Copper and the Skin

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DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES

COPPER and the SKIN

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

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

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

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

DERMATOLOGY: CLINICAL & BASIC SCIENCE SERIES

COPPER and the SKIN Edited by

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

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

New York London

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

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

Preface Preface

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

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Preface

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

Acknowledgment

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

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Contents

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

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

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

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

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

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Contributors

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

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

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

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

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

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

Copper and Copper Alloys

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Electromotive force

Ionization potential

Metallic elements

Oxidation

Oxidation potential

Penetration

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

ABBREVIATIONS AA L mEq/L SC

amino acids ligand milliequivalents per liter stratum corneum

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

ð1Þ

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

Kp ¼

ð% lossÞ5 1570

J ðfraction lostÞ CA VA ¼ DC ½timeðhrÞ A CA

ð2Þ

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

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

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

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

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

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

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

Men Women Men Women

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

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

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

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

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

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

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

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

Amount absorbed (mg)

Percentage of dose absorbed

632 56 130 15 30 3 5.0 0.9 <1.0

6.3 1.3 0.30 0.05 <0.001

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transdermally that led to the death of a university scientist having handled the chemical in the laboratory (81,104). Valence Ionic radii change with the number of outer electrons (i.e., with their valence), and thereby also the ion’s electrophilicity and reactivity toward proteins. These variables also determine the rate of metal diffusion. The negatively charged chromate and dichromate ions as oxo-complexes of hexavalent chromium do not bind to organic substances, whereas the electrophilic chromic ion [Cr(III)] shows a strong afﬁnity for epithelial and dermal tissues, forming stable complexes that slow the rate of diffusion (73) or prevent it altogether (96). Occurrence (induction or elicitation) of allergic reactions in the skin due to allergenic agents may be considered to be an indicator of skin diffusivity (i.e., does an allergen penetrate the SC to reach the Langerhans’ cells in the viable epidermis). In animals, epicutaneous sensitization with trivalent chromium could be achieved only with difﬁculty. A 20-fold increase in the concentration of chromic sulfate [Cr(III)] was required to achieve the same degree of sensitization as with potassium chromate [Cr(VI)]. On the other hand, the same degree of contact sensitivity resulted from the intradermal application of either potassium dichromate or chromic sulfate, demonstrating that the SC is the barrier inhibiting percutaneous diffusion of trivalent chromium (105). In a study in which chromium sensitive volunteers were patch tested with a Cr(III)-containing detergent as well as with dichromate [Cr(VI)], those identiﬁed as chromium sensitive showed a positive response to dichromate, but none were sensitized to the detergent bar containing trivalent chromium (106). Differences in diffusivity due to protein reactivity between dichromate and chromic ions were also noted in vitro with human fullthickness abdominal skin. The permeability constant for hexavalent chromium as dichromate was an order of magnitude greater than that of trivalent chromic salts, overriding the retarding effect of dichromate’s larger molecular volume (106). Iron occurs mainly in its trivalent state (ferric ion) in nature, which is biologically unavailable. As a consequence, several mechanisms have evolved in the course of evolution to make its absorption by living organisms possible, and in mammals, iron is primarily absorbed in the duodenum as the divalent ferrous ion. Nutritionally most important is Fe2þ bound in hemoglobin, occurring in organs and muscle tissues, and absorbable through gastrointestinal membranes. Once it reaches its target organ, iron is enzymatically oxidized back to Fe3þ (107). pH and Solubility Changes in pH can have an effect on the skin penetration by electrolytes. From a dermatotoxicological perspective chromic [Cr(III)] salts are the least

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problematic because of their poor aqueous solubility, which also decreases further with rising pH. Diffusivity of chromium as the oxo-complex dichromate can vary widely, ranging over more than an order of magnitude. Depending on pH, Cr(VI) exists in either the chromate (pH > 6) or dichromate form (pH ¼ 2–6). In either form, Cr(VI) is the greater hazard for human health, due to its heightened membrane diffusivity. The permeability constants measured for chromate–dichromate were higher in the pH range 8 to 12.7 than in the lower range of pH 1.4 to 5.6. Chromium in the higher pH range exists as the smaller CrO42 ion, whereas in the lower pH range, the larger, less diffusible dichromate ion Cr2O72 predominates (108). The pH dependence of zinc-oxide absorption was demonstrated by ˚ gren, where at a pH of 5.4 in vitro, uptake of the compound through A human skin was 21 times greater than at pH 7.4 (8). Protein Reactivity and Depot Formation The electrophilic nature of many metals creates pronounced protein reactivity that can result in depot formation in the SC. Such protein–metal binding typically occurs with aluminum(III), silver(I), mercury(II), chromium(III), nickel(II), and the metalloid arsenic(III); it can take place in all strata of the skin to the extent of building up a secondary barrier and inhibiting further diffusion (109,110). Such deposits can also be reversible, however, as is the case for essential elements subject to homeostatic control (e.g., zinc or copper), which are retained by speciﬁc storage proteins, such as MT or coeruloplasmin, and released again upon physiological demand (111). Chromium is an example of the metals that, through nonspeciﬁc binding, causes protein denaturation with formation of permanent depots in the epidermis; these depots appear to be subject to the countercurrent effect of continuous sloughing of the outer SC layers, with release of signiﬁcant amounts of the metal originally absorbed. Traces of chromium(III) are tenaciously retained, following exposure to chromium-containing dermatologicals. Elevated levels of the metal were seen in the skin up to three weeks, following a single inunction of 0.5% potassium dichromate in petrolatum (112). In an in vitro diffusion experiment by Gammelgaard, no chromium was detected in the recipient phase following the application of Cr(III) as the chloride or nitrate on full-thickness skin for 168 hours, or following application of dichromate over 48 hours (105). The avidity with which aluminum(III) forms complexes with skin and shunt protein means that there is only superﬁcial penetration into the epidermis. This is the basis of the antiperspirant activity of certain aluminum salts, such as aluminum chlorohydrate, which, at the pH of the skin (or sweat), form insoluble salts and precipitates to effectively form a diffusion barrier (113).

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Dermal or systemic long-term exposure to a number of metals through occupation, therapy, lifestyle, or diet can result in their accumulation in the body, and their preferential deposition in dermal tissues can become visible as a characteristic discoloration of the skin. As these metals are not subject to signiﬁcant natural processes of elimination, they accumulate over a lifetime and form permanent deposits in their elemental state, visualized histologically as metal particles. With the exception of arsenic, however, that phenomenon does not seem to have any untoward effects other than to alter the natural coloring of the skin. Time Postapplication Deposits formed by electrophilic metals appear as the cause for considerable lag times of several hours or days as observed in in vitro diffusion experiments. Before any permeant appears in the receptor compartment, in some cases, such as with chromium as observed by Gammelgaard in the previous section, the metal fails to appear at all. According to other observations, diffusion rates will increase with increases in donor concentration to a point, level off, and ultimately decrease. With mercuric chloride in vitro on whole-thickness skin, postapplication time was the most inﬂuential variable for absorption rate, indicating secondary barrier buildup. The percutaneous absorption of mercury was greatest in the ﬁrst period (zero to ﬁve hours); it decreased in each successive period to the lowest rate, usually in the ﬁnal period (36–48 hours), or sometimes in the nextto-the last period (24–36 hours). By the end of the experiments, the absorption rate was 40% of the initial rate, often 25% to 35% and sometimes as little as 1%. This trend was seen with both guinea pig skin and human skin (114). Commercial products (emulsions and ointments) containing copper have been studied over a period of 72 hours (9,61). Whether formulated with copper sulfate or the organic salt copper 2-pyrrolidone 5 carboxylate, apparent permeability coefﬁcients over 72 hours were in the range 0.015 104 to 0.13 104 cm/hr. Noteworthy differences in permeability coefﬁcients were associated with the period of exposure; the values decreased over successive time periods from near 1 104 cm/hr (zero to two hours) to 0.1 104 cm/hr or less (25–48 hours) and ﬁnally to undetectable values in two cases (49–72 hours). As for percutaneous zinc, absorption rates from commercial emulsions and ointments, zinc 2-pyrrolidone 5-carboxylate, ZnO and ZnSO4, measured in vitro with human skin were highest in the ﬁrst two hours after application, then decreased to less than a 10th of the initial rates (9,61). Endogenous Factors Regional Variation Skin absorption by chemicals, investigated with steroids and pesticides, is subject to variation among different sites of the body, decreasing in the

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

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

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

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

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

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

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

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

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

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technique followed by inductively coupled plasma–mass spectrometry analysis was useful in visualizing SC penetration by nickel, in particular (78,101). The outstanding biological characteristic of nickel, from a public health perspective, is its immunotoxicity. Nickel started out as a prime occupational hazard reported from the metalworking and reﬁning industry in the late 19th century and the ﬁrst part of the 20th century due to its allergenicity (139,148,149), but since the Second World War it has become a consumers’ afﬂiction as well, because the metal is now part of most alloys used in the manufacture of common materials in tools and articles of daily contact. In a number of dermatotoxicity studies, the incidence of nickel hypersensitivity noted for women formerly ranged from 10% to 20%, that for men from 1% to 4%. In more recent studies from a number of countries, a striking increase in these numbers has been recorded, particularly among women and children of school age: to 31.9% among schoolgirls in Italy (150), to 43.7% among dermatological clinic patients in Poland (151), and to 30% among an unselected group of schoolgirls in Finland (152). To gain insight into the dermatopharmacokinetics of nickel as allergen, the protocol of sequential adhesive tape stripping was implemented to examine the penetration characteristics of both the nickel salts and nickel in its metallic state in human SC and its potential to reach the viable epidermis, following their application on the skin volunteers. Inductively coupled plasma-mass spectroscopy (ICP-MS) was used to analyze tape strips (78,101). For nickel chloride, sulfate, nitrate, and acetate, the depth-penetration proﬁles obtained by tape stripping and analysis led to a number of conclusions: 1. Up to 24 hours, most of the nickel dose applied remained on the SC surface 2. Nickel adsorbed accumulated in the uppermost SC layers, and the concentration gradient between the superﬁcial and deeper layers increased commensurate with exposure time and concentration. 3. In a number of experiments, mass balance based on amounts retrieved from the skin surface and cumulative nickel analysis to the level of the glistening layer showed a deﬁcit. Within 24 hours nickel salts thus appeared to penetrate beyond the SC to a minor degree, possibly via the skin shunts, to become systemically available. 4. While the concentration gradients of nickel adsorbed varied with counterion, anatomical site, dose, and exposure time, toward the level of the glistening layer for all variables tested, the depth proﬁles converged toward nondetectable levels (<20 ppb). Occluded application of nickel as powder on the skin conﬁrmed that nickel containing metals are oxidized in contact with skin exudates to yield lipophilic, diffusible salts, sufﬁcient to elicit allergic reactions. Within the superﬁcial layers of the skin, diffusion of nickel became evident from time-dependent concentration gradients of the metal. The gradients of the

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distribution proﬁles increased proportionally with occlusion time but leveled off with increasing depth after the 10th strip, to continue at constant levels to the 20th strip. From application (of a large excess) of metal powder on a surface of 1.15 cm2, the total nickel removed with 20 SC strips after maximum occlusion of 96 hours was 41.6 mg/cm2 (101). The method of SC stripping as applied in nickel research could be used to investigate skin diffusivity of other metals, such as copper. It would bring insight into the interaction of that metal brought in contact with the microenvironment on live skin. In Vivo–In Vitro Correlation In validation studies comparing absorption values, good agreement between in vivo and in vitro methods have been obtained if the studies were conducted along generally accepted guidelines. The application of a permeant to the skin in vivo may be more physiologically relevant than the use of in vitro methods, and in case of disagreement, precedence goes to in vivo data, although in vivo execution may be subject to error. The results of in vivo studies are reported as the percent of dose absorbed, whereby appropriate transformation becomes necessary if a Kp is required. In vitro absorption values are generally good predictors of the rate or extent of percutaneous absorption in the intact animal. However, the factors described may affect the predictive accuracy of in vitro absorption methods, especially for compounds of high hydrophilicity or lipophilicity. Failure to account for these variables will lead to poor correlation between in vitro and in vivo percutaneous absorption. If Kp data are used to estimate percutaneous absorption, it is necessary to examine the experimental conditions used if in vitro Kps are to correlate with in vivo data. An example is given with the skin absorption experiment conducted with boron compounds. Parallel in vitro and in vivo studies conducted by Wester et al. on the skin absorption of a number of boron compounds identiﬁed substantial and unexpected discrepancies between the results obtained (153). In the in vitro study, dosing corresponded to inﬁnite (1 mL/cm) and ﬁnite (2 mL/cm) amounts. These doses correspond to inﬁnite amounts available for absorption as a theoretical setting to deﬁne diffusivity, versus a dose remaining on the skin to convey realistic in vivo short-term exposure. The latter dose was also applied on the skin in vivo, which was then allowed to dry. The Kp values calculated from the inﬁnite dose exposure was 1000-fold higher than the one from the in vivo study, while the ﬁnite in vitro dosing mode was only 10-fold higher. The authors concluded that the in vivo and the in vitro ﬁnite dose data appeared more relevant for a real-life exposure scenario, reﬂecting that under inﬁnite dosing over 24 hours in vitro the skin membrane was progressively becoming more permeable. The inﬁnite in vitro dosing protocol would only be relevant for a full-body exposure over an extended period.

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ANALYTICAL METHODS FOR METAL DETECTION The toxicology of metal compounds is becoming increasingly important in the ﬁelds of environmental, occupational, and clinical medicine. The basis for studying metal toxicity lies in the application of sensitive and accurate analytical techniques, and tissue localization of metals in tissue (skin) contributes signiﬁcantly to the understanding of toxic mechanisms. Going beyond conventional chemical analysis, physical methods now afford sensitivities in metal detection that are lower by several orders of magnitude, often reaching below the ppb level, and the quantitative tools of atomic spectroscopy have brought the most signiﬁcant advances. Adapted to monitoring of toxic elements in biological substrates, such as tissues or body ﬂuids, such sensitivity is essential for the assessment of risk to toxic metals without the need to resort to radionuclides. Thus, monitoring of metal levels in skin and body ﬂuids by physical methods can provide adequate estimates of dose absorbed, indicating the need for corrective intervention in cases of potential exposure hazard, especially in the work environment. Inductively Coupled Plasma–Atomic Emission Spectroscopy ICP-atomic emission spectroscopy (ICP-AES) permits detection of metals at the trace amount level, obviating the use of radioisotopes. For the detection of copper, the current quantitation limit falls in the 5 to 10 ppb (mg/L) range, a factor of 5 above the true instrumental detection limit as deﬁned by the U.S. Environmental Protection Agency (EPA) (22). Quantitative detection of metals is accomplished by ionization of elements in inductively coupled argon plasma maintained by the interaction of a radiofrequency ﬁeld and ionized argon gas. In a sample aerosol (e.g., a vaporized metal salt solution) atoms and ions are activated at 6000 C to an unstable energy state, and as they revert to their ground state again, they emit light of characteristic wavelength and intensity that can be measured. Inductively Coupled Plasma–Mass Spectroscopy ICP-MS is a technique applicable to mg/L (ppb) concentrations of many elements in aqueous medium upon appropriate sample preparation of biological materials. Reliability of the method for elemental analysis is based upon multilaboratory performance compared with that of either furnace atomic absorption spectroscopy or ICP-AES. Normal instrumental detection limit for copper, as for a number of other transition metals, falls in the 0.5 ppb range. The method measures ions produced by radiofrequency inductively coupled plasma. Compound to be analyzed, present in liquid form, is nebulized and the resulting aerosol transported by argon gas into the plasma torch. The ions produced are entrained in the plasma gas and introduced, by means of a water-cooled interface, into a quadrupole mass spectrometer.

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The ions produced in the plasma are sorted according to their mass-tocharge ratios and quantiﬁed with a channel electron multiplier (142). Stable-Isotope Inductively Coupled Plasma–Mass Spectroscopy Analysis Assessment of the relevance of metal absorption data can be difﬁcult and is rendered ambiguous by the intake of naturally occurring elements in the normal diet, reﬂecting in the skin and in the overall organism. Thanks to recent developments in analytical techniques and the availability of artiﬁcially generated stable metal isotopes, it is now possible to overcome those earlier difﬁculties. By dosing stable isotopes in vivo, which pose no risk to volunteers, it is now possible to differentiate between the two kinds. Using ICP-MS for the analysis of the stable isotope 10B, the percutaneous absorption of boric acid, borax, and disodium octaborate over a 24-hour exposure period was determined in human volunteers, unambiguously differentiating between the amount absorbed through the skin and that absorbed with the diet (153). The stable-isotope approach was used to investigate lead metabolism in normal humans by replacing part of the dietary lead with lead-204 tracer. Kinetic and metabolic balance studies of the volunteers kept on controlled diets for six months indicated that approximately two-thirds of assimilated lead was dietary in origin; the remainder was inhaled (143). By that approach, absorption of copper and other metals in the skin now becomes measurable, independent from endogenous natural isotopes present in the organism. Atomic Absorption Spectrophotometry Atomic Absorption Spectrophotometry (AAS) with Zeeman background correction is the reference method accepted by the International Union of Pure and Applied Chemistry and the International Agency for Research on Cancer for trace element analysis (144). The method is based on absorption of radiation energy by free atoms, characteristic for each element. In an atomizer, thermal energy (air–acetylene ﬂame) converts the analyte to free atoms. In the ground state they absorb resonance radiation from a light source that emits characteristic radiation (i.e., the spectrum of the analyte element) whereby an electron transitions from the ground state to one of the empty orbitals at a higher energy level. A portion of this light is thus attenuated by such resonance absorption in the probe. A photomultiplier detector measures the change in radiation intensity and converts it into an absorbance signal. There is a linear relationship between absorbance and the concentration of the analyte. The technique is intrinsically speciﬁc,

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because the atoms of a particular element absorb only radiation of their own characteristic wavelength (154). AAS is the most common technique for nickel analysis in biological ﬂuids. The sample to be analyzed is digested in acid, the nickel then chelated with ammonium tetramethylene dithiocarbamate, the chelate extracted with 4-methyl-2-pentanone, and the nickel in the extract measured by AAS, or the initial acid solution analyzed as such by AAS directly (155). Currently, the detection range for nickel by AAS in body ﬂuids (urine and serum) is 0.4 to 0.05 ppb (156). Electron Spin Resonance Electron spin resonance (ESR) applies to transitions between electronic spin states in a magnetic ﬁeld. The ﬁeld of a probe is swept by a magnet, and resonance indicates presence of unpaired d-electrons in paramagnetic transition metals, the triplet state of two unpaired electrons in orthogonal orbitals, such as in elemental oxygen, or in isotopes, such as 13C or 17O. If an unpaired electron registers proximity of magnetic nuclei with a nuclear spin 1/2, the single absorption is split into hyperﬁne structures, identifying interacting nuclei and their number in the vicinity of the unpaired electron. ESR is applied in in vitro analysis of biological materials, and, most recently, in analogy with nuclear magnetic resonance (NMR), instruments have been developed that allow in vivo analysis of live tissue. ESR detects the presence of stable organic species, such as nitroxides; organometallic and inorganic species with unpaired electrons can be detected in liquid as well as solid materials. Transient, short-lived species, such as organic free radicals, must be continuously generated in the ESR probe to maintain a sufﬁcient steady-state concentration for detection. Low temperature (freezing) and spin trapping techniques are used to visualize transient radicals in the biological matrices. To stabilize short-lived radicals, these are trapped with alkyl nitroso compounds leading to long-lived adducts, making them amenable to ESR analysis. Identity and oxidation state of paramagnetic transition metals with unpaired electrons in their d-shell are detectable by ESR (e.g., Mn4þ and Mn2þ, Cu2þ, Ni1þ and Ni3þ, Ti3þ, and Fe3þ). The nuclear spin of the transition metal and the natural abundance of the magnetic isotopes assist in this intent by causing different signal splitting and patterns. Changes in the structure and properties of biological membranes (e.g., skin) under the inﬂuence of changing physical parameters or under the inﬂuence of xenobiotic penetrants have been studied by ESR. Perturbation and conformational changes in natural constituents in the skin, due to experimental penetration enhancers applied, have been described by incorporating various (ESR-detectable) stearic acids as spin labels in the lipid bilayers of human skin (157).

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Using skin biopsies, the effects of ultraviolet (UV) irradiation on epidermal tissue have been studied by ESR spectroscopy and ESR imaging, providing evidence for the generation of hydroxyl and lipid radicals that potentially result in oxidative injury in the SC and epidermis (158). Particle-Induced X-Ray Emission PIXE analysis with a proton microprobe allows the determination of trace elements in epidermal strata prepared by cryosection (159). In PIXE a proton beam activates an atomic electron, lifting it into a higher orbital. When an outer shell electron falls back to ﬁll the vacancy created, the transition is measured as the emission of an X-ray photon, characteristic of the excited atom. The method with an elemental sensitivity approaching 0.1 mg/kg (0.1 ppm) is also useful in bulk analysis of alloys with multielement detection capability, and in spatial analysis to localize elements present in a sample (160). Using this analytical method in their research into skin penetration by nickel, Forslind et al. and Malmqvist et al. have been able to localize nickel in the superﬁcial strata of human skin, with a spacial resolution of 3 mm by 15 mm and a detection limit of about 30 ppm. The concentration of nickel following exposure to a nickel solution was found to be highest in the outer parts of the epidermis, mainly in the outermost SC (161,162), an indication of the metal ion’s reactivity. SUMMARY AND CONCLUSIONS While there is a considerable volume of medical and toxicological literature addressing the topical and systemic effects of metal absorption, the data available on the rate of their skin diffusivity are few, vary considerably, and are fragmented and hard to reconcile. In reviewing the determinants for the diffusion of metals, both exogenous and endogenous, the intent here was to delineate the numerous interrelated factors involved in that process; a closer examination of the multitude of variables may serve to explain the difﬁculties encountered by investigators in this line of research, and underscores the need for continued endeavors in that ﬁeld and for the development of reliable methods by which to determine exposure toward toxicological risk assessment. The diffusion process of metals, particularly of the transition metals, is different from that of small-molecular-weight, lipophilic organic compounds, such as drugs, solvents, perfumes, etc., which follow Fick’s law of diffusion and whose Kps can be predicted applying structure–activity relationships. Development of a mathematical algorithm with which to assess dermal absorption of metals by a uniﬁed approach on the other hand has not been possible, due to the myriad of variables described here. For a small number of metals, diffusion has been thoroughly investigated, primarily motivated by morbidity and mortality, as a consequence

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of skin exposure occurring intentionally for the purpose of personal embellishment, or accidentally in the work environment. The process of diffusion examined from the anatomical viewpoint of the skin barrier and the metallurgical and physicochemical properties of the permeants are nature given but, as documented in this review, they present only part of the difﬁculties to be overcome when investigating metal diffusion. The other set of difﬁculties stems from the experimental approach in examining the process, either in vitro or in vivo. In determining skin diffusivity of metals in vitro for toxicological risk assessment or therapeutic beneﬁt, for instance, it would appear important not to limit the focus solely on permeant reaching the receptor phase. Analysis should also include permeant retained in the strata of the skin for topical bioavailability purposes. In the case of some metals, the more electrophilic ions in particular, that value may be the preponderant quantity, and a potentially signiﬁcant factor for consideration in exposure risk analysis, as it may be mobilized subsequently through homeostasis. Some metals, such as copper, zinc, or calcium, form a reservoir that can be mobilized on homeostatic demand; others again penetrate into the strata by passive diffusion and can albeit slowly, reach the systemic compartment. On the other hand, metals of slow diffusivity, such as transition metals, also encounter a countercurrent effect in the strata of the skin. As that barrier is characterized by continuous desquamation, with a total turnover over two to three weeks (corresponding to an approximate ‘‘inverse’’ Kp of 106), bioavailability of the metal may be less than indicated by absorption in skin tissue, because epidermal turnover can signiﬁcantly reduce further diffusion into the systemic circulation of highly electrophilic compounds. As illustrated here, the process of skin barrier diffusion by metal ions is multifactorial, and the rate of their absorption, both in vitro and in vivo, remains imponderable a priori due to a number of potentially promoting and retarding processes acting simultaneously.

ABBREVIATIONS EPA ESR GI HPLC MT NMR QSAR RA SC UV

Environmental Protection Agency electron spin resonance gastrointestinal high pressure liquid chromatography metallothionein nuclear magnetic resonance quantitative structure–activity relationships rheumatoid arthritis stratum corneum ultraviolet

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64. Wahlberg JE, Skog E. Nickel allergy and atopy. Threshold of nickel sensitivity and immunoglobulin E determinations. Br J Dermatol 1971; 85:97. ¨ berempﬁndlichkeit gegen hartgeld. Dermatol Wochenschr 1930; 65. Rothman S. U 90:98. ¨ berempﬁndlichkeit gegen nickelgeld. Dermatol Wochenschr 66. Preininger T. U 1934; 99:1082. 67. Samitz MH, Pomerantz H. Studies of the effects on the skin of nickel and chromium salts. AMA Arch Ind Health 1958; 18:473. 68. Gollhausen R, Ring J. Allergy to coined money: nickel contact dermatitis in cashiers. J Am Acad Dermatol 1991; 25:365. 69. Lide´n C, et al. Nickel release from tools on the Swedish market. Contact Dermat 1998; 39:127. 70. Lide´n C, Carter S. Nickel release from coins. Contact Dermat 2001; 44:160. 71. Fisher AA. Allergic contact urticaria from various metals. 3rd ed. Philadelphia: Lea and Febiger, 1986. 72. Fregert S. Occupational dermatitis in a 10-year material. Contact Dermat 1975; 1:96. 73. Wass U, Wahlberg JE. Chromated steel and contact allergy. Contact Dermat 1991; 24:114. 74. Flint GN. A metallurgical approach to metal contact dermatitis. Contact Dermat 1998; 39:213. 75. Bergman M, Bergman B, So¨remark R. Tissue accumulation of nickel released due to electrochemical corrosion of non-precious dental casting alloys. J Oral Rehab 1980; 7:325. 76. Hosty´nek JJ. Nickel-induced hypersensitivity: etiology, immune reactions, prevention, and therapy. Arch Dermatol Res 2002; 294:249. 77. Menne´ T, et al. Patch test reactivity to nickel alloys. Contact Dermat 1987; 16:255. 78. Hosty´nek JJ, et al. Human stratum corneum penetration by nickel: in vivo study of depth distribution after occlusive application of the metal as powder. Acta Derm Venereol 2001; 212(suppl):5. 79. Sens DA, Simmons MA, Spicer SS. The analysis of human sweat proteins by isoelectric focusing. I. Sweat collection utilizing the macroduct system demonstrates the presence of previously unrecognized sex-related proteins. Pediat Res 1985; 19:873. 80. Rasetti L, Cappellaro F, Gaido P. Contribution to the study of saturnism by inhibited oils. Rass Med Ind Ig Lav 1961; 30:71. 81. Toribara TY, Clarkson TW, Nierenberg DW. Chemical safety: more on working with dimethylmercury. Chem Eng News 1997; 75:6. 82. Geigy Scientiﬁc Tables. 7th ed. Geigy Pharmaceutical, New York: Ardsley, 1975. 83. Jensen O. ‘‘Rusters’’. The corrosive action of palmar sweat: II. Physical and chemical factors in palmar hyperhidrosis. Acta Derm Venereol (Stockh) 1979; 59:139. 84. Lind PO, et al. Amalgam-related oral lichenoid reaction. Scand J Dental Res 1986; 94:448. 85. Hier SW, Cornbleet T, Bergheim O. The amino acids of human sweat. J Biol Chem 1946; 166:327.

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86. Coltman CA, Rowe NJ, Atwell RJ. The amino acid content of sweat in normal adults. J Clin Nutr 1966; 18:373. ¨ hman H, Vahlquist A. The pH gradient over the stratum corneum differs in 87. O X-linked recessive and autosomal dominant ichthyosis: a clue to the molecular origin of the ‘‘Acid Skin Mantle’’. J Invest Dermatol 1998; 111:674. 88. Schurer NY, Elias PM. The biochemistry and function of stratum corneum lipids. Adv Lipid Res 1991; 24:27. 89. Feingold KR. The regulation of epidermal lipid synthesis by permeability barrier requirements. Crit Rev Ther Drug Carrier Syst 1991; 193. 90. Fluhr JW, et al. Impact of anatomical location on barrier recovery, surface pH and stratum corneum hydration after acute barrier disruption. Br J Dermatol 2002; 146:770. 91. Bennett BG. Environmental nickel pathways to man. In: Sunderman FW, Aitio A, eds. Nickel in the Human Environment: Proceedings of a Joint Symposium Held at the International Agency for Research on Cancer, Lyon, France, 8–11 March 1983. Oxford University Press, New York, 1984:487. 92. Ermolli M, et al. Nickel, cobalt and chromium-induced cytotoxicity and intracellular accumulation in human hacat keratinocytes. Toxicol 2001; 159:23. 93. Fullerton A, et al. Permeation of nickel salts through human skin in vitro. Contact Dermat 1986; 15:173. 94. Wahlberg JE. Percutaneous absorption of trivalent and hexavalent chromium. Arch Dermat 1965; 92:315. 95. Fitzgerald JJ, Brooks T. A new cell for in vitro skin permeability studies— chromium (III)/(VI) human epidermis investigations. J Invest Dermatol 1979; 72:198. 96. Gammelgaard B, et al. Permeation of chromium salts through human skin in vitro. Contact Dermat 1992; 27:302. 97. Wahlberg JE. Nickel: the search for alternative, optimal and non-irritant patch test preparations. Assessment based on laser Doppler ﬂowmetry. Skin Res Technol 1996; 2:136. 98. Hadgraft J, Green PG, Wotton PK. Facilitated percutaneous absorption of charged drugs. In: Bronaugh R, Maibach HI, eds. Percutaneous Absorption. New York: Marcel Dekker, 1989:555. 99. Mazzenga GC, Berner B. The transdermal delivery of zwitterionic drugs I: the solubility of zwitterionic salts. J Contr Rel 1991; 6:77. 100. Mazzenga GC, Berner B, Jordan F. The transdermal delivery of zwitterionic drugs II: the ﬂux of zwitterionic salts. J Contr Rel 1992; 20:163. 101. Hosty´nek JJ, et al. Human stratum corneum adsorption of nickel salts: investigation of depth proﬁles by tape stripping in vivo. Acta Derm Venereol 2001; 212(suppl):11. 102. Wahlberg JE. Nickel chloride or nickel sulfate? Irritation from patch-test preparations as assessed by laser Doppler ﬂowmetry. Dermatol Clin 1990; 8:41. 103. Bress WC, Bidanset JH. Percutaneous in vivo and in vitro absorption of lead. Vet Human Toxicol 1991; 33:212. 104. Smith SL. An avoidable tragedy. Occup Hazards 1997; 59:32. 105. Wahlberg JE. Percutaneous absorption from chromium (51Cr) solutions of different pH, 1.4–12.8. Dermatologica 1968; 137:17.

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106. Dupuis D, et al. In vivo relationship between horny layer reservoir effect and percutaneous absorption in human and rat. J Invest Dermatol 1984; 82:353. 107. Hallberg L. Bioavailability of iron in man. Annu Rev Nutr 1981; 123:1981. 108. Wahlberg JE. Percutaneous toxicity of metal compounds. Arch Environ Health 1965; 11:201–204. 109. Alder JF, et al. Depth concentration proﬁles obtained by carbon furnace atomic absorption spectrometry for nickel and aluminium in human skin. J Anal Atomic Spectrom 1986; 1:365. 110. Santucci B, et al. The inﬂuence exerted by cutaneous ligands in subjects reacting to nickel sulfate alone and in those reacting to more transition metals. Exp Dermatol 1998; 7:162. 111. Burch RE, Hahn HKJ, Sullivan JF. Newer aspects of the roles of zinc, manganese and copper in human nutrition. Clin Chem 1975; 21:501. 112. Christie OHJ, et al. Spark source mass spectrographic study of metal allergenic substances on the skin. J Invest Dermatol 1976; 67:587. 113. Quatrale RP, Thomas EL, Birnbaum JE. The site of antiperspirant action by aluminum salts in the eccrine sweat glands of the axilla. J Soc Cosm Chem 1985; 36:435. 114. Wahlberg JE. Percutaneous absorption of sodium chromate (51Cr) cobaltous (58Co), and mercuric (203Hg) chlorides through excised human and guinea pig skin. Acta Derm Venereol 1965; 45:415. 115. Maibach HI, et al. Regional variation in percutaneous penetration in man. Arch Environ Health 1971; 23:208. 116. Rougier A, et al. Regional variation in percutaneous absorption in man: measurement by the stripping method. Arch Dermatol Res 1986; 278:465. 117. Lotte C, et al. In vivo relationship between transepidermal water loss and percutaneous penetration of some organic compounds in man: effect of anatomic site. Arch Dermatol Res 1987; 279:351. 118. Loth H, et al. Statistical testing of drug accumulation in skin tissues by linear regression versus contents of stratum corneum lipids. Int J Pharm 2000; 209:95. 119. Feldmann RJ, Maibach HI. Regional variation in percutaneous penetration of C-14 cortisol in man. J Invest Dermatol 1967; 48:181. 120. Kligman AM. Perspectives and problems in cutaneous gerontology. J Invest Dermatol 1979; 73:39. 121. Hosty´nek JJ. Flux of nickel salts vs. a nickel soap across human skin. In: Brain KR, Walters KA, eds. Perspectives in Percutaneous Penetration. Cardiff: STS Publishing, 2002:99. 122. Warner RR, Myers MC, Taylor DA. Electron probe analysis of human skin: element concentration proﬁles. J Invest Dermatol 1988; 90:78. 123. Menon GK, Elias PM. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol (Chicago) 1991; 127:57. 124. Cousins RJ. Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol Rev 1985; 65:238. 125. Rugstad NE, Norseth T. Cadmium resistance and content of cadmiumbinding protein in cultured human cells. Nature 1975; 257:136.

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126. Weismann K. Zinc Deﬁciency and Effects of Systemic Zinc Therapy. Dissertation, FADL’s Forlag, Kobenhavn, 1980. 127. Holmqvist I. Occupational arsenical dermatitis. A study among employees at a copper ore smelting work including investigations of skin reactions to contact with arsenic compounds. Acta Derm Venereol 1951; 31(suppl 26):1–214. 128. Artik S, et al. Nickel allergy in mice: enhanced sensitization capacity of nickel at higher oxidation states. J Immunol 1999; 163:1143. 129. van Neer FCJ. Reacties op intracutane injecties van drie en zeswaardige chroomverbindgen bij gesen-sibiliseerde mensen, vackens en caviae. Nederlands Tijdschrift Geneeskunde 1965; 109:1684. 130. Little MC, Gawkrodger DJ, Macneil S. Chromium- and nickel-induced cytotoxicity in normal and transformed human keratinocytes: an investigation of pharmacological approaches to the prevention of Cr(VI)-induced cytotoxicity. Br J Dermatol 1996; 134:199. 131. Requena CL, Sanchez LM, Coca MS. Argiria generalizada. Estudio clinico-histologico, histoquimico y ultrastructural. Actas Derm Sif 1985; 76:533. 132. Granstein RD, Sober AJ. Drug- and heavy metal-induced hyperpigmentation. J Am Acad Dermatol 1981; 5:1. 133. Gummer CL, Hinz RS, Maibach HI. The skin penetration cell: an update. Int J Pharm 1987; 40:101. 134. Franz TJ. Percutaneous absorption: on the relevance of in vitro data. J Invest Dermatol 1975; 64:190. 135. Franz TJ. On the relevance of in vitro data. J Invest Dermatol 1975; 64:190. 136. Bronaugh RL, Maibach HI. Percutaneous absorption of nitroaromatic compounds: in vivo and in vitro studies in the human and monkey. J Invest Dermatol 1985; 84:180. 137. Spencer TS, et al. Evaporation of diethyltoluamide from human skin in vivo and in vitro. J Invest Dermatol 1979; 72:317. 138. Reifenrath WG, Robinson PB. In vitro skin evaporation and penetration characteristics of mosquito repellents. J Pharm Sci 1982; 71:1014. 139. Bulmer FMR, Mackenzie EA. Studies in the control and treatment of nickel rash. J Industr Hyg 1926; 8:517. 140. Shah PV, Guthrie FE. Dermal absorption of benzidine derivatives in rats. Bull Environ Contam Toxicol 1983; 31:73. 141. Yang JJ, Roy TA, Mackerer CR. Percutaneous absorption of benzo[a]pyrene in the rat: comparison of in vivo and in vitro results. Toxicol Ind Health 1986; 2:409. 142. EPAU. Inductively Coupled Plasma—Mass Spectrometry. Washington D.C.: US EPA, 1986. 143. Rabinowitz MB, Wetherill GW, Kopple JD. Studies of human lead metabolism by use of stable isotope tracers. Environ Health Persp 1974; 7:145. 144. Brown SS, et al. IUPAC reference method for analysis of nickel in serum and urine by electrothermal atomic absorption spectrophotometry. Clin Biochem 1981; 14:295. 145. Derry JE, McLean WM, Freeman JB. A study of the percutaneous absorption from topically applied zinc oxide ointment. J Parent Bulmer Ent Nutr 1983; 7:131.

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146. Miller SJ. Nutritional deﬁciency and the skin. J Am Acad Dermatol 1989; 21:1. 147. Tsai JC, et al. Drug and vehicle deposition from topical applications: use of in vitro mass balance technique with minoxidil solutions. J Pharm Sci 1992; 81:736. 148. Blaschko A. Die Berufsdermatosen der arbeiter, ein beitrag zur gewerbehygiene (I). Deutsche Med Wochenschrift 1889; 15:925. 149. Cronin E, ed. Nickel, Contact Dermatitis. Edinburgh: Churchill Livingstone, 1980:338. 150. Angelini G, Ven˜a GA. Allergia da contatto al nickel. Considerazioni su vecchie e nuove acquisizioni. Bollettino Dermatol Allergol Professionale 1989; 4:5. 151. Kiec-Swierczynska M. Allergy to chromate, cobalt and nickel in Lodz 1977–1988. Contact Dermat 1990; 22:229. 152. Kanerva L, Jolanki R, Toikkanen J. Frequencies of occupational allergic diseases and gender differences in Finland. Int Arch Occup Environ Health 1994; 66:111. 153. Wester RC, et al. In vivo percutaneous absorption of boric acid, borax, and disodium octaborate tetrahydrate in humans compared to in vitro absorption in human skin from inﬁnite and ﬁnite doses. Toxicol Sci 1998; 45:42. 154. Reynolds RJ. Atomic Absorption Spectroscopy: A Practical Guide by R.J. Reynolds and K. Aldous, with a Chapter by K.C. Thompson. New York: Barnes & Noble, 1970. 155. Sunderman FW Jr. Chemistry, analysis and monitoring. In: Maibach HI, Menne´ed T, eds. Nickel and the Skin: Immunology and Toxicology. Boca Raton, Florida: CRC Press, Inc., 1989:2. 156. Sunderman FW Jr. Biological monitoring of nickel in humans. Scand J Worker Environ Health 1993; 19(suppl 1):34. 157. Quan D, Maibach HI. Electronic spin resonance. In: EW Smith, Maibach HI, eds. Percutaneous Penetration Enhancers. Boca Raton: CRC Press, 1995:427. 158. Herrling T, et al. Electron spin resonance detection of UVA-induced free radicals. Skin Pharmacol Appl Skin Physiol 2002; 15:381. 159. Cullander C, et al. A quantitative minimally invasive assay for the detection of metals in the stratum corneum. J Pharm Biomed Anal 2000; 22:265. 160. Johansson SAE, Campbell JL. PIXE—a Novel Technique for Elemental Analysis. Chichester: Wiley, 1988. 161. Forslind B, et al. Recent advances in X-ray microanalysis in dermatology. Scan Electron Micros 1985; (pt 2):687. 162. Malmqvist KG, et al. The use of PIXE in experimental studies of the physiology of human skin epidermis. Biol Trace Element Res 1987; 12:297.

4 Percutaneous Absorption of Copper Compounds Jurij J. Hosty´nek and Howard I. Maibach Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

INTRODUCTION Published and unpublished qualitative, semi-quantitative, and quantitative data on cutaneous absorption of this essential trace element brought in contact with the skin in its metallic form and its organic and inorganic species are reviewed. The different approaches that have been used to investigate copper diffusion and analysis of results are dicussed, as well as problems associated with their interpretation. Diffusion constants Kp registered range from 107 to 103 cm/h. Recognized as a critical endogenous anti-inﬂammatory agent in the normal organism, inﬂammatory conditions induce an increased demand in the organism for copper; such demand could potentially be satisﬁed by dermal exposure. Role and importance of copper’s anti-inﬂammatory activity and the possibility of transdermal dosing is viewed in light of the metal’s skin diffusivity data now available. The largest part of the literature published on the biology of copper addresses its importance and function as an essential trace element (ETE) in living organisms, its biochemistry, and role in nutrition and clinical medicine. Little has been published on its potential for skin diffusivity. So far, the only quantitative cutaneous diffusion rates given in the literature refer to experiments performed with copper compounds on the cat in vitro and in vivo, and with human skin in vitro as part of dermatological formulations. 67

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Transformation of data from those earlier studies, based on certain assumptions, lead to estimated Kp values of 106–105 cm/hr for the copper salts tested, values which lie at the lower end of skin diffusivity rates measured for transition metal salts. Interpretation of absorption data so far must rely on clinical observations. Quantitative data are difﬁcult to interpret due to the facile and dynamic changes in speciation potentially occurring in the membrane environment. Copper can exert its biological role thanks to the lability of its outer electrons in the 3d and 4s orbital shells, energy levels which overlap, allowing the element to switch readily from the monovalent to the divalent oxidation state. The transition thus requires minimal activation energy, and the shift between Cu(I) and Cu(II) is rendered favorable. Such facile changes in valence, noted also for chromium and iron, play a decisive role for the ions’ rate of transfer across cell membranes and epithelia, including the skin. Copper absorbed from the gastrointestinal tract is bound to albumin as a Cu(II) ion or as a Cu(II)-amino acid-albumin complex, and circulates in plasma bound to ceruloplasmin, albumin, or transcuprein. At the cellular interphase, copper dissociates from the carrier and is reduced to Cu(I) by plasma membrane reductase; once it has crossed the plasma-membrane barrier, it continues in the Cu(I) state (1). Such changes in speciations are presumed to occur in transit through the skin also. QUALITATIVE DIFFUSION DATA Earlier data on skin penetration by copper are circumstantial and qualitative only, derived from clinical observations, pharmacological activity, reports from dermatological practice, and observations made through use of spectroscopic methods (Table 1). In those studies, parameters that would allow calculation of diffusion rates are not available. Reaction of Metallic Copper in Contact with the Skin and Absorption of Derivatives Formed There In prolonged contact with the skin, metallic copper is oxidized, forming copper salts with amino acids, or soaps with fatty acids naturally present on the skin surface. This becomes apparent through the discoloration of the skin and of the copper-releasing objects in the areas of contact. These copper complexes formed on sustained skin contact appear to penetrate the skin, and the anti-inﬂammatory (AI) activity attributed to wearing of copper metal is attributed to their action, as Cu(II) in these complexes is considered to be essential for such an effect (11,12). Corrosion of copper articles, worn as bracelets, in contact with the skin or on immersion in natural or synthetic sweat was investigated by Walker and Grifﬁn; weight losses of 0.1% to 0.8% per month were recorded (4).

Percutaneous Absorption of Copper Compounds

69

Table 1 Clinical Signs and Analytical Data Demonstrating Cutaneous Copper Absorption References

Experiment

2 3 4 5

In In In In

Warner R.R., personal communication, 1993 6

In vitro, Cu sulfate

Human

Rat

9

In vivo, copper salicylate (AlcusalTM) In vivo, copper salicylate (DermcusalTM) In vivo, lipophilic copper complexes In vivo

10

In vivo

Cu metal

7

8

vivo, copper oleate vivo, copper metal vivo, copper metal vitro

Species Human Human Human Human

Detection Urine levels Serum level Metal loss Electron microscopy Spectral analysis

Rat

Inﬂammation arthritis Inﬂammation

Rat

Inﬂammation

Cu metal in ointment

Serum, scalp hair, urine; AA, ICPMS Inﬂammation, urine level

Abbreviations: ICP-MS, inductively coupled plasma-mass spectrometry; AA, atomic absorption.

Absorption of copper as reﬂected in serum levels has been observed following the accidental lodging of the ﬁnely divided metal in the skin. From an initial value of 13.0 mmol/L, copper serum levels increased over four days to 19.4 mmol/L (average control: 17.34 mmol/L), then gradually decreased again, with an apparent half-life of over 100 days. The shape of the serum copper plot suggests prolonged, extensive absorption (3). Absorption of the metal was determined qualitatively on men and women after a four-week application of an ointment containing 20% elementary copper. Serum and urine concentrations were determined by atomic activation spectroscopy and concentrations in hair by inductively coupled plasmamass spectrometry (ICP-MS). Serum concentration was seen to have increased signiﬁcantly, in urine it decreased, and in hair it remained constant (9). Diffusion of Copper Compounds Following in vivo application of copper oleate in a lanolin-petroleum base over 24 hours to human back skin, a signiﬁcant increase in urinary copper

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levels was observed over several days, to a maximum of 0.32 mg/L; determined for a normal control, that value was 0.11 mg/L (2). AlcusalTM (copper salicylate ethanolate) in ethanol-glycerin applied on rat skin in vivo showed AI and anti-arthritic activity (6). DermcusalTM (copper salicylate) in dimethyl sulfoxide applied on the skin of rats with adjuvant-induced polyarthritis was three times as effective as a similar Alcusal formulation (7). AI activity for a number of lipophilic copper complexes and phenols in dimethyl sulfoxide was studied by Beveridge et al.; particularly miﬂumic acid applied dermally on rats was found to be effective in reducing experimental arthritis in those animals (8). Electron microscopy of skin treated topically with copper acetate revealed that copper initially was localized in the intercellular spaces. Subsequently, the membranes of viable cells were also penetrated, with accumulation of the metal in and around the cell nucleus (5). By electron probe analysis and analytical electron microscopy, the occurrence of copper was followed across human skin in vitro following application of copper sulfate using Franz-type diffusion cells. Copper was observed to enter the outer stratum corneum (SC) cells, then, due to an apparent barrier occurring approximately halfway within the SC, the level fell below the detection limit (0.1% by weight), only to reappear again at the granular interface. There, copper was seen to diffuse by the intercellular route through the stratum granulosum to reach the stratum spinosum (Warner R.R., personal communication, 1993). Analysis of coppercontaining organic compounds applied by those authors on human cadaver skin by iontophoresis showed high and uniform penetration into the viable epithelial cells, with approximately half that level present in the dermis. SEMIQUANTITATIVE DATA Semiquantitative evidence for the formation of copper complexes and their diffusion into the superﬁcial skin strata (epidermis) was obtained by occlusion of ﬁnely divided copper powder on the arm of volunteers, followed by stripping of the exposed areas. Evidence was thus obtained that, over time, copper derivatives formed with skin exudates were absorbed beyond the SC and became available to systemic absorption (Hostynek, unpublished data). Under occlusion with exclusion of air, up to 72-hour exposure, copper values detected in the strippings were low, reverting to naturally present levels in those individuals. Under semiocclusion allowing access of air by covering the skin with ‘‘breathable’’ tape, however, after 72 hours, 0.4– 1.4 mg/cm2 was seen at the level of the stratum lucidum, and in individual volunteers cumulative amounts of up to 84 mg/cm2 copper were present (area under the curve [AUC], determined by ICP-MS analysis; Table 2). These observations conﬁrm the occurrence of chemical action of skin exudates (sweat and sebum) on contacting metals, such as copper, to form in situ

Percutaneous Absorption of Copper Compounds

71

Table 2 Copper (AUC) Removed with Tape Strips After Semiocclusive Application of Copper Powder on Human Skin Mean AUC (ng/cm2)

Occlusion time (hr) 0 (control) 24 48 72

38033 45845 55960 83974

Abbreviation: AUC, area under the curve.

diffusible salts, such as the chloride, pyruvate or lactate, and lipophilic derivatives (soaps) of likely diffusivity, as demonstrated earlier for nickel (13,14). QUANTITATIVE DATA Bis(glycinato) Cu(II) complex was the ﬁrst copper compound for which quantitative absorption data became available (15). A radioactive (64Cu) 0.05 M solution in physiological saline applied to excised cat skin revealed, after a lag time, a steady-state transport rate described as Kp ¼ 24 104 cm/hr. The percutaneous absorption of copper from copper chloride and copper sulfate has been investigated in vitro with human skin (16). Formulated for therapeutic purposes, these salts were administered together with zinc chloride and zinc sulfate, incorporated in the vehicles petrolatum and two aqueous gels. For CuSO4 from petrolatum or from CarbopolTM gel and for CuCl2 from MetoloseTM gel, the apparent permeability coefﬁcients were similar, 0.032–0.045 104 cm/hr (Table 3). For CuCl2 from petrolatum, the permeability coefﬁcient was somewhat higher, 0.023–0.16 104 cm/hr. Skin penetration of commercial products (emulsions and ointments) containing copper and zinc was investigated in human, dermatomed abdominal skin (400 mm) in vitro for periods of 72 hours (16). Whether formulated with copper sulfate or the organic salt copper 2-pyrrolidone 5-carboxylate,

Table 3 Diffusivity of Copper (with Zinc) Salts Through Human Skin In Vitro Salt CuSO4 (þZnSO4) CuSO4 (þZnSO4) CuCl2 (þZnCl2) CuCl2 (þZnCl2)

Formulation

104 Kpa (cm/hr) SD

Petrolatum Carbopol 940TM gel Petrolatum Metolose 60 SHTM gel

0.032 0.031 0.045 0.057 0.16 0.12 0.023 0.010

a Permeability coefﬁcient Kp calculated from copper in the receptor ﬂuid. Source: From Ref. 16.

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Table 4 Percutaneous Absorption of Copper Compounds from Various Vehicles Through Human Skin In Vitro

Formulation Experiment No. 1 Emulsion A Emulsion B Emulsion C Experiment No. 2 Emulsion A Ointment D Ointment E

Exposure (hr) 104 Kpa

Compound

Concentration (mg/cm3)

0–2

24–48

48–72

CuPC CuSO4 CuSO4

1 1.3 0.5

0.57 0.44 1.2

0.02 0.010 0.03

0.06 0.015 0.08

CuPC CuSO4 CuSO4

1 0.9 0.5

1.2 0.8 0.9

0.06 0.05 0.10

0.13 0.09 0.12

a

Permeability coefﬁcient Kp (cm/hr). Source: From Ref. 17.

apparent permeability coefﬁcients, were in the range 0.015 104– 0.13 104 cm/hr (Table 4). Permeability coefﬁcient values decreased over successive time periods from near 1 104 cm/hr (zero to two hours) to 0.1 104 cm/hr or less (25–48 hours) and ﬁnally to undetectable values in two cases (49–72 hours). The vehicles were three emulsions (two commercial products and a custom-made variant of one of the former) and two commercial ointments. All contained both zinc and copper compounds. Each formulation was applied at the rate of 16 mg/cm2. 1. Emulsion A: water/oil, containing ZnO, ZnPC, and CuPC 2. Emulsion B: same as A except that CuSO4 and ZnSO4 replaced CuPC and ZnPC 3. Emulsion C: water/oil, containing ZnSO4, ZnO, and CuSO4 4. Ointment D: containing ZnSO4, ZnO, and CuSO4 5. Ointment E: containing ZnO and CuSO4 The absorption rate of copper generally decreased as the experiments progressed, and for formulations B, C, and E there was little or no absorption in the ﬁnal 24 hours. After 72 hours exposure, there was a signiﬁcant increase in the average copper concentrations (140–430% of control values) in the epidermis for all the formulations. In the dermis, where control concentrations averaged just 2% to 3% of the epidermal control values, the treatments signiﬁcantly increased the copper contents. Using human abdominal epidermis, the diffusion of copper sulfate and copper acetate as 1% aqueous donor solutions was compared in vitro (Hosty´nek, unpublished data). The receptor ﬂuid, high pressure liquid chromatography–pure water, was collected up to 96 hours after the application

Percutaneous Absorption of Copper Compounds

73

Table 5 Coppera Diffusion Constants Kp Through Human Skin In Vitro Compound Sulfate (þZnSO4) Sulfate Sulfate Chloride (þZnCl2) Acetate Acetate Histidinate Gluconate Glycinate Glycinate GHKd GHK GHK

104 Kp (cm/hr)

Comments

0.032–0.045 8.84 0.027 0.023–0.16 0.019 0.066 1.03 2.28 24 2.43 0.76e 25.1e 0.002

Split thickness Split thickness Epidermis Split thickness Split thickness Epidermis Split thickness Split thickness Full thicknessc Split thickness Split thickness Split thickness Epidermis

References 16 Unpublishedb Unpublished 16 Unpublished Unpublished Unpublished Unpublished 15 Unpublished Unpublished Unpublished Unpublished

a

Cu(II) compounds. Unpublished data, Hostynek et al. c Cat skin. d Cu-glycine-histidine-lysine. e Kps from separate sets of cells in the same diffusion experiment. b

of the donor solutions. The copper concentrations in the receptor ﬂuid were analyzed using ICP-MS. The Kps measured at steady-state ﬂux were 2.73 0.29 106 cm/hr for the sulfate and 6.05 1.19 106 cm/hr for the acetate. Permeability coefﬁcients measured for aqueous copper sulfate and acetate through human epidermis in vitro are of the order of 106 cm/hr. For copper compounds formulated together with zinc compounds for therapeutic purposes, applied on dermatomed human skin in vitro in various vehicles, the apparent penetration coefﬁcients were in the range of 3.2 106–1.6 105 cm/hr (Table 5). DISCUSSION AND CONCLUSIONS Toxicological considerations and pharmacological evidence for the effects of exposure to exogenous copper: Aspects of safety of exogenous copper need to be put in proper perspective to allay concerns on the part of clinicians not accustomed with the action of copper compounds in the therapy of rheumatoid arthritis and other degenerative-inﬂammatory human diseases. Data available indicate that copper may have noxious effects only following chronic oral (or parenteral) exposure to high amounts of the metalloelement, particularly upon chronic oral ingestion with food (e.g., water) that supplies the organism with more than 5 mg/kg of copper per day (18) or in the case of prolonged hemodialysis with systems that introduce into the circulation the metal from copper-containing, semipermeable membranes or

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Table 6 Acute Toxicity and Therapeutic Indices of Copper Complexes TI Copper complex Cupric acetate Cupric anthranilate Cupric aspirinate

LD50 (SC) mg/kg

CFE

PA

350 750 106 760 100

70 50 150

– 750 760

Abbreviations: TI, therapeutic index; CFE, carrageenan foot edema; PA, polyarthritis. Source: From Refs. 20 and 21.

copper tubing (19). Thus, a real risk of copper toxicity may exist only if the administering amounts of the metallo-element certainly are far greater than those that can be actually used for therapeutic purposes, by transdermal exposure in particular. Sorenson investigated the acute toxicity of AI copper complexes injected subcutaneously in rats (partly listed in Table 6) (20,21). Comparison with the chelating agent demonstrated that the copper complexes were less toxic and damaging to the organism than the parent drug. Cupric sulfate has been used medicinally as emetic, astringent, and anthelmintic. The intestinal mucosa acts as an efﬁcient barrier; however, excessive amounts of oral copper salts may lead to death. In the normal human organism, an efﬁcient homeostatic mechanism controls copper activity, as most is ﬁrmly bound to alpha-ceruloplasmin, and heightened exposure does not result in disease. Investigating the cutaneous absorption of copper in the skin, applied as copper(I) oxide and in its elemental form in an ointment at 20% over a four-week period, the authors concluded that dermal exposure to copper at those levels does not present a risk of systemic toxicity, nor does it have any untoward effects in the skin exposed (9).

LIMITATIONS IN MEASURING COPPER ABSORPTION IN VIVO Homeostasis: Formation of depots and thus of a secondary barrier, which inhibits further diffusion can be reversible or irreversible. Examples of the ﬁrst kind are essential elements subject to homeostatic control, e.g., copper or zinc, which are retained by speciﬁc storage proteins, such as metallothionein or, for the former, ceruloplasmin. They are again released upon physiologic demand (22). Homeostasis may thus compromise the validity of experimentally determined Kp values for copper. Salts of copper or zinc present in the SC, epidermis, and dermis are kept in equilibrium by control mechanisms as part of the overall physiological dynamics of micronutrients, which regulate their status as free ions. Homeostasis maintains equilibria

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necessary for the optimal functioning of the organism in general, and the skin in particular, as absorption is increased in the copper-deﬁcient organism and decreases once optimal copper status is established. Dermal absorption experiments conducted in vivo thus are bound to reﬂect such controls, which may counteract passive diffusion and add a degree of uncertainty to permeability constants measured. INTERDEPENDENCE OF SYSTEMIC COPPER AND ZINC LEVELS Levels and activity of zinc in the mammalian organism are closely related to tissue levels of copper. By inference, it is likely that dermal copper absorption is also a variable in the function of prevailing zinc levels, and some observations made as to physiological dynamics of zinc by inference may reﬂect on copper also, based on such interrelationship of these two metals. Measurements of zinc absorption have been contradictory. Apparently its absorption does not take place by simple diffusion, being regulated by homeostasis, whereby uptake is inversely related to the individual’s nutritional status and the actual body burden of the metal. Because copper and zinc coexist in a competitive relationship, excessive levels of zinc as consequence of dietary supplementation for therapeutic purposes appear to determine deﬁciency in copper, resulting in untoward health effects. The normal serum copper/zinc ratio of 0.9 to 1.2, for example, decreased to 0.5 in consequence of long-term dietary supplementation with the latter (23). As an electrophilic metal, copper(II) is highly reactive with electronrich biological tissues (24) and as applies other transition elements, its in-depth penetration beyond the superﬁcial layers of the SC will be retarded by reactivity with sulfhydryl groups in cystein, glutathione, or thioglycolic acid, which abound in skin tissues. Limitations in the validity of percutaneous absorption data determined for copper in vivo are thus due to (i) depot formation, (ii) homeostasis, and (iii) the interdependence of copper and zinc. As with most transition metals, skin penetration by copper is subject to numerous interrelated factors, and its nature as an ETE makes diffusion experiments difﬁcult and interpretation of results uncertain. This may be an additional reason why in-depth investigation of copper’s dermatopharmacokinetics is virtually nonexistent. Overall evidence on skin penetration by metals available so far points to a few commonalities, be it in respect to the individual xenobiotic or to characteristics of the skin barrier and the underlying organism; this makes a few generalizations and anticipatory statements possible, putting copper diffusivity in perspective with other transition metals. A challenging determinant in transition metals is the variability in their speciation, which is critical for their diffusivity through biological membranes. Mineral salts of copper (electrolytes) in their ionized form, however, traverse the skin barrier

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with permeability coefﬁcients of the order of 104–106 cm/hr. For comparison, the Kp for water has been determined at 103 cm/hr. The present literature review covers only studies conducted by us and others on normal, healthy, and intact skin, not preconditioned in any way. The only quantitative data on skin penetration by copper or its compounds through human skin in vivo or in vitro so far is that acquired in our laboratory (Table 5). The information generated in our laboratory and by others earlier on diffusion characteristics permits the following conclusions. 1. Copper compounds and copper applied as the metal do penetrate the stratum corneum; however, data on their penetration as measured so far differ widely and, thus, rate and degree of absorption remain unpredictable. 2. Transepidermal (primarily intercellular) permeation pathways have been observed for copper compounds. Penetration via hair follicles, sebaceous glands, and sweat glands has also been established. 3. Lipophilic organo-copper compounds are more easily absorbed when compared with coordination complexes and salts. 4. Brought in contact with the skin in the elemental state, copper is oxidized by exudates present on the skin surface, and the resulting ion penetrates the SC (as the derivative salt or soap) in a timedependent fashion. 5. In terms of biological functions, the degree and rate of copper compound penetration is inﬂuenced by: (i) homeostatic mechanisms, (ii) formation and/or pre-existence of a reservoir, and (iii) regulation by speciﬁc binding proteins (metallothionein or ceruloplasmin).

RECOMMENDATIONS FOR RESEARCH TO FILL EXISTING DATA GAPS Evidence is well established for the various functions of copper as an ETE, AI activity among others. In consideration of the evidence concerning the beneﬁts resulting from transdermal AI therapy with copper compounds, it would appear useful to establish a database for the skin penetration of such compounds using rigorous experimental protocols. That becomes possible by implementing in vitro diffusion experiments through human skin by accurately measuring the absorption of copper and its compounds under carefully controlled conditions of skin membrane selection and preparation, ‘‘permeant’’ concentration, vehicle, area and time of exposure, donor and receptor medium, and also by analysis of diffusion membrane analysis for retained permeant. There appears to be a need for research to establish the potential for dermal absorption of copper through skin-diffusible forms of the metal. Supplementation in the copper-deﬁcient organism may present much needed

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relief for a number of conditions associated with the deﬁciency in this ETE: integumentary and skeletal abnormalities, defects in growth and development, abnormalities in sensory perception (22), the myriad of manifestations of inﬂammation and connective tissue disease, such as rheumatoid arthritis (25). While copper(II) has been administered by traditional drug delivery systems, intravenously as salts or by intramuscular injection of Cu(II) complexes, there still appears to be a need for a suitable Cu(II) complex of ready transdermal transport in order to deliver the element to intracellular sites where it is needed. Parenteral administration is hazardous because of local toxicity when long-term therapy is necessary (26). Oral administration of copper complexes in turn is problematic due to their breakdown at the low gastric pH, whereby copper ions released from the complex will be subject to excretion and the highly efﬁcient homeostatic control mechanism designed to prevent much of the metal to be assimilated (27,28). While the scarce data reviewed here indicate that copper in the ionized form may be a permeant too poor to effectively reach the target tissue in adequate dose, results obtained by Pirot et al. and in our laboratory point to somewhat higher diffusion rates when skin is exposed to copper complexed with organic moieties. For a better understanding of the pharmacodynamics of copper skin penetration, further investigation of its diffusivity in its different chemical forms would appear useful. In analogy with results obtained for nickel applied as the metal (14), particularly when associated with amino acids or skin-identical ligands, topical application of copper as ﬁnely disperse powder for maximum surface activity, e.g., already points to therapeutically effective penetration rates of lipophilic oxidation products formed with skin exudates, although oxidation of the metal on the skin to yield diffusible derivatives may appear to be an ineffective dosage form. CONCLUSIONS Observations made in vivo serve to conﬁrm that copper metal on prolonged contact with skin exudates (sweat and sebum), and in the presence of air forms in situ diffusible salts such as the chloride, pyruvate or lactate, and lipophilic derivatives (soaps) of likely diffusivity. The role of the skin as a toxicologically important route of exposure to environmental agents in general and metal compounds in particular is hereby underscored. It represents an important port of entry not only to hazardous materials such as readily oxidized metals which harbor the potential for serious health effects, but also to essential elements such as copper, whose release and diffusivity through skin contact has the potential for beneﬁcial and therapeutic action in the inﬂamed organism. These ﬁndings may contribute to the acceptance of the long-held belief in the AI effects of copper metal in direct contact with the skin, in particular, and also may promote the concept of external AI therapy by patches as alternative to systemic dosing such as intravenous, intraarticular, or intraperitoneal.

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Critical for AI activity may be the supplementation of endogenous copper with exogenous sources, irrespective of the agent’s nature (i.e., elemental, salt, complex, or as a covalent derivative), because endogenous formation of the chelate of maximum biological activity will be formed under homeostatic control. The mechanism of copper complexes acting as AI agents is not known in all its details, yet evidence so far points to the possible formation of a unique, as yet undeﬁned metabolite responsible for the observed clinical AI effect. GLOSSARY Absorption Adsorption Corrosion Electromotive series Ionization potential

Ligand

Oxidation

Oxidation potential

Penetration Permeation Reduction

Speciation

Uptake into the organism. Material adhering to the skin surface. Electrochemical process involving redox reactions in the presence of electrolytes. Arrangement of metals by decreasing order in their ability to oxidize. Energy (electron volts, ev) required to remove an electron from its atomic orbit, with the value for the Standard Hydrogen Electrode set at 0.00 ev as an arbitrarily selected standard reference. Atom, ion or functional group bonded to a central atom, usually a metal, to form a complex. Process of electron removal from an atom or ion (e.g., the increase in the proportion of oxygen in a compound). Electrical driving force toward electron loss, expressed as a potential value (in electron volts, ev). Passive diffusion process of a solute through skin. Diffusion through one or several layers. Process of electron gain by an atom or an ion (e.g., the increase in the proportion of hydrogen in a compound). Valence state; organic vs. inorganic ligand bonds.

ABBREVIATIONS AA AAS AI

atomic absorption atomic activation spectroscopy anti-inﬂammatory

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AUC ETE HPLC NMR SC UV

79

area under the curve essential trace element high pressure liquid chromatography nuclear magnetic resonance stratum corneum ultraviolet

REFERENCES 1. Elam JS, et al. Copper chaperones. Adv Protein Chem 2002; 60:151. ¨ ber die Kutane Kupferresorption aus einer Kupfer 2. Schmid R, Winkler J. U Enthaltenden Salbe. Klin Wochenschr 1938; 17:559. 3. Bentur Y, et al. An unusual skin exposure to copper; clinical and pharmacokinetic evaluation. J Toxicol Clin Toxicol 1988; 26:371. 4. Walker WR, Grifﬁn BJ. The solubility of copper in human sweat. Search 1976; 7:100. 5. Odintsova NA. Permeability of human skin to potassium and copper ions and their ultrastructural localization. Chem Abstr 1978; 89:360. 6. Walker WR, Beveridge SJ, Whitehouse MW. Antiinﬂammatory activity of a dermally applied copper salicylate preparation (Alcusal). Agents Actions 1980; 10:1. 7. Beveridge SJ, Walker WR, Whitehouse MW. Anti-inﬂammatory activity of copper salicylates applied to rats percutaneously in dimethyl sulphoxide with glycerol. J Pharm Pharmacol 1980; 32:425. 8. Beveridge SJ, Whitehouse MW, Walker WR. Lipophilic copper(II) formulations: some correlations between their composition and anti-inﬂammatory/anti-arthritic activity when applied to the skin of rats. Agents Actions 1982; 12:225. 9. Gorter RW, Butorac M, Cobian EP. Examination of the cutaneous absorption of copper after the use of copper-containing ointments. Am J Ther 2004; 11: 453–458. 10. Fat L, Gyorffy L. Occupational dermatitis due to copper exposure. Proceedings of the OEESC conference, 2002:51–52. 11. Walker WR, Beveridge SJ, Whitehouse MW. Dermal copper drugs: the copper bracelet and Cu(II) salicylate complexes. Agents Actions (Suppl) 1981; 8:359. 12. Walker WR, Keats DM. An investigation of the therapeutic value of the ‘‘Copper Bracelet’’—dermal assimilation of copper in arthritic/rheumatoid conditions. Agents Actions 1976; 6:454. 13. Hostynek JJ. Flux of nickel (II) salts vs. a nickel (II) soap across human skin, in vitro. Exog Dermatol 2003; 2:216–222. 14. Hostynek JJ, et al. Human stratum corneum penetration by nickel: in vivo study of depth distribution after occlusive application of the metal as powder. Acta Derm Venereol (Suppl) 2001; 212:5. 15. Walker WR, et al. Perfusion of intact skin by a saline solution of bis(glycinato) copper(II). Bioinorg Chem 1977; 7:271. 16. Pirot F, et al. Simultaneous absorption of Cu and Zn through human skin in vitro. Skin Pharmacol 1996; 9:43.

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17. Pirot F, et al. In vitro study of percutaneous absorption, cutaneous bioavailability and bioequivalence of zinc and copper from ﬁve topical formulations. Skin Pharmacol 1996; 9:259. 18. Aggett PJ, Fairweather-Tait S. Adaptation to high and low copper intakes: its relevance to estimate safe and adequate daily dietary intakes. Am J Clin Nutr 1998; 6:1061S–1063S. 19. Scheinberg HI, Sternlieb I. Copper toxicity and Wilson’s disease. Trace Elements in Human Health and Disease, Vol. 1; Zinc and Copper, New York: Academic Press, 1976:415–438. 20. Sorenson RJ. Some copper coordination compounds and their antiinﬂammatory and antiulcer activities. Inﬂammation 1976; 1:317–331. 21. Sorenson RJ. Copper chelates as possible active forms of the antiarthritic agents. J Med Chem 1976; 19:135–148. 22. Burch RE, Hahn HKJ, Sullivan JF. Newer aspects of the roles of zinc, manganese and copper in human nutrition. Clin Chem 1975; 21:501. 23. Abdulla M. Copper levels after oral zinc. Lancet 1979; 1:616. 24. Oster G, Salgo MP. The copper intrauterine device and its mode of action. N Engl J Med 1975; 293:432–438. 25. Rafter GW. Rheumatoid arthritis: a disturbance in copper homeostasis. Med Hypoth 1987; 22:245. 26. Perrin DD, Whitehouse MW. Metal ion therapy: some fundamental considerations. In: Rainsford KD, Brune K, Whitehouse MW, eds. Trace Elements in the Pathogenesis and Treatment of Inﬂammation. Basel: Birkhauser, 1981. 27. Underwood EJ. Trace Elements in Human and Animal Nutrition. 4th ed. New York: Academic Press, 1977. 28. Williams DR, Furnival C, May PM. Computer analysis of low molecular weight copper complexes in bioﬂuids. In: Sorenson JRJ, ed. Inﬂammatory Diseases and Copper. Clifton: Humana Press, 1982; 45:45–55.

5 Diffusion of Copper Through Human Skin In Vivo Jurij J. Hosty´nek and Howard I. Maibach Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

Frank Dreher Neocutis, Inc., San Francisco, California, U.S.A.

INTRODUCTION Sequential tape stripping was implemented on healthy volunteers to examine the diffusion of copper through human stratum corneum in vivo following application of the metal as powder on the volar forearm for periods of up to 72 hours. Exposure sites were stripped 20 times and the strips analyzed for metal content by inductively coupled plasma–mass spectroscopy with a detection limit for copper of 0.5 ppb. Untreated skin was stripped in the same fashion to determine baseline copper levels for comparison with exposure values resulting from exposure in respective volunteers.

This chapter, in part, was reprinted from Hosty´nek JJ, Maibach HI, Dreher F. Human stratum corneum penetration by copper: in vivo study after occlusive and semiocclusive application of the metal as powder. Food Chem Toxicol (in press), with permission of Elsevier.

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Under occlusion with exclusion of air, up to 72 hours copper values decreased from the superﬁcial to the deeper layers of the stratum corneum with gradients increasing commensurately with occlusion time, characteristic of passive diffusion processes. From the 10th strip on, however, levels reverted to background values. Under semiocclusion allowing access of air by covering the skin with ‘‘breathable’’ tape, initial copper values lay signiﬁcantly above baseline values and concentration gradients increased proportionally with occlusion time. At 72 hours, from the 10th to the 20th strip reaching the glistening epidermal layer, copper values continued at constant levels, signiﬁcantly above baseline values. The results indicate that, in contact with skin, copper will oxidize and may penetrate the stratum corneum after forming an ion pair with skin exudates. The rate of reaction seems to depend on contact time and availability of oxygen. A marked interindividual difference was observed in baseline values and amounts of copper absorbed. Arthritis patients have worn copper jewelry of various types for thousands of years for the relief of inﬂammation and musculoskeletal disorders. Their use continues today as folk remedy. Data documenting statistical signiﬁcant amelioration of arthritic conditions in patients wearing copper objects in intimate contact with skin as compared with the effect of placebo objects, however, are missing to date. Indicative of the process of skin penetration by certain metals is the short-term onset of skin reactions, primarily immunologic or nonimmunologic irritation, resulting from contact with coinage, tools, jewelry, or other articles of daily use. Major skin reactions to metals involve nickel and chromium (1,2). Few reports suggest skin reactions to copper also (3–6). It remains to be demonstrated that copper metal in contact with the skin does diffuse through the SC to reach the nucleated epidermis, becoming locally or systemically available. The critical factor in determining if a metal will penetrate the SC is believed to be the formation of soluble compounds. In the presence of oxygen, skin exudates may lead to electrochemical reactions resulting in metal oxidation (corrosion) to form potentially skin-diffusible compounds with naturally present skin-identical anions such as chloride ion, with amino acids or fatty acids present on the skin surface (7,8). Such reaction usually becomes apparent through the discoloration of the skin of the wearer and of the copper-releasing objects in the areas of contact. Besides metallurgical properties of the metal (composition, surface structure, and ﬁnish), occurrence and rate of such corrosion will depend on individual variables: amount and composition of exudates, pH, temperature, and the availability of oxygen. So far, this phenomenon of copper metal dissolution, its uptake into the SC, and its clinical effects has been supported by a number of investigations. After immersion of copper turnings in human sweat over 24 hours, the metal concentration increased by an average of two orders of magnitude from the

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natural level: from 1–3.5 105 to 0.6–3.4 103 M (9). Copper complexes generated on sustained contact of the metal with animal skin appeared to exert anti-inﬂammatory (AI) activity in the animals; that was attributed to the action of copper compounds formed in situ and their cutaneous absorption (10). By electron microscopy, Odintsova (11) observed the presence of copper in intercellular spaces, and ultimately around the cell nucleus in human skin upon exposure to Cu(II) acetate. Absorption of copper has been observed indirectly following the accidental lodging of the ﬁnely divided metal in human skin dermatoglyphics. Serum levels were found to increase over four days, and then gradually decrease with an apparent half-life of over 100 days (12). A strong argument in favor of the therapeutic activity of elemental copper brought in intimate contact with the organism was provided earlier in an animal study that demonstrated the AI properties of copper metal present in the organism: a clear prophylactic effect was achieved when experimental inﬂammation failed to be induced in rats in which copper metal had been implanted two months prior to the experiment (13). It remains to be demonstrated that copper penetrates the skin upon exposure to the metal in vivo, and thus potential AI effects could be achieved by dermal exposure also in humans. In order to arrive at a conclusive assessment of skin penetration by copper, it appears useful to obtain evidence of cutaneous penetration on contact by use of quantitative metal analysis. The purpose of the present investigation into the fate of metallic copper brought in intimate contact with the skin was (i) to obtain (semiquantitative) evidence for the formation of copper complexes that diffuse and are detectable by the presence of copper in the SC, (ii) to trace the path and kinetics of copper diffusion through the SC, (iii) to assess adsorption and reservoir formation by the penetrating metal in the SC, and (iv) obtain evidence that if copper absorbed over time it could penetrate beyond the SC and become available to systemic absorption. Credence could thus be conferred to the contended beneﬁts of relief from musculoskeletal disorders attributed to the wearing of copper jewelry, as the potential for the metal’s activity as an AI agent by dermal exposure in humans so far is still subject of controversy. This report illustrates the process of copper diffusion following application of ﬁnely distributed (micronized) copper metal on the skin, through intimate contact under occlusion and semiocclusion. It thus becomes possible to estimate actual copper concentration proﬁles, to the level of the living epidermal tissue and presumed subsequent uptake by dermal microcirculation. Determining kinetics and penetration depth of permeants by tracing the concentration proﬁles in SC has been rendered facile by using the virtually noninvasive method of SC stripping with adhesive tape and by the possibility to detect minute quantities of metals using ICP-MS analysis (14–19). It thus becomes possible to analyze for the presence of elements such as copper in skin and other biological materials, with detection limits on the order of 0.5 ppb (20), making the use of radioisotopes unnecessary.

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Investigated by Schwindt et al. (21), on average each tape strip removes one layer of corneocytes, of approximate 0.5 mm thickness, and after the ﬁrst 2–3 strips, for a given skin site and test subject each subsequent tape strip removes the same amount of SC, down to approximately the 20th strip (r2 ¼ 0.99). Although furrows in the SC account for the uncertainty in singlelayer stripping (22), by progressive SC removal with 20 strips it nevertheless becomes possible to estimate actual copper concentration proﬁles to the level of the living epidermal tissue. EXPERIMENTAL Subjects Healthy Caucasian volunteers between the ages of 34 and 69 without evident dermatological disease participated after giving informed consent. The study was approved by the University of California Committee on Human Research. The volar forearm was selected as study site. Three replicates on adjacent sites were conducted on the same volunteer. Copper Application, Occlusion, Decontamination, and Stripping For the purpose of determining baseline copper values, the naturally occurring copper in the skin of the volunteers was determined by stripping untreated skin, with three iterations for each occlusion period, on the volar forearm of the participating volunteers. For occlusion of the SC, plastic chambers of 12 mm diameter (1.15 cm2) were placed on the premarked area of the ﬂexor surface on the arm, the chamber covered with the dressing and left for the predetermined length of time (24, 48, and 72 hours). The occlusive and semiocclusive application systems consisted of a plastic chamber (Hill-Top Res., Inc., Cincinnati, U.S.A.) and micropore semiocclusive tape (3M Health Care, St. Paul, Minnesota, U.S.A.), respectively. Copper powder [99.7% 3 mm particle size (Aldrich Chem. Co., Milwaukee, Wisconsin, U.S.A.)] was applied in triplicate on three volunteers. Prior to application, skin sites were cleansed with deionized water and dried using cotton swabs. Copper powder was applied on the volar forearm between wrist and antecubital fossa. The experiment was conducted during the months of September through April, whereby the exposed skin of the volunteers exhibited no noticeable tanning. Twenty-ﬁve milligrams of copper powder were placed on a plastic chamber; the chamber then placed on the premarked area of the ﬂexor surface of the arm of the volunteer, and left undisturbed for the predetermined length of time. At the end of the exposure period, the occlusive materials were removed, the site carefully washed with a metal-complexing detergent containing a 5% solution of Extran 300TM (EM Science, Gibbstown, New Jersey, U.S.A.), using cotton swabs to remove all traces of metal left on the skin surface, then rinsed

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with water. The skin site was dried by tapping with cotton balls and ﬁnally by passing an air stream over the surface for one minute. Before tape stripping, the area of metal contact was marked with a pen and a template (TransporeTM tape with a 1.2 cm diameter hole) was applied, to guarantee that SC was removed only from the application site. After increasing intervals of occlusion, the area of application was stripped sequentially 20 times after covering the 1.15 cm2 treated area with a 1 in (6.45 cm2) of the precut adhesive tape strips. Polypropylene tape with a backing of pressure-sensitive acrylate adhesive (Transpore, 3M Health Care, St. Paul, Minnesota, U.S.A.) of 2.54 cm width was used for stripping. Constant and uniform pressure (100 g/cm2) was applied on the tape for ﬁve seconds, which was then gradually removed in one slow draw (17). The tape with adhering SC were placed individually in scintillation vials, 5 mL of concentrated nitric acid (70%; EM Science, Gibbstown, New Jersey, U.S.A.) was added and the vials agitated vigorously for three hours on a rotary shaker (GyrotoryTM water bath model G76; Edison, New Jersey, U.S.A.). The acid solution was then diluted 20-fold with water for ICP-MS analysis. Metal Analysis Samples were prepared by diluting the samples from the stripping experiment at 1:10 (volume/volume) using 2% HNO3 immediately prior to analysis. Standard solutions were prepared by diluting a 1000 g/mL Cu stock solution with 2% HNO3. A six-point external calibration curve (r2 > 0.999) was used for quantitation for both Cu isotopes (63Cu and 65Cu), with 89Y being used as an internal standard. The ICP-MS was an Agilent 7500c spectrometer operated under He collision gas mode. The instrumental detection limit was <100 ppt Cu. For those samples that were high in Cu concentration (>1000 ng/mL), a Perkin-Elmer 4300 dual view ICP optical emission spectrometer was used for analysis. Statistical Analysis Analyses of the AUC were performed using two-sided, paired Student t-test. The probability value, P < 0.05, was considered signiﬁcant. RESULTS General Due to the passive nature of the diffusion process, for all subjects a decreasing concentration gradient from the horny layer to the subcutaneous tissue became evident (Figs. 1 and 2). This concentration gradient was steep in the outermost layers of the SC due to its barrier function (Schaefer and Redelmeier, 1996) (39) and became less so towards the viable layers of the epidermis.

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

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

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

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

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

Mean AUCa (ng/cm2)

SD

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

18,086

p-value 0.11 0.03b 0.08

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

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

ð1Þ

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

ð2Þ

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

ð3Þ

Cuð0Þ ! Cuþ2 þ 2e 0:34 ev

ð4Þ

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

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

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

LD50 (sc) (mg/kg)

CFE

PA

350 750 106 760 100

70 50 150

750 760

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

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

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GLOSSARY Absorption Coinage metals

Corrosion Electromotive series Ionization potential

Metallic elements

Noble metals

Oxidation

Oxidation potential

Penetration Permeation Reduction

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

ABBREVIATIONS AI AUC ev SC

anti-inﬂammatory area under the curve electron volt stratum corneum

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sc ICP-MS

subcutaneous inductively coupled plasma–mass spectroscopy

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

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

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

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

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

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

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

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

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

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

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

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

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

Au

Pt

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

76.9 50.0 5.7 44.9

1

a

1.0 0.1

Pd

Ag

Cu

1.4 9.0 25.0 3.0

9.6 4.0 67.2 39.0 67.6 69.6

11.1 34.0 12 5 2.8

Sn

Zn 2.9

1.0 1.0 26.2 26.9

0.26 0.96

Experimental alloy.

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

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

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

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

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

Zn SD (mg/mL)

Cu SD (mg/mL)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Ni, wt% (other metals)

Cu, wt%

Copper (0) JSC—gold alloy

100 11.5

Degussa Training Metal Trindium

87.5

Duracast MS

81.6

Goldent

76.0

Modulay

77.0

ANA 68

5.0

87.0

DispersalloyTM

12.4

ValiantTM

20.0

Gallium alloy GF

15.0

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

Abbreviation: N/R, none reported.

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

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

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

Adhesive pads

Occup. exposure to copper

1

1

Diagnostic test

Urticaria

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

Copper metal 2% aq. CuSO4

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

Scratch, CuSO4

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

Visible signs and symptoms

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

2 1

1

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

Cohort/etiology

1 1 1

Cases

Table 2 Immunologic Contact Urticaria Due to Copper

1–2 1?

2

1? 1 2

CR

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

Positive Punctate wheals

Positive

None Dyspnea Erythema

Challenge reaction

50

76

80 47

79

77 78 2

References

Copper Hypersensitivity 127

Metal contact Dermatol. patients Teleph. lineman Jewelry

Metalworker

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

1 2 1 1

1

10 1 6 4 2 140

Abbreviation: CR, clinical relevance.

1 1 2 1 3 1 5 3 1

Dermatol. patients

Cohort/etiology

3

Cases

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

Dermatitis Eczema Eczema Dermatitis Dermatitis Dermatitis

Eczema

Dermatitis Dermatitis Dermatitis Eczema

Hand eczema

Visible signs and symptoms Patch test

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

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

Table 3 Allergic Contact Dermatitis Due to Copper

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

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

Challenge reaction

1 0–1 1 0–1 0–1 1–2 0–1 0–1 2–3

50 87 88 89 90 91 92 93 53

84 74 85 19 86 49

83

0–1 1? 2 0 0 1? 0–1

81 82 44 45

43

References

2–3 0–1 1 1–2

0–1

CR

128 Hosty´nek and Maibach

1

1 2

3 1 2

1 1

Multiple metal sensitivities

Nephritis Oral lesions

Eczema, edema Edema, erythema None

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

3

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

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

1

1

1 4

Visible signs and symptoms

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

Cohort/etiology

1

Cases

Patch test

97 98 99

1? 1 0–1 1 1

Pos. to CuSO4 and NiSO4 Positive patch at 72 hr

Cu and other metal patches

Eczema

95 96

2 0–1

CuSO4 5% aq. CuSO4

48

0–1

(Continued )

51

100 67

94 4 1

1 2

3

2

33 46

2–3

Positive at 96 hr, negative

References

0–1

CR

Challenge reaction

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

Cu metal, CuO 1% aq. CuSO4 Cu metal

Table 4 Systemic Allergic Contact Dermatitis Due to Copper

Copper Hypersensitivity 129

Visible signs and symptoms

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

Lychen planus

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

Cohort/etiology

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

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

1% CuSO4 pet.

Patch test

Systemic Allergic Contact Dermatitis Due to Copper (Continued )

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

3

1

1

1

40 1

1

Cases

Table 4

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

Positive to copper and silver salt Positive

Positive patch Positive patch, positive

Positive patch

Challenge reaction

68 106

0–1

105

104

102,103 52

101

References

0–1

1?

0–1

0 1

0–1

CR

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131

Recommended Screening Procedure in Suspected Copper Urticaria

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

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

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

60 Dermatol. patients

1 2 32 5 5 7 3 1

1

1

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

Cohort/etiology

10 0 0 4 140

Cases

Dermatitis

Dermatitis None Metal allergies Dermatitis Allergies Metal allergies Oral symptoms Dermatitis

Allergy

Dermatitis Dermatitis Eczema Dermatitis Dermatitis

Visible signs and symptoms

Table 5 Population Studies of Copper Hypersensitivity

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

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

Patch test

89 88 102,103 92 115 116 93 52 40

2

114

84 113 24 19 49

References

0–1 1 0 0–1 0 0 0–1 2–3

1–2

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

1? – – 0 0–1

CR

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

Challenge reaction

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

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

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

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

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

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

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

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

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

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

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

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

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26. Basketter DA, Gerberick GF, Kimber I, Loveless SE. The local lymph node assay: a viable alternative to currently accepted skin sensitization tests. Food Chem Toxicol 1996; 34:985–997. 27. Ikarashi Y, Ohno K, Tsuchiya T, Nakamura A. Differences of draining lymph node cell proliferation among mice, rats and guinea pigs following exposure to metal allergens. Toxicology 1992; 76:283–292. 28. Ikarashi Y, Tsuchiya T, Nakamura A. Detection of contact sensitivity of metal salts using the murine local lymph node assay. Toxicol Lett 1992; 62:53–61. 29. Haneke KE, Tice RR, Carson BL, Margolin BH, Stokes WS. ICCVAM evaluation of the murine local lymph node assay. Regul Toxicol Pharm 2001; 34:274–86. 30. Amin S, Engasser P, Maibach HI. Sensitive skin: what is it? In: Baran R, Maibach HI, eds. Textbook of Cosmetic Dermatology. London: Martin Dunitz, Ltd., 1998:343–349. 31. Wide L, Bennich H, Johansson SGO. Diagnosis of allergy by an in vitro test for allergen antibodies. Lancet 1967; 2:1105–1107. 32. Halmepuro L, Lovenstein H. Immunological investigation of possible structural similarities between pollen antigens and antigens in apple, carrot and celery tuber. Allergy 1985; 40:264–272. ˚ IB, et al. Allergy to copper derived 33. Frykholm KO, Frithiof L, Fernstro¨m A from dental alloys as a possible cause of oral lesions of lichen planus. Acta Derm Venereol (Stockh) 1969; 49:268–281. 34. Cousins RJ. Absorption, transport, and hepatic metabolism of copper and zinc: special reference to metallothionein and ceruloplasmin. Physiol Rev 1985; 65:238–309. 35. Denko CW, Petricevic M, Whitehouse MW. Inﬂammation in relation to dietary intake of zinc and copper. Int J Tissue React 1981; 3:73–76. 36. Hosty´nek JJ. Gold: an allergen of growing signiﬁcance. Food Chem Toxicol 1997; 35:839–844. 37. Hosty´nek JJ. Metals. In: Amin S, Lahti A, Maibach HI, eds. Contact Urticaria Syndrome. Boca Raton, Florida: CRC Press LLC, 1997:189–211. 38. Milanino R, Conforti A, Franco L, Marrella M, Velo G. Copper and inﬂammation—a possible rationale for the pharmacological manipulation of inﬂammatory disorders. Agents Actions 1985; 16:504–513. 39. Powanda MC. Systemic alterations in metal metabolism during inﬂammation as part of an integrated response to inﬂammation. Agents Actions 1981(suppl 8): 121–136. 40. Lisi P, Brunelli L, Stingeni L. Co-sensitivity between cobalt and other transition metals. Contact Dermatitis 2003; 48:172–173. 41. Uter W, Fuchs T, Ha¨usser M, Ippen H. Patch test results with serial dilution of nickel sulfate (with and without detergent), palladium chloride, and nickel and palladium metal plates. Contact Dermatitis 1995; 32:135–142. 42. Ale SI, Maibach HI. Operational deﬁnition of allergic contact dermatitis. In: Maibach HI, ed. Toxicology of Skin. Philadelphia: Taylor & Francis, 2001: 345–356. 43. Gaul LE. Metal sensitivity in eczema of the hands. Ann Allergy 1953; 11: 758–762.

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

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

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

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106. Santosh V, Ranjith K, Shrutakirthi DS, Sachin V, Balachandran C. Results of patch testing with dental materials. Contact Dermatitis 1999; 40:50–51. 107. Hannuksela M, Salo H. The repeated open application test (ROAT). Contact Dermatitis 1986; 14:221–227. 108. Nakada T, Hosty´nek JJ, Maibach HI. Use tests: ROAT (repeated open application test)/PUT (provocative use test): an overview. Contact Dermatitis 2000; 43:1–3. 109. Wilkinson SM, Heagerty AHM, English JSC. A prospective study into the value of patch and intradermal tests in identifying topical corticosteroid allergy. Br J Dermatol 1992; 127:22–25. 110. Herbst R, Lauerma A, Maibach HI. Intradermal testing in the diagnosis of allergic contact dermatitis—a reappraisal. Contact Dermatitis 1993; 29:1–5. 111. Lahti A, Maibach HI. Contact urticaria syndrome. In: Moschella SL, Hurley HJ, eds. Dermatology. 3rd eds. Philadelphia: WB Saunders Co., Harcourt Brace Jovanovich, Inc., 1992:433–440. 112. Hannuksela M, Lahti A. Contact urticaria from foods. In: Roe D, ed. Nutrition and the Skin. New York: Alan R.: Riss, 1986. 113. Joules H. Asthma from sensitisation to chromium. Lancet 1932; 2:182–183. 114. Zabel M, Lindscheid KR, Mark H. Kupfersulfatallergie unter besonderer Berucksichtigung der internen Exposition. Z Hautkr 1990; 65:481–486. 115. Tschernitschek H, Wolter S, Korner M. Allergien auf Zahnersatzmaterialien. Dermatosen 1998; 46:244–248. 116. Candura SM, Verni P, Dellabianca A, et al. Sensibilizzazione epicutanea a metalli e dermatite allergica da contatto: analisi di una casistica ambulatoriale. Giornale Italiano di Medicina del Lavoro ed Ergonometria 1999; 21:40–45. 117. Moulon C, Vollmer J, Weltzien HU. Characterization of processing requirements and metal cross-reactivities in T cell clones from patients with allergic contact dermatitis to nickel. Eur J Immunol 1995; 25:3308–3315. 118. Pistoor FHM, Kapsenberg ML, Bos JD, Meinardi MMHM, von Blomberg BME, Scheper RJ. Cross-reactivity of human nickel-reactive T-lymphocyte clones with copper and palladium. J Invest Dermatol 1995; 105:92–95. 119. Jerschow E, Hosty´nek JJ, Maibach HI. Allergic contact dermatitis elicitation thresholds of potent allergens in humans. Food Chem Toxicol 2001; 39:1095–1108. 120. Mitchell JC. Egregious blunder of maximization by the angry back and a note on unconﬁrmed ergodata. Contact Dermatitis 1996; 35:131–132. 121. Mitchell J, Maibach HI. Managing the excited skin syndrome: patch testing hyperirritable skin. Contact Dermatitis 1997; 37:193–199. 122. van der Burg CKH, Bruynzeel DP, Vreeburg KJJ, von Blomberg BME, Scheper RJ. Hand eczema in hairdressers and nurses: a prospective study. Contact Dermatitis 1986; 14:275–279. 123. Benezra C, Sigman CC, Perry LR, Helmes CT, Maibach HI. A systematic search for structure–activity relationships of skin contact sensitizers. I. Methodology. J Invest Dermatol 1985; 85:351–356. 124. Jouppila P, Niinima¨ki A, Mikkonen M. Copper allergy and copper IUD. Contraception 1979; 19:631–637. 125. Aro T, Kanerva L, Hayrinenimmonen R, Silvennoinenkassinen S. Long-lasting allergic patch test reaction caused by gold. Contact Dermatitis 1993; 28:276–281.

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˚ , Bjo¨rkner B, Bruze M. The histological and immunohis126. Mo¨ller H, Larsson A tochemical pattern of positive patch test reactions to gold sodium thiosulfate. Acta Derm Venereol (Stockh) 1994; 74:417–423. 127. Samitz MH, Katz SA. Nickel-epidermal interactions: diffusion and binding. Environ Res 1976; 11:34–39. 128. Dupuis D, Rougier A, Roguet R, Lotte C, Kalopissis G. In vivo relationship between hotny layer reservoir effect and percutaneous absorption in human and rat. J Invest Dermatol 1984; 82:353–356. 129. Alder JF, Batoreu MCC, Pearse AD, Marks R. Depth concentration proﬁles obtained by carbon furnace atomic absorption spectrometry for nickel and aluminium in human skin. J Anal Atom Spectrom 1986; 1:365–367. 130. Fullerton A, Hoelgaard A. Binding of nickel to human epidermis in vitro. Br J Dermatol 1988; 119:675–682. 131. Santucci B, Cannistraci C, Cristaudo A, Camera E, Picardo M. The inﬂuence exerted by cutaneous ligands in subjects reacting to nickel sulfate alone and in those reacting to more transition metals. Exp Dermatol 1998; 7:162–167. 132. Frentz G, Teilum D. Cutaneous eruptions and intrauterine contraceptive copper device. Acta Derm Venereol (Stockh) 1980; 60:69–71.

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

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

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

B.C.

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

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

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THE BABYLONIAN–ASSYRIAN CULTURE: CIRCA 1750–539

B.C.

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

B.C.

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

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

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

B.C.

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

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

B.C.

TO 476

A.D.

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

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

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

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

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

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

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

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

ABBREVIATIONS L R&P

taxonomic classiﬁcation by Linne´ taxonomic classiﬁcation by Ruiz and Pavon

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

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

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

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

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

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

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

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

Days of diet 30

90

Total serum copper (mg/dL SD)

Paw edema volume (plethysmographic units SD)

Hours after challenge

Normal

Deﬁcient

Normal

Deﬁcient

0 3 5 20 0 3 5 20

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

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

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

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

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

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

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

Inﬂammatory model CPE

CP

Total serum copper (md/dL SD)

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

Hours after challenge

Normal

Deﬁcient

Normal

Deﬁcient

0 3 5 22 0 6 22

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

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

0 18.2 1.6 24.3 2.1 10.1 3.3 0 0.83 0.3 —

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

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

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

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

Inﬂammatory model CPE

CP

Total serum copper (md/dL SD)

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

Hours after challenge

Normal

Deﬁcient

Normal

Deﬁcient

0 3 5 22 0 6 22

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

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

0 18.9 6.0 29.3 8.2 13.8 5.1 0 1.0 0.2 —

0 20.3 5.7 27.9 8.4 12.7 4.9 0 1.1 0.4 —

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

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

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

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

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35

*

Arthritic score

28

*

Cu-normal

*

21 14 7 Cu-deficient 0 0

7

14

21

28

Days after the inoculum

Serum Cu (µg/dL)

350

*

*

*

*

Cu-normal

280 210 140 70 Cu-deficient 0 0

7

14

21

28

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

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

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

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

Disease

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

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

U — — — I U U — — — — — —

— — I D U U U — — — — — —

— — — — U — — — — — — — —

— — — — — P I — I I — — —

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

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

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

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

Dog

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

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

— — — —

I I I I

D — — —

— — — —

4 33 46 47

I I I I

I U — —

I I I —

D — U —

I — — —

48 49 50 51

I

—

—

—

—

49

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

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

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

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

Total serum Cu (mg/dL)

Liver weight (g)

Liver copper Cu (mg/g)

Total liver Cu (mg)

151.3 þ8% þ0% þ3

6.6 4% 12%a 21%b

4.31 þ5% þ16%b þ25%b

33.03 þ2% þ5% þ3%

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

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

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

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

Cu (mg/g) a

þ26% þ38%a þ32%a

Severe disease

Cu total (mg) a

þ92% þ50%a þ47%a

Cu (mg/g) a,b

þ39% þ136%a,b þ106%a,b

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

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

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

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

Edema weight (g)

Total plasma copper (mg/g)

Paw copper (mg/g)

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

0 4 2 3 þ74b þ63b þ55b þ8 2

0 þ12a þ20b þ19b þ21b þ26b þ25b þ25b þ21b

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

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

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

56

42

28

a,b

a a

14

0

Acute inflamm.

Chronic inflamm. Asymptom.

Chronic inflamm. Mild dis.

Chronic inflamm. Severe dis.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Source

Edema inhibition (%)

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

None 73 44 35 53 37 72

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

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

6–10 infusions at intervals of 2–4 days

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

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

Source: From Ref. 116.

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

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

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

Administration route

Reference

sc sc sc sc

118 119 7 120

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

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

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

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

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

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

Vehicle

Reference

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

131 143 144 144 92 145 141 146 146 62

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

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

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

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

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

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

30 days

58 days

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

þ36%a None None

þ17%a None None

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

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

None

None

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

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

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

35

% inhibition of arthritic score

* 28 Cu = 200 ppm 21

14

7

Cu = 100 ppm

0

Cu = 50 ppm 14

21

28

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

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

150 AA day 14

% copper increase

120 90 60 30 0 plasma

hind paws

150 AA day 28 % copper increase

120 90 60 30

* 0 plasma AA rats on copper normal diet

hind paws AA rats on copper 40 times supplemented diet

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

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

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

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

þ58 53 47 þ41 þ46

þ30a 34a 16a þ12a þ13a

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

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

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

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

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

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

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

Cu,Zn superoxide dismutase (SOD 1) Ceruloplasmin

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

In vivo main functions

References

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

17, 183

Melanin biosynthesis

192

Regulation of glucose uptake and cell adhesion Bioactivation of neuroactive peptides

193

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

195

17, 184 17, 185–187

153, 188 184, 189 190, 191

184, 194

182 196–198

182, 199

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

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

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

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

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

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

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

Activity on oxygen radicals scavenging, and other PMNLs reactions

References

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

223 127, 224

Scavenging of oxygen radicals Inhibition of superoxide production

227 210

Scavenging of superoxide; inhibition of superoxide production Scavenging of superoxide

139, 140, 228

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

225 158, 213

226

229 230 231, 232

128 233 140 63

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

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

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

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

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

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

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

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40

AA not suppl.

Neutrophil adhesion (%)

30

20

*

*

AA Cu suppl.

Healthy Cu suppl. 10 Healthy not suppl.

0

Diet 30 d Diet 44 d AA inoculum AA 14 d

Diet 58 d AA 28 d

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

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

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

References

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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act as a bactericide and fungicide on the skin environment of the metallic copper. At the same time, it cannot cause any harmful general effects to the individual, since the amount of copper released will only constitute a small percent ( <1%) of the general copper intake via the diet (30). Copper bracelets as a remedy for arthritis were mentioned in a 1995 Copper Development Association publication: Evidence has been presented to suggest that metallic copper in the form of copper bracelets may be effective in relieving arthritic discomfort; whilst much more evidence on efﬁcacy is required, it is now accepted that dissolution of copper in sweat can be followed by absorption through the skin and that this provides a mechanism for direct, and possible local, copper supplementation and thereby a plausible explanation of the beneﬁt of copper bracelets (31). Similar information also appeared more recently in a book available from the Scandinavian Copper Development Association: A number of observations have been reported indicating that metallic copper, e.g. in the form of ‘‘copper bracelets,’’ may alleviate the pains in rheumatic patients (31). It is well known that copper dissolved by sweat from metallic objects can penetrate the skin. Thus, it is possible that it is the direct, topical copper absorption that could be an explanation of the experienced beneﬁt of ‘‘copper bracelets’’ and ‘‘copper rings’’ (32). The studies that shifted the focus of use of copper bracelets from an antibiotic to an antiarthritic application were conducted 30 years ago in Australia. They remain the only serious attempt by qualiﬁed scientists to look at this possible medical application of copper, and they are still cited (e.g., Refs. 33,34). The next section will critically review their ﬁndings. The Clinical Trial of Copper Bracelets Revisited or Just What Did the Early Studies Prove? In 1974, Dr. J.R.J. Sorenson posed a question for the scientiﬁc community: ‘‘Is there anything to the folk myth that copper bracelets have great therapeutic value?’’ (35). This was taken up as a challenge by Dr. W. Ray Walker of the Department of Chemistry, University of Newcastle, NSW, Australia, who was intrigued also by an interest in the interaction between copper and aspirin. The studies conducted by Dr. Walker and colleagues (35–37) remain the only systematic attempt to evaluate the efﬁcacy of copper bracelets. The original trial (35) is often cited in the scientiﬁc literature and occasionally by sellers of copper bracelets in support of their product’s advertised usefulness.

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A word of caution about efﬁcacy—proposed medical therapies must be subject to three tests: efﬁcacy, or the ‘‘extent to which an intervention does more good than harm under ideal circumstances’’ (can it work?); effectiveness, ‘‘whether an intervention does more good than harm under usual circumstances of healthcare practice’’ (does it work in practice?); and efﬁciency, ‘‘effect of an intervention in relation to the resources it consumes’’ (is it worth it?) (38). It must be noted that it is not necessary for any therapy to be fully understood (only that it works). In 1974, Ray Walker was a university researcher with an interest in coordination chemistry. For the initial copper bracelets trial, volunteers with self-declared ‘‘arthritis’’ were recruited by publishing letters in several Australian newspapers. A substantial number of volunteers (60 of the 300 who answered a questionnaire) were rejected by a physician on the grounds that they did not ﬁt an arthritic proﬁle (based on an examination of their questionnaire responses); there was no direct assessment of their health and so we cannot be certain of the diagnosis of the trial participants (39). Roughly half reported having worn copper bracelets for arthritis. For the trial, the remaining 240 volunteers were randomly assigned (randomization method not speciﬁed) to one of three groups: Group I wore a copper bracelet for one month followed by an anodized aluminum bracelet (placebo) for one month; Group II did the reverse; and Group III (control) wore no bracelet for two months. Bracelets were weighed at the beginning and end of the period and the groups comprised equal numbers of experienced and inexperienced users. A subjective assessment was made by questionnaire after the end of the two-month trial—the evaluation categories were ‘‘better’’ (including both ‘‘much better’’ and ‘‘little better’’), ‘‘same,’’ and ‘‘worse’’ (including ‘‘little worse’’ and ‘‘much worse’’) (35). In the evaluation, a signiﬁcantly greater number of the patients who expressed a preference reported that they ‘‘were better’’ while wearing the copper bracelet than while wearing the aluminum bracelet. The effect was attributed to the supply of a steady supplemental amount of copper from the bracelets; copper bracelets were reported to have lost an average of 13 mg in a month of use and the lost copper was assumed to have dissolved into the wearers’ sweat and then to have been absorbed into their skin. Subsequent laboratory experiments showed that copper from turnings could dissolve into sweat and that copper complexed to an amino acid perfused cat skin (40,41). The trial results were reported in preliminary form in 1976, and the data were reported at least twice again (35–37). It is difﬁcult to make a conﬁdent assessment of what was proved by the Walker and Keats bracelets trial, and since its publication the study has been criticized on several grounds: 1. It was poorly controlled (15,42). Because copper bracelets discolor and turn the wearer’s skin green during use, it is doubtful that the

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experienced users (and even a proportion of the inexperienced ones) could not distinguish the real thing from the placebo. This calls into question the ‘‘blinding’’ of the subjects in the trial since the patient must be unable to distinguish the treatment from the placebo (43). If either the researcher or patient can guess which is the real treatment and which is the sham, the study results may be biased (44). Further, such inadequate concealment in pain research and complementary and alternative medicine (CAM) trials results in the treatment effects being overestimated (45,46). There is no indication in the three papers about the Walker and Keats trial that the investigators were blinded to the treatment versus placebo bracelets when weights were taken—in fact there was no indication that the placebo bracelets were weighed at all (if they were, no data were presented for them). To establish an effect of a treatment ‘‘above and beyond natural history of the condition and non-specific effects,’’ it is essential that the trial is randomized, controlled and (preferably) double-blinded (44). The Walker and Keats trial did not meet this standard. 2. The Walker and Keats trial used a vaguely deﬁned outcome (‘‘perceived effectiveness’’) determined by a subjective evaluation by questionnaire at the end of each one-month treatment period and a comparison of the relative effectiveness of the bracelets at the end of the second period. The data presented are in categories (‘‘a little worse,’’ ‘‘much better,’’ etc.) but exact criteria for these evaluations are not speciﬁed. It would appear that ‘‘better’’ or ‘‘worse’’ refers to a superﬁcial judgment of the ‘‘effectiveness’’ of the copper bracelet versus the placebo bracelet, not to the symptoms of the wearer per se and may be based on the wearers’ appraisal of the ‘‘state of their condition’’ (37). Combining ‘‘a little better’’ and ‘‘much better’’ into a single category may exaggerate the apparent effectiveness of the treatment bracelets. 3. In conditions such as arthritis that are characterized by periods of remission and ﬂare, ‘‘it is not uncommon for two out of every three patients to report subjective improvement after a treatment that, unknown to them, contains no active agent’’ (47). There is a strong suggestion of this in the Walker and Keats (35) data. A statistical test of the effect of previous experience on the experimental outcome showed that it was signiﬁcantly related to the evaluation provided by the volunteers (35). The largest group of volunteers overall (30 of 77) perceived no difference in effectiveness between the copper and aluminum bracelets. The natural history of their conditions over the two-month trial period could be expected to signiﬁcantly affect their perception of the effectiveness of the bracelet they were wearing during that part of the trial.

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This phenomenon is common to many pain-causing conditions and greatly complicates the design of trials in pain research (44). Moreover, patients often enroll in clinical trials when their symptoms are at their worst—and ‘‘the next change is likely to be an improvement’’ (44). 4. The placebo effect, ‘‘a change in a patient’s illness attributable to the symbolic import of the treatment rather than a speciﬁc pharmacologic or physiological property’’ (44), can be powerful in pain research. Paracelsus understood the importance of the placebo effect (or wishful thinking): ‘‘It is not the curse or the blessing that works, but the idea. The imagination produces the effect’’ (29). The data for the Walker and Keats trial volunteers who wore no bracelet at all for the two months (control group) were even more interesting and suggestive that a strong placebo effect might have operated in this trial. While 11 of 19 of the previous users said they were ‘‘worse’’ during the trial period (seven reported ‘‘same’’ and one ‘‘better’’), the previous nonusers were evenly split, with six reporting ‘‘worse,’’ six ‘‘better,’’ and 10 reporting no difference (35). The authors also observed that, of the previous users, 14 dropped out of the trial because they could not be without their bracelets. Assuming an initial pool of 40 volunteers in that part of the control group, seven other testers dropped out for other (unspeciﬁed) reasons. The attrition loss in the ‘‘previous nonusers group’’ was 18—for reasons also not speciﬁed in the paper. Did many ‘‘control’’ subjects just get better anyway and so lose interest in completing the trial? Patient expectations of success of the treatment are an important component of the placebo effect (44,48) and highly compliant patients may show better outcomes, just as they did in the Walker and Keats trial. While all participants were instructed to carry on normal routines, diet, and medications for the duration of the trial (35), no data are given on the use of medications during the trial that might have had a signiﬁcant inﬂuence on the outcome. 5. Bracelet weight loss was claimed by the authors to supply up to 13 mg/month supplemental copper. It was argued that this copper dissolved into the sweat beneath the bracelet and perfused the skin of the wearer. There are some difﬁculties with the weight loss claim, however. No error estimates for the calculated mean weight loss are given in the original Walker and Keats report or subsequent republications. The bracelets were weighed before the trial and then sent in plastic bags to the subjects. They were sent back to the researchers at the end of the trial, ‘‘usually’’ in plastic bags, at which time they were again weighed. Importantly, an unspeciﬁed number of bracelets were observed to have gained weight over

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the two-month trial period. The researchers took this as evidence that some bracelets had not been worn—those bracelet weights and the questionnaire responses of those participants were then eliminated from the analyses—a substantial source of bias for both the test and the bracelet weight loss claim. We are not told how many bracelets were in this group, but of the 160 testers, only 77 wore the bracelets for the full trial, ﬁlled out all questionnaires, supplied classiﬁable responses, and had bracelets that did not gain weight. It would have been interesting to know what weight change was observed in the placebo bracelets and if the trial participants were able to guess which bracelet (copper or placebo) they were wearing. In a review discussing the results of the Walker and Keats trial, the authors admit that the bracelet weight loss observed might have been caused by mechanical wear, although in an earlier publication they reported that the used bracelets did not appear abraded (39,40). Walker et al. (36) also remarked that the appearance of a green stain on the skin below bracelets [from copper (II) salt build-up] could be seen as evidence that some copper must be lost from the bracelet during wear. 6. In a subsequent experiment, copper was shown to dissolve from turnings that had been submerged and shaken in human sweat for 24 hours (40). Extrapolating from this result to the dissolution of copper from a solid bracelet into a thin ﬁlm of sweat on a human arm would seem problematic. To begin with, the turnings present a substantially greater surface area for dissolution compared with a solid bracelet. Since study participants would have bathed regularly, it is likely that an undetermined amount of the copper (green stain) under their bracelets would simply wash off the skin’s surface and not be absorbed. 7. The trial questionnaires included a semiquantitative assessment of the diet of study participants. Copper-rich foods (liver, shellﬁsh, mushrooms, and nuts) were an insigniﬁcant part of the diet of the study participants (35). This is not surprising, since for most Western diets, these copper-rich sources do not play a large part; in fact, grains and vegetables commonly provide up to 60% of copper intake (49). The observation that most of the participants’ diets were in the ‘‘low’’ category for intake of copper-rich foods is typical of populations as a whole and does not indicate anything useful about what the overall dietary copper intake of study subjects might have been—it certainly cannot be taken as robust evidence that the participants were copper deﬁcient. Walker and Keats were initially tentative in their support for the therapeutic value of the bracelets, and suggested in their original paper that the

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subjects who had not been bracelet wearers before the trial may have been ‘‘less ready to commit’’ themselves. In a 1977 letter in The Medical Journal of Australia, Whitehouse and Walker (50) mention that the ‘‘Pharmacy Board in Victoria is currently reconsidering its ban on the sale of copper bracelets’’ and that there had been discussion of the issue in the lay press. It is possible that people who volunteered for the trial might have felt quite passionate about the issue before the trial and that their enthusiasm could have affected the trial’s outcome, as the investigators themselves indicated. In calling for trials to fully validate the role of the bracelet, the researchers concluded, ‘‘even if it has little value, except as a placebo, it does have the merit of being of little harm (by virtue of being the most inefﬁcient source of a potential toxin)’’ (50). Further well-designed and properly conducted clinical trials, rigorously controlled for placebo effects, and double blinded, would be necessary to conﬁrm or refute the ﬁndings of the Walker and Keats trial and prove or disprove ﬁnally the claims of the promoters of copper bracelets. The study of ‘‘the copper bracelet myth’’ and the science behind it led to the development of a topical anti-inﬂammatory copper-salicylate gel (36,37,51) that is available as an over-the-counter product in several countries. An independent, double-blind, placebo-controlled, randomized trial of the gel failed to conﬁrm its effectiveness: applied to the forearm it was no better than the placebo gel for pain relief in patients with osteoarthritis of the hip or knee (52). The treatment gel also produced signiﬁcantly more skin rashes than the placebo. In 1998, The Medical Journal of Australia published a letter critical of the trial by one of the gel’s developers (and a colleague of Dr. Walker’s). Dr. Boettcher criticized the gel study on the grounds that the gel should have been applied (as directed) at or near the hip or knee and the composition of the gel was incorrectly described in the article (53). Dr. Boettcher also took exception to the statistical analysis used. Drs. Brooks and Kellett (54) countered these objections—the trial was designed to investigate systemic effects on distant joints—and they refuted Dr. Boettcher’s statistical argument. Investigation of the effectiveness of topical therapies for arthritis pain relief continues—results of several substantial studies have been published recently. A meta-analysis of randomized controlled trials of topical NSAIDs (popular over-the-counter drugs) used for the relief of osteoarthritis pain concluded that they are no more effective than a placebo beyond two weeks of treatment (55). A systematic review of double-blind, placebo-controlled trials for efﬁcacy concluded, ‘‘topical NSAIDs were effective and safe in treating acute painful conditions for one week’’ (56). A New Bracelet Study A new study of the effects of copper/zinc bracelets on joint and muscle pain was conducted at the Mayo Clinic and published by them in 2002 (57). The

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study was designed as a randomized, double-blind, placebo-controlled trial of ‘‘ionized’’ wrist bracelets that were advertised and marketed as effective for the treatment of pain. Because the bracelets (both ‘‘ionized’’ and placebo) used in the trial were made mostly of copper (85% copper/15% zinc) they are relevant to the discussion of the Walker and Keats bracelets trial. There are interesting similarities between the two studies: 1. The subjects for both studies were volunteers with self-reported pain (in the Mayo Clinic study they were recruited by posters in the clinic). 2. The patients in both studies wore bracelets for a one-month test period. 3. In both studies, patients replied to questionnaires on which (among other questions) they rated their own conditions subjectively. 4. The placebo effect was evident in both trials. 5. In both trials, a proportion of the patients believed in the power of the remedy beforehand. In the Mayo Clinic study, participants were asked in advance if they believed the bracelets would be effective and, of the participants who answered the question, 80% replied in the afﬁrmative; however, only 4.4% reported having ever used a bracelet before the trial. Patients wearing both placebo and ‘‘ionized’’ bracelets reported statistically signiﬁcantly lower pain scores during the course of the trial, but the two groups showed the same degree of improvement, leading the authors to conclude that the treatment bracelets were no better than the placebos. The new study differed in methodology in signiﬁcant ways from the Walker and Keats trial. Firstly, the Mayo Clinic study used a more clearly deﬁned outcome (pain rated on a scale of 1–10) and they evaluated pain at several points during the month (days 1, 3, 7, 14, 21, 28). Remarkably, on day 1, 63.3% of the ‘‘ionized’’ bracelet wearers and 68.5% of the placebo bracelet wearers reported an improvement in maximum pain scores—from the study’s description, there is good reason to expect that an especially strong placebo effect may be evidenced. As part of the Mayo Clinic study design, patients were instructed in the correct placement of the bracelets according ‘‘to the manufacturer’s recommendations’’ (57)—a procedure that very likely induced additional conﬁdence in the patient that the remedy was likely to be powerful, judging by the manufacturer’s instructions available on their website—in particular, the speciﬁc instructions for changing the orientation of the terminals if the bracelet is worn on the left instead of (recommended) right wrist; the implication of the term ‘‘terminal’’ for the bobble on the open end of the bracelet; the instruction that the ‘‘terminals’’ should be 1⁄2 to 11⁄2 inches apart during use; and the warning, ‘‘Do not let the terminals directly touch each other.’’ As Bratton et al. (57) point out, going through these instructions with patients is an excellent example of the ‘‘healing

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ritual’’ described by Kaptchuk (48) as producing an ‘‘exaggerated placebo effect’’ and it could well explain the observation that 2/3 of the participants reported an improvement after just one day of wearing the bracelets. Interestingly, although 80% of the participants answering the question reported ahead of time that they believed the remedy would work, the highest proportion reporting an improvement in pain scores during the trial was 77.7%. Unfortunately, the Mayo Clinic study did not have a control group that wore no bracelet at all during the trial—this could have provided valuable information about the signiﬁcance of the pain improvements (which were not large) reported by both groups of study participants. In 2003, the U.S. Federal Trade Commission (58), citing the Mayo Clinic study, charged the marketers of the Q-Ray Ionized Bracelets with making false and unsubstantiated claims, speciﬁcally ‘‘deceptively claiming that the Q-Ray Bracelet is a fast-acting effective treatment for various types of pain and that tests prove that the Q-Ray Bracelet relieves pain.’’ HealthScout News reporter Ed Edelson concluded an online article about the study (HealthScout News, November 12, 2002) with this wry comment from Dr. Robert Bratton, the study’s lead author: It’s all a placebo effect and ‘‘based on the study results, you may be just as well off wearing a rubber band around your wrist and saving the money spent on the bracelet.’’ The Gold Ring Study Gold treatments (by injection) are used in the treatment of rheumatoid arthritis and an intriguing study published in 1997 provided some evidence that gold from rings may delay articular erosion in rheumatoid patients (59). In the study, conﬁrmed rheumatoid arthritis patients were divided into two groups—non-ring wearers and ring wearers—and their hands and wrists examined by standard radiographic technique. While the two groups had statistically indistinguishable overall articular erosion, the ﬁngers of the ring-bearing hands were signiﬁcantly less deformed. While the study was small (55 subjects in total), and, as the authors admit, it is possible that the effect could relate to the different pattern of use between the left (ringbearing) and right hand, it does suggest that gold from the rings might provide a protective effect. THE PRESENT STATE OF THE COPPER BRACELETS ‘‘ISSUE’’ More Recent Studies Have Been Less Promising or Just How Satisfied Are Bracelet Users? Two features of the most common arthritic conditions make arthritis patients vulnerable to the lure of alternative remedies: their conditions have no known cause or proven cure and they are characterized by chronic pain (33). In 1997, American consumers spent approximately $27 billion on alternative

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therapies (60). Alternative therapies are ‘‘aggressively promoted’’ and, in the opinion of D.M. Marcus (61), MD, Baylor College of Medicine, ‘‘most AM [alternative medicine] information that is readily available to lay persons is misleading or wrong.’’ This point of view is supported by a survey made in May 1998 of Internet information for patients with rheumatoid arthritis. After evaluating the quality and source of materials offered its authors concluded, ‘‘over two-thirds of the sites with overt ﬁnancial aims promoted the use of alternative therapy, often claiming that their products were effective for arthritis and other conditions . . . Although some alternative therapies may be efﬁcacious, most have not been adequately studied, and often, positive studies have major methodological ﬂaws’’ (62). Surveys of patterns of usage of copper bracelets have been conducted in Australia/New Zealand, North America, and the United Kingdom. Let us suppose that it is true that copper bracelets provide great symptomatic relief for arthritis sufferers. Since they are widely available, inexpensive, and heavily promoted, one would expect to observe high rates of bracelet use among patients, and patients should show high rates of satisfaction (and continued use). There is evidence to suggest that patients are generally aware of the copper bracelet claims. For example, 75% of the 51 Hawaiian patients with inﬂammatory arthropathies in a Hawaiian survey had heard of copper bracelets as an alternative therapy (63). Findings from this and several other patient surveys are summarized in Table 1. Patients reported that they turn to CAMs to relieve pain; many discontinued conventional medications because they found medication schedules complicated (71). Patients also perceive the inability of conventional therapies to cure or adequately control their disease and the potentially serious adverse effects they may cause as reasons to turn to alternative treatments (72). Well-publicized

Table 1 Use of Copper Bracelets/Bands by Arthritis Patients Location Australia Australia/ New Zealand Australia Canada Canada England England United States United States United States

No. of patients using

% of survey population

Age of patients (yrs)

Citation

45 21

61 70

15–65þ 12.1–27.2

(64) (65)

3 4 16 75 45 17 42 11

3 57 11 38 28 13 29 21.6

Mean ¼ 61.1 9.3–19.3 Mean ¼ 10.4–10.9 N/A N/A Adults Mean ¼ 55.5 Mean ¼ 51

(66) (65) (67) (68) (69) (70) (60) (63)

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side effects of conventional medicine (in contrast with the natural/safe/ gentle claims for CAMs) provoke an ‘‘exaggerated fear of conventional medications’’ among arthritis patients (61). Sometimes, new studies show that patients’ concerns about the potential side effects of conventional drug therapies are justiﬁed. For example, in September 2004, it was revealed that the popular Merck arthritis and acute pain medication VIOXX1 (rofecoxib), with worldwide sales in 2003 of $2.5 billion, increased the relative risk of heart attack and stroke when used for more than 18 months. The press coverage of this announcement and subsequent negative studies on VIOXX was extensive and featured such frightening headlines as this one that appeared in The Times (London) online edition on January 25, 2005: ‘‘VIOXX may have killed thousands of UK patients study warns.’’ CAM users tend to be female, ‘‘better educated,’’ and employed. Many arthritis patients use CAMs to supplement their conventional treatment and report that friends and relatives and the mass media are their main source of information on alternative treatments. In a recent survey of arthritis patients in New Mexico, for example, the most frequently cited sources of information on CAMs were family and friends (66.1%), medical doctors (56.1%), magazines and books (34.6%), radio/TV/newspapers (22.6%), and the Internet (11.3%) (73). As for effectiveness, in one survey, 74% of adult Americans polled expressed a belief that copper bracelets would help treat arthritis; tellingly, only 13% of arthritis patients reported having used them (70). Several small studies have reported on the success rate claimed by arthritis patients for various alternative and complementary therapies (Table 2). Overall, copper bracelets did not fare well in these surveys. Among the therapies with a

Table 2 Patient Satisfaction Level with Bracelet Use: Proportion of Patients Using or Having Used Copper Bracelets Reported to Have Found Them to Be ‘‘Helpful’’ Patients reporting a beneﬁt from using bracelets

Location

No. (%) using bracelets

Australia England United States United States United States

45 (61) 75 (38) 11 (21.6) 42 (29)a N/A

a

No. (% of users) reporting beneﬁt or currently using 6 6 4 9

(13) (8) (36) (21)b (3.9)

% total patient population 8 3 7.8 6 N/A

n

Citation

74 199 51 146 612

(64) (68) (63) (60) (73)

‘‘Ever used’’ includes both presently using and used in the past. Compare with clinical improvement observed in 30% to 35% of patients treated with a placebo in clinical drug trials (42). b

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higher reported ‘‘usefulness’’ or success rate are chiropractic, herbal therapies, nutritional supplements, medication/relaxation, electrical stimulators, special diets (especially avoidance of certain foods), acupuncture/acupressure, osteopathy, and faith healing. Most unproven remedies, including copper bracelets, are considered ‘‘harmless,’’ with the caution that even innocuous therapies could be harmful if diagnosis is delayed (especially with the advent of aggressive therapy early in the course of the disease that has been shown to prevent permanent joint damage) or proven therapy neglected (74). Rao and coworkers (75) conducted a longitudinal study on the same group of rheumatology patients they reported on in 1999, with follow-up surveys at six and 12 months. Over the one-year period, three patients (out of 174) started using a copper bracelet, three maintained their use, and ﬁve stopped using a bracelet. Of the 28 patients in their study who reported having ‘‘ever used’’ a copper bracelet but had stopped, 24 gave ‘‘did not help’’ as a reason, one claimed to have been made worse by wearing a copper bracelet (two said ‘‘other’’ as a reason, and one declined to say) (75). In a recent study, a group of 40 Asian and 40 Caucasian rheumatoid arthritis patients in the United Kingdom were asked to provide a subjective ranking of effectiveness of complementary therapies on a ﬁve-point scale; ‘‘The two groups rated copper bracelets and magnets very similarly, only just above useless’’ (76). Additional Research and Scientific Data Needed to Resolve the Copper Bracelets Issue The precise ‘‘ . . . role of copper in inﬂammatory processes remains quite obscure. Both pro- and anti-inﬂammatory effects of copper have been documented’’ (15). The studies by Ray Walker and colleagues were visionary, but inadequate to establish or refute the efﬁcacy of copper bracelets for arthritis therapy. The comment is made repeatedly, in the scientiﬁc and other literature, that the available data are not sufﬁcient to constitute a proof of the effectiveness of externally worn copper as therapy for arthritis. The original trials could certainly be repeated and improved on by: recruiting known patients with clearly deﬁned and medically veriﬁed disease; better screening patients for prior bias about the therapy; improving placebo design and control; using objective measures of pain and disease activity as measurable study outcomes; rigorously measuring bracelet weight changes during wear, establishing a rate and accounting for loss of weight due to abrasion and/ or increase in weight, and using a statistically reliable method of determining weight change;

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modeling corrosion of copper from bracelets and complex formation in sweat during use; and using a properly double-blinded study design.

Recent surveys of the use of complementary and alternative therapies by arthritis patients do not lend strong support to the belief that bracelets are found to be of much use and this must call into question any impulse to do more research on them. As discussed above, convincing evidence that bracelets exert more than a placebo effect is lacking. A survey of 2146 primary care physicians and rheumatologists ranked copper jewellery (at 25%) ﬁfth on the list of ‘‘top 10 alternatives doctors advise against.’’ In a parallel survey of 790 people with arthritis who read Arthritis Today, ‘‘metal jewellery’’ was an alternative reported as used by only 15% of the survey respondents (77). A search of the new National Institutes of Health (NIH) Clinical Trials.gov on October 20, 2004, found 136 ongoing studies on ‘‘arthritis’’ but none involving copper bracelets. The nonproﬁt agencies and foundations dedicated to the well-being of arthritis sufferers and governmental regulatory agencies are similarly unenthusiastic about copper bracelets as a folk remedy; their position statements are compiled in Appendix A. On the other hand, there is a deﬁnite need to establish the extent of dermal absorption of copper from a metallic source. ‘‘Dermal exposure to copper can result from the use of consumer products containing copper pigments, through the use of copper as an agicide in swimming pools and the use of copper jewellery. No quantitative exposure levels could be found’’ (78). There remains a need for objective, stringent human studies involving copper and arthritis. Several lines of investigation that have been proposed over the years seemed interesting and yet, to my knowledge, remain to be fully studied: 1. The role of dietary copper deﬁciency in the etiology of arthritis and the use of supplemental copper (or copper-rich diets) in arthritis treatment:

A small double-blind, double placebo controlled factorial trial of copper (in a complexed form at 3 mg/day) and/or omega-3 ﬁsh oil supplements showed no signiﬁcant effect of copper on systemic lupus erythematosus disease activity—a signiﬁcant decline in disease activity was demonstrated for the ﬁsh oil supplement (79).

2. Quantiﬁcation of the supplemental supply of copper from an intrauterine device (IUD) and an epidemiological study of arthritis among copper IUD users. 3. The mechanism of formation of copper complexes with salicylates from arthritis analgesic products in vivo and how that increases their effectiveness.

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IS THERE LIKELY TO BE A FUTURE FOR COPPER BRACELETS IN ARTHRITIS CARE? In Geneva in January 2000, the World Health Organization launched The Bone and Joint Decade: 2000–2010 (80). In recognition of the immense burden of the disease, the U.S. Congress initiated a national arthritis prevention program with a budget of $12 million for the year 2000 (2). One of its goals was to strengthen the science base for arthritis treatment options, especially the roles of good nutrition, appropriate exercise, evaluation of intervention strategies including self-help patient education programs, and promoting good communication. There is good reason to suppose that the therapeutic use of copper for arthritis will be a recurring issue. Copper bracelets are not the only copper product to be touted as therapeutic. America’s ﬁrst great medical quack claimed to cure disease with copper rods passed over the body (81). The Internet is a rich source of unproven and quack remedies involving copper, and it is likely that more will spring up from time to time (Appendix B). The Internet makes it much simpler and less expensive for unproven claims to be widely promoted. In 1999, the establishment of the National Center for Complementary and Alternative Medicine (NCCAM) under the U.S. National Institutes of Health signaled a shift in the American medical establishment’s approach to alternative therapies. Scientists had been understandably reluctant to risk ridicule by attempting serious study of therapies not recognized by traditional medicine. Ray Walker experienced this ﬁrst hand while embarking on the investigation of the copper bracelet by publishing the initial invitation to study participants in the popular press: ‘‘The letter caused some concern to several of my colleagues and raised the eyebrows of my medical friends. Although I had some qualms about my action, these were somewhat allayed by the work of Hagenfeldt . . . and Okereke et al.’’ [(37); citing Hagenfeldt’s 1972 study on the mode of action of the copper-T IUD and Okereke, Sternlieb, Morell, and Scheinberg’s 1972 paper in Science on the systemic absorption of copper from an IUD]. NCCAM has become a formidable force in health research and for 2006 requested a budget of $122.1 million (82). Its activities include funding research projects and clinical trials (it identiﬁes conditions affecting the elderly as a priority area for investigation), developing a comprehensive communications program, and training young scientists in CAM. In its ﬁrst ﬁve years it supported more than 1200 projects on various CAMs, including clinical trials on acupuncture, mind–body medicine, and herbal products. The NCCAM is not without its critics (e.g., Ref. 83), and the conﬂict between conventional and alternative/complementary medicine is a hot topic in journals such as Journal of the American Medical Association, British Medical Journal, and the Medical Journal of Australia. The substantial research award amounts now available for studies on CAMs mean that

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they are likely to continue to enjoy a public proﬁle, particularly as more studies are completed, published, and covered in the popular press—as the example of the Mayo Clinic Q-Ray bracelet study demonstrated. The methods used to study CAMs are also likely to excite ongoing controversy. There is even an interesting discussion of whether CAMs should be evaluated in a fundamentally different way from conventional therapies. The placebo effect, for example, that played such a prominent role in the bracelet trials seems to play an important role in many alternative therapies. In a highly entertaining essay in the Annals of Internal Medicine, Doctor of Oriental Medicine (OMD) Ted Kaptchuk examines the possibility that there is some real value for the placebo effect in clinical practice. Kaptchuk’s (48) essay illustrates vividly a growing gulf between CAM proponents and practitioners of traditional science and medicine: ‘‘Some may dismiss these types of investigation as useless. After all, a placebo is just a placebo. Others would argue that such avoidance impoverishes and narrows the understanding of what patients receive from alternative medicine . . . ’’ Examination of complementary and alternative therapeutic approaches (especially emphasizing longitudinal studies) is a focus of the new ‘‘National Arthritis Action Plan’’ (74). There clearly needs to be much better patient education and public education about arthritis, and there is a deﬁnite need on the Internet for additional high-proﬁle, reputable sites that meet the highest standards of scientiﬁc evidence. This might help to counteract the clamor of the lesscredible websites.

Considering the scant evidence of the metal’s systemic absorption and anti-inﬂammatory effects in laboratory rats, one can safely say that nothing has been learned in the past 3,500 years that would explain the longevity of the belief in copper’s powers. —Sharon L. Kolasinski (84)

APPENDIX A: POSITION STATEMENTS OF SUPPORT ORGANIZATIONS, GOVERNMENT AGENCIES, ETC. Arthritis Foundation (USA)

From Arthritis Today magazine (available on the society’s website): ‘‘there is no scientiﬁc research proving they provide any therapeutic beneﬁt for arthritis. Nor is there any research proving they don’t.’’ From their new guide to alternative therapies: ‘‘Expert Opinion: Wearing a copper bracelet won’t hurt, but there’s not enough scientiﬁc evidence that it helps’’ (85).

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The Arthritis Society (Canada) ‘‘On the hardware side of the store, things get even stranger: ‘‘inductoscopes’’ (allegedly of magnetic induction), ‘‘solarama boards’’ (to align mixed-up electrons), ‘‘earthboards’’ or ‘‘vitalators’’ (to produce rejuvenating electrons), ‘‘oxydonors’’ (to reverse the death process into the life process as a cure for arthritis) and, oh yes, copper bracelets. None of them has ever been proven effective in treating arthritis and relieving its pain’’ (86). Arthritis Foundation of Victoria (Australia) Under unproven remedies: ‘‘The use of copper has little scientiﬁc support. Copper bracelets are a popular alternative treatment for arthritis, but there have been no proper trials of copper bracelets in rheumatoid arthritis’’ (87). National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH (USA) ‘‘Some of these alternative therapies have included wearing copper bracelets, drinking herbal teas, and taking mud baths. While these practices are not harmful, some can be expensive. They also cause delays in seeking medical treatment. To date, no scientiﬁc research shows these approaches to be helpful in treating osteoarthritis’’ (88). National Arthritis Action Plan (USA) ‘‘Using unproven remedies for arthritis can waste time and money . . . may cause people to delay seeking early diagnosis and appropriate management. Because of their ongoing pain and lack of awareness of helpful intervention strategies, people with arthritis are particularly vulnerable to promoters of unproven remedies’’ (74). Federal Trade Commission—FTC (USA) ‘‘Consumers spend an estimated $2 billion a year on unproven arthritis remedies—thousands of dietary and so-called natural cures, like . . . magnets and copper bracelets. But these remedies are not backed by adequate science to show that they offer long-term relief’’ (89). Better Business Bureau of Eastern Massachusetts, Maine, and Vermont (USA) ‘‘Quack arthritis cure . . . Doctors say uranium is useless against arthritis, and a copper bracelet does nothing but turn your wrist green’’ (90).

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Food and Drug Administration—FDA (USA)

Under unproven remedies: ‘‘Some remedies, such as vinegar and honey or copper bracelets, seem harmless. But they can become harmful if they cause people to abandon conventional therapy’’ (27).

APPENDIX B: MISCELLANY Another Copper Bracelet Myth Native North Americans recount a charming myth about a girl and a bear. It exists in several different versions and is a long and richly detailed tale. Brieﬂy, a girl out gathering food slips on bear excrement (violating the bear’s mythical power, which is believed to be associated with its excrement). She utters an oath, further insulting the bear. The bear was considered the most powerful of animals—and thought to have the mystical powers of a shaman. The bear kidnaps the girl and leads her to a mystical world where it keeps the girl as its wife. The version collected at Bella Bella, British Columbia, includes this lovely detail: ‘‘He asks her what sort of excrement she has that would give her the right to scold him, and she says her excrements are abalone shells and copper. He tells her to sit down and show him, which she does, slipping off one of her copper bracelets’’ (25). Other Copper-Crafted ‘‘Healing Products’’ Found On the Internet:

Copper cup (add warm water, let stand for 8–10 hours and drink the healing liquid). A healing wand of copper, brass, silver, quartz, and polished gemstones, with a hollow handle for addition of herbs, etc. A small copper disk to wear on the bottom of a wristwatch as an alternative to a bracelet. Copper patches as an alternative to copper bracelets. Mineral water enriched with copper (100 ppm) for joint problems. Copper coil placed in a warm water foot bath to draw toxins from the body through the feet and so treat a variety of ailments and restore energy.

In the daily newspaper:

Copper wires to insert in the nose to cure the common cold, described in a Reuters story carried by The Vancouver Sun on February 16, 2000.

In the scientiﬁc literature:

A copper bed sheet for treatment of the pain of ﬁbromyalgia (91).

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Early Copper Quackery from a Classic Book on Pseudoscience America’s ﬁrst great quack was Dr. Elisha Perkins (1740–1799). The doctor had a theory that metals draw diseases out of the body, and in 1796 patented a device consisting of two rods, each three inches long. One rod was supposed to be an alloy of copper, zinc, and gold; the other—iron, silver, and platinum. By drawing ‘‘Perkins’ Patented Metallic Tractor’’ downward over the ailing part, the disease was yanked out. Perkins sold his tractors for ﬁve guineas each to such notables as George Washington, whose entire family used it, and Chief Justice Oliver Ellsworth. His son, Benjamin D. Perkins (Yale, class of ’94) made a fortune selling the tractors in England. In Copenhagen, twelve doctors published a learned volume defending ‘‘Perkinism.’’ Benjamin himself wrote a book about it in 1796, containing hundreds of stirring testimonials by well-educated people. They included doctors, ministers, university professors, and members of Congress. Most historians of the subject think the old man actually believed in his tractors, but that his son—who retired in New York City as a wealthy man—was simply a crook promoter. It is worth noting that orthodox medical opinion, by and large, ignored Perkinism, regarding it as not worthy of serious refutation. One doctor, however, did trouble to make some tests with phony tractors. They looked like the genuine article, but actually were non-metallic. His results, of course, were excellent. Oliver Wendell Holmes, in an amusing discussion of Perkinism, relates that one woman was quickly cured of pains in her arm and shoulder by using a fake tractor made of wood. ‘‘Bless me!’’ the woman exclaimed. ‘‘Why, who could have thought it, that them little things could pull the pain from one!’’ (81). Web Resources: Sites with Reliable Information on Arthritis

American College of Rheumatology: http://www.rheumatology.org Arthritis Care (United Kingdom): http://www.arthritiscare.org.uk/ Arthritis Foundation (United States): http://www.arthritis.org/ Arthritis Foundation of Victoria (Australia): http://www.arthritisvic. org.au/ Mayo Clinic: http://www.mayoclinic.com/ National Center for Complementary and Alternative Medicine (NCCAM)(NIH): http://nccam.nih.gov/ National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH): http://www.nih.gov/niams/ NLM—National Library of Medicine: http://www.nlm.nih.gov/ medlineplus/arthritis.html The Arthritis Society (Canada): http://www.arthritis.ca/home.html U.S. Food and Drug Administration: http://www.fda.gov/

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A Final Word on Questionable Health Claims An intensive sweep of the Internet in 1998 targeted over 1200 sites making potentially false or deceptive advertising claims regarding the prevention, treatment, or cure of arthritis, cancer, diabetes, heart disease, AIDS, and multiple sclerosis. The ‘‘Health Claim Surf Day’’ spotlighted the tremendous power of the Internet as an information resource for consumers. It also uncovered the Web’s dark side: ‘‘it also provides promoters of fraudulent health products and treatments easy access to consumers from all over the world’’ (92). REFERENCES 1. Sagan C. The demon-haunted world: Science as a Candle in the Dark. New York: Ballantine, 1996:20. 2. CDC Coordinating Center for Health Promotion. Targeting arthritis: reducing disability for 43 million Americans. Atlanta: Centers for Disease Control and Prevention, March 2005. (http://www.cdc.gov/nccdphp/aag/aag_arthritis.htm). 3. Lewis C. Arthritis: timely treatments for an ageless disease. FDA Consumer, May–June 2000. (http://www.fda.gov/fdac/features/2000/300_arth.html). 4. Barceloux DG. Copper. Clin Toxicol 1999; 37:217. 5. Milanino R, Marrella M, Velo G. Copper in the treatment of arthritic conditions: myth or reality? Chim Indust (Milan, Italy) 1994; 76:138. 6. Sorenson JRJ. Copper complexes—a unique class of anti-arthritic drugs. Prog Med Chem 1978; 15:211. 7. Mussalo-Rauhamma H, Konttinen YT, Lehto J, et al. Predictive clinical and laboratory parameters for serum zinc and copper in rheumatoid arthritis. Ann Rheum Dis 1988;47:816. 8. Milanino R, Frigo A, Bambara LM, et al. Copper and zinc status in rheumatoid arthritis: studies of plasma, erythrocytes, and urine, and their relationship to disease activity markers and pharmacological treatment. Clin Exp Rheumatol 1993; 11:271. 9. Milanino R, Marrella U, Moretti U, et al. Copper and zinc status in rats with acute inﬂammation: focus on the inﬂamed area. Agents Actions 1988; 24:356. 10. Milanino R, Moretti U, Concari E, et al. Copper and zinc status in adjuvantarthritic rat: studies on blood, liver, kidneys, spleen and inﬂamed paws. Agents Actions 1988; 24:365. 11. Neve J, Fontaine J, Peretz A, et al. Changes in zinc, copper and selenium status during adjuvant-induced arthritis in rats. Agents Actions 1988; 25:146. 12. Kishore V. Effects of copper aspirinate and aspirin on tissue copper, zinc, and iron concentrations following chronic oral treatment in the adjuvant arthritic rat. Biol Trace Elem Res 1990; 25:123. 13. Aaseth J, Haugen M, Førre Ø. Rheumatoid arthritis and metal compounds— perspectives on the role of oxygen radical detoxiﬁcation. Analyst 1998; 123:3. 14. Sorenson JRJ. Copper-potentiation of non-steroidal antiinﬂammatory drugs. In: Berthon G, ed. Handbook of Metal–Ligand Interactions in Biological Fluids. Bioinorganic Medicine. New York: Marcel Dekker, 1995; 2:1318–1321.

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15. Rosenstein ED, Caldwell JR. Trace elements in the treatment of rheumatic conditions. Rheum Dis Clin North Am 1999; 25:929. 16. Thunus L, Dauphin JF, Moiny G, et al. Anti-inﬂammatory properties of copper, gold and silver, individually and as mixtures. Analyst 1995; 120:967. 17. Ackerknecht EH. A Short History of Medicine. Rev. ed. Chap. 1. Baltimore: Johns Hopkins University Press, 1982. 18. Majno G. The Healing Hand: Man and Wound in the Ancient World. Cambridge, MA.: Harvard University Press, 1975. 19. Dollwet HHA, Sorenson JRJ. Historic uses of copper compounds in medicine. Trace Elem Med 1985; 2:80. 20. Dresher WH. Copper in my medicine chest. Innovations: the online magazine of the Copper Development Association, June 2000. (http://www.copper.org/ innovations/2000/06/medicine-chest.html). 21. An onlooker’s notebook. Copper on the up and up? Pharm J 1974; Oct 5:318. 22. Sorenson JRJ. Therapeutic uses of copper. In: Nriagu JO, ed. Copper in the Environment, Part II: Health Effects. New York: John Wiley & Sons, 1979:83–162. 23. Whitehouse MW. Ambivalent role of copper in inﬂammatory disorders. Agents Actions 1976; 6:201. 24. Sharples NM. English Heritage Book of Maiden Castle. London: B.T. Batsford, 1991. 25. Loucks G. The girl and the bear facts: a cross-cultural comparison. Can J Native Stud 1985; 5:218. 26. Weissmann G. A fashion in metals. Hosp Pract (Hosp Ed) 1982; 17:33. 27. Strange CJ. Coping with arthritis in its many forms. FDA Consumer, March 1996. (http://www.fda.gov/fdac/features/296_art.html). 28. Hostynek JJ, Maibach HI. Copper hypersensitivity: dermatologic aspects—an overview. Rev Environ Health 2003; 18:153. 29. Livingston JD. Magnetic therapy: plausible attraction. Skeptical Inquirer Magazine (online), July 1998. (http://www.csicop.org/si/9807/magnet.html). ¨ sterberg R, Sjo¨berg B, Branega˚rd B. A critical review of copper in medicine. 30. O Final report. INCRA Project No. 234. New York: International Copper Research Association, 1975:44. 31. Shorrocks VM, Alloway BJ. Copper in plant, animal and human nutrition. Potters Bar, UK: Copper Development Association, 1985:75. 32. Landner L, Lindestro¨m L. Copper in society and in the environment: an account of the facts on ﬂuxes, amounts and effects of copper in Sweden. 2nd rev. ed. Translated by Lars Landner. Va¨stera˚s, Sweden: Swedish Environmental Research Group (MFG), 1999:292. 33. Gaby AR. Alternative treatments for rheumatoid arthritis. Altern Med Rev 1999; 4:392. 34. Resman-Targoff BH. Alternative therapeutic approaches to the treatment of arthritis. J Pharm Prac 1999; 12:346. 35. Walker WR, Keats DM. An investigation of the therapeutic value of the ‘copper bracelet’—dermal assimilation of copper in arthritic/rheumatoid conditions. Agents Actions 1976; 6:454. 36. Walker WR, Beveridge SJ, Whitehouse MW. Dermal copper drugs: the copper bracelet and Cu(II) salicylate complexes. Agents Actions Suppl 1981; 8:359.

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37. Walker WR. The results of a copper bracelet clinical trial and subsequent studies. In: Sorenson JRJ, ed. Inﬂammatory Diseases and Copper: The Metabolic and Therapeutic Roles of Copper and Other Essential Metalloelements in Humans. Clifton, NJ: Humana, 1982:469–478. 38. Haynes B. Can it work? Does it work? Is it worth it? The testing of health care interventions is evolving. Br Med J 1999; 319:652. 39. Ferna´ndez-Madrid F. Treating Arthritis: Medicine, Myth, and Magic. New York: Plenum, 1989:108. 40. Walker WR, Grifﬁn BJ. The solubility of copper in human sweat. Search 1976; 7:100. 41. Walker Wr, Reeves RR, Brosnan M, et al. Perfusion of intact skin by a saline solution of bis(glycinato) copper(II). Bioinorg Chem 1977; 7:271. 42. Caldwell JR. Venoms, copper, and zinc in the treatment of arthritis. Rheum Dis Clin North Am 1999; 25:919. 43. HealthWatch. Folklore arthritis remedies: copper bracelets. HealthWatch Newsletter (online), 4, April 1990. (http://www.healthwatch-uk.org/nlett04.html). 44. Turner JA, Deyo RA, Loeser JD, et al. The importance of placebo effects in pain treatment and research. JAMA 1994; 271:1609. 45. Jadad AR, Moore RA, Carroll D, et al. Assessing the quality of reports of randomized clinical trials: is blinding necessary? Controlled Clin Trials 1996; 17:1. 46. Moher D, Sampson M, Campbell K, et al. Assessing the quality of reports of randomized trials in pediatric complementary and alternative medicine. BMC Pediatr 2002; 2:2. 47. The HealthWatch Committee. The placebo effect: let’s unwrap it and then tap it. HealthWatch Newsletter (online), 13, Autumn 1993. (http://www.healthwatch-uk. org/nlett13.html). 48. Kaptchuk TJ. The placebo effect in alternative medicine: can the performance of a healing ritual have clinical signiﬁcance? Ann Intern Med 2002; 136:817. 49. Ralph A, McArdle H. Copper metabolism and requirements in the pregnant mother, her fetus and children: a critical review. Chap. 2. New York: International Copper Association, 2001. 50. Whitehouse MW, Walker WR. The ‘‘copper bracelet’’ for arthritis. Med J Aust 1977; 1:938. 51. Walker WR, Whitehouse MW. Facilitated transdermal absorption of copper (II) suppresses experimental inﬂammation. Aust N Z J Med 1979; 9:112. 52. Shackel NA, Day RO, Kellett B, et al. Copper-salicylate gel for pain relief in osteoarthritis: a randomised controlled trial. Med J Aust 1997; 167:134. 53. Boettcher B. Copper-salicylate gel for pain relief in osteoarthritis [letter]. Med J Aust 1998; 168:312. 54. Brooks PM, Kellett B. Reply to letter by B. Boettcher. Med J Aust 1998; 168:312. 55. Lin J, Zhang W, Jones A, et al. Efﬁcacy of topical non-steroidal anti-inﬂammatory drugs in the treatment of osteoarthritis: meta-analysis of randomised controlled trials. Br Med J 2004; 329:324. 56. Mason L, Moore Ra, Edwards JE, et al. Topical NSAIDs for acute pain: a metaanalysis. BMC Fam Pract 2004; 5:10. 57. Bratton RL, Montero DP, Adams KS, et al. Effect of ‘‘ionized’’ wrist bracelets on musculoskeletal pain: a randomized, double-blind, placebo-controlled trial. Mayo Clin Proc 2002; 77:1164.

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58. Federal Trade Commission (USA). Marketers of Q-Ray Ionized Bracelet charged by FTC: FTC seeks to halt deceptive pain relief claims and provide consumer refunds. Press release, June 2, 2003. 59. Mulherin DM, Struthers GR, Situnayake RD. Do gold rings protect against articular erosion in rheumatoid arthritis? Ann Rheum Dis 1997; 56:497. 60. Rao JK, Mihaliak K, Kroenke K, et al. Use of complementary therapies for arthritis among patients of rheumatologists. Ann Intern Med 1999; 131:409. 61. Marcus DM. Alternative medicine and the Arthritis Foundation [editorial]. Arthritis Rheum 2002; 47:5. 62. Suarez-Almazor ME, Kendall CJ, Dorgan M. Surﬁng the Net—information on the World Wide Web for persons with arthritis: patient empowerment or patient deceit? J Rheumatol 2001; 28:185. 63. Camara K, Danao-Camara T. Awareness of, use and perception of efﬁcacy of alternative therapies by patients with inﬂammatory arthropathies. Hawaii Med J 1999; 58:329. 64. Kestin M, et al. The use of unproven remedies for rheumatoid arthritis in Australia. Med J Aust 1985; 143:516. 65. Southwood TR, Malleson PN, Roberts-Thomson PJ, et al. Unconventional remedies used for patients with juvenile arthritis. Pediatrics 1990; 85:150. 66. Buchbinder R, Gingold M, Hall S, et al. Non-prescription complementary treatments used by rheumatoid arthritis patients attending a community-based rheumatology practice. Intern Med J 2002; 32:208. 67. Hagen LEM, Schneider R, Stephens D, et al. Use of complementary and alternative medicine by pediatric rheumatology patients. Arthritis Rheum 2003; 49:3. 68. Struthers GR, Scott DL, Scott DGI. The use of ‘alternative treatments’ by patients with rheumatoid arthritis. Rheumatol Int 1983; 3:151. 69. Seymour A, Mayes C, Young A, et al. The use of complimentary and alternative medicine in patients with rheumatoid arthritis. Abstr. 221. British Society for Rheumatology XIX Annual General Meeting, Brighton, United Kingdom, April 23–26, 2002. Rheumatology 2002; 41(suppl 2):88. 70. Price JH, Hillman KS, Toral ME, et al. The public’s perceptions and misperceptions of arthritis. Arthritis Rheum 1983; 26:1023. 71. Keysor JJ, Currey SS, Callahan LF. Behavioral aspects of arthritis and rheumatic disease self-management. Dis Manage Health Outcomes 2001; 9:89. 72. Barnes J. Quality, efﬁcacy and safety of complementary medicines: fashions, facts and the future. Part I. Regulation and quality. Br J Clin Pharmacol 2003; 55:226. 73. Herman CJ, Allen P, Hunt WC, et al. Use of complementary therapies among primary care clinic patients with arthritis. Prev Chronic Dis 2004; 1:A12. 74. Arthritis Foundation, Association of State and Territorial Health Ofﬁcials and CDC. National Arthritis Action Plan: a public health strategy. Atlanta: Arthritis Foundation, 1999. (http://www.cdc.gov/nccdphp/pdf/naap.pdf). 75. Rao JK, Kroenke K, Mihaliak KA, et al. Rheumatology patients’ use of complementary therapies: results from a one-year longitudinal study. Arthritis Rheum 2003; 49:619. 76. Neville CE, Hassan W. Complimentary therapy use in different ethnic groups with rheumatoid arthritis. Abstr. 237. British Society for Rheumatology XX Annual General Meeting. A joint meeting with La Socie´te´ Franc¸aise de Rhumatologie. Manchester, United Kingdom, April 1–4, 2003. Rheumatology 2003; 42(suppl 1):94.

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77. Horstman J. Why doctors aren’t asking and you aren’t telling. Arthritis Today, November–December 1999. (http://www.arthritis.org/resources/arthritistoday/ 1999_archives/1999_11_12alternatives.asp). 78. IPCS (International Programme on Chemical Safety). Environmental Health Criteria 200. Copper. Geneva: World Health Organization, 1998:71. 79. Duffy EM, Meenagh GK, McMillan SA, et al. The clinical effect of dietary supplementation with omega-3 ﬁsh oils and/or copper in systemic lupus erythematosus. J Rheumatol 2004; 31:1551. 80. Brooks PM, Hart JAL. The bone and joint decade: 2000–2010. Med J Aust 2000; 172:307. 81. Gardner M. Fads and Fallacies in the Name of Science. 2nd rev. ed. New York: Dover, 1957:204. 82. Straus SE, Beldon W. Fiscal year 2006 budget request, statement to the House Subcommittee on Labor-HHS-Education Appropriations. Bethesda, MD: National Center for Complementary and Alternative Medicine, NIH, March 9, 2005. (http://www.hhs.gov/budget/06budget/documents/NCCAM.doc). 83. Sampson WI. Why the National Center for Complementary and Alternative Medicine (NCCAM) should be defunded Quackwatch. (http://www.quackwatch.org/01QuackeryRelatedTopics/nccam.html). 84. Kolasinski SL. Complementary and alternative therapies for rheumatic disease. Hosp Pract 2001; 36:31. 85. Horstman J. The Arthritis Foundation’s Guide to Alternative Therapies. Atlanta: Longstreet Press, 1999:151. 86. The Arthritis Society (Canada). Living well with arthritis: unproven remedies; potions, elixirs, gadgets and devices. Toronto: The Arthritis Society (Canada), 1999. 87. Arthritis Foundation of Victoria (Australia). What can be done? Elsternwick, Australia: Arthritis Foundation of Victoria (Australia), 1998. 88. National Institute of Arthritis and Musculoskeletal and Skin Diseases. Handout on health: osteoarthritis. National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Health (USA), 2002. 89. Federal Trade Commission (USA), ‘Miracle’ health claims: add a dose of skepticism. Federal Trade Commission (USA), produced in cooperation with the Food and Drug Association (USA), 2001. (http://www.ftc.gov/bcp/conline/pubs/ health/frdheal.htm). 90. Better Business Bureau. Health quackery. Natick, MA: Better Business Bureau of Eastern Massachusetts, Maine and Vermont, 1996, (http://www.bosbb.org/ literature/lit_0011.asp). 91. Biasi G, Badii F, Magaldi M, et al. Un nuovo approccio al trattamento della syndrome ﬁbromialgica: L’uso del Telo Cypro1. [A new approach in the treatment of ﬁbromyalgia: The use of a copper wire sheet (‘‘Telo Cypro1’’)]. Minerva Med 1999; 90:39. 92. Federal Trade Commission (USA). Remedies targeted in International Health Claim Surf Day; consumer protection, public health, privated agencies from 24 countries assess over 1,200 Internet sites. Washington, DC: Federal Trade Commission press release. November 10, 1998. (http://www.ftc.gov/opa/ 1998/11/intlhlth.htm).

11 Role of Copper in Anti-inflammatory Therapy and the Potential for Its Transdermal Application Jurij J. Hosty´nek Department of Dermatology, University of California at San Francisco School of Medicine, San Francisco, California, U.S.A.

Roberto Milanino Facolta` di Medicina e Chirurgia, Sezione di Farmacologia, Dipartimento di Medicina e Salute Pubblica, Universita` di Verona, Verona, Italy

INTRODUCTION Copper is an essential trace element (ETE), critical for a variety of biological processes, for example hemoglobin synthesis, enzyme activation, particularly that of superoxide dismutase, and, more generally, as a key component of mitochondrial, cytoplasmic, and nuclear enzyme systems. Absorbed mainly from dietary sources (organ meats, shellﬁsh, nuts, whole-grain cereals), copper absorption and utilization is interdependent with that of other metals, including zinc, iron, cadmium, and molybdenum. Signiﬁcant stores of copper are found in liver, muscle, and bone. It is required for bone formation, cardiac function, keratinization, and tissue pigmentation, where it binds to proteins, preferentially sulfur- and nitrogen-containing ligands. In mammals, ceruloplasmin (CP), a multifunctional endogenous plasma glycoprotein, is responsible for copper transport in the blood where it may bind some 60% to 70% of the metal present. Physiological factors that require involvement 267

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of copper as a remedial factor induce CP production in the liver, thus assuring an adequate transport medium in response to increased translocation requirements (1). Total plasma copper is a reliable indicator of circulating CP, as a highly signiﬁcant correlation exists between the two values (r ¼ 0.8), in rats as well as humans (2,3). Copper plays an active role in inﬂammation, and animal models show that the anti-inﬂammatory activity of the metal is ampliﬁed when complexed with nonsteroidal anti-inﬂammatory drugs (NSAIDs). As a rule, such complexes also become more effective and less irritant than the parent ligands (4–9). It has been hypothesized that supplementation with exogenous copper may bring remedial action in inﬂammatory disease such as arthritis (A), and it was widely demonstrated that intravenous (IV) and subcutaneous (SC) administration of copper solutions is effective in controlling edema induced in a variety of animal screens (6,10–12). The inﬂammatory condition comprises a wide spectrum of diseases that include rheumatoid arthritis (RA), childhood arthritis, carpal tunnel syndrome, osteoarthritis, lupus, and gout—conditions associated with pain, stiffness, and swelling in and around the joints. It affects one out of every three adults and is the most common cause of disability in the United States. It results in 75,000 hospitalizations and 39 million outpatient visits annually (13). The majority of those affected by inﬂammatory diseases, of all racial and ethnic groups, are less than 65 years of age, as onset is most often seen between the ages of 25 and 50 years. RA and osteoarthritis, the most common forms of A, are characterized by chronic joint inﬂammation eroding cartilage and bone. Some forms of A, such as RA, psoriatic arthritis, and lupus, can affect multiple organs and cause widespread symptoms. RA is characterized by nonspeciﬁc, usually symmetric inﬂammation of the peripheral joints, and may lead to destruction of articular and periarticular structures. It is a chronic syndrome generally classiﬁed as an autoimmune disease, and a genetic predisposition has been identiﬁed. Control of the condition in humans is achieved through use of immunosuppressive drugs, and symptoms can be reduced by treatment with anti-inﬂammatory compounds. The majority of those afﬂicted improve symptomatically with conservative treatment in the early stages of the disease, but many are severely disabled over the course of their lives, as a cure is not known (14,15).

TRADITIONAL AND MODERN THERAPIES FOR RA AND RELATED DISORDERS Copper Metal—Bracelets and Rings Historically, use of copper in the form of the metal, as verdigris (basic cupric acetate) or vitriol (cupric sulfate), has been a time-honored practice since antiquity, recognized for its anti-inﬂammatory activity (16). The potential

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for its activity as an anti-inﬂammatory agent by transdermal delivery, however, remains subject to controversy because scientiﬁc studies designed to demonstrate therapeutic beneﬁts for arthritic conditions through dermal contact with metallic copper or its compounds have been inadequate. Quantitative data for percutaneous penetration of the metal’s putative oxidation products, which may be generated in contact with skin in humans, are not available. Because no beneﬁcial effect could be statistically documented for skin contact with copper devices, their use was and still is relegated to ‘‘quack’’ medicine. It is unfortunate that this term has been used when the term ‘‘not understood’’ would have been more appropriate. Persisting lay use of copper devices to the present day and assertions of beneﬁcial effects obtained seem to warrant an independent study of the metal to objectively evaluate its effectiveness, separating activity from placebo effect, even if the current state of anti-inﬂammatory medicine casts a doubt over the economic value of such studies. A ﬁrst step in reinstating the metal in the armamentarium of anti-inﬂammatory remedies would be a quantitative, controlled study of copper release from the metal brought in intimate contact with human skin in vivo. Release of Copper in Contact with the Skin In order to conﬁrm the long-standing assertions that skin contact with copper bangles has an AI effect due to skin penetration of solubilized metal, Walker and Keats performed experiments to verify that copper metal was oxidized and dissolves in artiﬁcial sweat. In samples where initial copper concentration was of the order of 2 105 M, after 24 hours the samples turned blue and the concentration had increased to 2 103 M Cu. The same authors also measured the weight loss from copper bracelets due to corrosion worn by volunteers. Bracelets measuring 22 1.3 0.1 cm lost an average of 13 mg per month (17). Weight losses of 0.1% to 0.8% were observed again in copper bracelets weighing 14 g used in a trial involving volunteers and lasting one month (18). Such variation can be explained by differential mechanical wear. An alternative explanation is the variability in subjective sweat composition and sweating rate. Skin Penetration by Copper Compounds The only experiment that allows calculation of a diffusion constant Kp for a copper compound was performed by Walker. Bis(Glycinato) copper (II) complex, a copper compound likely to be formed with skin exudates, was applied to cat skin in vitro (19). A radioactive (64Cu) 0.05 M solution in physiological saline applied to excised cat skin resulted in a steady-state transport rate Kp ¼ 24 104 cm/hr. All other diffusivity data for copper compounds had to be deduced from clinical observations or physical analytical methods on animals and humans following exposure.

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In vivo application of copper oleate in a lanolin-petroleum base over 24 hours to human back skin resulted in a signiﬁcant increase in urinary copper levels over several days (20). Following topical application of lipophilic Cu(II) salicylate and -phenylbutazone complexes, designed to study their penetration and biodistribution in rats,64Cu was eliminated almost quantitatively, primarily in feces, an indication that Cu(II) rapidly permeates the dermal barrier (21). Electron microscopy of skin treated topically with copper acetate led to the observation that copper initially localizes in the intercellular spaces; subsequently, the cell membranes of viable cells are penetrated as well, and the metal accumulates in and around the cell nucleus (22). By electron probe analysis and analytical electron microscopy, occurrence and levels of copper were traced across human cadaver skin following application of CuSO4 using Franz cells. Copper was observed to enter the outer sc cells at high concentrations; it then encounters an apparent barrier approximately halfway within the epidermis, falling below the detection limit (0.1% by weight), to reappear at the granular interface. There, it was primarily seen traversing an intercellular route through the granular layer, to reach the spinosum layer (Warner, personal communication). Scope and Limitations of the Quantitative Structure Activity Relationships Approach to Diffusivity of Metal Compounds Principles that govern diffusion of exogenous compounds through biological membranes such as the skin are prejudicial towards penetration by metal complexes in general, governed as they are by numerous factors difﬁcult to evaluate a priori. Mathematically derived (mechanistic) models predictive of percutaneous penetration have been developed based on in vitro and in vivo data through regression analysis of experimental results, and serve to predict diffusivity of untested structures. Within limits of molecular parameters such as size, lipid solubility, or hydrogen bonding, these models apply to organic compounds such as drugs, pesticides, cosmetic ingredients, etc., and can predict their dermal absorption with increasing accuracy (23–26). The elements required for construction of such predictive models can be reduced to the criteria of structure and physical properties (27). No such reduction is feasible when modeling diffusion of electrolytes, however. Attempts to model the dermal absorption process of metal compounds, and those of transition metals in particular, have been thwarted owing to a number of confounding factors, as became evident from in vivo and in vitro experiments. Because movement through biological membranes is highly element and chemical species speciﬁc, molecular physicochemical parameters alone do not sufﬁce to model their migration into and through the strata of the skin. A number of factors are closely inter-related and their combined effects are neither entirely understood nor predictable. Unless the dynamics of in situ changes

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in valence (oxidation state), or electrophilic reactivity, among others, can be factored in, the diffusivity of metallic elements eludes modeling. Furthermore, the limited experimental data available so far from in vitro and in vivo experimentation for metal compounds have been acquired under disparate conditions and are too scarce considering the number of metals and metalloids of variable valence existing as free ions or forming chelates, coordination compounds, or complexes with electron donors such as oxygen, sulfur, or phosphorus, abundant in biological systems. The resulting database is too limited for the development of predictive algorithms. Computer calculations have been used to predict skin diffusivity for chemical structures, and of conventional AI agents in particular (28–30), but not for cupriphores. DRUG THERAPY Aspirin, Salicylates, and Other Anti-inflammatory Drugs Aspirin, ﬁrst used as an antipyretic, has also been recognized for its analgesic activity, and it was elaborated in a plethora of derivatives (salicylates). Relatively safe, these derivatives, as well as aspirin itself, continue in use as the cornerstone of AI drug therapy to the present day. Introduced to replace cortisone, an AI hormonal agent ﬁrst isolated in the 1930s, they act on the swelling, heat, and pain of inﬂammation that is suffered by A victims. They are relatively safe and inexpensive. Given PO they can induce GI symptoms affecting the gastric mucosa, however, and erosion and bleeding gastric ulcers may result. Besides derivatives of salicylic acid, NSAIDs from other chemical classes have been evaluated for their anti-inﬂammatory activity: N-arylanthranilic acids, e.g., mefenamic acid, derivatives of arylacetic acid, e.g., ibuprofen, diclofenac, piroxicam, aniline and p-aminophenol derivatives, acetaminophen, etc. (31). Given PO, their dosage is still limited, however, as they also carry the risk of GI symptoms. Recent anti-inﬂammatories are selective NSAIDs, such as the family of COX-2 cyclo-oxygenase inhibitors Vioxx (rofecoxib), Bextra (valdecoxib), or Celebrex (celecoxib). In contrast to aspirin, however, they do not impair platelet adhesiveness, which may lead to cardiovascular disorders, and in addition are suspected to induce psoriasis (32). Gold Systemic treatment with soluble gold (I) salts (chrysotherapy) represents a continuum in the armamentarium of rheumatologists since the beginning of drug therapy to combat arthritis, as it affords rapid symptomatic relief. The different aspects of gold therapy, positive and negative, are therefore reviewed here in greater depth. Initially used for the treatment of tuberculosis until the 1930s, promoted by Forestier chrysotherapy found its principal therapeutic application in the treatment of RA (33). Use continues in addition to

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salicylates or other NSAIDs if the latter do not bring sufﬁcient relief or suppress active joint inﬂammation. A serious limiting factor in systemic application of soluble gold salts has been the high incidence of adverse reactions, as some degree of toxicity appears inevitable at effective dose levels. The most common toxic effects involve the skin and mucous membranes, including pruritus, allergic contact dermatitis (ACD), and stomatitis, but gold compounds can also be toxic to the hematopoietic organs and the kidney. Gold therapy must be discontinued when any of those manifestations appear, which occurs in up to one-third of patients (34,35). Parenteral preparations include gold sodium thiomalate, gold thioglucose, or gold thiosulfate, and are administered IM at weekly intervals: 10 mg the ﬁrst week, 25 mg the second, and 50 mg/wk thereafter until a total of 1 g has been given or signiﬁcant improvement is apparent. When maximum improvement is achieved, dosage is decreased to 50 mg every 2–4 weeks (14). In active RA, chrysotherapy provides rapid pain relief, and can induce partial or complete remission in as high as 80% of cases, as long as therapeutic regime is maintained. Even with sustained therapy, however, there is roentgenological evidence of progression of the disease. Eventually, all patients relapse in 3–6 months when therapy is discontinued; the condition then returns to the pretherapeutic status and the disease continues on its natural course (36). Despite the high incidence of serious adverse effects, chrysotherapy is still widely accepted, since for many forms of arthritis, given parenterally, orally, or more recently, intra-articularly, gold (I) salts still represent the most effective therapy (37). Thanks to its anti-inﬂammatory properties, over the past half century gold has found a number of applications, such as treatment of juvenile and adult rheumatoid arthritis via intramuscular injection of the water-soluble thiomalate, thioglucose, or thiosulfate (38). Other new uses include the treatment of such skin diseases as pemphigus and psoriatic arthritis, which involve an immunological component (39). Such use is no longer practiced in the United States. Following parenteral therapy, which involves relatively high doses, gold was seen to persist in the organism for several months. Carried by plasma, bound to alpha-globulin, it is carried to practically every tissue, including the skin where it becomes evident in characteristic blue-gray discoloration, or chrysiasis, most often appearing on the face and hands, areas exposed to sunlight, hair, and nails (36,38,40). While for nearly three-quarters of a century gold salts have been used to relieve the symptoms of arthritis, just how the metal exerts its antiarthritic effect is not clear, as it does not have a known biological function itself. Stoyanov and Brown (41) propose an indirect role by gold since they ﬁnd that it speciﬁcally binds to a copper-activating protein, thus turning on production of a protein pump involved in copper transport, mobilizing endogenous copper reserves. The biological effect of gold antiarthritic drugs may thereby consist in their effects on copper management in eukaryotic systems.

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Immune Reactions in Chrysotherapy Due to increasing systemic exposure of patients for therapeutic purposes over the past 50 years, gold is now recognized as a signiﬁcant factor in the etiology of cellular and humoral immunity (39,42). It is also an inducer of circulating immune complexes (43,44). Systemically, gold compounds have modulatory effects on immediate and delayed immune responses, both in vitro and in vivo (42), causing type I hypersensitivity (contact urticaria syndrome), cell-mediated (type IV) sensitization or ACD, as well as type III or Arthus reactions involving antigen– antibody complexes. Hypersensitivity reactions to both the monovalent and trivalent forms of the metal have been described. While ACD is not uncommon due to the widely practiced systemic therapy using gold compounds, or the intimate skin contact with metallic gold, urticarial reactions are extremely rare. The relatively high incidence of delayed skin reactions has been noted since the introduction of gold compounds for the treatment of rheumatoid arthritis, and is seen in more than 50% of patients so treated (45,46). Although not clinically relevant to the most part, the rate of sensitization to gold seems to be on a steady increase, with comparable prevalence in the United States, Europe, and Japan since routine screening for gold allergy was started in those countries (47–51). Injection of water-soluble gold complexes is associated with a high risk of sensitization in the form of a type I hypersensitivity response (52). Repeated exposure to gold salts in sensitized persons does not necessarily lead to recurrence of dermatitis, however. This indicates a gradual buildup of tolerance (53). Abnormally high serum IgE levels together with eosinophilia have been associated with gold therapy, but remit on cessation of such therapy (54,55). Nephrotic syndrome, and glomerulonephritis in particular, is noted as a consequence of parenteral or oral administration of gold in its organic form. Certain gold compounds, such as Ridaura (auranoﬁn), given orally in which gold is complexed to trialkylphosphines, are suspected of giving rise to circulating immune complexes (type III hypersensitivity) (42). As an electrophile reacting with native protein, the metal forms a complete antigen that induces speciﬁc antibody formation, which, in turn, gives rise to the glomerular injury observed (43,44,56,57). In patients treated parenterally with a variety of gold compounds, the metal is seen to be retained in signiﬁcant amounts (over 80% of the amount injected over 39 days of administration) bound to the plasma component alpha-globulin as a gold protein complex. The amounts excreted (primarily in urine) were not related quantitatively to the amounts injected (38), and retention may increase the risk of immune complex glomerulonephritis in patients receiving gold preparations. In a large Swedish study involving 823 dermatology patients, the incidence of 8.6% positive reactions to gold was recorded, without any signs of an irritant etiology (58). Recent conﬁrmation of this result now makes gold

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the second most common contact allergen (in the Swedish population) after nickel. Thus, routine patch testing of dermatitis patients in Europe has revealed a rate of positive readings approximating 10% and 14.3% in North America (59,60). Based on test results by the North American Contact Dermatitis Group, with 9.5% positive, gold is the sixth most common allergen among dermatology patients in the United States (61). A similar prevalence is noted in Europe and Japan (50,51). Systemically provoked ﬂare-ups of contact allergy to gold appear as another characteristic of response to that allergen. Intramuscular injection of gold sodium thiomalate was reported to reactivate previously involved but clinically healed skin. It also led to corresponding pathobiochemical changes in the venous blood of the patients (i.e., activation of neutrophil granulocytes and release of several cytokines). Similarly, intradermal test sites that had healed 9 to 24 months previously were reactivated (62–64). The diverse skin reactions do not appear to depend on concentrations of the metal in the skin. Patients receiving cumulative doses of 1 to 50 g of gold (I) salts may not experience adverse reactions (65), and there is no consensus on optimal or limit in dosage. Thus, tolerance for gold salts is highly individual, and patients require constant monitoring for toxic manifestations. In summary, while contact of intact skin with metallic gold is not known to induce hypersensitivity, its occurrence in context with systemic exposure through injection of soluble salts for therapeutic purposes is now considered to be an unavoidable side effect in the risk–beneﬁt balance of anti-inﬂammatory therapy. Corticosteroids Corticosteroids are effective oral and intra-articular anti-inﬂammatories, but their efﬁcacy diminishes with time and they do not prevent joint destruction. Their use is also associated with various side effects, and steroid therapy is only recommended on failure of less hazardous drugs (14). Superoxide Dismutase Anti-inﬂammatory copper-dependent metalloenzymes, superoxide dismutase, have been developed for the treatment of arthritic diseases (66). Two such preparations, Ontosein and Orgotein, have been shown to be safe and effective for the treatment of established rheumatoid and osteoarthritis when given IV or IA into knee and hip joints in single or multiple doses. They cannot be dosed PO, however, due to gastric inactivation. Immunosuppressants Methotrexate, cyclosporine, and others are used for severe RA, but bring the risk of major side effects: liver disease, bone marrow suppression, and potentially, malignancy (14).

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The search for new anti-inﬂammatory agents has had limited success so far, owing to the lack of insight into the causes and mechanisms of inﬂammatory diseases. Physiotherapy Other than surgical intervention, relief from arthritic pain was sought in a number of noninvasive approaches: ultrasound, thermotherapy, balneotherapy, electrotherapy, laser therapy, acupuncture, and even tai chi. PRECEDENTS IN TOPICAL DELIVERY OF ANTI-INFLAMMATORY AGENTS Topical anti-inﬂammatory formulations have a history of successful application. An early precedent for topical treatment of rheumatic discomfort were thermogenic ointments based on methyl salicylate (wintergreen) and menthol, at present marketed as Ben-Gay by Pﬁzer. Also some of the drugs mentioned above, such as NSAIDs, corticosteroids, immunosuppressives, and also dimethylsulfoxide and capsaicin were reported to be clinically effective in placebo-controlled trials (67). The argument for developing copper complexes with proven analgesics can be made invoking the ﬁndings by Sorenson and others, cited elsewhere, that copper–ligand chelates show anti-inﬂammatory activity superior to that of the ligands themselves. ROLE OF COPPER IN AI ACTIVITY Knowledge sent down from early historic times has been combined with modern pharmacology. Recognition of the essential and therapeutic role that endogenous copper plays in the control of inﬂammatory processes led to the concept of introducing the ETE into state-of-the-art pharmacology, thus combining the two elements. A hypothesis ﬁrst: the idea of using exogenous copper in combination with analgesics and anti-inﬂammatories may turn out to be a successful concept with augmenting effects in modulation and control of inﬂammatory processes. Inﬂammation is tissues’ response to injury and represents the ﬁrst step of the repair process towards normalization. While many anti-inﬂammatory agents may reduce some aspects of inﬂammation, they do not necessarily involve repair processes towards correcting the cause of the disease. Copper complexes, on the other hand, were demonstrated to promote tissue repair processes, and they involve activity in a number of diseases with inﬂammatory components other than arthritis. Their therapeutic use includes infection, cancer, diabetes, and epilepsy (7,8). Copper as an ETE is present in the mammalian organism only in minimal concentration as free ionic copper (1018 mol, with a range between 1011 and 1019 mol) and is predominantly complexed with proteins and amino acids, ligands that are subject to dynamic

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exchange in response to physiological requirements and homeostatic equilibria (8,68). In animals and humans, copper levels change in response to inﬂammation. Heilmeyer and Stuwe (69) had ﬁrst observed that serum copper rose with arthritic disease, and fell again on remission. Stores of copper normally immobilized are bound to metallothionein, released, and bound to CP that binds over 60% of total serum copper. CP was shown to possess remarkable anti-inﬂammatory characteristics (70) in vivo. Experimental induction of either acute or chronic inﬂammation in the rat promotes accumulation of copper in some body compartments that are known to be directly or indirectly involved in the development (and remission) of the inﬂammatory processes (2). Thus, inﬂammation appears to determine an increased demand for copper by the organism, a requirement fulﬁlled by adjusting the intestinal absorption without depleting the major body stores (11,71). A copper-supplemented diet exhibits an antiarthritic action, and, conversely, copper deﬁciency appears associated with proinﬂammatory effects, as observed in animals on a copper-deﬁcient diet (3). In the 1960s and 1970s, publications reported on the anti-inﬂammatory activity in animal models for copper compounds in different forms: as mineral salts, as enzymes, or complexes, when given IM, intraperitoneal, SC, and, most of all IV. It was also suggested that active copper complexes were formed within the organism, as their activity in animal models of inﬂammation was found to be greater than either treatment with inorganic copper salts or the complexing agents alone, while the parent compounds or ligands were inactive in test models of inﬂammation even at larger screening doses, in coordination with copper they were found to be active (4,5). Thus, the therapeutic activity of a ligand appears to depend on its association with (endogenous or exogenous) copper. This may be attributed to the ligand acting as the carrier for the metal, or copper in the complex acting as a catalyst, interfering with the inﬂammatory process. As reviewed by Bonta, occasional publications reported that sodium-3(N-allylcuprothiouredo)-l-benzoate and cuprous iodide had antiinﬂammatory activity and that Cu(II)(salicylate)2 had fever-lowering effects in various animal models of inﬂammation or fever. In addition, copper complexes of antiarthritic drugs, including salicylic acid, acetylsalicylic acid, penicillamine, and several corticoids, were found to be more active than the parent drugs. A comparison of copper, gold, and silver thiomalate and thiosulfate complexes in models of inﬂammation revealed that the copper complexes were effective, while the gold and silver complexes were virtually inactive. As a general rule, copper complexes are less toxic and suppress the ulcerogenic effect of many of the ligand drugs themselves. Those early reports of anti-inﬂammatory activity of copper complexed with various ligands were based on IV or parenteral administration (72).

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Based on this hypothesis, the compounds investigated subsequently included copper complexes of well-known antiarthritic drugs, including steroidal and nonsteroidal anti-inﬂammatory drugs: niﬂumic acid, D-penicillamine, hydrocortisone, dexamethasone, dimethylsulfoxide, clopirac, ketoprofen, (þ)-naproxen, indomethacin, mefenamic acid, diclofenac, ibuprofen among a number of others. All the copper complexes of those drugs have been found to be active or more effective anti-inﬂammatory agents than the parent drug (8,73). Oral and local administration of Cu(I) and Cu(II) complexes in animals mostly led to equivalent responses in the edema model. Since the two forms of the metal undergo different reactions in vitro, a catalytic function is attributed to exogenous copper ion, also suggesting that both Cu(I) and Cu(II) act by the same anti-inﬂammatory mechanism involving a common metabolite (74). The ETE, normally absorbed as Cu(II) ion, is carried in the plasma by CP, albumin, transcuprein or low molecular weight complexes such as those with different amino acids in that oxidation state, since, as is the case for Fe(II), Cu(I) can potentially prime the harmful Fenton reaction. As in the cell interior, the metal seems to exist almost exclusively in the Cu(I) form, the necessary reduction step is performed by plasma membrane reductase, an enzyme likely to be expressed by all body cell types. The cells themselves are well equipped with copper chaperones for the control of intrinsic Cu(I) toxicity. These metallochaperone proteins escort copper ions directly to enzymes that require the metal to function, such as the antioxidant copper–zinc superoxide dismutase. In a review of anti-inﬂammatory activity of exogenous copper, Milanino et al. (11) also concluded that copper indeed is active as an acute anti-inﬂammatory agent irrespective of chemical form given, including inorganic copper salts. Administration of exogenous copper is effective in controlling inﬂammation associated with arthritis, and may lead to complete (albeit temporary) remission. Sorenson (8) reviewed the AI activity of several dozen copper complexes, mostly exploratory and several as commercial drugs, the categories of ligands including non-anti-inﬂammatory compounds such as amino acids, heterocyclic carboxylic acids and amines, among others. The AI activities reported were based on oral or parenteral administration. However, copper salts compounded with salicylic acid or ethyl salicylate have also been shown to have AI activity in animal models of inﬂammation following topical application (75). Another indication of the anti-inﬂammatory action of copper is the rise of serum copper in pregnancy, in tandem with remission of RA, which is attributed to the protective AI action due to an increase in CP levels. This is followed by a postpartum relapse to pre-existing RA (76).

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PAST USE OF COPPER CHELATES IN THE TREATMENT OF RHEUMATOID ARTHRITIS Mixtures of copper salts and salicylic acid or ethyl salicylate in aqueous or glycerol-dimethyl sulfoxide solutions were tested successfully for antiinﬂammatory activity following topical application in a variety of animal models of inﬂammation. During the period 1940–1950, the copper-containing compounds sodium 3-(N-allylcuprothiouredo)benzoate (Cupralene) and Alcuprin, given IV, sodium meta-(N-allylcuprothiocarbamide), given IM, and Cu(II) (4,6diethylammonium-8-hydroxyquinoline)2 sulfonate (Dicuprene or Cuprimyl), given IM, were reported by Forestier to be effective in treating a variety of arthritic diseases, including acute and chronic RA, chronic polyarticular gout, ankylosing spondylitis, and disseminated spondylitis. Forestier et al. (77) provided evidence in support of the suggestion that these copper complexes were superior to gold therapy. Work linking copper and inﬂammation has been done by Sorenson (4,5), who demonstrated that copper (II) acetate alone has an AI activity in experimental models of inﬂammation, with the suggestion that the active metabolites for AI activity are copper chelates. Sorenson and Hangarter (6) reported on clinical data obtained for some 1500 patients suffering from diffuse connective tissue disease over 30 years, noting that toxic side effects such as those occurring with chrysotherapy were not observed with copper therapy. Although these results were conﬁrmed by others, the advent of hydrocortisone and the belief that it was going to ‘‘cure’’ arthritis led to the disuse of these preparations. Hangarter’s preparation of aq. copper salicylate, an aqueous mixture of 12.5 mM of sodium salicylate and 0.04 mM Cu(II) chloride (Permalon), given IV, was more effective than the other clinically used complexes in the treatment of A and other degenerative diseases. That complex was effective in treating acute rheumatic fever, acute and chronic RA, erythema nodosum, and sciatica either with or without lower back spinal degenerative disease. From a total of 620 patients treated over a 20-year period, 65% beneﬁted by remission through this therapy, with a mean remission time of three years, 23% experienced improvements in their condition, while 12% remained unchanged (7). Hangarter mentions a lack of gastrointestinal complaints in the cohort of patients, which were common when only sodium salicylate was used in therapy. In 1970, the manufacture of Permalon was discontinued for economic reasons, however, and Hangarter’s approach to therapy came to an end. TRANSDERMAL DELIVERY OF ANTI-INFLAMMATORY COPPER CHELATES VS. CONVENTIONAL (SYSTEMIC) ANTI-INFLAMMATORY THERAPY Since it appears important to maintain a needed minimal level of copper in tissues for the control of inﬂammation (78), the skin may be an adequate site

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for the delivery of cupriphores at maintenance levels. The case can therefore be made in favor of dermatologicals for copper-based therapy in the treatment of inﬂammation, since it may avoid patient discomfort and potential disadvantages such as the ﬁrst-pass metabolism or idiosyncratic drug reactions and GI side effects inherent in conventional routes of administration (PO, ID, IP, IV, IA) (79–81). Due to lessened systemic clearance, local tissue accumulation of the AI agent would be possible in the treatment of conditions such as muscle pain and inﬂammation by low-level, controlled, and sustained release. As computer simulation of metal–ligand equilibria reveals that salicylates and similar complexes are thermodynamically unstable in plasma (82,83), this implies that the exogenous form of copper administered is different from the complex formed with endogenous ligands, and has no direct bearing on the anti-inﬂammatory activity. It thus appears fortuitous that most AI activity of copper has been studied using salicylate derivatives, in animals and humans, as AI activity of copper was exhibited by a wide variety of other ligands also. It is the increased availability of endogenous copper per se, which affords protection against inﬂammation. This is particularly encouraging in the search for skin-diffusible copper compounds, since the mechanism by which copper acts as the actual anti-inﬂammatory and antiarthritic agent, and what its bioactive moieties are, is still under discussion. In topical therapy, substances can be applied in concentrations that would be too toxic for systemic exposure. Given orally, compounds may contribute to gastric irritation (e.g., the salicylates), and the pharmacologically active form of copper complexes is also likely to be compromised (84). Copper complexes dissociate at the varying pH prevailing in the GI tract and become subject to sequestration and biliary excretion. Diffusion through the skin on the other hand avoids irritation in the GI tract and ﬁrst-pass inactivation by the liver, and avoids discontinuity in therapy due to lack of patient compliance. Several theoretical and practical obstacles need to be overcome. With data presently available, progress in the development of copper-based dermatologicals for inﬂammatory disease therapy is severely limited. One key element awaiting development is data on the penetration of the ETE through human skin in any of its forms: as a polar mineral salt, or as a low molecular weight hydrophilic or larger lipophilic complex. Although dermatological copper preparations are, or were available, e.g., the topical anti-inﬂammatory copper-salicylate gels Alcusal1 or Dermcusal1, found to be effective in animals, they are not commonly employed for the treatment of RA in humans. This can be attributed to skepticism on the part of the medical profession due to the folkloristic component attached to such therapy, and also due to a lack of adequate controlled human studies. New treatments must be shown to be effective through unbiased, objective testing before the medical community acknowledges its potential beneﬁts. Thus, an independent randomized placebo-controlled trial of the copper

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salicylate–ethanol complex failed to show the effectiveness of the product: applied to the forearm it was no better than the placebo gel for (subjectively evaluated) pain relief in patients with osteoarthritis of the hip or knee (85). Skepticism prevails vis-a`-vis copper-based therapy because its mode of anti-inﬂammatory action in the organism is insufﬁciently understood, and stability and fate of complexes delivered is unknown. For the electrophilic copper ion in particular, primarily binding sites in proteins may interfere with the dermal diffusion process. A major effort should be dedicated to the cutaneous delivery of cupriphores in quantities signiﬁcant for local therapeutical activity. A ﬁrst step is a de minimis approach: certain types of compounds could be deﬁned as priority candidates for topical application of cupriphores for which Kps can be determined using standard in vitro permeation protocols, in order to put them on a relative scale of diffusivity. Such priority would go to small molecular weight complexes with ready solubility in polar media, which do not exhibit skin toxicity, bound to ligands of established safety. A prime such category that comes to mind are: Reaction products with low molecular weight essential amino acids such as glycine or histidine. Molecular size is a key determinant for optimal ﬂux across the skin, and low molecular weight complexes will also determine the ability of plasma copper to diffuse into tissues. Unstable copper complexes with toxicologically safe proﬁles, which may release the metal to endogenous protein-binding sites in signiﬁcant amounts, such as citrate, gluconate, histidinate, or sebacate. The latter category may also enhance the amount of copper absorbed, overall thanks to signiﬁcant SC diffusion. The case can be made in favor of dermatologicals for copper-based therapy in the treatment of localized musculoskeletal disorders, as it allows targeted application of higher local drug concentrations too toxic systemically, avoiding or minimizing patient discomfort and untoward effects potentially incurred using conventional forms (IM, PO, IP, IV, IA) of treatment. Targeted exposure focusing on speciﬁc areas of inﬂammation becomes possible, providing higher local tissue concentration, rather than the blunderbuss approach through systemic therapy, which renders the agent subject to the vagaries of enterohepatic circulation and biliary excretion (81). As an instance, given orally compounds may contribute to gastric irritation or worse (e.g., the salicylates), and the pharmacological activity of copper complexes is also likely to be compromised (84). The compounds dissociate at the varying pH prevailing in the GI tract, and become subject to sequestration and excretion. Also, when a localized condition such as arthritis is treated systemically, the drug reaches the intended target only after resorption, passage through the liver, and distribution throughout the entire system. Apart from

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the potential for selective accumulation in the target area upon systemic dosing, the quantity of drug, which eventually becomes available on the intended target, is thus limited. Following systemic application, the pharmacokinetics are entirely different from those resulting from topical exposure. In the latter case, the drug can be applied directly targeting the afﬂicted part of the anatomy, i.e., the sites of clinical signiﬁcance, with the potential for local skin reaction as the worst case. Since dissipation of the therapeutic agent due to transport throughout the system does not occur, topical, local therapy affords higher tissue concentrations, especially in the outer skin layers, which would be too toxic systemically. The potential for copper activity as an anti-inﬂammatory agent by transdermal delivery, however, is subject to controversy. The studies designed to demonstrate therapeutic beneﬁts of topical therapy for arthritic conditions with either metallic copper or dermaceuticals such as Alcusal or Dermcusal1 by Walker et al. were not conducted lege artis. They were based on subjective patient assessment, obscured by placebo effect. Also, quantitative data for percutaneous penetration of copper’s putative oxidation products generated in contact of the metal with skin in humans, a critical factor in accrediting beneﬁcial effects of copper bangles, have not been determined. Walker measured such absorption, albeit by one degree of separation, by gravimetrically determining the amount of copper lost in function of contact time only, and evaluating the resulting beneﬁcial effect in afﬂicted patients by using questionnaires and psychologic parameters (18). Shackel et al. assessed the efﬁcacy and safety of a copper-salicylate gel in osteoarthritis patients in a randomized, double-blind, placebo-controlled study, to be sure. But they were again based on self-assessment of pain, scores decreasing in both the treated and placebo groups, with no signiﬁcant difference between the two groups for decrease in pain (85). Aside from the copper bangles issue, one missing, important factor for a convincing case of copper’s potential beneﬁts is thus the deﬁciency in adequate, systematic research into the beneﬁts of topical application of cupriphores. A measure of copper penetration through human skin in any of its forms, as polar mineral salts or as the more lipophilic chelates, is missing. Subcutaneous Administration of Cupriphores Favorable for transdermal therapy is evidence of anti-inﬂammatory activity obtained when bypassing the epidermal barrier through sc injection of cupriphores. Transdermal therapy as an effective route was documented repeatedly in animal models by sc administration of anti-inﬂammatory copper compounds (74,86,87). Notably, one of those investigations concluded that sc administration of some copper complexes was more effective than if they were given orally, while noting also that no difference was observed between the activities of copper (I) and copper (II) compounds in those

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experiments (74). The latter serves to counter the argument that, in applying copper compounds, speciation of that ETE may be of importance. That levels of copper introduced into plasma strongly correlate with the anti-inﬂammatory effect was demonstrated when a wide range of copper–ligand complexes, administered sc, i.e., reaching the plasma without having to traverse the skin, were effective in controlling edema induced in animal screens (4,5,88). Positive such results were also obtained through sc administration of copper complexes by Lewis et al. An AI effect by sc dosing of copper aspirinate and other copper compounds in different animal species has been described (86). Precedents in Effective Transdermal Cupriphore Delivery in Animals and Humans High levels of 64Cu radioactivity localized in loci of inﬂammation following dermal application of copper chelates by Beveridge indicated that exogenous copper was sequestered at inﬂammatory sites, and provides evidence of percutaneously absorbed copper complexes (75). Two lipophilic formulations of Cu(II) salicylate, Alcusal1 and Dermcusal1, were developed by Walker et al. for dermal treatment of inﬂammatory conditions. Alcusal1 is a copper salicylate–ethanol complex prepared by reﬂuxing Cu(II) hydroxide with salicylic acid in anhydrous ethanol, Dermcusal1 from copper salicylate prepared in a dimethyl sulfoxide– glycerol solution. Applied on the shaved dorsal skin of rats, they signiﬁcantly reduced artiﬁcially induced paw edema and suppressed experimental arthritis, thus demonstrating facile cutaneous penetration (21,89,90). When the copper salicylate–ethanol complex was applied topically on the skin of human volunteers, analysis of elevated plasma salicylate levels did indicate that penetration had occurred. However, upon application on self-diagnosed arthritis and rheumatism patients, upon subjective evaluation no statistically signiﬁcant results in pain relief could be documented (18). Overall, organic copper compounds to treat RA, albeit promising in the hands of many clinicians over time, led to inconsistent, and sometimes contradictory results. Pharmaceutical copper preparations are available but are not widely employed for the treatment of RA due to the effective and continuing use of chrysotherapy and the application of steroidal preparations, and also due to skepticism on the part of the medical profession in consideration of the folkloristic component attached to therapy based on copper. Further research proved inconclusive because of deﬁcient experimental design in selection and evaluation/classiﬁcation of patient cohorts, in the diagnostic criteria applied, the lack of controls, the confounding placebo effect, and to subjective evaluation by patients themselves, which was based on perception. Adding to such procedural deﬁciencies came the lack

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of marketing incentives at the pharmaceutical industry level. Thus, use of copper derivatives to treat RA and related inﬂammatory diseases gave way to other emerging anti-inﬂammatories such as Cox-2 inhibitors, which inhibit inducible cyclo-oxygenase enzymes and thereby inhibit prostaglandins, important mediators of inﬂammation. Health complications have become associated with that class of drugs now: aggravation of pre-existing ulcers, increase in the incidence of thrombosis, rise in blood pressure among others (32,91,92). They thus give rise to criticism and prompt inquiries on the part of health ofﬁcials, favoring efforts to ﬁnd alternatives for the treatment of inﬂammation. Should the epidermal barrier prove too formidable for diffusion of copper chelates to reach concentrations required for clinically meaningful analgesic and anti-inﬂammatory effect, however, several alternative techniques are now are available to carry drugs into or through the skin. Percutaneous Drug Delivery Systems and Methods Chemical Adjuvants There are limitations in the selection of drugs that may qualify for passive transdermal delivery (93). Passive percutaneous penetration, whether transcellular, via intercellular lipids or shunts, depends on several limiting factors, including molecular weight, optimally below 500 Da, or volume, lipophilicity, charge, and drug potency. To overcome some of the obstacles to passage of drugs through the SC, the ﬁrst and most restricting barrier of the skin, research has endeavored to ﬁnd chemical adjuvants, which would overcome the relatively low SC permeability to enhance penetration. Enhancement to deliver therapeutic levels has been achieved with formulations in which the active ingredients were compounded with terpene derivatives, e.g., linalool, limonene, menthone, or eugenol (94,95). Enhancers leading to higher drug levels in the dermis have chemical characteristics, which have the potential of disrupting the arrangement of the intercellular lipid bilayers and liqueﬁng lipid sheets, structural changes that paradoxically are the very prerequisites for enhanced diffusion. Because exogenous penetration enhancers can exhibit overt toxic or irritant effects, their continued use for improved drug delivery is put in doubt. Dermal absorption of copper complexes by simple passive diffusion, particularly of those with higher molecular weight, e.g., Cu(II)-diclofenac or Cu(II)-ibuprofenato complex with AI effect, may be problematic (96,97). Methods of entrapment (liposomes) or active delivery by physical methods have been developed, however, and could also be successfully applied. Liposomes Liposomes (LSs) have been shown to efﬁciently deliver the large molecular weight class of proteins, such as antigens for immunization studies (98).

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Depending on the molecular moiety to be delivered, LSs can be formulated to incorporate a wide array of substances as a payload, either in the water or in the lipid compartments, to contain antibodies, large molecular weight ligands, or polysaccharides. LSs are spherical uni- or multilamellar lipid/water vesicles with diameters in the micron-to-nanometer range. They are formed when thin lipid ﬁlms or lipid cakes of natural, nontoxic phospholipids and cholesterol are hydrated and stacks of liquid crystalline bilayers become ﬂuid and swell. The hydrated lipid sheets detach during agitation and self-assembly to form large, multilamellar vesicles that prevents interaction of water with the hydrocarbon core of the bilayer at the edges. The size of the particles can be reduced by sonication or extrusion. Properties of lipid formulations can vary depending on the composition (cationic, anionic, or neutral species) and degree of fatty acid saturation; the same preparation method can be used for all lipid vesicles regardless of composition (99). Biological particulate materials can be incorporated in LSs without compromising their structure or activity by exposing them to organic solvents, or light-sensitive compounds can be protected from radiation by incorporating a light-absorbing compound in the liposomal structure. Also, sensitive pharmaceuticals or labile bioactive materials can be encapsulated in an LS designed for controlled or sustained release, to degrade only at a target-speciﬁc application site. The LS structure is very similar, physiologically, to the material of cell membranes. When a formulation containing liposomes is applied to the skin, for example, the LSs are deposited on the skin and begin to merge with the cellular membranes. In the process, the LSs release their payload of active materials into the cells. As a consequence, not only is delivery of the actives very speciﬁc—directly into the intended cells—but the delivery takes place over a longer period of time. Thus, LSs as a delivery system can be designed for slow or fast release of both a hydrophilic or hydrophobic payload (100). The SC is recognized as the main barrier to skin penetration, as well as the major pathway for intracellular and intercellular drug delivery. Going a step further, Scheuplein (101–104) suggested that hair follicles also act as shunts, permitting rapid transport of charged and large polar molecules. Feldmann and Maibach (105) observed increased percutaneous transport through skin areas with high follicular density, also suggestive of follicular delivery. Reﬁnements in methods of observation later conﬁrmed this route for a wide range of molecules, particularly identifying particulate moieties such as LSs, localizing them in follicular and sebaceous areas to act as drug reservoirs (106,107). The potential for targeted percutaneous delivery of copper complexes of differing polarity and large molecular weight in LSs as carriers to the hair follicle may also offer an alternative in achieving therapeutically effective levels of that anti-inﬂammatory agent. Particularly attractive appears liposomal delivery of copper complexed with conventional

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anti-inﬂammatories that exhibit an augmenting activity, such as ibuprofen or diclofenac (96,97). These complexes appear unsuitable for conventional passive diffusion due to molecular size and limited solubility. Potentially nontoxic, degradable, and nonimmunogenic (108,109) LS acting as slow release vehicles could thus create a copper reservoir in the SC. Physical Methods Enhancement in skin absorption can be achieved by occlusion or different forms of energy input, characteristic of active transfer of drugs through the skin. These approaches utilize patch techniques, iontophoresis, sonophoresis, or electroporation to overcome the severe restrictions imposed on passage through the skin. Patches Transdermal absorption is a slow process of passive diffusion, driven by the gradient between the given concentration in the external delivery system and the zero concentration present in the skin. The delivery system using patches keeps the penetrant in continuous intimate contact with the skin with occlusion over days. Given adequate diffusion rates, steady blood levels can thereby be achieved through such continuous and even delivery over extended periods, which is preferable to the spikes and troughs occurring when administration occurs by other routes such as PO or IV. As an instance in delivery of analgesics, the fentanyl patch acts for 72 hours, providing long-lasting pain relief. Some other major drugs currently qualiﬁed for marketing as transdermal patches, intended for daily or weekly application, are clonidine (hypertension), estradiol (estrogen deﬁciency), estrogen-progestin (contraception), fentanyl (analgesic), lidocaine (anesthetic), nicotine (smoking cessation), nitroglycerine (angina pectoris), scopolamine (motion sickness), and testosterone. Iontophoresis Iontophoresis facilitates diffusion of ionic drugs and higher molecular weight compounds such as peptides. By anodal or cathodal electrophoresis negatively or positively charged drugs can be delivered respectively through the patient’s skin; under application of electric current one electrode serves to deliver the drug, the other to close the circuit. By this method luteinizing hormone-releasing hormone, a decapeptide of 1200 Da molecular weight was successfully dosed (110). Electroporation Electroporation is a technique that delivers high voltage pulses to the skin, causing transient changes in cell membranes or lipid bilayers (111).

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Such pretreatment of the skin can facilitate delivery of large, polar molecules such as peptides. Sonophoresis Low-frequency ultrasound treatment of the skin, or sonophoresis, has been developed as another physical method to promote diffusion; this is for highly hydrophilic drugs like mannitol (112).

CONCLUSIONS Copper as an essential trace element and its role in current anti-inﬂammatory therapies are reviewed, from the controversial, age-old practice of wearing copper bangles to the copper complexes as anti-inﬂammatory drugs. Oxidation products formed by copper in contact with the skin and their potential for their skin diffusion are discussed. Considering the potentially negative side effects as well as the discomfort associated with anti-inﬂammatory therapies using current methods of application, transdermal delivery of cupriphores appears to be an attractive alternative worth exploring, although the present lack of data on the skin diffusivity for all categories of copper compounds is a shortcoming, which must ﬁrst be overcome. Should transdermal delivery of copper chelates lessen or suppress inﬂammation, untoward systemic effects associated with conventional modes of anti-inﬂammatory therapy could be lessened or avoided. Aside from general principles of percutaneous absorption and prerequisites for skin penetration by chemicals, the potential for topical delivery of copper complexes is reviewed with the intent to impart renewed impetus to the evaluation of transdermal anti-inﬂammatory therapy with cupriphores. Strategies are identiﬁed for their passive or active diffusion in order to penetrate the stratum corneum barrier, with the purpose to compensate with exogenous sources delivered through the skin potential, local shortfalls in endogenous reserves of copper. Evidence gathered in animals and humans affected by inﬂammatory processes suggests that percutaneous administration of copper may represent an appealing therapeutic approach, particularly in consideration of the unfavorable aspects of adverse side effects and discomfort associated with more conventional therapies. Also, applied dermally, the drug in the target tissues can be expected to exceed the concentrations achievable through more conventional, systemic avenues. At this time, several theoretical and practical obstacles prejudice endeavors in the development of copper-based dermatologicals for the therapy of inﬂammatory diseases, regardless of the form in which that metal may be applied.

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Although dermatological copper preparations are available, they are not commonly employed for the treatment of RA in humans. Skepticism on the part of the medical profession persists due to a lack of controlled efﬁcacy studies. A missing building block towards making a convincing case for the potential beneﬁts in transdermal therapy is the lack of quantitative data on the penetration rate of copper through human skin, in any of its forms: derivatives of skin-identical amino acids, prepared in analogy with peptides potentially formed in contact of the metal with the skin (glycinate, histidinate, etc.), as polar mineral salts, or as the more lipophilic chelates (e.g., as ibuprofen or diclofenac complexes) applied as liposomes. Principles governing the transfer of metal compounds through biological membranes, including the skin, are prejudiced by numerous factors that are difﬁcult to anticipate a priori. For copper in particular as an ETE, binding sites in proteins may interfere with the dermal diffusion process, resulting in degradation or blockage (homeostatic controls). Studies designed to demonstrate signiﬁcant therapeutic beneﬁts for arthritic conditions through skin contact with metallic copper or its complexes in humans, e.g., with oxidation products that may be generated in contact of the metal with the skin have not been conducted in a convincing manner.

That transdermal AI therapy can be effective has already been demonstrated in animal models by administration of a number of copper compounds (74,86,87). The investigation by Brown et al. (74) concluded that administration of some of the complexes was more effective than when given orally, and no obvious differences were observed between the activities of copper (I) and copper (II) compounds. Endeavors should focus on achieving transport of copper compounds through the skin in pharmacologically signiﬁcant quantity. To overcome some of the obstacles, as a ﬁrst step in a de minimis approach, small molecular weight amino acids could be investigated for skin diffusivity. Such copper chelates may release the metal to protein binding sites in signiﬁcant amounts. Technical arguments elaborated at this time in favor of transdermal cupriphores indicate that: 1. Regardless of the ligand with which copper reaches the organism, the metal in the initial complex recombines with endogenous proteins and is homeostatically directed to the target locus in the organism where the metal exercises its function. 2. Both Cu(I) and Cu(II) in the exogenous complex are equally effective in reducing inﬂammation, speaking for a common

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(recombinant) transition state of that highly reactive metal within the organism, a characteristic shared by other transition metals. Copper exerts its role as ETE, thanks to that ability of the outer electrons in the 3d and 4s orbital shells, which allow it to ﬂuctuate between the monovalent and the divalent oxidation state. Such facile changes in valence play a decisive role for their transfer across cell membranes and epithelia, including the skin. OUTLOOK Summing up the factors enumerated, the essential groundwork for the transdermal delivery of cupriphores appears to have been laid. An array of technologies for their delivery are available; preliminary results obtained so far in animals and humans warrant pursuit of that form of therapy and make a resurgence in the use of copper as anti-inﬂammatory possible. Albeit dermal diffusion of copper complexes formed by the metal in contact with the skin have been demonstrated (not published), transdermal application in form of patches would appear to be a more efﬁcient method of dosing exogenous copper. Closer deﬁnition of copper complex stability is not necessary, since homeostasis takes control of the ETE in whatever form exogenous copper reaches the organism and converts it to endogenous complexes appropriate for anti-inﬂammatory function. Should cupriphores be adapted as patches for needle-free transdermal delivery, by use of chemical adjuvants, physical preconditioning of the skin, or imbedded in LS, some of the traditional remedies for RA (e.g., gold therapy or copper bracelets) may be replaced by effective and less controversial approaches. Transdermal therapy would bring with it the advantages of lesser immunotoxicity, of selfadministration, avoidance of discomfort, and economy. ABBREVIATIONS A AI CP ETE GI IA IM IV L LS MT NSAIDs

arthritis anti-inﬂammatory ceruloplasmin essential trace element gastrointestinal intra-articular intramuscular intravenous ligand liposomes metallothionein nonsteroidal anti-inﬂammatory drugs

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per os quantitative structure activity relationships rheumatoid arthritis subcutaneous stratum corneum

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Index

Absorption, 22 degree of, 76 dermal, 22 measuring percutaneous, 45 limitations and problems in, 46 radioactivity method for, 48 in vitro method for, 45–47, 52 in vivo methods for, 47–52 of metal compounds, 22, 46 skin, 22 Acute inﬂammation, 162, 163, 166 Acute periodontal disease, 161 Adjuvants, chemical, 283 Alcusal1, 200 Allergic contact dermatitis (ACD), 116, 272 copper, 128 test concentrations for, 123 diagnostic tool for, 120 predictive test for, 126 See also Allergy, delayed-type Allergy delayed-type, 116, 120, 139 immediate-type, 119 Alloys brass, 5 bronze, 5–6 cast, 4

[Alloys] chromium coppers, 5 compositions of, 104 copper, wrought, 4 copper–nickel, 2, 6 nickel–silver, 6 solubility of particulate, 107–108 Alpha-ceruloplasmin, 74 Aluminum bronzes, 5 applications of, 6 characteristics of, 6 American Conference of Governmental and Industrial Hygienists (ACGIH), 98 Amino acids, 11 Analgesic–narcotic principles, 152 Analytical electron microscopy, 69 Angry back syndrome, 131 Anti-inﬂammatory (AI) activity, 68, 167 copper chelates as, 243 topical delivery of, 275 Anti-inﬂammatory drugs, nonsteroidal, 238 Anti-inﬂammatory potentials, 150 Anti-inﬂammatory properties, 150 Antiarthritic agents, 162 application, 244 295

296 [Antiarthritic] copper potential, 159 drugs, 162 Antirheumatic agents, 187 Antiseptic properties, 151 Area under the curve (AUC), of triplicate stripping experiments, 88 Arthritic deformation, 240 Arthritis analgesic products, 255 pain relief, 249 patients with copper jewelry, 82 remedy for, 238 rheumatoid, 238, 250 treatment, 243 Artiﬁcial sweat copper ion in, 13 skin penetration data for, 13 Aspirin, 190, 271 for analgesic activity, 271 an antipyretic, 271 Atomic absorption analysis, 14

Biliary excretion, 279 Blue copper oxidases, 186 Bracelets, 240 copper, 239 therapeutic, 240 weight loss, 247–248 Brass, (red or yellow), 5 Breathable tape, 82 Bronze alloy, 5–6 aluminum, 5 silicon, 5 tin, properties of, 5 Bronze Age, 5

Carrageenan-induced paw edema (CPE), 163, 165 Carrageenan-induced pleurisy (CP), 165 Cast alloys, 4 Ceruloplasmin, 164, 167 concentrations, 172

Index [Ceruloplasmin] in serum, 181 as markers, 172 transcription of, 171 Chemical adjuvants, 283 Chemical irritants, 98 Chlorophyllin copper complex, 102 Cholinergic urticaria reaction, 139 Chondrocytes, sensitivity of, 207 Chromatography, ion exchange, 11 Chromium copper alloys, applications for, 5 Chronic inﬂammation, 162, 168–170, 171 Chronic inﬂammatory agents, 152, 162 Chronic inﬂammatory disease, 238 Chronic inﬂammatory processes, 163 Chronic systemic pathologies, 199 Chrysotherapy, 272 applications of, 282 immune reactions in, 273 Complementary and alternative medicine (CAM), 246 Connective tissue metabolism, 206 Contact urticaria syndrome, 116 Copper absorption, 67, 74, 72, 83, 244, 267 cutaneous, 67, 74 dermal, 255, 270, 283 administration exogenous effects, 184–203 percutaneous, 199–203 routes of, 184–186 subcutaneous and intraperitoneal, 188–191 allergy, 121, 123, 128 alloy applications of, 2 biofouling resistance of, 3 castability of, 3 characteristics of, 2–3 corrosion resistance of, 2 fabricability of, 3 families, 3 friction rates of, 3 wear rates of, 3 ANA 68 alloy, 104 animal models for, 276

Index [Copper] anti-inﬂammatory activity of, 67, 188, 203–216, 268, 275 anti-inﬂammatory effects, 91–92 antimicrobials, 102 as applications, 84–85 articles, corrosion of, 68 baseline values of, 82–84, 88 blood clotting factors, 8 bracelet, 239, 255, 269 myth, study of, 249 weight loss, 13, 269 chelates in rheumatoid arthritis (RA), 278 chemicals, 3 chlorophyllin, 102 comparison of nickel and, 87, 89 complexes, 7–8, 158 compounds, 67 concentration, 172, 181, 182 content of hair and skin, 89 contraceptive effects, 14 corrosion in environment, 8 in physiologic media, 9 cross-reactivity between palladium and, 133 decontamination, 84–85 deﬁciency, 101, 163, 255 alimentary regimen, 181 experimental animals study, 163–170 feeding, 163 organism, 75, 76 dental alloys, released from, 100 in dental materials, 124 depletion, proinﬂammatory effect of, 163 deprivation, 163 derivatives, used as pesticides, 118 dermal irritation by, 111 in dermatology, 118 dermatopharmacokinetics, 75 detection of limits for, 81–82 diffusible compounds, 118 diffusion, 67, 69, 81, 83, 269, 288 diffusivity, 269, 270 electron conﬁguration and reactivity of, 8

297 [Copper] electron microscopy for, 270 electron probe analysis for, 270 electrophilic reactivity of, 271 endogenous, 102 epicutaneous tests for, 135 exogenous, 103, 277 effects of, 91 exposure, 97 types, 125 formation of, 99, 255 and hemolytic anemia, 101 in human organism, 14 human stratum corneum penetration by, 81 hypersensitivity, 115, 132 immune reactions case reports, 138 individual reports, 138 reports of, 115–116 immune response, 125 as immunogen, 117 immunogenic potential of, 123 immunologic contact urticaria due to, 127 in intrauterine devices (IUDs), 100, 115, 125 intravenous administration of, 186–188 ionic, 243 irritation epidemiology of, 100 incidence of, 100 in skin and mucosa, 103 in jewelry, 82, 98 machinability of, 3 mechanical properties of, 3 properties of, 2–4 thermal and electrical conductivity of, 3 types of, 6 wrought, 3 leaching of, 10 LG-1/ANA 68, 105 LM-hard gold alloy, 104 in mammalian organism, 275 in medicine, 243 application, 244

298 [Copper] development of, 240 metabolism disorder, 101 metal dissolution, 82 solubilization, 98 in metallic bonding, 8 metallurgy of, 117 micro/ANA 68, 104 midi/ANA 68, 105 noxious effects on, 91 occlusion, 84–85 oleate, in vivo application of, 118 oral administration of, 191–198 oxidases, blue, 186 oxidation in body ﬂuids, 117 states for, 8 oxidation potential of nickel and, 90 parameters for, 270 patch test material, 133 penetration under occlusion, 87–88 under semiocclusion, 88–89 PGE2 released, 109–110 pharmaceutical, 282 pharmacological activity of, 280 pharmacology of, 155 physiological factors for, 267 plasma, 268 polystyrene, 104 population-based studies, 134–140 powder, 84 predictive immunology test results for, 119 properties of, 1–2, 83, 240 protein, multifunctional, 158 quantitative data for, 102 radioactivity of, 282 removal by sequential tape stripping, 86–87 salicylate gel, 281 salicylate–ethanol complex, 282 scope and limitations of, 270 sensitivity, 126 sensitization potential of, 115 as sensitizer, 117 skin contact with, 83, 269

Index [Copper] skin penetration by, 84, 86, 269 skin reactions to, 82 skin-diffusible, 279 sources of exposure to, 9 status in blood and urine, 170–173 in solid tissues and inﬂamed areas, 173–179 stems, 97 stripping, 84–85 in synthetic sweat, 99 systemic allergic contact dermatitis due to, 129–130 systemic lupus erythematosus disease activity in, 254 T-cell-mediated reactions to, 123 as therapeutic agent, 238 therapeutic uses of, 240 in therapy, 150 threshold limit values applicable to, 98 toxicity, 72 and therapeutic indices of, 91–92 toxicological considerations of, 91 transdermal anti-inﬂammatory action of, 102–103 transdermal therapy by, 278, 279 urticaria, screening procedure, 131 utilization, 267 in vitro assays, 109 in vivo assays agarose overlay test in, 106 comparison of the different tests in, 108 erythrocyte lysis assay of, 107 implantation test of, 103–105 toxicity test using murine macrophages in, 107 Copper metabolism, studies on endogenous, in acute chronic inﬂammation, 170–179, 179–184 Copper sulfate diffusion of, 72 pharmacological use of, 153 Copper-dependent enzymes, 186, 211 lysyl oxidase, 199 role of, 206 Copper–nickel alloys, 6

Index Copper/zinc bracelets, study of, 249 Corneocytes, 23 Corpus hippocraticum, 154 Corrosion-resistant materials, 3 Corticosteroids, 274 CP. See Carrageenan-induced pleurisy CPE. See Carrageenan-induced paw edema Cranial trepanation procedure, 153 Cupric ion, testing of, 119 Cupriphores effective transdermal, 282 subcutaneous administration of, 281 Cutaneous treatment, efﬁcacy of, 199 Cyclo-oxygenase (COX) activities, 205 pathways, 205–206 Cytochrome-c oxidase activity, 183 Cytolysis, degree of, 106

Delayed contact stomatitis, 124 Dental alloy contact dermatitis, 124 Dental materials copper alloys used in, 124 copper sensitivity induced by, 126 Dermal absorption experiments, 22–27, 75 approaches for, 25 descriptors of, 25 percent of, 26 permeability coefﬁcient of, 25–26 Dermatitis dental alloy contact, 124 systemic contact, 117 Dermatome, use of, 44 Dermatopharmacokinetics, 26 Dermatosis, 116 Dermatotoxicology, 26 Dermcusal1, 200 Dermis, 22 Diagnostic equivocation, 133 Diet, semiquantitative assessment of, 248 Dietary copper deﬁciency, effects of, 168 Diffusion characteristics of, 76 in copper, 269, 270 experiments, in vitro, 76

299 [Diffusion] Fick’s law of, 25 Kalia’s equation, 23 membrane analysis, 76 passive, 75 rates of, 68 Dimethyl acetamide, 37 Dimethyl formamide, 37 Dimethyl mercury, 22 Dimethylsulfoxide (DMSO), 37 applications of, 122 skin penetration by, 122 as vehicle, 122 Divalent oxidation state, 68 D-penicillamine, 162 Drug delivery systems, 76 intercellular, 284 percutaneous, 283 ionic, 285 therapy, 271

Elastic connective tissue, 24 Electric resistance, 29 Electrolyte composition, 10 concentration, 11 Electromotive forces and potential, 10 Electron probe analysis, 69 Electron-dense granules, 24 Electrophilic chromic ion [Cr(III)], 39 Electrophilic metals, 31 Electroporation, 286 Electropositivity, 8 Endogenous origin of skin exudates, 33 Energy dispersion X-ray (EDAX) analysis, disadvantage of, 105–106 Enterohepatic circulation, 280 Enzyme, 209 lipolytic, 12 as marker, 183 Epidermis, constitution of, 22, 23 Epithelial cells, 23, 69 Erythrocytes, copper concentration, 181 Erythropoiesis, 203 Escherichia coli, 3

300 Essential trace element (ETE), 67 for biological processes, 294 elimination of, 22 Excited skin syndrome, 133 Exogenous copper administration, effects of, 184–203 Extracellular-matrix macromolecules, 199 Femur fracture, 174 Fertility-related phenomenon, 15 Fibroblast–keratinocyte cocultures, 109 Fick’s law of diffusion, 25 Fluid fraction, 166 Food and Drug Administration (FDA), 242

Gastric irritation, 279 Generic therapeutic remedies, 152 GHK-Cu. See Glycyl-L-histidyl-Llysine-Cu(II) Glomerulonephritis, immune complex of, 273 Glycyl-L-histidyl-L-lysine-Cu(II) (GHK-Cu), 198, 199, 206 Gold, 271 antiarthritic drugs, biological effect of, 272 ring study, 250 therapy anti-inﬂammatory properties for, 272 soluble gold salts for, 272 therapeutic application, 271 Gold sodium thiomalate, 272 intramuscular injection of, 274 pathobiochemical effects of, 274 Gold sulfhydryl compounds, 239 Gold thioglucose, 272 Gold thiosulfate, 272 contact allergy to, 274 modulatory effects on, 273 tolerance for, 274 GPMT on 20 guinea pigs, 119 as predictor of skin sensitization, 119 GyrotoryTM water bath model, 85

Index HaCaT cells, cytotoxic potential in, 110–111 Hair follicles, 33 Hapten–protein conjugate, 120 Haruspecism, deﬁnition of, 152 Hematochemical serum markers, 163 Hemoglobin, synthesis of, 157 Histamine activity, 203–205 release, 203–205 Histopathology, 105 Homeostasis, 42, 74 Homeostatic mechanisms, 74, 76 Human abdominal epidermis, 72 inﬂammatory process, 171 plasma or serum, 118 rheumatoid arthritis, 158, 184 serum albumin (HAS), 120 skeleton, 156 skin dermatoglyphics, 83 skin features, 12 stratum corneum penetration by copper, 81 metal analysis of, 85 statistical analysis of, 85 Hypercupremia, 172 Hypersensitivity asymptomatic contact, 124 copper, 115 copper allergic, 121 population studies of, 132 diagnostic tests for, 119 immediate allergic, 120 open test for, 119 radioallergosorbent test (RAST) for, 120

ICAMs. See Intercellular adhesion molecules ICU. See Immunologic contact urticaria IgE antibodies, 120 production of, 124 Immune system development and reactivity, 212–216

Index Immunologic contact stomatitis, 124 Immunologic contact urticaria (ICU), 115–116, 119 due to copper, 127 See also Allergy, immediate-type Immunosuppressants, 274 In vivo methods, 47–51 advantages of, 49 approaches for, 48 conclusions of, 50 semiquantitative, 50 skin stripping in, 50 Inductively coupled plasma-mass spectrometry, 69 Inductively coupled plasma–mass spectroscopy (ICP-MS) analysis, 85 Inﬂammatory arthropathies, 252 Inﬂammatory reaction, 165, 207 Intercellular adhesion molecules (ICAMs), 211, 212 Intrathoracic injection, 165 Intrauterine devices (IUDs), 98 Ion, cupric, testing of, 119 Ion exchange chromatography, 11 Ionic copper, release of, 243 Ionic drugs, 285 Iontophoresis, 285 Irritant contact dermatitis (ICD), incidence of, 101 Irritant chemicals, 98

Kalia’s diffusion equation, 23 Keratinocytes, 23

Lactic dehydrogenase (LDH), 107 Leukocyte activity, 207–212 migration, 166, 207–212 Lipid bilayers, intercellular, 283 Lipolytic enzymes, 12 Lipophilic compounds, 28 Lipophilic formulations, 282 properties of, 284

301 Liposomes (LS), 284 biological particulate materials in, 284 oxidation, 76 particle size of, 284 Local lymph node assay (LLNA), cuprous chloride in, 119

Mammalian sweat, 11 Membrane transport, 8 Membrane-bounded organelles, 24 Menkes’ syndrome, 101–102 Metabolism, of connective tissue, 206 Metal(s) corrosion rates between two, 90 oxidation of, 89 Metal detection, 53–56 analytical methods for, 53 atomic absorption spectrophotometry (AAS) for, 54–55 electron spin resonance (ESR) for, 55 inductively coupled plasma–atomic emission spectroscopy (ICP-AES) technique for, 53 inductively coupled plasma–mass spectroscopy (ICP-MS) technique for, 50, 53–54 particle-induced X-ray emission for, 56 stable-isotope inductively coupled plasma–mass spectroscopy technique for, 54 Metal surfaces, sebum and sweat in, 36 Metal–ligand equilibria, computer simulation of, 279 Metallo-element–deﬁcient animals, 163 Metallothionein, 32 Micronutrients, physiological dynamics of, 75 Microparticle-induced X-ray emission (PIXE), 29 Mononuclear leukocytes, activity of, 207 Musculoskeletal disorders, localized, 280 Mycobacterium butyricum, 168

302 Nephrotic syndrome, 273 Neurotoxic antimicrobial hexachlorophene (1–3), 22 Nickel cross-reactivity between copper and, 133 cross-reactivity between palladium and, 133 dermatopharmacokinetics of, 50 epicutaneous tests for, 135 ion-speciﬁc T-cell clones, 133 reactivity of, 133 Nickel ion–speciﬁc T-cell clones, types of reactivity of, 133 Nickel–silver, 2 alloys, 6 Nitric oxide synthase activity, regulation of, 206 Nonarthritic copper deﬁciency, 170 Nonsteroidal anti-inﬂammatory drugs (NSAIDs), 187, 188, 191, 200, 238, 268 piroxicam, 210 NSAIDs. See Nonsteroidal antiinﬂammatory drugs

Optimal ﬂux, 280 Orbital shells, 68 Osteoarthritis, 249

Patch testing copper sulfate under, 121 for delayed-type allergy, 120–121 detection of allergens by, 121 diagnostic, 37 optimal solvent in suspected delayed-type copper allergy, 123, 126 positive reactions, 90 role of vehicle in, 121–122 Periodontal disease, 158 acute, 161 Perkin-Elmer 4300 dual view ICP optical emission spectrometer, 85

Index Permeability coefﬁcients, measurement of, 72 Permeation protocols, in vitro, 280 Petrolatum, 122–123 Pharmacokinetics, 281 principles, 47 Pharmacological therapy, 159 Phorbol miristate acetate (PMA), 209 Physiotherapy, 275 Pilosebaceous glands, 12 Placebo effect, 247 Plasma copper concentration, 172 Plasma glycoprotein, multifunctional endogenous, 267 Plasma-membrane barrier, 68 PMNLs. See Polymorphonuclear (neutrophils) and mononuclear leukocytes Polymorphonuclear (neutrophils), activity of, 207 Polymorphonuclear (neutrophils) and mononuclear leukocytes (PMNLs), 207, 209 migration, 210–211 phagocytosis, 207 phagosomes, 208 respiratory burst, 209 Polypropylene tape, 85 Proinﬂammatory effect, 163 Prostaglandin E2 (PGE2), 109 Prostaglandin-synthetase activity, 169 Provocative use test (PUT), 126 Pseudoscience, survey of, 243

Quantitative cutaneous diffusion rates, 67 Quantitative structure–activity relationships, 28

RA. See Rheumatoid arthritis Radioactivity, 26 Radioallergosorbent test (RAST) inhibition test, 120

Index Reactive oxygen species (ROS)–induced reactions, 209 Red blood cell, copper level, 181 Repeat open application test (ROAT), 126 Rheumatic patients, 243 articular erosion in, 250 Rheumatoid arthritis (RA), 76, 171, 179–184, 268 autoimmune disease, 268 chronic syndrome, 268 copper chelates in, 278 inﬂammatory disease, 268 patients, 161 spontaneous remission of, 162 symmetric inﬂammation of, 268 therapy of, 72 traditional and modern therapies for, 268 treatment of, 238, 250 ROS. See Reactive oxygen species– induced reactions ‘‘Rusters’’, 11, 34

Sebaceous glands, 12 Sebum, classes of, 12, 36 Serum copper levels, determination of, 167 Silicon bronzes, 5 Skin absorption, 22 by different sites of the body, 42 methods for measuring percutaneous, 45 afﬂictions, 243 age of, 42 allergic reactions in, 39 arsenic in, 45 aspects of metals, 23 chemical microenvironment on, 9 chromium applied on, 44 compounds formed by metals in contact with, 31–35 diffusion of metal compounds, 35

303 [Skin] nonelectrolytes, 28 paths of, 28 discoloration of, 44 electrolytes across, 29–31 principal conditions of, 29 electron micrographs of, 14 function as diffusion barrier, 23 gold in contact with, 90 immune reactions to chemicals in, 116 as membrane, 22 metabolism (red/ox), 44 nickel as powder on, 50 in contact with, 90 organic compounds, 28–29 penetration, 21 by DMSO, 122 by nickel chloride, 43 by xenobiotics, 22, 33 permeant categories of, 28 pH effect on, 39 reactions to copper, 82–83 sensitization, 119 structure of, 23 Surface, formation of free acids on, 99 sweat and sebum, 9, 90 in vitro, in function of polarity, 38 Skin-diffusible derivatives, 90 Skin-diffusible oxidation products, 32 Skin diffusion endogenous factors for, 41 age of skin as, 42 homeostatic control mechanisms, 43–43 regional variation, 41 exogenous factors for, 35 counterion and molecular volume, 37 dose, 35–36 nature of chemical bond, 38 solubility, 39 time postapplication, 41 valence, 39 vehicle as, 36–37

304 [Skin diffusion] polarity determinates for, 38 variables determining, 35 Skin diffusivity rates, 67 Skin prick device, sterile, 120 Skin prick test (SPT), 119 for immediate-type allergy, 119–120 positives, 120 Solubility, in polar media, 280 Sonophoresis, 286 Sorenson’s hypothesis, soundness of, 191 Staphylococcus aureus, methicillinresistant, 3 Steady-state transport rate, 14 Steroid therapy, 274 Stomatitis delayed contact, 124 immunologic contact, 124 systemic anaphylactic, 124 Stratum corneum (SC), 23, 98 absorption of copper into, 91 barrier, 286 components of, 24 copper diffusion through, 81, 83, 89 depots in, 90, 40–41 intercellular lipid domains in, 30 occlusion of, 84 protein reactivity, 40–41 stripping, 83 swelling of basal, 116, 122 Student t-test, 85 Superoxide dismutase, 243 anti-inﬂammatory copper-dependent metalloenzymes, 274 Superoxide metabolism, regulation of, 168 Sweat components, 10, 33 duct, 29 glands, 33 mammalian, 11 mean body, 11 mean levels of, 33 proteins and amino acids in, 34 Sweating rate, 12

Index Syndrome contact urticaria, 116 nephrotic, 273 Systemic anaphylactic stomatitis, 124 Systemic contact dermatitis, 117, 129–130

Talcum powder, formulation of, 22 Therapeutic agent, dissipation of, 281 Therapeutic properties of copper, 150 Therapy drug, 271 effectiveness of topical, 249 gold, 271 pharmacological, 159 steroid, 274 Thermal stimulation, 12 Thermogenic ointments, 275 Threshold limit values (TLV), 98 Throat inﬂammation, 155 Tin bronzes, 5 Topical therapy, 279 Total serum copper, increase of, 171 Transdermal absorption, 285 applications, 288 Transepidermal water loss (TEWL), 23 Transition metals in biological systems, 8 characteristic for, 8 TransporeTM tape, 85 Tuberculosis and syphilis, infection of, 157

Ulcerogenic effect, 276

VAP-1. See Vascular adhesion protein-1 Vascular adhesion molecule-1 (VCAM-1), 211 Vascular adhesion protein-1 (VAP-1), 211, 212 VCAM-1. See Vascular adhesion molecule-1

Index Wilson’s disease (WD), 101, 162 Wilson’s disease protein (WND), 185 Wounds, treatment of postoperative, 151 Wrought alloys, 4 Wrought copper alloys, 3

305 Xenobiotics, Fick’s law applied to, 22, 25

Zinc absorption, measurement of, 75