ATLA 28, 119–131, 2000 119Biomarkers as Predictive Tools in ToxicityTestingThe Report and Recommendations of ECVAM Workshop 40 1,2Diane J. Benford,3 A. Bryan Hanley,4 Krys Bottrill,5 Sarah Oehlschlager,4Michael Balls, 6 Francesco Branca,7 Jean Jaques Castegnaro,8 JaquesDescotes, 9 Kari Hemminiki,10 David Lindsay11 and Benoit Schilter123School of Biological Science, University of Surrey, Guildford, Surrey GU2 5XH, UK;4Central Science Laboratory, Sand Hutton, York Y041 1LZ, UK; 5FRAME, Russell andBurch House, 96–98 North Sherwood Street, Nottingham NG1 4EE, UK; 6ECVAM, Institutefor Health & Consumer Protection, Joint Research Centre, European Commission, 21020Ispra (VA), Italy; 7Unitadi Nutrizione Umana, Instituto Nazionale della Nutrizione, Rome,Italy; 8Unit of Gene Environment Interactions, IARC, 150 Cours Albert Thomas, 69008Lyon, France; 9Department of Pharmacology, Medical Toxicology and Medicine, INSERM U98-X, Faculté de Médecine Lyon RTH Laennec, 69372 Lyon Cedex 98, France; 10Departmentof Molecular Epidemiology, Centre of Nutritional Toxicology, Karolinska Institute, Novum,141 57 Huddinge, Sweden; 11Euro Science Perspectives Ltd, 3 Patcham Grange, BrightonBN1 8UR, UK; 12Nestle Research Safety Centre, Verschezles Blanc, 1000 Lausanne 26,Switzerland.Preface tion of alternative tests into regulatory pro- cedures. It was decided that this would beThis is the report of the fortieth of a series of best achieved by the organisation of ECVAMworkshops organised by the European Cen- workshops on specific topics, at which smalltre for the Validation of Alternative Methods groups of invited experts would review the(ECVAM). ECVAM’s main goal, as defined in current status of various types of in vitro1993 by its Scientific Advisory Committee, is tests and their potential uses, and make rec-to promote the scientific and regulatory ommendations about the best ways forwardacceptance of alternative methods which are (1). In addition, other topics relevant to theof importance to the biosciences and which Three Rs concept of alternatives to animalreduce, refine or replace the use of labora- experiments have been considered in severaltory animals. One of the first priorities set by ECVAM workshops.ECVAM was the implementation of proce- This ECVAM workshop on Biomarkers asdures which would enable it to become well- Predictive Tools in Toxicity Testing was heldinformed about the state-of-the-art of in Burnham Market (Norfolk, UK) on 5–8non-animal test development and validation, October 1998, under the co-chairmanship ofand the potential for the possible incorpora- Diane Benford (University of Surrey, UK)Address for correspondence: Diane Benford, School of Biological Science, University of Surrey, Guildford, Sur-rey GU2 5XH, UK.Address for reprints: ECVAM, TP 580, JRC Institute for Health & Consumer Protection, 21020 Ispra (Va), Italy.1European Centre for the Validation of Alternative Methods. 2This document represents the agreed report of theparticipants as individual scientists.
120 D.J. Benford et al.and Bryan Hanley (Central Science Labora- system that can be related to an expo-tory, UK). The participants, whose expertise sure to, or effect from, a specific xenobi-included toxicology, biomarker research, the otic or type of toxic material.” (4)Three Rs and methods of validation, came 4. “Biomarkers, broadly defined, are indi-from industry, universities and other cators of variation in cellular or bio-research institutions. The aim of the work- chemical components or processes,shop was to provide a forum for discussing structures, or functions, that are mea-the utility of biomarkers as predictive tools surable in biological systems or sam-for toxicity testing. A major focus of theworkshop was to examine the possibilities ples.” (5)afforded by the use of biomarkers for the 5. A biomarker is “an indicator that sig-reduction, refinement or replacement of ani- nals events in biological systems or sam-mal use in toxicity testing. This report sum- ples, and it is generally taken to be anymarises the outcome of the discussions and biochemical, genetic, or immunologicalincludes a number of conclusions and recom- indicator that can be measured in a bio-mendations. logical specimen.” (6)Introduction 6. “The term biomarker has been used toThe term biomarker has grown in popularity describe measurements in the sequencein recent years. With this growth has come a of events leading from exposure . . . togreat increase in applications and a corre- disease. At each step, persons may differspondingly increased diffuseness in the in susceptibility, thus a biomarker maymeaning of the term, to the extent that one also refer to an indicator of susceptibil-of the major difficulties in the area of bio- ity.” (7)marker research is reconciling differing 7. “A biomarker is a xenobioticallyviews on what constitutes an acceptable def- induced variation in cellular or bio-inition. This is reflected in the large number chemical components or processes,of definitions appearing in the literature, a structures, or functions, that is measur-selection of which are given below in chrono- able in a biological system or sample.”logical order. This is not designed to be (J.F. McCarthy, R.S. Halbrook & L.R.either comprehensive or a critically evalu- Shugart, 1991, Conceptual strategy forated collection, but it does represent the design, implementation and validationrange of uses to which biomarkers are put of a biomarker-based biomonitoringand the justifications for the use of the word capability. Internal document of thein different applications. They range from Environmental Sciences Division of theexposure measures, and biological indices US EPA, used as the discussion docu-which support a mechanistic postulate, to ment for the NATO Advanced Researchclinical markers with diagnostic implica- Workshop on Strategy for Biomarkertions. Research and Application in the Assess-1. “Biological markers are indicators sig- ment of Environmental Health). nalling events in biological systems or 8. “. . . the biomarkers are any of a series samples”, and “Biological markers are of biochemical or molecular responses measurements of body fluids, cells or to compounds that have entered an tissues that indicate in biochemical or organism, reached sites of toxic action, cellular terms the presence and magni- and are exerting an effect on the organ- tude of toxicants or of host responses.” ism.” (Proposal document for the (2) NATO Advanced Research Workshop2. Biomarkers are “cellular, biochemical, on Strategy for Biomarker Research or molecular alterations which are mea - and Application in the Assessment of surable in biological media such as Environmental Health, Texel, NL, May human tissues, cells or fluids.” (3) 1991)3. “The term biomarker refers to the use 9. Biomarkers are “parameters that puta- made of a piece of information, rather tively represent some step along the than to a specific type of information. A causal pathway between exposure and biomarker is a change in a biological effect.” (8)
ECVAM Workshop 40: biomarkers 12110. Biomarkers are “markers of biologic It is apparent from the breadth of these def- activity that reflect not evidence of, but initions that any use of the term “bio- the potential for, neoplastic progression marker” must also include a definition of . . . Unlike tumour markers, which are what is meant by it within the context of a biological indicators found in neo- specific discussion. This workshop high- plasms, prevention biomarkers are lighted the great difficulties associated with specifically related to earlier stages of the imprecise use of this word. A realistic carcinogenesis. These intermediate end- appraisal of the biomarker area is required points can be defined as measurements in order to clarify between scientists and of a particular biologic factor associated clinicians the different ways in which the with the evolution of neoplasia and term may be used. occurring with increased frequency in abnormal cells.” (9) Genesis/Taxonomy of Biomarkers11. “The term biomarker is used in a broad sense to include almost any measure- The term biomarker has a significant and ment reflecting an interaction between lengthy history. The meaning for which it is a biological system and an environmen- used clearly depends upon the context and tal agent, which may be chemical, phys- this is reflected most clearly in the parame- ical or biological.” (10) ters of the database which is used as the12. “A biomarker is a measurable event basis for any search. Since the topic of this occurring in a biological system, such workshop was the use of biomarkers as pre- as the human body. In environmental dictive tools in toxicity testing, the focus was epidemiology, a biomarker represents on biological, medical and toxicological uses a sub-clinical and reversible change; it of the term. Therefore, an appropriate data- is not a diagnostic test, but an indica- base set was likely to be found in Medline tor that an early change has occurred and Toxline, so these databases were that could later lead to clinical dis- searched for the term “biomarker”, with the ease.” (11) following results:13. A biomarker is “a measurement made 1966–1975: Only one record, which related on body tissue, body fluid or excretion to the measurement of porphyrin in soil sam- to give a quantitative indication of expo- ples (16). sure to a chemical and which may give 1976–1985: 51 records, of which the majority an estimate of the risks consequent on were concerned with the use of tumour bio- the exposure.” (12) markers to diagnose cancer, obtain a progno-14. “Biomarkers are observable endpoints sis or monitor therapy. Other topics included in a continuum of events leading from ageing, monitoring wildlife rabies vaccina- exposure to toxic agents to diseases that tion, rat pituitary enzymes, ecotoxicology, ultimately result from exposure.” (13) assessment of toxic exposure, and the molec- ular epidemiology of cancer.15. “The term biomarker is used in a broad sense to describe parameters reflecting 1986–1990: 308 records, in which the major an interaction between a biological sys- subjects were the use of tumour biomarkers tem and a potential hazard of chemical, for diagnosis, prognosis, monitoring of ther- biological and physical nature. The apy and chemoprevention programmes, measured response may be functional, assessment of toxic effects, and ecotoxicol- physiological and biochemical at a cellu- ogy. Other topics included ageing, assess- lar or molecular level.” (14) ment of toxic exposure, use of biomarkers in risk assessment, assessment of susceptibility16. “A biomarker is a parameter which can to toxic insult, monitoring wildlife rabies be evaluated quantitatively, semi-quan- vaccination, and studies on other diseases. titatively or qualitatively, and which provides information on exposure to a 1991–1995: 3579 records, which included the xenobiotic, or on the actual or potential first references to validation (17, 18). The sub- effects of that exposure in an individual jects covered included all those mentioned or in a group.” (15) above, but with far less emphasis on ageing.
122 D.J. Benford et al.There was increased use of biomarkers for the ageing. These investigations have little over-monitoring of chemoprevention programmes, all relevance to classical toxicity testing,the monitoring of specific groups of workers though they may contribute to the assess-for exposure, and the assessment of suscepti- ment of cumulative toxic insult in humans, ifbility. There was a new subject for discussion, ageing is the result of a predominantlynamely, the ethical aspects associated with exogenous process.the biomonitoring of individuals, especially In the period 1986–1990, the total numberwith respect to their susceptibility to disease. of references to biomarkers increased sub-Other new topics included the use of bio- stantially, particularly in relation to toxicol-markers in epidemiology and in the assess- ogy. There was a gradual increase in studiesment of health risks within the general on biomarkers of occupational exposure,population, the use of biomarkers in studies which is likely to reflect a change in termi-on the mechanisms of carcinogenesis, and the nology from “biological monitoring” to “bio-identification of biomarkers of dietary intake. markers”, as well as an actual increase inThere was more theoretical consideration of research. The primary use of biomarkers ofmethods to be used in the analysis of bio- exposure is to establish the adequacy of con-marker data, possible sources of bias, and the trol measures with respect to chemical expo-validation of biomarkers. sure in the workplace, i.e. occupational exposure. They are not intended to be pre-1996–November 1998: 3107 records covering dictive of an adverse effect, but since occupa-topics very similar to those of the previous tional exposure limits are intended to protectperiod. the workforce from a known effect, infringe-This breakdown of the uses of the term ment of the control measures clearly has theshows a clear progression in the ideas associ- potential to cause harm. (Studies relating toated with biomarkers. The earliest reference smoking aimed to investigate specific causalgiven above refers to the measurement of a agents and mechanisms of carcinogenicity.)biological compound (porphyrin) in a non- A further area of development was that ofbiological matrix (shale), and as such, has no biomarkers of effect. These are parameterstoxicological relevance. that change in response to exposure and Between 1976 and 1985, many of the stud- reflect a biological consequence to that expo-ies in which the term biomarker was used sure. For the first time, some studies whichwere concerned with tumour biomarkers as related biomarkers to risk assessment werediagnostic/prognostic tools. This use of the carried out.term remains current. Most of these diag- In the period 1991–1995, a number of def-nostic biomarkers are concerned with indi- initions of biomarkers were published, five ofcating current status and measuring the which are listed above (11–15). A number ofeffectiveness of treatment. Such biomarkers papers referred to biomarkers as a means ofare not strictly predictive of outcome, monitoring the effects of interventions. Thisalthough they may contribute to an overall usage is based on the hypothesis that a par-prognosis given by the physician. ticular exposure is associated with a particu- A second use of the term biomarker in this lar disease. Intervention methods may beperiod was in the detection of exposure to used to modify the exposure under thetoxic agents. In some cases, biomarkers were assumption that this will reduce the risk ofmeasured in humans. But a major usage at that disease. Biomarkers of exposure arethis time was related to the use of biological used to verify that the intervention has thesystems as bioindicators of environmental anticipated effect on the internal exposure;pollutants. For the purposes of such studies, biomarkers of effect, if available, may give anthe most biologically relevant systems were earlier indication of whether the interven-those which were most sensitive in relation tion is likely to result in reduced incidence ofto the pollutants of interest. This type of the disease. If such relationships are estab-study is not intended to be predictive of lished, this helps to strengthen the evidenceeffects in other species, but is indicative of of causality. Because of their increasingexposure that may represent a potential haz- availability and sensitivity, biomarkers areard to our species. increasingly being used to explore the links The final area of active research in this between exposure and effect in order toperiod was the development of biomarkers of establish causality. Because biomarkers can
ECVAM Workshop 40: biomarkers 123measure internal exposure in an individual, animal use in toxicology studies. In particu-the importance of individual susceptibility lar, the ultimate aim of toxicology is to eval-and vulnerability become more clearly uate the potential risk to humans, where thedefined. Biomarkers that reflect these indi- risk is a function of exposure as well as thevidual differences are referred to as bio- hazard.markers of susceptibility; they need not Biomarkers of exposure could be predic-relate to specific exposures. In addition, tive of risk, if sufficient information weresome awareness of the ethical issues involved available on the dose-response relationship.in biomarker research and monitoring popu- Biomarkers of effect cover a range of mea-lations began to emerge. surements which may be indicative of expo- In the most recent period (1996–Novem- sure to a particular agent, although theber 1998), the numbers of biomarker-related specificity is generally less than for biomark-records in Medline and Toxline increased ers of exposure. They may also show correla-dramatically, although the topics being dealt tions with possible clinical outcomes, butwith were broadly similar to those noted in cannot be assumed to be predictive unlessthe previous period. they are shown to reflect a significant step In conclusion, work on biomarkers over along the causal pathway between exposurethe past 30 years or so has progressed, such and effect. The extreme example of a bio-that, currently, a range of definitions have marker that reflects a current clinical condi-been adopted, and this diversity is repre- tion, is actually a diagnostic marker and,sented in Figure 1. However the most com- because it is rarely related to a chemicalmonly used terms are biomarkers of exposure, it does not meet the above defini-exposure, biomarkers of effect and biomark- tion of a biomarker. Biomarkers of suscepti-ers of susceptibility. bility most notably include genetic For the context of this report, we will susceptibility. The concept of susceptibilitydefine a biomarker as “a parameter which will not be dealt with in this report per se,can be measured in a biological sample, and but it must be recognised that individual sus-which provides information on an exposure, ceptibility and population susceptibility areor on the actual or potential effects of that important issues, which should be taken intoexposure in an individual or in a group”. account and may be appropriate for a future This report aims to consider the use of bio- workshop, once the establishment of a coher-markers as predictive tools in toxicity test- ent meaning to the use of biomarkers in pre-ing, so we must also consider what is meant dictive toxicology has been better defined.by predictivity. The purpose of regulatory It is important to distinguish between usetoxicity testing is to define the inherent haz- of biomarkers in toxicological studies inardous properties of a substance. Previous experimental animals and in humans. Tradi-ECVAM workshops have considered the tional toxicological endpoints are generallyvalue of in vitro test systems for the predic- empirical observations of the consequencestion of various hazardous properties, and of exposure to specific agents. However, pro-important elements of the discussion include vided that a study is conducted under strictlythe required level of accuracy of predictivity, controlled conditions, with appropriate sta-in terms of false negatives and false posi- tistical analysis, it is reasonable to assumetives, and how to achieve acceptance as that the observed effects were caused by thereplacement tests for regulatory toxicity exposure under investigation. Exposure totesting. The point of reference is usually the the animal is well defined but, nevertheless,results produced by in vivo animal toxicity there may be requirements for internal expo-studies; comparison with human data would sure to be verified by toxicokinetic measure-be preferable, but sufficient human data of ments, such as blood levels of test material,sufficiently high quality are rarely available. in guidelines for some types of toxicity test.Clearly, the uses of biomarkers listed above These are not normally considered to be bio-do not include prediction of hazardous prop- marker studies, although they clearly meeterties or of “toxicity”. We therefore need to many of the definitions of a biomarker. Bio-consider the broader aspects of toxicology markers of effect (although again not termed(rather than toxicity testing) and the value of as such) are frequently included in a batterybiomarkers as predictive tools that might of clinical chemistry assays on blood andalso allow for refinement and reduction of liver, and possibly other tissues, depending
124 D.J. Benford et al.upon the nature of the effect under investi- assay is chromosomal damage, this could begation. In contrast, human studies, even considered to be the use of a biomarker of (aapparently well controlled volunteer studies, secondary) effect as a biomarker of exposure.involve many other variables and inter-indi- In principle, biomarkers of effect could bevidual differences that mean effects cannot valuable tools in toxicity testing protocols.be so readily ascribed to the exposure under Biomarkers that develop early in the courseinvestigation. With investigations on “free- of repeat exposure, but that can be reliablyliving” human populations, the potential for correlated to the subsequent development ofconfounding factors increases enormously. It a lesion or an effect, would allow animal test-therefore becomes essential to establish the ing protocols to be refined and animal testingspecificity of a “biomarker” for the exposure to be reduced. However, the mechanistic linkand/or effect of interest. For a biomarker of to the lesion needs to be understood in orderexposure, this relates to the measurement to define the uses and limitations of a partic-techniques and the toxicokinetics of the sub- ular biomarker. At the current time, there isstance but, for a biomarker of effect, it must insufficient understanding of mechanisms ofbe based on understanding of the mechanism toxicity to permit the identification of mean-of action. This leads to two key questions. ingful biomarkers for inclusion in routine1. What are the current status and future toxicity testing protocols. For a very small prospects for the use of biomarker number of compounds, some associations are methodology in toxicity testing? known, such as liver glutathione content as an indicator of paracetamol hepatotoxicity2. What are the prospects for use of bio- (19), but these cannot be used in a general markers leading to refinement, reduction sense. Some of the “biomarkers” now used in and replacement of animal procedures in clinical chemistry, such as serum levels of toxicology? liver transaminases, are more appropriatelyAnswers to these questions form the basis of termed “organ function tests”, and arguablythe rest of this report. should be defined as diagnostic markers rather than as biomarkers. Nevertheless, they are valuable because they demonstrateBiomarkers in Toxicity Testing an important link between in vivo and in vitro testing, i.e. that the same endpoints canToxicity tests are designed to identify the be used.hazardous properties of a chemical sub- Biomarkers have the potential either to bestance, tested in an isolated form in labora- used as indicators of events at various stagestory animals. Because they are primarily in the progression of a toxic lesion, or to pro-intended for the testing of novel substances, vide tools to test the relevance of proposedthe protocols have to be generic and cannot toxic mechanisms. The extent to which a bio-be related to specific mechanisms of action. marker is a predictive tool depends on itsThere is little opportunity for biomarkers of connection to the causal pathway leadingexposure to be included in such studies. As from exposure to effect, and on the quantita-noted above, there are some exceptions. One tive assessment of the consequences ofexample is the requirement for plasma levels changes in the biomarker in terms of toxico-of compound in carcinogenicity studies of logical endpoints.Our current understandingpharmaceutical agents, which are compared of mechanisms of toxicity is generally notwith data on human plasma levels at the sufficient to support development of bio-therapeutic dose, in order to demonstrate markers that could be early predictors ofthat sufficiently high doses of drug have been toxic effect. However, as our understandingtested. A second example is the mouse bone increases, and with the enormous growth inmarrow micronucleus test, for which a nega- the use of new techniques such as genomicstive result requires evidence that the com- and proteomics, which seek to determine thepound is available to the bone marrow, link between gene expression, gene function,either by specific chemical analysis (bio- protein synthesis and protein function andmarker of exposure), or by changes in the exposure to environmental and chemicalratio of normal to polychromatic erythro- agents, there is the potential to develop suchcytes. Because this reflects toxicity to the predictive biomarkers in the future. The suc-bone marrow, whereas the endpoint of the cess of a genomics/proteomics based
ECVAM Workshop 40: biomarkers 125approach to biomarkers relies on the use of enzymes at much lower (and probably moreintelligent data manipulation, and on corre- physiologically relevant) levels. At normallation between measurable changes in the consumption levels, the benefits of the latterfunctional properties of biological macromol- effect may outweigh the risks associatedecules and the properties of the cell, organ or with the former; however, until directly com-organism. parable human studies are carried out, it is In addition to traditional toxicity tests, not possible to make a net toxicity/benefitpredictive models are available for risk prediction. The dose responsiveness of bioac-assessment, ranging from structure/activity tive constituents, the way that a biomarkermodels (i.e. QSAR) and knowledge based sys- can reflect this, and the choice of appropriatetems (for example, DEREK) to in vitro test systems, are clearly keys to establishingassays. The relevance, reliability and overall the validity of biomarkers as predictive tools.value of any predictive model system is cru-cially dependent on the quality of the data-base used in its development and the method Exposure and Effectused for assessing the reliability of variouspieces of information. It is possible that any The relationship between exposure andbiomarker which has been shown to fit into effect is crucial to any consideration of thean established mechanistic pathway could risk associated with a given chemical. Whenprovide information which could be used by considering experimental toxicology, it hasan established predictive model. Such predic- been assumed that there is a linear progres-tive systems should also be capable of assess- sion from exposure to effect, with the likeli-ing the effects of substances which may hood of increasing severity of toxicity withreduce or limit toxicity (in dietary terms, the increasing exposure. Therefore, it is possibleconcept of “protective factors”). to envisage a number of biomarkers indica- Clearly, the expression of a toxic effect is tive of the magnitude of exposure/responsedetermined by the magnitude of exposure, and/or the progression along the pathwayi.e. the dose. There are a growing number of (Figure 2).examples where the validity of extrapolating The control exerted over the process in thefrom high doses to low doses can be ques- laboratory helps to assure of the linktioned, especially where this extrapolation is between exposure and effect. In contrast,not based on detailed knowledge of the mech- when considering the majority of humananisms of toxicity. (However, the concepts of health problems, it is possible to identify aminimum effective dose and biological differ- large number of potential exposures and aences between high and low dose scenarios large number of diseases for a given popula-must also be included. The dose level at tion, but the links between them are poorlywhich a biomarker is measured in response defined, if at all. If, on the basis of knowledgeto a chemical challenge must be realistic in of mechanisms of toxicity, biomarkers can beterms of “normal” human exposure. There is developed that match those defined in Figurelittle purpose in developing effective bio- 2, there is the potential to:markers (in terms of their relevance to a 1. unravel the links between exposure andtoxic endpoint), if they are of little relevance effect, i.e. establish causality;to quantifiable measurements at humanexposure levels, or cannot be measured at 2. use biomarkers of internal exposure andthese levels. In addition, it is important to be early biomarkers of effect to determineable to compare conflicting data on the the efficacy of interventionary measures;effects of compounds by working in similar andtest systems. For example, consumption of 3. use biomarkers of effect as predictiveover-boiled coffee has been found to increase tools in investigating agents that mayblood cholesterol (20) in a human study; exert a similar toxic effect.however, the level of exposure was higherthan would normally be encountered outside Biomarkers of exposure can be defineda testing regime. The same test material has strictly by chemical analysis. The simplestbeen demonstrated to have a chemoprotec- forms involve measurement either of thetive effect against genotoxicity (21) in a non- agent in question, or of a metabolite; how-human test system by stimulating phase 2 ever, they must be specific to the exposure of
ECVAM Workshop 40: biomarkers 127Figure 2: The pathway from exposure to overt clinical effect Exposure Internal Effective exposure Dose Cumulative exposure Early marker of effect Late marker of effect Overt Diagnostic toxicity markersinterest. For example, hippuric acid has been half-life of lead in the blood is 36 days, and itused as a urinary biomarker of exposure to is therefore most useful as a measure oftoluene. While hippuric acid is a metabolite recent exposure (24). Estimates of cumula-of toluene, and therefore could be expected tive lead exposure can be made by measuringto reflect exposure in a coherent way, it is bone lead, where the half-life of lead in corti-non-specific, because it may also be derived cal bone is more than ten years, while that infrom a number of food constituents (22). The the more metabolically active trabecularvalue of urinary hippuric acid as a biomarker bone is less. However, although techniquestherefore depends upon the relative sources that permit in vivo measurement of lead infrom dietary or occupational origins in a par- bone are available, they are not suitable forticular individual. In contrast, the presence routine monitoring purposes. A further com-of the urinary metabolite, equol, is com- plication is introduced by the fact that bonepletely specific for the ingestion of lead can be mobilised in various circum-isoflavones. However, some individuals are stances, such as pregnancy, and may there-unable to produce this metabolite, so its fore be a significant contributor to blood leadabsence in urine may not be reflective of levels (24). Urinary lead levels may also benon-exposure (23). Therefore, knowledge of used as a marker of recent exposure, but arethe toxicokinetics of the agent is required, in subject to considerable inter-individual vari-order to determine the most appropriate ation.metabolite, tissue, and sampling times, and Once considerations go beyond a directthe relevance of the results obtained. In measurement of chemical exposure, theysome instances, it may be possible to estab- begin to include an assessment of effect andlish different biomarkers as measures of lose specificity for the chemical in question.recent and cumulative exposure. An example For example, other biomarkers that haveof this can be seen in studies on lead. The been used in measuring lead exposure
128 D.J. Benford et al.involve the monitoring of effects upon the in investigations of causal links and intohaem synthesis pathway. This is based on mechanisms of toxicity. Once causality hasthe known haematotoxicity of lead, which is been established, a biomarker should fulfilmediated via the inhibition of enzymes of the criteria discussed below, if it is to behaem synthesis, leading to the accumulation used in a predictive sense, whether in exper-of the intermediates δ-aminolaevulinic acid imental toxicology, or in human populations.(ALA), zinc protoporphyrin (ZPP) and copro-porphorin (24). These biomarkers of effectare not specific to lead exposure, as they may Validation Issuesbe influenced by other haematotoxic agentsor by nutritional or sub-clinical disease sta- The validation of biomarkers is not directlytus. In addition, they are not connected to comparable to validation of in vitro tests, butthe disease endpoints now considered to be must follow the basic principle of demon-of most concern (because haematoxicity strating reproducibility, reliability and fit-occurs at higher levels than are now likely to ness-for-purpose. The actual process willoccur, whereas reproductive and neurobe- vary, depending on the type of biomarkerhavioural effects may occur at lower levels of and its intended use. It was beyond the scopeexposure). of this workshop to consider this issue in Provided there is sufficient evidence to detail, but some general comments can beassume that a given disease is associated made.with a specific exposure, it is not necessary Validation of biomarkers of exposurefor the mechanism of effect to be understood should include consideration of: a) the toxi-in order for biomarkers of exposure to be of cokinetics of the substance, i.e. the quantita-value. Thus, it can be assumed that increas- tive and time relationship of levels in bodying exposure increases risk, and decreasing fluids and tissues when exposure is of short,exposure decreases risk, even though these medium or long duration; b) specificity, i.e.increases or decreases may not be quantifi- whether the substance or its metabolite mayable. However, in order to identify meaning- result from sources other than the exposureful biomarkers of effect, as indicated in of interest; c) analytical quality control, i.e.Figure 2, it is necessary to have an estab- the use of appropriate reference materials tolished, mechanistic causal pathway which establish the accuracy of results; d) stabilitylinks measurements of exposure, intermedi- in appropriate biological fluids; and e) poten-ate effects and the final outcome. At the far tial confounding factors.end of the chain of events are diagnostic It is notable that a number of quality con-markers. Just as exposure markers are tied trol schemes are in operation, at regional,to specific chemical exposures, diagnostic national and international levels, for meth-markers relate to specific clinical events. It is ods used in biological monitoring of occupa-axiomatic that the further along the pathway tional exposure (25). Such schemes couldfrom exposure to effect, the more difficult it provide a model for other biomarker applica-is to define precisely the extent to which the tions.exposure contributes to a specific clinical Validation of biomarkers of effect shouldoutcome, and vice versa. However, measure- include consideration of: a) relevance to thements of biological indices which are indica- effect of interest; b) reliability of predictiontive of a causal pathway between exposure of the associated endpoint; c) interlaboratoryand outcome (i.e. biomarkers of effect) may reproducibility; d) analytical quality control;be considered to have a degree of predictiv- e) stability in appropriate biological fluids;ity. Following the definition of the scope and and f) potential confounding factors.limitations of such biomarkers, they could beincorporated into in vivo and in vitro toxicitystudies. Future Considerations In conclusion, biomarkers of exposurealone cannot be predictive of a toxic effect, Although biomarkers currently cannotbut have established value in monitoring and replace or reduce the use of animals in toxic-controlling exposure to chemicals that are ity testing, future technological develop-recognised or assumed hazards to health. ments show great promise for making thisBiomarkers of effect might be able to be used possible. In vitro techniques may play an
ECVAM Workshop 40: biomarkers 129important role in the development of bio- decreased risk of developing particular dis-markers of effect, as the greater control eases) are potentially of great value.which is possible in in vitro studies facilitates The importance of the establishment of ainvestigation of mechanisms of toxicity, and causal pathway was a recurring theme of thetherefore the identification of key events. A workshop. Currently, there are few examplesmechanistic understanding of the progres- of situations where this has been achieved.sion of a toxic effect is a necessary prerequi- However, this reflects the lack of informa-site for the use of biomarker approaches for tion on the mechanisms which underpin tox-the prediction of toxicity. It is possible to icity. At present, too little emphasis is placedenvisage a natural progression, in which a on this issue in toxicological studies.potential biomarker is identified during invitro mechanistic studies, and its valuetested in animal models, is then incorporated Conclusions and Recommendationsin in vivo testing protocols (refinement ofanimal testing), and is subsequently vali- 1. The term “biomarkers” is employed verydated for use in in vitro toxicity tests (lead- loosely. There are many differences ofing to reduction and replacement of animal opinion about the definition, uses andtesting). Once an acceptable level of use has potential value of biomarkers.been established for a new chemical, there is 2. Most controversy relates to the use of thea potential for biomarkers to provide the term “predictive biomarker”. A bio-continuity between initial toxicity testing marker is, by definition, a reflection ofand post-marketing surveillance, to ensure the current status of the biological sam-that the acceptable levels are adequate to ple or system at the time of analysis. Forprotect the human population against a biomarker of exposure, that statusadverse effects. could reflect either recent or longer term Biomarkers of exposure are not directly exposure, i.e. a past or present situation.relevant to toxicity testing per se, but they Provided there is a known associationmay become relevant outside the laboratory with a disease, the biomarker of exposureenvironment in the determination of expo- can predict a change in risk of that dis-sure and dose (molecular dosimetry) and in ease, but it cannot predict a toxic effect. Amonitoring to check that acceptable expo- biomarker of effect might reflect an earlysure levels are not exceeded. They are also stage in the development of a disease, andrelevant to risk assessment and to investi- therefore may be predictive of eventualgating mechanistic causes of human dis- disease. However, the development ofeases. such biomarkers is dependent upon If biomarker techniques can be readily understanding of the aetiology of the dis-automated, they have the potential to facili- ease in question, and few examples aretate the development of high throughput available as yet.assays, which permit the testing of com-pounds which have not already been tested, 3. The use of biomarkers as research toolsbut to which populations are exposed. This is to illuminate a postulated pathway or toparticularly relevant to dietary exposure, measure imprecise endpoints is an impor-partly because of the vast number of chemi- tant aspect of their role.cal components of food, but also because the 4. Traditional toxicity testing is not predic-complex nature of whole foods makes delin- tive of the range of effects which mayeation of the effects of individual compo- occur in a real-life exposure situation.nents both difficult and misleading. For This incomplete picture of the effects ofexample, dietary oestrogens interact with exposure may be clarified by the develop-each other and with the consumer in unpre- ment of biomarkers which have specificdictable ways. Therefore, markers which can relevance to human disease. Such rele-combine the different active and protective vant biomarkers could ultimately be usedcomponents into an overall biological respon- to help establish the causal links betweensiveness (for example, increases in levels of exposure and outcome and/or be indica-sex hormone-binding protein or changes in tive of a potential effect in humans.the expression of specific genes) linked to There is a need to establish the criteriaclinical endpoints (for example, increased or by which biomarkers can be considered to
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