protected]

Thomas Clausen PhD Vice President R&D, Wella AG, Berliner Allee 65, D – 64274 Darmstadt, Germany.

Vladimir Bencko, MD, PhD Professor and Head, Institute of Hygiene & Epidemiology, First Faculty of Medicine, Charles University of Prague, Studnickova 7, CZ 128 00 Prague 2 Czech Republic. [email protected]

P. Raniero De Stasio PhD Proctor & Gamble, Rusham Park Technical Centre, Whitehall Lane, Egham, Surrey TW20 9NW UK. [email protected] xx

Contributors

xxi

G. Frank Gerberick, PhD The Procter & Gamble Company Miami Valley Laboratories P.O. Box 538707 Cincinnati, OH 45253–8707 USA. [email protected]

Lindsay Morton Division of Environmental Health Sciences, Yale School of Public Health 60 College Street, New Haven C7 065 20, USA.

Janusz Jachowicz PhD 1361 Alps Rd., Wayne, NJ 07470, USA. [email protected]

Tamsin O’Connell PhD McDonald Institute for Archaeological Research, University of Cambridge, Downing Street, Cambridge CB2 3ER, UK. [email protected]

Stefanos N. Kales, MD Professor and Medical Director, Employee Health & Industrial Medicine, Cambridge Health Alliance & Assistant Professor, Harvard Medical School & Harvard School of Public Health, Dept. of Environmental Health, Macht Bldg., Room 427, The Cambridge Hospital, 1493 Cambridge Street, Cambridge, MA 02139, USA. [email protected] Pascal Kintz PhD Laboratoire ChemTox, 3 rue Gruninger, F-67400 Illkirch France. [email protected] Barbara C. Levin Analytical Chemistry Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technolgy, Gaithersburg, MD 20899, USA.

Cindy A. Ryan PhD The Proctor & Gamble Company, Miami Valley Laboratories, P.O. Box 538707 Cincinnati, OH 45253–8707 USA.

Desmond J. Tobin PhD. Professor of Cell Biology, Biomedical Sciences, School of Life Sciences, University of Bradford Bradford, West Yorkshire, BD7 1DP, UK. [email protected]

Marion Villain PhD Laboratoire ChemTox, 3 rue Gruinger, F-67400 Illkirch France. [email protected]

xxii

Contributors

Andrew S. Wilson PhD Wellcome Trust Research Fellow in Bioarchaeology, Dept. of Archaeological Sciences & Dept. of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK. [email protected]

Yawei Zhang Division of Environmental Health Sciences Yale school of Public Health 60 College Street New Haven, CT 06520, USA.

Tongzhang Zheng, BMed, ScD, ScM Associate Professor and Head, Division of Environmental Health Sciences, Yale School of Public Health, 60 College Street, New Haven, CT 06520, USA. [email protected]

Yong Zhu Division of Environmental Health Sciences Yale school of Public Health 60 College Street New Haven, CT 06520, USA.

Part 1 Biology of Hair

CHAPTER 1

The Biogenesis and Growth of Human Hair DESMOND J. TOBIN

1.1 The Hair Follicle Mini-organ 1.1.1 Introduction As social beings we communicate significantly via our physical appearance and so together with epidermal pigmentation the hair fibre-producing mini-organ accounts for most of the variation in the phenotype of different mammals and between different human population groupings. Although commonly dismissed as being of superficial importance, the hair follicle(s) (HF) is truly one of human biology’s most fascinating structures.1 Hair growth, one of only two uniquely mammalian traits (in addition to mammary glands), serves several important functions. These include thermal insulation, camouflage (melanin affords significant protective value, e.g. change of coat colour in the arctic fox with season), social and sexual communication (involving visual stimuli, odorant dispersal etc.), sensory perception (e.g. whiskers), and protection against trauma, noxious insults, insects, etc. These features have clearly facilitated evolutionary success in animals, but it is not immediately clear how these may have proved critical for human survival. That said, one should not diminish the role of hair in social and sexual communications among humans. Because of our relative nakedness most attention and study is focused on scalp hair that, uniquely amongst primates, can be very thick, very long and very pigmented. Conversely, its absence from the human scalp can result in significant psychological trauma2, e.g. in cases of androgenetic alopecia, alopecia areata and chemotherapy-induced alopecia. Our ancient pre-occupation with hair is further heightened today as our increasing longevity inevitably fuels our desire to extend youthfulness. This increasing attention to hair-care is reflected in the unremitting growth of the hair-care market, already a multi-billion euro enterprise world-wide (Euromonitor). Unlike most other mammals, we humans have all but lost our ability to grow hair synchronously or as a wave. Instead, our hair grows in a mosaic pattern where significant autonomy of growth and pigmentation resides in individual HF. 3

4

Chapter 1

The evolutionary selective pressure for why humans developed such a luxurious growth of pigmented scalp hair is more perplexing. One possible explanation may relate to the hair fibre as a dispersal conduit for pigment – melanin is an avid binder of a broad range of toxins and metals (for further discussion see Chapter 3 in this volume). This view, advanced by Hardy, derives from the evolution of early humans along riverbanks and seacoasts.3 As such, a diet rich in fish, concentrators of heavy metals, could have had significant health implications. A mechanism to quickly remove these toxic metals, thereby preventing their build-up in the body, may have been exploited by melanin’s capacity to bind these compounds into a rapidly dividing tissue that ultimately keratinises to form the hair fibre. The hair bulb exhibits the body’s second highest rate of proliferation (after hematopoietic tissue) and so could swiftly incorporate metals and toxins into a pigmented and cornified hair shaft,4 and in this way limit their access to the living tissue of the highly vascularised scalp.

1.1.2 A Unique Mammalian Epithelial-MesenchymalNeuroectodermal Interactive System The HF or more accurately the ‘pilo-sebaceous unit’ encapsulates all the important physiological processes found in the human body, namely controlled cell growth/ death, interactions between cells of different histologic type, cell differentiation and migration, hormone responsitivity etc. Thus, the value of the HF as a model for biological scientific research goes way beyond its scope for cutaneous biology or dermatology alone. Indeed, the recent and dramatic upturn in interest in HF biology has focused principally on the pursuit of two of biology’s holy grails: postembryonic morphogenesis and control of cyclical tissue activity. The HF mini-organ is formed from a bewilderingly complex set of interactions involving ectodermal, mesodermal and neuroectodermal components, which go to elaborate five or six concentric cylinders of at least fifteen distinct interacting cell sub-populations. These together provide a truly exceptional tissue5 that rivals the vertebrate limb-bud6 as a model for studies of the genetic regulation of development. An important consideration for the remit of this book is that the formation of the HF product, its fibre, occurs in a highly time-resolved manner and so locks in a snap-shot of the individual’s physiology and chemistry at the time of the hair fibre’s formation. Thus, the hair fibre does not undergo further biogenic change.

1.1.3 Comparison with Other Keratinised Skin Appendages – The Nail Hair, scales, feathers, claws, horns and nails are all derived from skin and so all consist of keratinised modified epidermal cells.7 Like the hair fibre, the biological and chemical composition of the nail is not altered by changes in the blood chemistry or by exposure to toxins, chemicals etc. occurring after these structures were formed (i.e. no post-biogenic change). Therefore, both hair and nails are of

The Biogenesis and Growth of Human Hair

5

major interest to toxicologists and to those interested in forensic and medico-legal investigations. The slower growth of nail (toenail, 0.05 mm per day: finger nail, 0.1 mm per day) compared with human scalp hair fibres ( 235.3 2.09e4 Area

% −1 9.17;7005

281003_4 Sm (SG, 2x2) 100 %

MRM of 16 Channels ES+ 290.1 > 154.1 7.21e4 Area

−1 4.00 4.50 5.00 5.50 6.00 6.50

Time 7.00 7.50 8.00

8.50 9.00 9.50 10.00 10.50 11.00 11.50 12.00

Figure 4.1 Chromatogram of a hair extract. Top: two daughter ions of zolpidem (at 4.4 pg/mg), Bottom: daughter ion of diazepam-d5, used as internal standard

zolpidem at concentrations of 22, 47, 67 and 9 pg/mg from the root to the tip. This demonstrated repetitive exposure to zolpidem before the alleged assault and therefore made the blood result inconclusive.

4.7.4 Case 4 During a party, a 42-year-old man was offered an alcoholic drink. Soon after, he lost all recollection of events and awoke four hours later in a bed with a woman. Terrified, as he was married, he privately requested us to perform some analyses in an attempt to identify the sedative drug. Hair was collected at the laboratory 21 days after the event. 7-Amino-flunitrazepam was identified in the proximal segment (root to 2 cm) at 5.2 pg/mg, while the proximal segment (2 to 4 cm) remained negative. No flunitrazepam, the parent drug, was detected.

4.7.5 Case 5 A 39-year-old woman, experiencing marital difficulties with her husband, felt sleepy for 24 hours after having drunk a coffee, at home. A blood sample, collected 20 hours later, revealed the presence of 51 ng/ml of bromazepam, whereas hair sampled at the same time was bromazepam-free. Another strand of hair was collected one month after the event and the proximal 2 cm long segment was positive for bromazepam at 10.3 pg/mg, the other segments (2–4 and 4–6 cm) remaining negative. These results were in accordance with a single exposure to this drug. The positive analysis of bromazepam in the coffee resulted in an admission by the husband, who did not challenge the biological conclusions.

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4.7.6 Case 6 A young woman was victim of a sexual assault in a highway gas station. She declared that the perpetrator forced her to absorb a white quadri-divisible tablet before abusing her. A blood sample, collected 18 hours after the offence, revealed the presence of 151 ng/ml of bromazepam. A strand of hair was collected three weeks after the event and the proximal segment (0–2 cm) was positive for bromazepam at 5.7 pg/mg (Figure 4.2), the consecutive segment (2–4 cm) was positive at 0.9 pg/mg and the last segment remained bromazepam-free. These results were in accordance with a single exposure to this drug.

4.7.7 Case 7 A 16-year-old girl claimed to have been raped during an afternoon, while sedated. Due to the long delay (six days) between the alleged event and her deposition to the police, there was no value in sampling blood or urine. We were requested to analyse the victim’s hair, collected nine weeks after the offense. Benzodiazepines and hypnotics were tested by LC-MS/MS and the first 3 cm segment was positive for zopiclone at a concentration of 4.2 pg/mg (Figure 4.3), the second (3–5 cm) at 1.0 pg/mg, whereas the last segment (5–7 cm) was zopiclone free. This appeared to be consistent with a single exposure to the drug. 03/724,0-2cm,46mg ech_chvx_011003_27 Sm (SG, 2x2)

MRM of 18 Channels ES+ 316 > 209.3 1.41e3 Area

7.59;111

100

%

8 ech_chvx_011003_27 Sm (SG, 2x2) 100

MRM of 18 Channels ES+ 316 > 182.2 1.87e3 Area

7.59;151

%

8 ech_chvx_011003_27 Sm (SG, 2x2)

9.12;1499

100

MRM of 18 Channels ES+ 290.1 > 154.1 1.37e4 Area

%

−2

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Figure 4.2

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Chromatogram of a hair extract from a victim of an alleged assault. Bromazepam concentration is 5.7 pg/mg. Top: two daughter ions of bromazepam, Bottom: daughter ion of diazepam-d5

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03/1170, 0-3cm, 45.8mg B+H-051203-8 Sm (SG, 2x2)

MRM of 18 Channels ES+ 389 > 345.1 1.15e3 Area

6.57;106

100

%

9 B+H-051203-8 Sm (SG, 2x2) 100

MRM of 18 Channels ES+ 389 > 245.1 2.74e3 Area

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1 9.12;2499 MRM of 18 Channels ES+ 290.1 > 154.1 1.97e4 Area

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Figure 4.3 Chromatogram of a hair extract from a victim of a alleged assault. Zopiclone concentration is 4.2 pg/mg. Top: two daughter ions of zopiclone, Bottom: daughter ion of diazepam-d5

4.7.8 Case 8 A 42-year-old man was offered a drink by a relative during a party. Several hours later, he noticed that his money was missing, but he had no recollection of events during the previous period. He went to the police, but no specimen was collected at that time. After several similar cases in the same region of France, the judge in charge of the case requested a hair analysis. 7-Amino-flunitrazepam, the major metabolite of flunitrazepam and its marker in hair, was detected (Figure 4.4) in the corresponding segment of hair at 31.7 pg/mg, while the distal segment was negative.

4.7.9 Case 9 A 21-year-old woman was confined illegally for 12 days and continuously raped by three men. To the police, she claimed that she had no recollection of the event due to incoherent behaviour and excessive sedation. Analysis of the proximal segment (root to 3 cm) of a strand of her hair demonstrated exposure to clonazepam, an antiepileptic drug with sedative and amnesic properties. Analysis revealed the identification of 7-aminoclonazepam, its marker, at 135 pg/mg, while the distal segment (3–6 cm) remained negative (Figure 4.5).

Hair in Forensic Toxicology with a Special Focus on Drug-Facilitated Crimes 04-134, 0-3cm, 28.1mg 220304-24 Sm (SG, 2x2)

8.60;3062

99

MRM of 8 Channels ES+ 287.1 > 138.1 3.61e4 Area

100

%

0 220304-24 Sm (SG, 2x2) 100

MRM of 8 Channels ES+ 284.1 > 135.1 3.09e4 Area

8.63;2729

%

1

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Figure 4.4

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Chromatogram of a hair extract from a victim of a robber. 7-Aminoflunitrazepam concentration is 31.7 pg/mg. Top daughter ion of 7-aminoflunitrazepam-d3, Bottom: daughter ion of 7-aminoflunitrazepam

4.7.10 Case 10 A 19-year-old woman went to the police to declare a rape after having a drink that may have been laced with ecstasy (MDMA). At the medico-legal unit of the hospital, a urine sample was collected (about ten hours after the rape) that revealed the presence of MDMA and its metabolite MDA at 1852 and 241 ng/ml, respectively, confirming her previous declarations. To the judge in charge of the case, she claimed that she never took ecstasy and directly gave the name of the rapist, who was rapidly arrested and send to jail. As the circumstances were unclear, the judge requested a hair analysis that demonstrated the simultaneous presence of various stimulants, with the following concentrations: 21.3, 31.6 and 6.7 ng/mg for MDMA, MDEA and MDA, respectively. These results were inconsistent with the claim of being drug-free. During a later confrontation with the judge, she admitted that it was a false notification, that no rape occurred and that it was a revenge on the alleged rapist.

4.8 Discussion Despite late sampling or even lack of collection of traditional biological fluids, such as blood and/or urine, results of hair testing allow us to document the use of hypnotics in drug-facilitated sexual assault (DFSA). The literature in this area is growing very rapidly. Frison et al.28 detected thiopental (150 to 300 pg/mg) and its metabolite pentobarbital (200 to 400 pg/mg)

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ML, 0-3cm, 24mg 090104-2 Sm (SG, 2x2) 100

MRM of 8 Channels ES+ 286.1 > 250.2 4.22e4 Area

6.76;3703

%

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MRM of 8 Channels ES+ 290.1 > 254.2 1.52e4 Area

6.75;1143

%

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9.59 6.77

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%

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3.08

0.45 0.85 1.25

1.67

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2.48

2.32

2.92 2.76

4.51 3.63 3.27 3.92 4.33 4.08

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8.25

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2 090104-3 Sm (SG, 2x2)

MRM of 8 Channels ES+ 290.1 > 254.2 9.98e3 Area

6.76;703

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%

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Figure 4.5

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Chromatogram of a hair extract from a victim of an alleged assault. A: Proximal segment, positive for 7-aminoclonazepam at 135 pg/mg; Top: daughter ion of 7-aminoclonazepam, Bottom: daughter ion of 7-aminoclonazepam–d4. B: Distal segment, negative for 7-aminoclonazepam. The peaks are the background noise of the machine, see the very low abundance. This pattern is classic from a chromatogram without a drug peak

in three different proximal segments, corresponding to the time of the assault, while distal segments remained negative. In two separate cases, Pe´pin et al.29 detected 7-aminoflunitrazepam (19 pg/mg) and zopiclone (13 pg/mg). The same authors30 demonstrated GHB exposure by comparing the concentrations along the hair shaft. Basal GHB concentrations were about 0.7 ng/mg, in comparison with a 5.3 ng/mg concentration in the segment corresponding to the time of the assault.

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From our own data and data from the literature, it is obvious that the target concentrations in hair after a single exposure are in the range of a few pg mg 1. To obtain the required ultra-low limits of detection together with suitable mass spectrometry information, tandem mass spectrometry appears to be a prerequisite. The selectivity and sensitivity of these methods have increased extraordinarily to almost completely suppressing the noise level. In comparison with the concentrations that are measured with drugs of abuse, such as heroin or cocaine, in cases of DFSA the concentrations are at least 1000 times lower. As is the case with other applications (survey of addicts, doping control, driving license regranting etc.) hair testing is a valuable approach to increase the window of drug detection. Embarrassment associated with urine collection, particularly after sexual assault, can be greatly mitigated through hair analysis. It is always possible to obtain a fresh, identical hair sample if there is any trouble during analysis, e.g. a specimen mix-up or a breach in the chain of custody. This makes hair analysis essentially fail-safe, in contrast to blood or urine analysis, since an identical blood or urine specimen cannot be obtained at a later date. The comparison between urine and hair is given in Table 4.2. The discrimination between a single exposure and long-term use can be documented by multi-sectional analysis. With the concept of absence of migration along the hair shaft, a single spot of exposure must be present in the segment corresponding to the period of the alleged event, using a growth rate for hair of 1 cm per month. As this growth rate can vary from 0.7 to 1.4 cm per month, the length of the hair section must be calculated accordingly. A delay of three to four weeks between the offence and hair collection and analysis of 2 cm sections is considered satisfactory in order to include the spot of exposurein the hair shaft sample. The hair must be cut as close as possible to the scalp. Particular care is also required to ensure that the individual’s hair strands retain the positions they originally had beside one another. Our typical collection procedure is presented in Figure 4.6. The unique possibility to demonstrate a single drug exposure through hair analysis has some additional interests. In case of late crime declaration, positive hair findings are of paramount importance for a victim, in order to start, under Table 4.2 Comparison between urine and hair for testing drugs of abuse and pharmaceuticals Parameters

Urine

Hair

Drugs Major compound Detection period Type of measure Screening Invasiveness Storage Risk of false negative Risk of false positive Risk of contamination Control material

all, except hormones metabolites 2–5 days, incremental yes high 20– C high low high yes

all, except hormones parent drug weeks, months cumulative no low ambient temperature low undetermined low needed

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Figure 4.6 Protocol for hair collection in case of drug-facilitated crimes

suitable conditions, a psychological follow-up. It can also help in the discrimination of false reports of assault, for example in the case of revenge. These cases are often sensitive with little other forensic evidence. Tedious interpretations, e.g. in cases of concomitant intake of hypnotics as a therapy for sleeping disorders, are avoided when investigations are done using hair instead of urine. Finally, in the absence of hair testing it is always possible for the advocate of the defendant, to claim during the criminal trial that the detected drug was ingested by the victim him/herself and had no connection with the crime.

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4.9 References 1. A.M. Baumgartner, P.F. Jones, W. Baumgartner and C.T. Black, J. Nuclear Med., 1979, 20, 748. 2. M. Saitoh, M. Uzaka, M. Sakamoto and T. Kobori in Advances in Biology of Skin, W. Montagna and R.L. Dobson (ed), Pergamon Press, Oxford, 1969, 183. 3. E. Cone, Ther. Drug Monit., 1996, 18, 438. 4. G.L. Henderson, Forensic Sci. Int., 1993, 63, 19. 5. R. Kronstrand, S. Fo¨rstberg-Peterson, B. Kagedal, J. Ahlner and G. Larson, Clin. Chem., 1999, 45, 1485. 6. G.L. Henderson, M.R. Harkey and C. Zhou, J. Anal. Toxicol., 1998, 22, 156. 7. W.A. Baumgartner, V. Hill and W. Blahd, J. Forensic Sci., 1989, 34, 1433. 8. L.W. Cartmell, A. Aufderhide and C. Weems, J. Okla. State Med. Assoc., 1991, 84, 11. 9. H. Sachs and P. Kintz, J. Chromatogr. B, 1998, 713, 147. 10. M.R. Moeller, J. Chromatogr., 1992, 580, 125. 11. M. Uhl, Forensic Sci. Int., 1997, 84, 281. 12. W.A. Baumgartner and V.A. Hill in Recent Developments in Therapeutic Drug Monitoring and Clinical Toxicology, I. Sunshine (ed), Marcel Dekker, New York, 1992, 577. 13. E.J. Cone, D. Yousefnejad, W.D. Darwin and T. Maguire, J. Anal. Toxicol., 1991, 15, 250. 14. D.L. Blank and D.A. Kidwell, Forensic Sci. Int., 1995, 70, 13. 15. D.A. Kidwell and D.L. Blank, in Drug Testing in Hair, P. Kintz (ed), CRC Press, Boca Raton, 1996, 17. 16. V. Cirimele, P. Kintz and P. Mangin, J. Anal. Toxicol., 1995, 19, 331. 17. P. Kintz, Toxicol. Letters, 1998, 102–103, 109. 18. H. Sachs, in Drug Testing in Hair, P. Kintz (ed), CRC Press, Boca Raton, 1996, 211. 19. M.R. Moeller, P. Fey and H. Sachs, Forensic Sci. Int., 1993, 63, 43. 20. R. Wennig, Forensic Sci. Int., 2000, 107, 5. 21. P. Kintz, V. Cirimele and B. Ludes, Forensic Sci. Int., 2000, 107, 325. 22. M. LeBeau, W. Andollo, W.L. Hearn et al., J. Forensic Sci., 1999, 44, 227. 23. P. Kintz, V. Cirimele, M. Villain and B. Ludes, Ann. Toxicol. Anal., 2002, 14, 361. 24. I. Hindmarch, M. Elsohly, J. Gambles and S. Salamone, J. Clin. Forensic Med., 2001, 8, 197. 25. F. Questel, G. Lagier, D. Fompeydie et al., Ann. Toxicol. Anal., 2002, 14, 371. 26. P. Kintz, V. Cirimele, C. Jamey and B. Ludes, Ann. Toxicol. Anal, 2002, 14, 129. 27. A. Verstraete, Ann. Toxicol. Anal., 2002, 14, 390. 28. G. Frison, D. Favretto, L. Tedeschi and S.D. Ferrara, Forensic Sci. Int., 2003, 133, 171. 29. G. Pe´pin, M. Che`ze, G Duffort and F. Vayssette, Ann. Toxicol. Anal., 2002, 14, 395. 30. G. Pe´pin, Y. Gaillard, M. Che`ze and J.P. Goulle´, J. Med. Leg. Droit. Me´d. 2003, 46, 93.

CHAPTER 5

Hair and Human Identification BRUCE A. BENNER, JR. and BARBARA C. LEVIN

5.1 Introduction Human beings are unique in many ways – externally and internally – and with society’s increasing emphasis on security, techniques and tools that can be used to identify individuals definitively have become part of our daily lives. Law enforcement personnel are particularly interested in identifying alleged perpetrators and distinguishing them from victims and have used both established (e.g. fingerprints) and creative (e.g. pet hair) comparisons for associating crime scene evidence with samples of known origin. Employers are continually evaluating the best way to ensure that only authorised personnel gain access to sensitive company locations. In addition to crime and work-place security challenges, identification is also needed for a uniquely personal and private concern – establishing blood relationships between adults and children. Over the last several years, there have been numerous examples of identification by deoxyribonucleic acid (DNA) sequencing techniques that have justified reuniting a child with his/her family as well as the release of an incarcerated yet innocent individual. One of the external characteristics that can make humans at least somewhat unique (depending upon the colour and style) is our hair, essentially a non-living tissue that we are continually growing and losing. Hair is one of the most common forms of forensic trace evidence, which explains why crime scene investigators are extremely interested in developing methods using hair for identifying and distinguishing people. Microscopic inspection of hair can suggest its origin (body location, ethnicity) and is typically the first step in forensic hair analysis. Tremendous progress in nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) sequence analysis during the last 18 years has enabled the characterisation of both minute amounts of DNA and degraded DNA. Included in these advancements would be Kary Mullis’s development of the polymerase chain reaction (PCR) whereby a specific segment of DNA from a minute amount of cells (e.g. a drop of blood, saliva, a small number of skin cells, a segment from a single hair) is amplified virtually without limits1 and then can be sequenced for identification purposes. Both nDNA and mtDNA analyses have been applied to hair samples.

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Plucked hairs usually have live follicle cells that can be used for nDNA sequence analysis, whereas cut hair or hair lost naturally during the telogen phase (quiescent) will likely not have live cells associated with the sample. For cut or shed hair, mtDNA sequencing methods can be used to associate or distinguish hair samples from different individuals. Given that hair is often retrieved from crime scenes and that it can be readily obtained in a noninvasive manner from alleged perpetrators and victims, it is a sample often used for forensic identification. This chapter will review the basic characteristics of hair, its composition and the current state-of-the-art analytical methods using hair for human identification. These methods include technologies based on physical (microscopy) and chemical (nDNA, mtDNA, internal and surface organic analysis) attributes.

5.2 Background 5.2.1 General Composition and Characteristics of Hair Mammals have hair growing from their skin, or more specifically from structures in the skin called follicles. This characteristic of mammals is one that distinguishes them from other animals. The general function of hair is to protect the mammal from extreme weather conditions and environmental risks.2 Human hair is composed of proteins (