Biomarkers and Risk Assessment:
    Concepts and Principles

    This report contains the collective views of an international group of
    experts and does not necessarily represent the decisions or the stated
    policy of the United Nations Environment Programme, the International
    Labour Organisation, or the World Health Organization.

    Published under the joint sponsorship of
    the United Nations Environment Programme,
    the International Labour Organisation,
    and the World Health Organization

    World Health Orgnization
    Geneva, 1993

         The International Programme on Chemical Safety (IPCS) is a
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    International Labour Organisation, and the World Health
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    and the quality of the environment. Supporting activities include
    the development of epidemiological, experimental laboratory, and
    risk-assessment methods that could produce internationally
    comparable results, and the development of manpower in the field of
    toxicology. Other activities carried out by the IPCS include the
    development of know-how for coping with chemical accidents,
    coordination of laboratory testing and epidemiological studies, and
    promotion of research on the mechanisms of the biological action of

    WHO Library Cataloguing in Publication Data

    Biomarkers and risk assessment : concepts and principles.

        (Environmental health criteria ; 155)

        1.Biological markers  2.Environmental exposure 
        3.Hazardous substances 4.Risk factors 

        ISBN 92 4 157155 1        (NLM Classification: QH 541.15.B615)
        ISSN 0250-863X

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        1.1. Biomarkers - concepts
        1.2. Definitions
        1.3. Biomarkers and the risk assessment process


        2.1. Use in health risk assessment
        2.2. Use for clinical diagnosis
        2.3. Use for monitoring purposes


        3.1. Selection - practical aspects
              3.1.1. General laboratory considerations
              3.1.2. Quality assurance and control
        3.2. Validation and characteristics of biomarkers




        6.1. Haematological biomarkers
        6.2. Nephrotoxicity biomarkers
        6.3. Liver toxicity biomarkers
        6.4. Biomarkers of immunotoxicity
        6.5. Biomarkers of pulmonary toxicity
        6.6. Biomarkers of reproductive and developmental toxicity
        6.7. Biomarkers of neurotoxicity


        7.1. Analysis of chemicals and metabolites
        7.2. Biomarkers for genotoxic carcinogens
              7.2.1. DNA adducts - general considerations
              7.2.2. DNA adducts in human samples
              7.2.3. Protein adducts
              7.2.4. Cytogenetic methods
              7.2.5. Chromosome damage
              7.2.6. Sister chromatid exchange

              7.2.7. Micronuclei
              7.2.8. Aneuploidy
              7.2.9. Mutation
        7.3. Biomarkers for non-genotoxic carcinogenesis


    9. SUMMARY


        10.1. General
        10.2. Research
        10.3. Applications







    Dr A. Aitio, Institute of Occupational Health, Helsinki, Finland

    Dr D. Anderson, British Industrial Biological Research Association,
         Carshalton, Surrey, United Kingdom  (Rapporteur)a,b

    Dr P. Blain, Division of Environmental and Occupational Medicine,
         The Medical School, University of Newcastle upon Tyne, United

    Dr J. Bond, Chemical Industry Institute of Toxicology, Research
         Triangle Park, North Carolina, USAb

    Dr M. Buratti, Clinica del Lavoro, Instituti Clinici di
         Perfezionamento, Milan, Italya

    Dr I. Calder, Occupational and Environmental Health, South
         Australian Health Commission, Adelaide, South Australia,

    Dr I. Chahoud, Institute of Toxicology and Embryo-pharmacology, Free
         University of Berlin, Berlin, Germanya

    Dr J.R. Fowle, Health Effects Research Laboratory, US Environmental
         Protection Agency, Research Triangle Park, North Carolina,

    Dr L. Gerhardsson, Department of Occupational and Environmental
         Medicine, Lund University Hospital, Lund, Swedenb

    Dr. R. Henderson, Lovelace Inhalation Toxicology Research Institute,
         Albuquerque, New Mexico  (Vice-Chairman)a,b

    Dr H.B.W.M. Koëter, TNO-CIVO Institute, AJ Zeist, The Netherlandsa

    Dr A. Nishikawa, Division of Pathology, National Institute of
         Hygienic Sciences, Tokyo, Japanb

    Dr C.L. Thompson, Laboratory of Biochemical Risk Analysis, National
         Institute of Environmental Health Sciences, Research Triangle
         Park, North Carolina, USAb

    Dr H. Zenick, Health Effects Research Laboratory, US Environmental
         Protection Agency, Research Triangle Park, North Carolina,


    Dr J. Lewalter, Institute of Biological Monitoring, Medical
         Department, Bayer AG, Leverkusen, Germany (attending on behalf
         of CECa and ECETOCb)

    Dr D. Howe, Unilever E.S.L., United Kingdom (attending on behalf of

    Mrs G. Richold, Unilever E.S.L., United Kingdom (attending on behalf
         of ECETOCb)


    Dr G.C. Becking, International Programme on Chemical Safety,
         Interregional Research Unit, World Health Organization,
         Research Triangle Park, North Carolina, USAa

    Dr J. Hall-Posner, Unit of Mechanisms of Carcinogenesis,
         International Agency for Research on Cancer, Lyon, Franceb

    Dr F. He, World Health Organization, Division of Health Protection
         and Promotion, Occupational Health, Geneva, Switzerlandb

    Dr A. Robinson, Ontario Ministry of Labour, Toronto, Ontario, Canada
          (Temporary Adviser)b


    a    Participant in Planning Meeting on Utilization of Biological
         Markers in Risk Assessment (Non-Carcinogenic End-Points), 25-27
         October 1989, Carshalton, UK

    b    Participant in Task Group on Biomarkers and Risk Assessment:
         Concepts and Principles, 16-20 November 1992, Carshalton, UK


         Every effort has been made to present information in the
    criteria monographs as accurately as possible without unduly
    delaying their publication. In the interest of all users of the
    Environmental Health Criteria monographs, readers are kindly
    requested to communicate any errors that may have occurred to the
    Director of the International Programme on Chemical Safety, World
    Health Organization, Geneva, Switzerland, in order that they may be
    included in corrigenda.

                            *   *   *

         A detailed data profile and a legal file can be obtained from
    the International Register of Potentially Toxic Chemicals, Case
    postale 356, 1219 Châtelaine, Geneva, Switzerland (Telephone No.

                             *   *   *

         This publication was made possible by grant number 5 U01
    ES02617-14 from the National Institute of Environmental Health
    Sciences, National Institutes of Health, USA.


         At the Sixth Meeting of the IPCS Programme Advisory Committee
    (31 October to 3 November 1989) it was recommended that the IPCS
    give priority to work on biomarkers, as outlined at an IPCS Planning
    Meeting (25-28 October 1989). One of the recommendations from the
    Planning Meeting was for the IPCS to prepare an Environmental Health
    Criteria monograph on the concepts and principles supporting the use
    of biomarkers in the assessment of human health risks from exposure
    to chemicals.

         The drafts of this monograph were prepared by Dr A. Robinson,
    Toronto, Canada. During the preparation of the monograph many
    scientists made constructive suggestions and their assistance is
    gratefully acknowledged.

         A WHO Task Group on Biomarkers and Risk Assessment: Concepts
    and Principles met in Carshalton, United Kingdom, from 16 to 20
    November 1992. Dr Robinson opened the meeting on behalf of the heads
    of the three cooperating organizations (UNEP/ILO/WHO), and Dr D.
    Anderson welcomed the participants on behalf of the British
    Industrial Biological Research Association, the host institution.
    The Task Group reviewed and revised the draft monograph.

         Following the Task Group Meeting, Dr Robinson collated the text
    with the assistance of Dr A. Aitio and Dr D. Anderson, Chairman and
    Rapporteur, respectively, of the Task Group. The Secretariat wishes
    to acknowledge their special contributions in finalizing this

         Dr A. Robinson was responsible for the overall scientific
    content, and Dr P.G. Jenkins (IPCS Central Unit) for the technical

         The efforts of all who helped in the preparation and
    finalization of the monograph are gratefully acknowledged. Special
    thanks are due to the United Kingdom Department of Health for its
    financial support of both the Planning and the Task Group Meetings.


         The purpose of this monograph is to examine the concepts and to
    identify the principles for the application of biomarkers to
    assessment of risk to human health from exposure to chemical agents,
    with special attention to criteria for selection and validation.

         Information and examples are provided to illustrate and assist
    the application of these principles to enhance human health risk
    assessment by reducing the uncertainties associated with the
    process. Biomarkers may be indicative of exposure, effect(s) or
    susceptibility of individuals to chemical agents, but their use must
    take account also of ethical and social considerations.

         Some guidance is provided for the selection of appropriate
    biomarkers to allow identification of individuals and
    sub-populations at increased risk, with consequent implications for
    administrative intervention, mitigation and health protection.

         A review has been made of biomarkers suitable for application
    to assessment of the risk of chemicals that are toxic to the
    hepatic, renal, haematological, immune, pulmonary, reproductive/
    developmental and nervous systems or are associated with
    carcinogenic mechanisms. However, greater detail is provided for
    biomarkers linked with carcinogenesis, reflecting the volume of
    scientific publications resulting from recent intensive studies of
    mechanisms, provoked by public attitudes and perceptions associated
    with diagnosis of the disease. This section serves to illustrate the
    complexity of the interactions and the many factors which will
    influence selection and application of biomarkers to improve further
    the process of health risk assessment.


    1.1  Biomarkers - concepts

         Analysis of tissues and body fluids for chemicals, metabolites
    of chemicals, enzymes and other biochemical substances has been used
    to document the interaction of chemicals with biological systems.
    Measurements of these substances, now referred to as "biomarkers",
    are recognized as providing data linking exposure to a chemical with
    internal dose and outcome and as relevant to the process of risk

         The term "biomarker" is used in this monograph, as it is in the
    US National Academy of Sciences report (US NRC, 1989b), in a broad
    sense to include almost any measurement reflecting an interaction
    between a biological system and a potential hazard, which may be
    chemical, physical or biological. The measured response may be
    functional and physiological, biochemical at the cellular level, or
    a molecular interaction. Various factors will apply in assessing
    risks to individuals and population subgroups compared with the
    general population.

         In the assessment of risk, biomarkers may be used in hazard
    identification, exposure assessment and to associate a response with
    the probability of a disease outcome. By examining the interactions
    between human host and chemical exposure, and comparable data for
    experimental studies of mammalian species, criteria for the
    selection of biomarkers indicative of exposure, effects,
    susceptibility and toxic response(s) to chemicals may be

         The reaction to exposure to a chemical depends on inherited and
    acquired characteristics and the life-style of the human subject (or
    other biological system), the properties and form of the chemical,
    and the circumstances of the contact. The outcome may be no effect,
    some adverse effect with recovery, or toxicity with morbidity.

         Human health is affected by all the activities of an
    individual, who is subject to a continuum of chemical exposures in
    the external environment, including air, water, soil and food. It
    should be noted that distinction of exposure to chemicals on the
    basis of context, such as recreational, residential or occupational,
    is often made for administrative convenience. The important
    considerations for assessment of risk are the dose rate, route,
    duration and frequency of exposure.

         The application of biomarkers, linked to toxic processes or
    mechanisms, to the risk assessment process, and particularly to
    quantitative risk assessment, has the potential to provide a more
    rational and less judgmental process, particularly when compared

    with methods that arbitrarily attach protection factors to doses
    assessed to minimize or avoid effects deemed adverse to health. 

         Selection of appropriate biomarkers is of critical importance
    because of the opportunity for greater precision in the assessment
    of risk to individuals or population sub-groups, with the consequent
    implications for mitigation and health protection. However,
    selection will depend upon the state of scientific knowledge and be
    influenced by social, ethical and economic factors.

         Subject to ethical considerations, the use of validated
    biomarkers to monitor exposed populations may provide the basis for
    early, health-protective intervention.

         Identification of practicable biomarkers associated with
    different toxic end-points or outcomes requires interdisciplinary
    cooperation and research, and this is evident in relation to
    carcinogenesis, neurotoxicity, pulmonary toxicity, immunotoxicity
    and human reproduction. While not all of these areas of interest are
    equally well developed, use of biomarkers linked with toxicity
    should enhance the process and reliability of predictions of risk.

         Improved definition of the risk associated with exposure to
    chemicals will permit effective preventive intervention to protect
    human health both in general and in particular circumstances.
    Protective measures may include avoidance of exposure to chemicals
    or protection of sensitive individuals.

    1.2  Definitions

         The term "biomarker" is used in a broad sense to include almost
    any measurement reflecting an interaction between a biological
    system and an environmental agent, which may be chemical, physical
    or biological. However, discussion in this monograph is limited to
    chemical agents. Three classes of biomarkers are identified:

    *     biomarker of exposure: an exogenous substance or its
         metabolite or the product of an interaction between a
         xenobiotic agent and some target molecule or cell that is
         measured in a compartment within an organism;

    *     biomarker of effect: a measurable biochemical, physiological,
         behavioural or other alteration within an organism that,
         depending upon the magnitude, can be recognized as associated
         with an established or possible health impairment or disease;

    *     biomarker of susceptibility - an indicator of an inherent or
         acquired ability of an organism to respond to the challenge of
         exposure to a specific xenobiotic substance.

    1.3  Biomarkers and the risk assessment process

         For a general discussion of concepts and principles underlying
    assessment of risk to human health associated with exposure to
    chemicals, the reader is referred to WHO (in press). 

         The process for assessment of human health risks associated
    with exposure to chemicals is multifaceted and incorporates the
    following major components:

    *     hazard identification: to confirm that the chemical is
         capable, subject to appropriate circumstances, of causing an
         adverse effect in humans;

    *     dose-response assessment: to establish the quantitative
         relationship between dose and effect in humans;

    *     exposure assessment: to identify and define the exposures
         that occur, or are anticipated to occur, in human populations.

         Risk characterization is the synthesis of the qualitative and
    quantitative information that describes the estimated risk to human
    health from the anticipated environmental exposure.

         Hazard identification and dose-response assessment make use of
    all available data for human and test species and, where
    appropriate, for  in vitro test systems.

         The relevance of biomarkers to the phases of the risk
    assessment process is discussed more fully in later sections that
    address biomarkers of effects, exposure and susceptibility. 


         Biomarkers may be used to assess the exposure (absorbed amount
    or internal dose) and effect(s) of chemicals and susceptibility of
    individuals, and they may be applied whether exposure has been from
    dietary, environmental or occupational sources. Biomarkers may be
    used to elucidate cause-effect and dose-effect relationships in
    health risk assessment, in clinical diagnosis and for monitoring

         Biomarkers of exposure can be used to confirm and assess the
    exposure of individuals or populations to a particular substance,
    providing a link between external exposures and internal dosimetry.
    Biomarkers of effect can be used to document either preclinical
    alterations or adverse health effects elicited by external exposure
    and absorption of a chemical. Thus the linkage of biomarkers between
    exposure and effect contributes to the definition of dose-response
    relationships. Biomarkers of susceptibility help elucidate the
    degree of the response to exposure elicited in individuals.

    2.1  Use in health risk assessment

         Measurements carried out for many years within the context of
    "biological monitoring" have been used to assess worker exposure
    and, in clinical settings, to evaluate the administration of
    therapeutic agents. These measurements, or biomarkers, provide the
    critical link between chemical exposure, internal dose and health
    impairment, and are of value in assessment of risk. However, there
    is a need to identify and validate for each organ system those
    characteristic parameter(s) that are indicative of induced
    dysfunction, clinical toxicity or pathological change, as well as to
    establish the specificity and sensitivity of each biomarker and its
    method of measurement.

    2.2  Use for clinical diagnosis

         Biomarkers may be used to:

         *    confirm diagnosis of acute or chronic poisoning;

         *    assess the effectiveness of treatment; and

         *    evaluate the prognosis of individual cases.

         For this purpose, a well-established relationship between
    biomarker(s) and outcome must be available. Assessment of exposure
    in short-term or long-term exposure situations can be evaluated on a
    more meaningful basis where previous exposure has been documented by
    consecutive measurements over a period of time. Although this may 

    not be possible in the circumstances of a major chemical release,
    biomarkers of effect may still find useful application to assess
    clinical outcome(s).

    2.3  Use for monitoring purposes

         Biomarkers may be used to confirm the exposure of individuals
    in a population to a particular substance, e.g., an organic solvent
    in exhaled breath, the cadmium burden of the kidney, lead in bone,
    or the fatty tissue storage of chlorinated hydrocarbons (see Table
    1, chapter 5). Quantitative measurements may facilitate the
    determination of dose-response relationships.

         Biomarkers are used for screening and for monitoring (repeated
    at timed intervals), and may be determined and applied on an
    individual basis or may be related to a population group. Population
    groups "at risk" may be identified by deviations from normal of mean
    values for biomarkers of exposure or effects; individual variations
    will be reflected in statistical terms.

         Some public and occupational health surveillance programmes
    include the use of biomarkers for screening and monitoring purposes.
    Although the terms "biological screening or monitoring" and "health
    monitoring" have been applied, there is no agreement that the terms
    are appropriate, and repeated measurement of biomarkers may be
    cost-effective methodologies to monitor disease development. In
    practice, however, ethical and social considerations, rather than
    cost, often preclude the widespread use of biomarkers for monitoring
    or surveillance purposes.

         Biomarkers of exposure or effect may be used to evaluate
    compliance with advice for minimizing exposure or for remedial
    measures in a public health context, e.g., to confirm reduced
    exposure to lead from environmental sources in a population group.
    In addition, biomarkers may be used to supplement environmental or
    ambient workplace measurements of chemicals with recognized
    potential adverse health effects that may be subject to regulatory

         Biomarkers may serve as a basis for assessing individual or
    population groups exposed to chemicals from any source, including
    life-style activities. In an occupational context, biomarkers will
    provide a supplementary means for reviewing the adequacy of
    protective measures, including work practices and working

         When the inter-individual variation of the biomarker is large
    in comparison to intra-individual variation, analysis of paired
    samples (before, during and after the exposure) may greatly enhance

    the power of the biomarker to detect exposure, e.g., serum
    acetylcholine esterase measurements in relation to exposure to an
    organophosphorus compound.

         Use of biomarkers reflecting genetically linked or acquired
    susceptibility to specific chemicals or their metabolites provides
    an opportunity for the recognition and protection of sensitive
    individuals. The classic example of genetically linked
    susceptibility is phenylketonuria in newborn infants. An example of
    acquired susceptibility is the development of hypersensitivity to
    certain inhaled gases or dusts (such as toluene diisocyanate,
    trimellitic acid anhydride or cotton dust) in the workplace.


         The process of selection and validation requires careful
    consideration of the specificity and sensitivity of the biomarker as
    a measure of the contribution of the exposure to an observed adverse
    health outcome. A similar process must be applied also to
    establishing the accuracy, precision and quality assurance of the
    analytical procedure for measurement of the selected biomarker.

         Before discussing the criteria for the selection and validation
    of biomarkers of exposure, effect and susceptibility, and their
    application to the risk assessment process, it is necessary to
    consider key factors that can influence the host reaction to
    xenobiotic chemicals. Fig. 1 summarizes some of the various factors
    that influence the interaction between host and chemical. 

         These factors may be considered in the context of a
    source-chemical-host response where the source of the specific
    chemical of concern may be the air, water, soil or food. It is
    important to consider the physico-chemical properties of the
    chemical (e.g., gas, vapour, particle) and whether the chemical is
    present in a complex chemical mixture or adsorbed on a particle. For
    example, the initial site of deposition (and perhaps the site of
    toxicity) of a chemical in the respiratory tract may be affected by
    the strength of the association between the chemical and particulate
    matter (which determines bioavailability), as well as by particle
    size, in the inhaled atmosphere (e.g., nose versus deep lung).
    Several exposure characteristics need also to be considered, such as
    the concentration of the chemical and the duration, frequency and
    magnitude of exposure. Exposure of the host can be through various
    routes including the respiratory tract (inhalation exposure), the
    gastrointestinal tract (oral exposure) and the skin (dermal
    exposure). Finally, there are a number of host characteristics that
    can influence response to chemical exposure, including age, race,
    gender, health status, genetic susceptibility, and previous exposure
    to the same or other chemicals. Information relating to these
    factors can provide clues as to the types of biomarkers that may be
    used to assess exposure, effect and susceptibility.

         Many factors require consideration in the process for selection
    and validation of a biomarker. To select the most appropriate
    biomarker requires several steps:

    (1)  the identification and definition of the end-point of interest;

    (2)  the assembly of the data base to document the relationship
         between the chemical exposure, the possible biomarkers and the
         end-point. This will include data from  in vitro, mammalian
         and human studies, with assessment of the validity of data and
         the study protocols;

    FIGURE 01

    (3)  selection of biomarker(s) specific to the outcome of interest
         with careful consideration of the biomarker to identify what is
         being quantified, to assess the sensitivity and specificity of
         the marker in relation to exposure, and the significance with
         respect to health outcome or pathological change over time;

    (4)  consideration of specimens potentially available for analysis,
         with emphasis on protecting the integrity of the specimen
         between collection and analysis, and a preference for
         non-invasive techniques;

    (5)  review of the analytical procedures available for
         quantification of biomarkers and their limitations with respect
         to detection limit, sensitivity, precision and accuracy;

    (6)  establishment of an appropriate analytical protocol with
         provision for quality assurance and quality control;

    (7)  evaluation of intra- and inter-individual variation for a
         non-exposed population;

    (8)  analysis of the data base to establish dose-effect and
         dose-response relationships and their variation, with special
         emphasis on susceptible individuals;

    (9)  calculation or prediction of risk to human health either for
         the general population or a sub-group; and

    (10) review of ethical and social considerations.

         These issues are discussed more fully in the following

         These steps may need to be carried out in an interactive and
    iterative manner before selection of the desired biomarker can be

    3.1  Selection - practical aspects

    3.1.1  General laboratory considerations

         Measurement of biomarkers may range from molecular events to
    functional outcomes such as behaviour or pulmonary function; the
    same consideration should be applied to all biomarkers.

         Analytical considerations include defined and appropriate
    precision and accuracy, and quality assurance and control, as well
    as the availability of automated instrumentation or alternative
    simple, but specific, methodology. Specimen collection, handling and

    storage should require the minimum of special precautions to avoid
    contamination and/or deterioration. The costs, in terms of skilled
    human resources, equipment and reagents should be reasonable. 

         Sampling and measurements should preferably be:

    *    non-invasive; 

    *    representative, i.e. the time of the exposure in relation to
         measurement should be taken into account; and 

    *    the stability of the analyte in the specimen should be

         In general terms, specimens available for analysis will include
    blood, urine, sputum, saliva, finger-nails, breath, hair, faeces and
    (shed) teeth. The clinic or hospital setting may provide the
    opportunity to collect unique fluid samples (e.g., follicular,
    amniotic, semen) or tissues accompanying examination of the patient
    (e.g., cytological material, pulmonary lavage), tissue biopsies
    (e.g., fat) or autopsy specimens.

         Specialized techniques for  in vivo determination may be
    available for some chemicals, e.g., cadmium in kidney or lead in
    bone, but such applications require exposure of individuals to
    radiation. In such instances, ethical considerations must also be
    taken into account.

    3.1.2  Quality assurance and control

         Critical to the successful and effective application of
    biomarkers is a well-documented quality assurance and control
    programme. It is beyond the scope of this monograph to discuss such
    programmes in detail, but these have been reviewed (Aitio, 1981;
    WHO, 1992b). It is important to note that good analytical
    performance does not necessarily guarantee accurate results in
    biomarker analyses since greater errors may be introduced during
    sampling. Thus, the quality assurance protocol has to cover the
    entire process. Major impediments to quality assurance of biomarker
    analyses include the lack of certified reference materials and
    external quality control programmes.

    3.2  Validation and characteristics of biomarkers

         Validation is a process to establish the qualitative and
    quantitative relationship of the biomarker (a) to exposure to a
    chemical, and (b) to the selected end-point. Desirable
    characteristics of biomarkers include that:

    (1)  the marker (measurement) 

         (a)  reflects the interaction (qualitative or quantitative) of
              the host biological system with the chemical of interest,

         (b)  has known and appropriate specificity and sensitivity to
              the interaction,

         (c)  is reproducible qualitatively and quantitatively with
              respect to time (short- and long-term);

    (2)  the analytical measurement has defined and appropriate accuracy
         and precision;

    (3)  the marker is common to individuals within a population or
         subgroup and is of defined variability within the normal,
         non-exposed population or group of interest; and

    (4)  the marker is common between species.


         It is important to recognize the ethical and social
    implications of the uses of biomarkers, in addition to the
    scientific and cost considerations. Ethical concerns may limit the
    extent of investigations of chemically exposed human individuals and
    populations, particularly those involving the living.

         Participation by individuals or groups will be influenced both
    by personal and scientific factors. Personal attitudes, ideals and
    beliefs will vary geographically with ethnic origin and cultural

         The process leading to participation is critically important
    and must respect the dignity, rights and freedom of choice of
    individuals; participation must be voluntary and based on full

         The freedom of choice of individuals will include the right of
    refusal to give blood or other biological samples for analysis of
    biomarkers. Personal decisions should be based on full information
    with the implications of a refusal being explained and understood. 

         Before measurement of a biomarker is undertaken, there should
    be consideration of how and to whom results should be provided, the
    interpretation of the results, and whether this should be on an
    individual or group basis, with or without protection of
    confidentiality. While practices will vary between countries, it is
    particularly important that the role of medical officers be defined
    in relation to their responsibility to the individual (patient) and
    in relation to administrative (company) management. 

         It is the ethical responsibility of medical officers to inform
    individuals fully of potentially hazardous exposures, recognizing
    that remedial action may involve administrative decisions. The
    latter may be taken in the context of prevailing economic and social
    considerations rather than of individual circumstances.

         Investigators need to recognize and accommodate the medical
    dilemma created by the conduct of biomarker research in terminally
    ill patients, since the data may clarify the use of the biomarker
    without contributing to improvement of the health status of the
    patient. Thus, careful consideration must be given to the desire to
    advance scientific understanding relative to meeting the needs of
    the patient.

         In addition to the general ethical issues associated with use
    of biomarkers, there are particular problems relating to biomarkers
    of susceptibility. 

         Identifying susceptible individuals may help to prevent their
    exposure to a specific harmful chemical(s) but may lead to
    discrimination in the employment of the susceptible individual when
    that chemical is known to be present in the workplace.

         It is also an ethical question as to whether individuals should
    be given information about their own susceptibility. This knowledge
    would allow them to make more informed choices, for example, the
    avoidance of exposure to specific substances. It is, however,
    important to realize that only for a few biomarkers of
    susceptibility is it well established that they are associated with
    the development of disease. If the individual does not fully
    understand this uncertainty, such information on biomarkers may
    cause unnecessary concern and anxiety. 


         The exposure assessment component of the risk assessment
    process is an attempt to provide qualitative and quantitative
    estimates of human exposure through the use of measurements and
    models. In this context, measurements may be made of chemical
    concentrations in food, water and air, selected environmental
    concentrations (e.g., occupational or residential settings) as well
    as measures of the actual exposures experienced by the individual or
    population. Exposure biomarkers extend this latter component of
    exposure assessment into the realm of data which provide the most
    direct evidence of human exposure to a given agent and the absorbed

         Adverse or toxic effects in a biological system are not
    produced by chemical agents unless that agent or its
    biotransformation products reach appropriate sites in the body at a
    concentration and for a length of time sufficient to produce the
    toxic manifestation. Thus, to characterize fully the potential
    hazard or toxicity of a specific chemical agent in an individual, it
    is necessary to identify not only the type of effect and the dose
    required to produce the effect but also information about the
    duration and frequency of exposure to the agent, and the
    susceptibility of the exposed individual.

         Methods for assessing exposure to a chemical fall into two

    1.   measurement of levels of chemical agents and their metabolites
         and/or derivatives in cells, tissue, body fluids or excreta;

    2.   measurement of biological responses such as cytogenetic and
         reversible physiological changes in the exposed individuals.

         Measurement of covalent adducts formed between chemical agents
    and cellular macromolecules (proteins, DNA), or their excretion
    products have characteristics of both category one and two above. 

         In evaluating exposure, distinction is made between the
     external dose, defined as the amount of a chemical agent in
    environmental contact with the organism, as determined by personal
    or area monitoring, and the  internal dose, which is the total
    amount of a chemical agent absorbed by the organism over a period of
    time. Biomarkers of exposure will reflect the distribution of the
    chemical or its metabolite throughout the organism. Theoretically,
    this distribution can be tracked through various biological levels
    (e.g., tissue, cell, etc.) to the ultimate target. The concept of
    biomarkers of exposure is illustrated in Fig. 2. 

         This figure illustrates that, of the total amount absorbed,
    only a portion will be delivered to a target tissue. A portion will
    reach internal macromolecules, and a smaller proportion will reach
    the critical site on the macromolecule, with only a fraction of the
    latter amount acting as the biologically effective dose. Biomarkers
    for each of these forms of internal dose would be useful for
    assessment of risk. The decreasing area of the triangle, from the
    total absorbed amount to each lower level as distribution and
    metabolism occur, illustrates the decreasing mass of the internal
    dose that reaches the target tissue, cell, or critical site. In
    progressing from biomarkers of total absorbed dose to markers of
    biologically effective dose, it becomes increasingly easy to relate
    the dose to the mechanism of the induced health effect. In the
    reverse process, from the dose for critical sites to the total
    amount absorbed, it becomes easier to relate the internal dose to
    the external exposure.

         The internal dose can be assessed by suitable analyses of
    biomarkers in biological samples (urine, faeces, blood and/or its
    components, expired air) (Alessio et al., 1983, 1984, 1986, 1987,
    1988, 1989; UK HSE, 1991; ACGIH, 1992; DFG, 1992; Bond et al.,
    1992). These biomarkers may be the unchanged chemical material, its
    known metabolites or biochemical markers affected by absorption of
    the chemical. It may be possible to estimate the dose quantitatively
    when the toxicokinetics of the chemical is well established and the
    sampling is conducted at appropriate points in time. The tissue dose
    may be further refined to a specific target dose which may be
    defined as the amount of chemical (or its metabolite) which, over a
    period of time, reaches the biologically significant site(s) within
    the target tissue.

         While such measurements may not equate to the biologically
    effective dose, the data can provide useful estimates of internal
    exposure (dose). Knowledge of the kinetics of formation and removal
    from the body of these types of biomarkers provides a link between
    exposure and internal dose.

         Specific measures of internal dose are the active chemical
    species (either parent compound or metabolite) delivered to target
    tissues or cells, the reactive chemical species delivered to target
    organelles or macromolecules, or the reactive chemical species that
    participates in biochemical reactions. For example, quantification
    of the generalized covalent binding of reactive species to
    macromolecules will provide a measure of absorbed dose delivered to
    target tissues or cells, while measurement of total DNA adducts is
    indicative of the dose delivered to target organelles or
    macromolecules. Finally, specific DNA adducts could be the
    biologically effective species that initiate the carcinogenic
    process. These issues are discussed in chapter 7. 

    FIGURE 02

         The potential impact of target tissue DNA-protein cross-links
    as a biomarker of the biologically effective dose is illustrated in
    the proposed risk assessment for formaldehyde put forward by US EPA
    (1991). The biological, mechanism-based model of formaldehyde
    carcinogenesis consists of three submodels (Conolly et al., 1992;
    Conolly & Andersen, in press). One of these is a tissue dosimetry
    submodel which incorporates formaldehyde-induced cross-links of DNA
    with adjacent proteins (Casanova et al., 1989, 1991). Although the
    role of DNA-protein cross-links in the mechanism of formaldehyde-
    induced nasal cancer is not known, their formation is used only as a
    biomarker of the "biologically effective" dose reaching target cells
    in the nasal cavity. An earlier proposal (US EPA, 1987) used
    external formaldehyde concentration as the measure of dose. The
    predicted quantitative human risk at low levels of exposure is lower
    when the interspecies extrapolation (from monkeys and rats) is based
    on the biomarker, i.e. DNA-protein cross-links, rather than on the
    concentration of formaldehyde inhaled. Although, there are several
    unresolved issues regarding the use of DNA-protein cross-links as a
    biomarker, this example illustrates that mechanistic data and a
    biomarker of delivered dose can be used in the risk assessment
    process for chemicals.

         A strategy to help relate biomarkers to prior exposures is to
    obtain quantitative information about the kinetics of formation and
    breakdown of the biomarker, as shown in Fig. 3. The information
    required includes the quantified correlation of the biomarker with a
    given exposure scenario and kinetic data for elimination of the
    biomarker. Biomarkers for chemicals that are cleared rapidly, such
    as vapours in exhaled breath or urinary metabolites, may be present
    in large amounts soon, during or immediately following an exposure,
    but are not detectable at later times.

         Other biomarkers, such as adducts formed with blood proteins,
    may represent only a small fraction of the total internal dose but,
    because they have a long half-life in the body (relative to exposure
    frequency), may accumulate to detectable levels with continued

         Use can be made of kinetic properties to define prior
    exposures. If a person has had only a single, recent exposure to a
    chemical, the level of biomarkers with short half-life will be high
    relative to those with a longer half-life. With continuing exposure,
    the levels of markers with both shorter and longer half-lives should
    be high. If a person was exposed in the more distant, rather than
    the more recent, past, only the biomarkers with the longer
    half-lives will be detectable. 

    FIGURE 03

         Thus by analysing several biomarkers with different half-lives
    (e.g., haemoglobin adducts in the blood, metabolites of the chemical
    in urine, parent compound in blood) from a single individual at a
    single point in time, more information may be obtained about the
    nature of the past exposure than from use of a single biomarker.

         Also of value in the interpretation of biomarker data is the
    use of mathematical models to describe the kinetics of formation and
    elimination of biomarkers of exposure. Absorbed chemicals are
    distributed between various compartments in the body, with the
    distribution being dependent on the nature of the compartment and
    the lipophilicity of the chemical. The most simple models use only
    one compartment; however, multicompartmental models are usually
    required to describe the disposition of most chemicals in the body
    (Gibaldi & Perrier, 1982). Multicompartmental models that
    incorporate biomarkers of exposure in relation to toxic end-points
    are well established. A biokinetics model for lead has been used to
    predict blood lead levels of individuals and communities (DeRosa et
    al., 1991).

         More recently, physiologically based toxicokinetic (PBTK)
    models have been developed that make use of the physico-chemical
    properties of a chemical (such as partition coefficients that
    indicate how the chemical or its metabolites become partitioned
    between different fluids in the body), the kinetics of metabolism of
    the chemical (such as Vmax and KM for metabolic pathways), and
    physiological parameters of the exposed individual (such as tissue
    blood flow, respiratory minute volume, cardiac output) to predict
    the actual concentrations of biomarkers that will occur after
    specific exposure regimens. These models are adapted for humans by
    changing the physiological and metabolic parameters to those
    appropriate for humans, and by testing for validity in limited human
    studies. These models can then be used to extrapolate between
    different exposure situations for the predicted levels of markers
    (Ramsay & Andersen, 1984; Droz et al., 1989).

         Another use of models is in defining the quantitative
    relationship between biomarkers in readily available biological
    samples (e.g., blood cells) and in those less readily available
    which might be more pertinent biomarkers for the health effect of
    concern (e.g., tissue DNA). An example is the use of haemoglobin
    adducts to predict the amount of DNA adducts at a critical site,
    e.g., after exposure to ethylene oxide (Passingham et al., 1988). To
    be able to make such predictions, one must know the kinetics of
    formation and breakdown of each of the markers and the factors that
    influence those kinetics. Based on such information a model can be
    developed to show the quantitative relationships between the markers
    under different exposure conditions or at different times after

         Biomarkers are used extensively in the surveillance of workers
    occupationally exposed to metals such as lead, cadmium, mercury,
    nickel, chromium and arsenic, and to organic chemicals such as
    aniline, benzene, carbon disulfide, styrene, chlorobenzene and
    chlorinated aliphatic hydrocarbon solvents (see Table 1). The
    examples in Table 1 are given as general information. Before
    applying them in specific circumstances, readers must consult the
    original references.

         These measures are used to indicate the absorbed dose. For a
    few chemicals, notably lead, mercury, cadmium and carbon monoxide,
    an approximate estimation of the associated health risk may be made.
    For other chemicals, exposure may be assessed in quantitative terms.
    It is noted that biomarkers are supplementary to environmental
    measurements rather than alternative or substitute measures of

         Considerable efforts are being made to develop biomarkers
    associated with exposure to chemical carcinogens and to establish
    the relationship between a marker and the future health risk. The
    use of animal models may facilitate this process; studies on the DNA

    adducts formed by vinyl chloride illustrate the types of strategies
    required to make each link (Swenberg et al., 1990). In rats, vinyl
    chloride induces liver tumours with a high incidence in young
    animals, and the DNA adducts (biomarkers) formed in the liver have
    been characterized and the half-lives determined. DNA fidelity
    replication assays were used to show one type of adduct that had
    both a long half-life and was capable of inducing mutations (Hall et
    al., 1981; Barbin et al., 1985; Singer et al., 1987; Swenberg et
    al., 1990). The level of this adduct was much higher in the livers
    of young rats exposed to vinyl chloride than in adults, and was
    characterized as the one most closely related to the health effect.
    To extend this animal research to predict human health risks, it
    would be necessary to determine if the concentration of the same
    adduct is associated with liver tumour formation in human tissue

        Table 1.  Some parameters proposed for biological monitoring by different organizations

    Exposure                                                           Measured parametera

                           American Conference of          Deutsche                   Finnish Institute of       United Kingdom Health
                           Governmental Industrial         Forschungsgemeinschaft     Occupational Health        and Safety Executive
                           Hygienists (ACGIH, 1992)        (DFG, 1992)                (FIOH, 1993)               (UK HSE, 1991)

    Acetylcholinesterase   E-acetylcholinesterase          E-acetylcholinesterase     E-acetylcholinesterase     E-acetylcholinesterase,
    inhibitors                                                                                                   P-cholinesterase

    Aluminium (Al)                                         U-Al                       U-Al

    Aniline                U-p-aminophenol,B-Met-Hb        U-aniline                  U-p-aminophenol

    Arsenic (As)                                           U-certain volatile         U-AsIII+, AsV+             U-AsIII+, AsV+
                                                           arsenic compounds                                     MMA+DMA
                                                           produced by direct 

    Benzene                U-phenol                        B-benzene, U-phenol        B-benzene                  Breath-, B-benzene

    p-tert-Butylphenol                                     U-p-tert-butylphenol

    Cadmium (Cd)           U-Cd, B-Cd                      U-Cd, B-Cd                 U-Cd, B-Cd                 U-Cd, B-Cd

    Carbon disulfide       U-TTCA                          U-TTCA                     U-TTCA                     U-TTCA

    Carbon monoxide        Breath-CO, B-COHb               B-COHb                     B-COHb                     B-COHb

    Chlorobenzene          U-4-chlorocatechol              U-4-chlorocatechol


    Table 1 (contd)

    Exposure                                                           Measured parametera

                           American Conference of          Deutsche                   Finnish Institute of       United Kingdom Health
                           Governmental Industrial         Forschungsgemeinschaft     Occupational Health        and Safety Executive
                           Hygienists (ACGIH, 1992)        (DFG, 1992)                (FIOH, 1993)               (UK HSE, 1991)

    Chlorophenols                                                                     U-tri+tetrapenta-

    Chlorophenoxy acids                                                               U-2,4-D+Dichloroprop+

    Chromium (Cr)          U-Cr                            U-Cr                       U-Cr                       B-Cr, U-Cr

    Cobalt (Co)                                            U-Co                       U-Co                       U-Co

    Dichloromethane                                        B-COHb, B-dichloromethane  B-COHb                     B-COHb, B-dichloromethane

    Dieldrin                                                                          P-dieldrin                 B-dieldrin

    Dimethylformamide      U-methylformamide                                          U-methylformamide          U-methylformamide

    Ethylbenzene           U-mandelic acid                                                                       U-mandelic acid

    2-Ethoxyethanol                                        U-ethoxyacetic acid        U-ethoxyacetic acid

    2-Ethoxyethyl acetate                                  U-ethoxyacetic acid        U-ethoxyacetic acid

    Ethylene oxide                                         Breath-b, B-ethylene oxide

    Fluoride (F)           U-F                             U-F                        U-F                        U-F


    Table 1 (contd)

    Exposure                                                           Measured parametera

                           American Conference of          Deutsche                   Finnish Institute of       United Kingdom Health
                           Governmental Industrial         Forschungsgemeinschaft     Occupational Health        and Safety Executive
                           Hygienists (ACGIH, 1992)        (DFG, 1992)                (FIOH, 1993)               (UK HSE, 1991)

    Furfural               U-furoic acid                                                                         U-furoic acid

    Halothane                                              U-trifluoroacetic acid

    Hexachorobenzene                                       P/S-hexachlorobenzene

    n-Hexane               U-hexanedione,                  U-hexanedione +            U-hexanedione
                           Breath-n-hexanec                dihydroxyhexanone

    Hydrazine                                              P-, U-hydrazine

    Lead (Pb)              B-Pb, U-Pb, B-ZPP               B-Pb, U-ALA                B-Pb, B-ZPP                B-Pb, U-ALA, B-ZPP

    Lindane                                                B(P,S)-lindane             B-lindane                  B-lindane

    Manganese (Mn)                                                                    U-Mn                       B-, U-Mn

    Mercury (Hg)                                           B-, U-Hg                   B-, U-Hg                   B-, U-Hg

    Methanol               U-methanol, U-formate           U-methanol                 U-formate

    Methylbromide                                                                                                B-Br

    Methyl butyl ketone                                    U-hexanedione +


    Table 1 (contd)

    Exposure                                                           Measured parametera

                           American Conference of          Deutsche                   Finnish Institute of       United Kingdom Health
                           Governmental Industrial         Forschungsgemeinschaft     Occupational Health        and Safety Executive
                           Hygienists (ACGIH, 1992)        (DFG, 1992)                (FIOH, 1993)               (UK HSE, 1991)

    Methylene                                                                                                    U-MOCA

    Methylene dianiline                                                                                          U-MDA

    Methyl ethyl ketone                                    U-MEK                      U-MEK

    2-Methoxyethanol                                                                  U-methoxyacetic acid

    2-Methoxyethyl acetate                                                            U-methoxyacetic acid

    Nickel (Ni)                                            U-Ni                       U-Ni                       U-Ni

    Nitrobenzene           U-p-nitrophenol, B-MetHb

    Parathion              U-p-nitrophenol,                U-p-nitrophenol,
                           E-cholinesterase                E-cholinesterase

    Pentachlorophenol      U-PCP, P-PCP                    U-PCP, P-PCP                                          U-PCP

    Phenol                 U-phenol                        U-phenol


    Table 1 (contd)

    Exposure                                                           Measured parametera

                           American Conference of          Deutsche                   Finnish Institute of       United Kingdom Health
                           Governmental Industrial         Forschungsgemeinschaft     Occupational Health        and Safety Executive
                           Hygienists (ACGIH, 1992)        (DFG, 1992)                (FIOH, 1993)               (UK HSE, 1991)

    Polychlorinated                                                                   S-PCB                      B-PCB
    biphenyls (PCB)

    2-Propanol                                             U-acetone, B-acetone

    Selenium (Se)                                                                     U-Se

    Styrene                U-mandelic acid, U-PGA          U-mandelic acid,           U-mandelic acid+PGA        U-mandelic acid
                                                           U-mandelic acid+U-PGA

    Thallium (Tl)                                                                                                U-Tl

    Tetrachloroethylene    Breath-,                        Breath-b,                                             B-tetrachloroethylene
                           B-tetrachloroethylene           B-tetrachloroethylene

    Tetrachloromethane                                     Breath-b, 

    Tin (Sn)                                                                                                     U-Sn

    Toluene                U-hippuric acid                 B-toluene                  B-toluene                  B-toluene

    1,1,1-Trichloroethane  Breath-1,1,1-trichloroethane,   Breath-b,                  B-1,1,1-trichloroethane    B-1,1,1-trichloroethane
                           B-trichloroethanol              B-1,1,1-trichloroethane


    Table 1 (contd)

    Exposure                                                           Measured parametera

                           American Conference of          Deutsche                   Finnish Institute of       United Kingdom Health
                           Governmental Industrial         Forschungsgemeinschaft     Occupational Health        and Safety Executive
                           Hygienists (ACGIH, 1992)        (DFG, 1992)                (FIOH, 1993)               (UK HSE, 1991)

    Trichloroethylene      U-TCA, B-trichloroethanol,      U-TCA, B-trichloroethanol  U-TCA, U-Trichloroethanol  U-TCA

    Vanadium (V)                                                                      U-V                        U-V

    Vinyl chloride                                         U-thiodiglycolic acid

    Xylenes                U-methylhippuric acids          U-methylhippuric acids, B- U-methylhippuric acids     U-methylhippuric acids


    a   The following abbreviations have been used:  E = erythrocyte; P = plasma; S = serum; U = urine; B = blood; ZPP = erythrocyte zinc
        protoporphyrin; ALA = delta-aminolevulinic acid; PGA = phenylglyoxylic acid; MMA = monomethylarsinic acid; DMA = dimethylarsinic acid;
        TCA = trichloroacetic acid; MCPA = chloromethylphenoxyacetic acid; 2,4-D = 2,4 dichlorophenoxyacetic acid;
        TTCA = 4-thio-4-thiazolidine carboxylic acid.
    b   DFG (1992) refers to alveolar air
    c   ACGIH (1992) refers to end-exhaled air

    Note: Readers must consult original references before applying the above examples to specific situations


         This section focuses on those human biomarkers that can be
    applied currently or will be in the near future. Biomarkers of
    effect may be used directly in hazard identification and
    dose-response assessment components of the risk assessment process.
    In hazard identification, biomarkers may facilitate screening and/or
    identification of a toxic agent and characterization of the
    associated toxicity. Biomarkers that are implicated in toxic
    mechanism(s) are preferred for quantitative dose-response
    assessments when extrapolating from existing data to a human
    situation of concern (e.g., from high to low dose or from test
    species to humans).

         There are wide inter-individual variations in the response to
    equivalent doses of chemicals. While the outcome of a chemical
    insult in an individual may be predicted more accurately from
    biomarkers of effect(s), such biomarkers may not be specific for a
    single causative agent. Many biomarkers of effect are used in
    everyday practice to assist in clinical diagnosis, but for
    preventive purposes an ideal biomarker of effect is one that
    measures change that is still reversible. Nevertheless, certain
    biomarkers of nonreversible effects may still be very useful in
    epidemiological studies or provide the opportunity for early
    clinical intervention.

         A limited range of tissues is available for routine analysis of
    biomarkers. The more accessible tissues are therefore used as
    surrogates for the known or putative target tissues. In some
    instances biomarkers of effect are not mechanistically related to
    chemically induced lesions, but may represent concomitant,
    independent changes. Therefore, although an effect (e.g., sister
    chromatid exchange) is being analysed, the use is conceptually close
    to assessment of exposure. 

    6.1  Haematological biomarkers

         Inhibition of the enzymes in the haem synthesis pathway (e.g.,
    ferrochelatase, levulinate dehydratase) has been used as a marker of
    effect of exposure to lead. This effect is reflected also in the
    levels of free erythrocyte protoporphyrin (FEP) and
    delta-aminolevulinate in the urine. Elevated levels of urinary
    delta-amino-levulinate are observed at higher lead exposures than
    changes in FEP, for example, while basophilic stippling of
    erythrocytes is an even less sensitive biomarker for the effects of
    lead. However, the effects on haem synthesis are not specific to
    lead as a causative agent; iron deficiency has a similar effect on
    FEP. The relationship of these effect biomarkers to toxicity
    requires further elucidation.

         Routine leucocyte, erythrocyte and thrombocyte counts have been
    used in the surveillance of patients treated with cytostatic drugs
    and in the monitoring of benzene-exposed workers. The predictive
    power in relation to benzene-induced aplastic anaemia or leukaemia
    is limited (Townsend et. al., 1978; Hancock et al., 1984; Lamm et
    al., 1989). Ferrokinetic measurements, such as plasma iron
    disappearance half-time, erythrocyte utilization of iron, plasma
    iron transport rate, or erythrocyte iron turnover rate, have been
    suggested as biomarkers of myelotoxicity (Rajamaki, 1984).

    6.2  Nephrotoxicity biomarkers

         Several different types of measures have been tested and used
    as biomarkers of renal damage. These have been classified as
    functional markers (e.g., serum creatinine and ß2-microglobulin),
    urinary proteins of low or high molecular weight (e.g., albumin,
    transferrin, retinol-binding globulin, rheumatoid factor,
    immunoglobulin G), cytotoxicity markers (tubular antigens, e.g.,
    BB50, BBA, HF5), enzymes (e.g.,  N-acetylglucosaminidase,
    ß-galactosidase) in urine, and biochemical markers (eicosanoids,
    e.g., 6-keto PGF2alpha, PGE2, PGF2alpha and TXB2, fibronectin,
    kallikrein activity, sialic acid and glycosaminoglycans in urine,
    and red blood cell negative charges) (Cardenas et al., 1993a,b;
    Roels et al., 1993).

         Biomarkers for nephrotoxicity were reviewed in WHO (1991) and
    are well validated in relation to exposure to cadmium (WHO, 1992a;
    Roels et al., 1993) but not in relation to exposure to mercury or
    lead (Cardenas et al., 1993a,b).

    6.3  Liver toxicity biomarkers

         Effects of chemicals on the liver have been estimated
    traditionally by measuring the activities of, for example,
    aminotransferase (most often aspartate or alanine aminotransferase)
    in the serum, where they are found when liver cells have been
    damaged and have leaked their contents. Many other enzymes have also
    been analysed for this purpose; they include 5-nucleotidase, alcohol
    dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase,
    leucine aminopeptidase, glutathione  S-transferase, ornithine
    carbamoyl transferase). Since tissues other than liver also contain
    these enzymes, their activities may be elevated in serum not only
    after liver damage but also when non-hepatic tissues have been
    damaged. To overcome this lack of specificity, analysis of specific
    isoenzymes has been used. Serum activities of enzymes such as
    alkaline phosphatase and gamma-glutamyl transpeptidase may be used
    as biomarkers of hepatic damage mainly involving biliary excretion.
    Several liver function tests can also be used as biomarkers of
    effects; these include the concentrations of serum proteins
    synthesized in the liver, e.g., albumin and clotting factors, or

    serum concentrations of bile acids, also synthesized in the liver,
    as well as tests for the hepatic excretory function such as
    bromsulfphthalein half-time. These parameters lack specificity since
    hepatic viral infections, alcohol and drug use affect these enzymes.
    Indirect measures of chemically induced change(s) in the cytochrome
    P-450 enzyme system, using provocation tests, have been proposed as
    sensitive indicators. However, the relationship to liver damage and
    disease is not established and the requirement for the
    administration of a drug limits the use of such tests.

         Hepatotoxicity is caused by a number of chemicals that are
    metabolized by the cytochrome P-450-dependent mixed-function oxidase
    system to reactive intermediates. For example, carbon tetrachloride
    has been studied extensively; it is metabolized to a reactive
    intermediate which initially depletes intracellular glutathione to a
    level that is no longer protective when the metabolite reacts with
    critical macromolecules leading to cell death and hepatoxicity. In
    this example, biomarkers of effect could include glutathione levels,
    lipid peroxidation or the number of necrotic cells.

    6.4  Biomarkers of immunotoxicity

         The immune system protects the organism against infectious
    microorganisms and the growth of at least some neoplasms. Reactions
    of the immune system are influenced by genetic factors, age,
    nutrition, life-style and health status. Xenobiotics may stimulate
    or suppress the immune system. After initial sensitization, even a
    minimal new exposure may lead to an anaphylactic reaction. The
    immune system may be more sensitive to chemical challenge than any
    other body system.

         Hypersensitivity reactions following inhalation exposure
    include asthma, rhinitis, pneumonitis and granulomatous pulmonary
    reactions (see section 6.5). Hypersensitive dermal reactions induced
    by chemicals include a wide variety of acute, subchronic and chronic
    changes. Patch testing has been used traditionally as a biomarker
    for identification of the causative agent of an allergic skin
    reaction. However, the possibility of inducing hypersensitivity by
    patch testing has been well documented and should not be overlooked
    (Adams & Fisher, 1990).

         Elevated levels of specific antibodies, usually of the IgE
    type, may indicate existing sensitization. However, not all
    individuals with elevated levels are symptomatic and not all
    symptomatic individuals exhibit elevated IgE levels (Horak, 1985;
    Sub-Committee on Skin Tests of the European Academy of Allergology
    and Clinical Immunology, 1989; Nielsen et al., 1992).

         Suppression of the immune system increases susceptibility to
    infections and neoplasia. Changes in the relative abundance of

    different lymphocyte subpopulations (suppressor and helper T-cells)
    have been used as biomarkers for the immune suppression (Jennings et
    al., 1988; Sullivan, 1989; Holsapple, et al., 1991). Individuals
    with asbestos-induced pleural or pulmonary changes, or
    asbestos-induced cancer, as well as those heavily exposed to
    asbestos but without apparent disease, have been reported to exhibit
    an altered immunological status (e.g., reductions in T-lymphocyte
    subsets) (Bekes et al., 1987).

         In view of the growing incidence of hypersensitivity reactions
    to chemicals, development and application of biomarkers for
    immunotoxic effects is important. However, this is made difficult by
    the current limited understanding of basic immunological mechanisms
    and the effects of chemicals thereon (US NRC 1992).

    6.5  Biomarkers of pulmonary toxicity

         The most frequently used markers of pulmonary toxicity measure
    gross effects on pulmonary function (e.g., peak expiratory flow,
    forced expiratory volume, transfer factors) rather than effects on
    cells or biochemical processes (US NRC, 1989a). These measures tend
    to be nonspecific with respect to the causative agent and may
    overlook effects specific to a certain cell type. Peak expiratory
    flow measurements can be performed by the exposed individuals
    themselves at the workplace, at home or elsewhere, and they provide
    information on the underlying causes of air-way obstruction,
    allowing a closer association between exposure, atmosphere and

         Air-way hyperactivity can be assessed by challenge tests using
    inhalation exposure. Although such tests may assist in identifying
    the factors causing hypersensitive pulmonary reactions, there is a
    clear risk of acute reactions, and testing should be carried out by
    qualified personnel in carefully controlled environments.

         Recently, analysis of bronchoalveolar lavage fluid (BALF) has
    been used to detect lung injury or to follow the progress of
    pulmonary disease or the efficacy of therapeutic treatment
    (Reynolds, 1987; Henderson, 1988).

         The use of cellular elements as markers of pulmonary disease
    state has been emphasized in human BALF analysis (Reynolds, 1987).

         Total cell counts and differential counts, including use of
    monoclonal antibody staining to distinguish T-cell subtypes, are
    used to detect alveolitis and to aid in the diagnosis of
    interstitial lung disease. High percentages of lymphocytes are
    indicators of granulomatous processes, such as sarcoidosis, or
    hypersensitivity pneumonitis. High percentages of neutrophils with
    some eosinophils indicate possible idiopathic pulmonary fibrosis.

    Other extracellular components, such as cytokines and other
    mediators of inflammation, have been used on an experimental basis
    to answer specific research questions.

         The analysis of BALF has been used to define the dose-response
    characteristics of inhaled or instilled toxins in animal toxicity
    studies (Henderson, 1988). The most sensitive biomarker of an
    inflammatory response in the bronchoalveolar region is the number of
    neutrophils in BALF. The levels of protein and of extracellular
    enzymatic activity are also useful markers of pulmonary toxicity.
    Increases in protein concentrations in BALF indicate increased
    permeability of the alveolar/capillary barrier. Lactate
    dehydrogenase (LDH) is a cytoplasmic enzyme that is found
    extracellularly only in the presence of lysed or damaged cells.
    Beta-glucuronidase or similar lysosomal hydrolytic enzymes are
    excellent markers for the toxicity of inhaled particles. These
    particles are phagocytosed by macrophages, and the enzymes are
    released from activated or lysed macrophages.

         The secretion of cytokines from pulmonary macrophages obtained
    by bronchoalveolar lavage provides markers of developing fibrosis.
    Recent studies by Piguet et al. (1990) demonstrate that the level of
    secretion of tumour necrosis factor (TNF) by pulmonary macrophages
    is associated with quartz-induced fibrotic processes. Lassalle et
    al. (1990) found elevated secretion of TNF by macrophages obtained
    from individuals with coal-workers pneumoconioses compared with
    macrophages from controls. The secretion of platelet-derived growth
    factor from pulmonary macrophage was elevated in patients with
    idiopathic pulmonary fibrosis (IPF).

         Glutathione (GSH), a tripeptide protective against oxidative
    stress, is present in BALF, and a decrease in GSH in BALF is a
    potential marker for oxidative stress. Decreased levels of GSH have
    been observed in patient with IPF (Cantin et al., 1989) and in
    animals exposed chronically to diesel exhaust, resulting in
    pulmonary fibrosis (Henderson, 1988).

         Nasal lavage fluid (NLF) also provides markers of response to
    inhaled toxins. The work of Graham et al. (1988) demonstrates the
    potential use of NLF analysis to document the influx of neutrophils
    into the nasal cavity in humans in response to inhaled ozone.

         Biomarkers in blood related to lung injury have not been
    validated. However, the work of Cavalleri et al. (1991) indicates
    that serum aminoterminal propeptide of type III procollagen (PIIINP)
    may become useful as an early marker for developing fibrosis. A
    dose-dependant increase in serum PIIINP was found in individuals
    exposed to low or high levels of asbestos. 

         Finally, urinary levels of amino acids associated with the
    connective tissue of the lung (hydroxyproline, hydroxylysine,
    desmosine and isodesmosine) have been used as markers of lung injury
    (Harel et al., 1980; Yanagisawa et al., 1986; Stone et al., 1991).
    However, such assays are not specific for lung injury and merely
    indicate the breakdown of connective tissue in any organ in the

    6.6  Biomarkers of reproductive and developmental toxicity

         Markers associated with an adverse outcome in reproduction may
    reflect toxic effects in the male or female or be associated with
    development during the embryonic, fetal, perinatal or neonatal
    period (US NRC, 1989b; Mattison, 1991).

         Biomarkers for the male reproductive system may include
    physiological indicators of impaired testicular function, or sperm
    number or characteristics (including cytogenetics). Measures of
    hormonal status (i.e. FSH, LH and testosterone) can also be readily
    obtained from blood and, in the case of testosterone, from urine and
    saliva. However, these levels are greatly influenced by circadian
    rhythms and demonstrate large inter- and intra-individual
    variability. A clearer picture of hormonal status can be obtained by
    administering GnRH or LH and examining the hormone response to these
    challenges. Biomarkers for the male reproductive system are rather
    easily accessible and some even reasonably well validated; such
    markers are less well developed for the female reproductive system.

         Biomarkers indicative of developmental toxicity should also be
    considered. As is the case for many biological markers of effects,
    it is often difficult to identify the causative agent in the absence
    of any specific exposure history. Biomarkers could include
    measurements of detrimental effects produced by chemical or other
    exposures during embryonic or fetal stages of development.
    Irreversible lesions can be embryolethal or result in functional
    anomalies in the offspring. Examples of biomarkers of developmental
    toxicity include low birth weight, chromosome anomalies, delayed
    growth of specific organ systems, mental retardation, and subtle
    behavioural changes. The changes associated with F1 male-mediated
    abnormalities have been discussed by Anderson (1990). Some of these
    biomarkers of developmental effects (malformations, mental
    retardation) are not biomarkers of effect as far as the individual
    is concerned, but rather represent the adverse health outcome
    itself. However, from the point of view of the exposed population,
    they may be considered as biomarkers since they show that within a
    population a harmful exposure has taken place. 

         Several biomarkers have been proposed for use during pregnancy,
    e.g., early pregnancy loss and assays for genetic defects of the
    conceptus. The latter comprise both classical cytogenetic studies,

    as well as specific DNA probes (US NRC, 1989b). The use of urinary
    human chorionic gonadotrophin (HCG) has been well documented as a
    biomarker for early fetal loss (US NRC, 1989b). Many different
    biomarkers have been used to follow the development of the pregnancy
    and the well-being of the conceptus, but they have not yet been
    applied to studies of effects of chemicals on pregnancy.

    6.7  Biomarkers of neurotoxicity

         The functions of the nervous system are complex and biomarkers
    may range from effects of chemicals on neural cellular and molecular
    processes to neurophysiological and neuro-behavioural measurements
    of complex functional entities.

         Inhibition of plasma and erythrocyte acetylcholine esterase
    (AchE) provides biomarkers of exposure to organophosphorus compounds
    and other cholinesterase inhibitors. While erythrocyte
    cholinesterase is similar to brain cholinesterase, and is therefore
    an effect biomarker, plasma nonspecific pseudocholinesterase only
    reflects exposure and is not a marker of CNS effects.

         Measures of the function of the peripheral nervous system
    (e.g., electroneuromyography, nerve conduction velocities, vibration
    sensitivity) are well defined. Assessment of peripheral nervous
    system dysfunction associated with exposure to chemicals can be
    carried out using electroneuromyography at the preclinical stage
    (Seppalainen et al., 1979).

         Some well-established neurophysiological (e.g., evoked
    potentials, electroencephalography) and neurobehavioural (e.g., the
    WHO Neurobehavioural Core Test Battery, Cassito et al., 1990)
    measures may be used as biomarkers to evaluate CNS dysfunction
    induced by neurotoxicants. These tests must be carried out under
    well-controlled conditions.

         Methods for assessing changes in higher cognitive function
    (e.g., learning and memory) have been used extensively, e.g., in
    workers exposed to solvents or heavy metals, but require further

         Available neuroimaging procedures, e.g., computed axial
    tomography (CAT), magnetic resonance imaging (MRI), nuclear magnetic
    resonance spectroscopy (MRS) and positron-emission tomography (PET),
    are considered non-invasive, but some of them require exposure to
    ionizing radiation. CAT and MRI can be carried out with current
    clinical techniques to assess chemically induced changes in the
    brain. The use of MRS and PET can provide a more detailed evaluation
    of the biochemical status (e.g., rate of energy generation, blood
    flow, L-glucose metabolism) in the central nervous system. They can
    be used as biomarkers for assessing exposure to neurotoxicants 

    inducing brain alterations. However, they are expensive, of little
    use in assessing spinal cord, nerve and muscle changes, and there is
    only minimal data validating their use in neurotoxicology (US NRC,

         Other promising biomarkers for neurotoxicity in animal studies
    include glial fibrillary acidic protein (localized in the
    astrocytes), which increases in localized areas within the brain
    where injury due to toxicants occurs (O'Callaghan, 1991).


         As new information about the multistep process of
    carcinogenesis unfolds, it is instructive to consider the various
    mechanisms by which chemicals induce cancer. Knowledge of mechanisms
    will enable the selection of appropriate biomarkers for use in risk
    assessment of carcinogens. Some chemicals are direct acting and
    others require metabolic activation. Once absorbed most chemicals
    undergo enzyme-mediated reactions that either detoxify them or
    activate them to reactive species. The balance between activating
    and detoxifying enzyme systems governs the rate of delivery of
    bioactive metabolites to the macromolecular site (Harris, 1991). The
    resulting macromolecular interaction could be a DNA adduct for
    carcinogens that are initiating agents or receptor occupancy for
    chemicals that are tumour promotors. Certain of the DNA adducts
    produced by such interactions are pro-mutagenic, and replication of
    the damaged DNA could lead to DNA sequence changes which may result
    in altered gene expression or mutated gene products. Weisburger &
    Williams (1981) have suggested that chemical carcinogens be
    classified as those that interact with DNA (genotoxic) and those
    that do not (epigenetic or non-genotoxic). The importance of mitotic
    activity in the latter group has recently been elaborated further
    (Cohen & Ellwein, 1990).

         The implications for invoking the "mechanistic" approach to the
    selection of appropriate biomarkers are significant. For example,
    chemicals that stimulate cell proliferation via mitogenesis or
    cytotoxicity (and subsequent proliferation) might require different
    biomarkers than chemicals whose major mechanism of action is based
    on DNA reactivity. In the latter case, measurements of DNA adducts
    or chromosome alterations may serve as suitable biomarkers, whereas
    in the former case alternate biomarkers (e.g., cell turnover
    measurements) may be more appropriate.

    7.1  Analysis of chemicals and metabolites

         Urinary or blood concentrations of several chemicals shown or
    suspected to be carcinogenic to humans (e.g., arsenic, cadmium,
    chromium, nickel, benzene, MOCA, polychlorinated biphenyls, styrene,
    tetrachloroethylene) have for long been used as biomarkers of
    exposure. Among people exposed to arsenic in a copper smelter, a
    dose relationship has been observed between the cumulative urinary
    excretion of arsenic and the risk of lung cancer (Enterline & Marsh,
    1982). For other carcinogenic chemicals, such data are not
    available, and the measured concentrations may only be interpreted
    in terms of exposure.

         Sensitive techniques based on physicochemical or immunochemical
    methods for the detection of a variety of carcinogen-modified DNA
    bases have been developed (Shuker & Farmer, 1992). These include the

    alkylated purines, aflatoxin-guanine adducts, cis-platinum adducts,
    thymine glycol, 8-hydroxydeoxyguanosine and PAH-derived adducts.

         The aflatoxin marker has been extensively used in both animal
    and human studies on the relationship between exposure and liver
    cancer induction. In an ongoing prospective study in Shanghai,
    China, Ross et al. (1992) reported that subjects with liver cancer
    were more likely than controls to have detectable concentrations of
    any of the known aflatoxin metabolites in their urine. Groopman et
    al. (1991) recently explored the relationship between dietary
    aflatoxin and excretion in the urine of aflatoxin metabolites and an
    aflatoxin-DNA adduct. This study was conducted on people living in
    the Guangxi Autonomous Region, China. These investigators found a
    positive correlation between aflatoxin N7-guanine and specific
    metabolites excreted in urine and aflatoxin B1 intake from the
    previous day.

         Exposure to chemical compounds capable of interacting with
    cellular macromolecules can originate from both exogenous and
    endogenous sources. Nitrite, nitrate and nitrosating agents can be
    synthesized endogenously in reactions mediated by bacteria and
    activated macrophage. In this way endogenous formation of
     N-nitroso compounds can occur at various sites in the body.
    Endogenously formed  N-nitroso compounds may be considered as
    biomarkers of susceptibility; they have been associated in humans
    with increased risk of cancer of the stomach, oesophagus and urinary
    bladder, although unequivocal epidemiological data are lacking
    (Bartsch & Montesano, 1984). The quantitative estimation of
    endogenous nitrosation in humans can be measured using the
     N-nitroso-proline test. L-proline is utilized as a probe for
    nitrosatable amines and  N-nitroso-proline excreted in the urine is
    determined as a marker. This assay has been applied in some
    population studies (Bartsch et al. 1991).

    7.2   Biomarkers for genotoxic carcinogens

    7.2.1  DNA adducts - general considerations

         DNA adducts are being used both as molecular dosimeters
    (biomarker of exposure) and to assess the genotoxic potential of
    chemicals (biomarker of effect). The biological significance of such
    adducts must be assessed on the basis of adduct heterogeneity and of
    cell and tissue specificity for adduct formation, persistence and
    repair. Some DNA adducts result in mutation whereas others do not.
    Mutational specificity in the p53 gene produced by a variety of
    chemical carcinogens provides evidence that DNA adduct location
    influences site-specific mutations (Hollstein et al., 1991). Some
    DNA sequence changes may lead to phenotypic alterations that can be
    selected, whereas others may not (Compton et al., 1991).

         Most tissues are comprised of multiple cell types, and cell
    types vary considerably in their capacity to convert chemicals to
    DNA reactive species. For example, lung is composed of multiple cell
    types in which the relative concentrations of various P-450
    isoenzymes and enzymes depends on the cell type. Thus, one compound
    may produce high concentrations of pro-mutagenic adducts in one cell
    type, but not in another, whereas the opposite might occur for a
    compound which is activated by a different P-450 isozyme. DNA adduct
    concentrations derived from a whole tissue homogenate may grossly
    overestimate or underestimate adduct concentrations in a given cell

         Some DNA adducts are repaired quickly, others hardly at all,
    the adduct loss correlating with cell turnover. Therefore, the
    concentration and gene location of DNA adducts will change with time
    after exposure to a genotoxic chemical. Furthermore, the existence
    of non-random repair in the genome makes it difficult to utilize
    total DNA repair capacity as an indicator of cell susceptibility to
    carcinogens (Hanawalt, 1987).  It is especially important in human
    studies to know the duration and timing of exposure for proper
    evaluation of the biological significance of a given adduct

         Many of the human studies described below have involved
    measuring metabolism, DNA adduct formation and repair in whole
    tissues. Techniques need to be refined in order that
    cell-type-specific variations can be monitored in human tissues, as
    well as experimental studies in animal models using
    immunohistochemical techniques for the cell type. Specific
    localization of DNA adducts has clearly demonstrated that such
    variations occur. Treatment of rats with the tobacco-specific
    nitrosamine, 4-( N-methyl- N-nitrosamine)-1-(3-pyridyl)-1-butanone
    (NNK), results in the induction of tumours in the nasal cavity,
    lung, liver and pancreas (Hoffman et al., 1984; Rivenson et al.,
    1988). At low doses of NNK, the prevalence of malignant lung tumours
    was higher than that observed in other tissues. Cell-type-specific
    differences have been observed within the lung, the highest
    concentration of O6-methylguanine having been found in the Clara
    cells. These cells have the highest levels of the P-450 metabolizing
    enzymes for NNK and low levels of the O6-methylguanine DNA
    methyltransferase repair enzymes (Belinsky et al., 1987).  Pulmonary
    tumours are also induced in mice and hamsters following either
    short- or long-term exposure to this carcinogen (Hecht et al.,

         As animals age, DNA adducts are detected in increasing amounts,
    and, although the relationship of these adducts to tumour
    development is unclear, they are believed to be derived from dietary
    constituents or endogenous chemicals such as hormones (Randerath &
    Randerath, 1991).

    7.2.2  DNA adducts in human samples

         In human studies, it is difficult to obtain non-tumourous
    target tissue for the quantification of DNA adducts. Lymphocytes are
    a readily accessible source of human cells that are known to contain
    DNA adducts. However, there is little information on the reliability
    of using lymphocyte adduct concentrations for the estimation of
    target cell or tissue adduct concentrations (Lucier & Thompson,

         Evaluation of dose-response relationships for chemical
    carcinogens in humans is more complex than in animal models.
    Radio-labelled carcinogens cannot be administered and the
    accessibility of tissues and cells is limited. Several approaches to
    detect DNA adducts in human samples have been evaluated (Wogan &
    Gorelick, 1985; Santella, 1988). The most frequently used methods
    are immunoassays and 32P-postlabelling. Other analytical
    techniques such as GC-MS and synchronous fluorescence spectroscopy
    are being used to measure DNA adducts (Weston & Bowman, 1991). In
    general, immunoassays are both specific and sensitive for alkylated
    adducts and aflatoxin adducts (Wild & Montesano, 1991; Groopman et
    al., 1991). However, these methods are not easily applied to
    quantification of adducts for bulky aromatic hydrocarbons such as
    benzo [a]pyrene-derived adducts. The main problem is the lack of
    specificity of the antibodies used in the assay which cross-react
    with a number of PAH-related adducts (Santella et al., 1985).

         The second assay frequently used to quantify DNA adducts in
    humans is the 32P postlabelling technique. For a complete
    description of this assay, see Randerath & Randerath (1991), Beach &
    Gupta (1992), IARC (1992). The assay is extraordinarily sensitive,
    being capable of detecting 1 adduct in 1010 normal nucleotides
    when appropriate modifications are made to the procedure. The assay
    is particularly useful for detecting adducts of non-polar polycyclic
    aromatic hydrocarbons such as 7,8-diol-9,10-oxide-benzo [a]pyrene
    deoxyguanosine (BPDE). Some DNA modifications such as alkylated DNA
    adducts, which cannot be easily detected by this assay due to the
    limitations of the chromatographic systems, can be quantified using
    a combined 32P immunochemical precipitation technique (Kang et
    al., 1993). Studies using the 32P labelling or immunological
    methods have been reviewed by Beach & Gupta (1992) and Wild &
    Montesano (1991). The groups of chemicals studied include alkylating
    agents, polycyclic aromatic hydrocarbons (PAHs), heterocyclic PAHs,
    nitro PAHs, cyclopenta-fused PAHs, aromatic amines, alkylbenzenes,
    quinones, mycotoxins, chemotherapeutic agents, pesticides and

    7.2.3  Protein adducts

         Ehrenberg and his associates pioneered the use of protein
    adducts as dose monitors for carcinogen exposure in humans, and this
    work has been reviewed by Hsia (1991). To date the class of proteins
    that have been most extensively studied are those found in
    circulating blood, i.e. haemoglobin and albumin. This is mainly
    because these proteins are relatively abundant and can be easily
    isolated for analysis.

         Haemoglobin has the unique biological property of having a life
    span equivalent to that of the erythrocyte, which in humans is
    approximately 120 days, and therefore adduct levels reflect
    exposures over several months. In contrast, albumin adducts can only
    be used for assessing recent exposure because of the faster turnover
    of albumin (half-life of 20-25 days). Protein adducts can be
    quantified using chemical methods, e.g., aromatic amine release by
    acid or basic hydrolysis from haemoglobin followed by derivatization
    and GC-MS analysis (Farmer, 1991), or immunological techniques
    (e.g., aflatoxin-albumin adducts, Wild et al., 1990).

         During the past few years, several human monitoring studies
    have demonstrated the usefulness of protein adducts as biomarkers of
    exposure. Examples of chemicals that have been detected as protein
    adducts in human studies include ethylene and propylene oxide,
    aniline, cigarette smoke, aromatic amines such as 4-amino-biphenyl,
    and aflatoxin (Wogan, 1989; Farmer, 1991). Albumin adducts of
    aflatoxin B1 have also been used in epidemiological studies of
    their role in the etiology of hepatocellular carcinoma in man. A
    significant correlation was observed, at the individual level,
    between dietary intake and the level of albumin-bound aflatoxin in a
    chronically exposed population in the Gambia (Wild et al., 1992).

    7.2.4  Cytogenetic methods

         Cytogenetic methods are used as biomarkers of exposure to
    DNA-damaging agents. 

         Many studies relating to cytogenetic changes in exposed human
    populations have been reviewed in a special issue of Mutation
    Research (Anderson, 1988). A second comprehensive review of more
    recent studies has also been published (Anderson, 1990), and a
    further review is in press (Anderson, in press). 

         All human monitoring studies suffer from variability of
    baseline frequencies (Carrano & Natarajan, 1988) due to the presence
    of endogenous (gender, age, medical history, etc.) and exogenous
    factors (life-style, smoking, drinking, eating habits, etc.).
    Anderson et al. (1991) have investigated the effect of these changes
    on baseline variability eight times over a two-year period.

         In contrast to studies on radiation, for which a marker
    (dicentric chromosome) has been identified, studies with chemicals
    have not yet identified a specific marker chromosome. For radiation
    a dicentric is a quantitative dosimeter. Therefore, after radiation
    exposure results can be used on an individual basis and a highly
    exposed individual removed from the radiation source. Results from
    chemical exposure studies, however, can only be used on a group
    basis, due to the lack of a specific marker chromosome.

    7.2.5  Chromosome damage

         Both chromosome and chromatid aberrations are induced in
    individuals exposed to chemical mutagens. The chromosome aberrations
    are thought to arise from misrepair of lesions in the G0 stage of
    circulating lymphocytes as well as from precursor cells in bone
    marrow and thymus (Carrano & Natarajan, 1988). Chromatid aberrations
    include chromatid breaks, intrachanges and exchanges, while
    chromosome aberrations include acentric fragments, dicentric
    chromosomes and ring chromosomes.  Balanced translocation and
    inversions can also arise and are difficult to quantify without
    banding analysis. Structural aberrations can be classified as
    unstable and stable depending on their ability to persist in
    dividing cell populations. Unstable aberrations consist of rings,
    acentric fragments and other asymmetrical rearrangements, and will
    lead to the death of the cell. Stable aberrations consist of
    balanced translocation inversions and other symmetrical
    rearrangements which can be transmitted to progeny cells at
    division. Therefore stable aberrations are more biologically
    significant than unstable ones and could be involved in the cancer
    process. Many human carcinogens have been shown to produce
    chromosome damage in populations exposed to them, although no causal
    relationship has been demonstrated (Sorsa et al., 1992). Proven
    human carcinogens for which cytogenetic endpoints have been measured
    in humans and corresponding animal data are available are listed in
    Table 2. 

         In a preliminary report of a prospective study among people
    whose lymphocytes were assayed for chromosome aberrations and SCE,
    high rates of chromosome aberrations were observed and appeared to
    be linked to cancer risk, but the finding was of borderline
    statistical significance (Sorsa et al., 1990).

    7.2.6  Sister chromatid exchange

         Sister chromatid exchange (SCE) is considered to be a more
    sensitive, rapid and simple cytogenetic end-point than chromosome
    aberrations for evaluating the genotoxic potential of a variety of
    mutagenic and carcinogenic agents. It is also used to detect and
    differentiate many chromosome fragility diseases that predispose to
    neoplasia. SCE is a DNA-replication-dependent phenomenon. Cellular

        Table 2.  Proven human carcinogens for which cytogenetic end-points have been measured in humans
              and corresponding data are available for experimental animalsa
    Agent/exposure                                             Cytogenetic findingsa
                                                         Humans                     Animals
                                                  CA      SCE    MN           CA      SCE    MN

    Human carcinogens (Group 1)

    Alcoholic beverages                           +       +                   _       +      ?
    Aluminium production                          _       _
    Arsenic and arsenic compounds                 ?       ?                   +              +
    Asbestos                                              ?                   _              _
    Azathioprine                                  ?       _                   +       _      +
    Benzene                                       +                           +       +      +
    Betel quid with tobacco                               +      +                           +
    Bis(chloromethyl)ether and                    (+)                         _       
      chloromethyl methyl ether 
      (technical grade)
    1,4-Butanediol dimethanesulfonate             (+)     +                   +              +
    Chlorambucil                                  ?       +                   +
    Cyclosporin                                   (+)                         _              _
    Coal-tars                                     +
    Coke production                                       +
    Combined oral contraceptives                  _       _
    Cyclophosphamide                              +       +                   +       +      +
    Hexavalent chromium compounds                 +       +                   +       +      +
    Melphalan                                     +       +                   +
    8-Methoxypsoralen plus                        _       _                           +
      ultraviolet A radiation
    Mineral oils, untreated and                   +
      mildly treated
    Nickel compounds                              +       _                   ?              _


    Table 2 (contd)
    Agent/exposure                                             Cytogenetic findingsa
                                                         Humans                     Animals
                                                  CA      SCE    MN           CA      SCE    MN

    Painter, occupational exposures as            _
    Radon                                         +                           _
    Rubber industry                               ?       ?
    Tobacco products, smokeless                                  +                           +
    Tobacco smoke                                 +       +      +                    +
    Tris(1-aziridinyl)phosphine sulfide           (+)                         +       +      +
    Vinyl chloride                                +       ?                   +       +      +


    a From: Sorsa et al. (1992); CA = chromosome aberrations; SCE = sister chromatid exchange;
      MN = micronuclei + = positive result; - = negative result; (+) = equivocal result; 
      ? = doubtful result

    factors such as nucleotide pools, repair and replication enzymes,
    and biorhythms can play an important role in its formation. A major
    source of variation can be attributed to the concentration of
    bromodeoxyuridine relative to the number of lymphocytes in the
    culture (Das, 1988; Morris, 1991; Morris et al., 1992). In a
    prospective cancer study (Sorsa et al., 1990), no relationship was
    observed between the frequency of SCE and the risk of cancer.

    7.2.7  Micronuclei

         Micronuclei are formed by condensation of acentric chromosomal
    fragments or by whole chromosomes that are left behind during
    anaphase movements (lagging chromosomes). The presence of
    micronuclei can therefore be taken as an indication of the previous
    existence of chromosomal aberrations. To visualize micronuclei,
    cells have to undergo mitosis. In peripheral lymphocyte cultures it
    is not easy to distinguish interface nuclei that have undergone a
    division from those that have not. This makes it difficult to
    quantify the frequencies of micronuclei for comparative purposes. A
    method using cytochalasin B can distinguish nuclei that have divided
    once (French & Morley, 1985).

    7.2.8  Aneuploidy

         Aneuploidy is a condition in which the number of chromosomes in
    cells of individuals is not an exact multiple of the typical haploid
    set for the species. Trisomy results when a single extra chromosome
    is added to a pair of homologous chromosomes. If one chromosome of a
    pair is missing, the result is monosomy. Absence of the pair is
    nullisomy. Two or more copies of a homologue result in tetrasomy or
    polysomy. Cells of individuals with missing or extra chromosomes are
    hypoploid or hyperploid (UK DH, 1989). The best-known numerical
    abnormalities result in the syndromes of Down (trisomy of chromosome
    21), Klinefelter (sex chromosome genotype is XXY), and Turner (sex
    chromosome genotype is X0). Aneuploidy is almost always found in
    human cancers (Dellarco et al., 1985). 

    7.2.9  Mutation

         Current somatic gene mutation assays used as biomarkers in
    human studies select for a change or loss of a normal protein
    produced by specific genes. Mutations at the X-linked hypoxanthine
    guanine phosphoribosyl transferase gene in cloned T-lymphocytes and
    in the autosomal locus for human leucocyte antigen-A (HLA-A) have
    provided information on frequency of mutation and molecular spectra
    of mutants. Detection of haemoglobin variants and loss of the cell
    surface glycoprotein glycophorin A measured in red blood cells have
    such limitations that analysis of the DNA mutations induced cannot
    be made (Compton et al., 1991). The background frequency of each of
    these specific locus assays varies greatly (Lambert, 1992) and is

    dependent on numerous confounding factors (e.g., age and smoking).
    Information on the mutation spectra at a particular locus will be
    extremely useful in elucidating the mechanisms by which mutations
    occur in human cells  in vivo. By comparing spontaneous and
    chemically induced mutational spectra in different populations, the
    etiological contributions of both exogenous and endogenous factors
    to human carcinogenesis could be assessed.

         An alternative approach for the measurement of induced base
    changes which does not require prior selection of a mutant
    population uses the restriction site mutation technique. This is
    based on the detection of DNA sequences resistant to the cutting
    action of specific restriction enzymes, and the amplification of
    these resistant sequences using the polymerase chain reaction. It
    may theoretically be applied to the study of DNA base changes in any
    gene for which the sequence has been determined (Parry et al.,

         More relevant biomarkers for chemically induced cancers would,
    however, preferably measure changes in genes thought to be important
    for cancer. Mutations that activate proto-oncogenes, which stimulate
    growth or inactivate suppressor genes to liberate cells from growth
    constraints, could lead to unregulated proliferation of cancer cells
    (Weinberg, 1991). For the most part, mutations in oncogenes and
    tumour suppressor genes have been characterized in tumour tissue. It
    remains to be determined whether the detection of mutant cells
    against a background of normal cells can be achieved prior to
    clinical diagnosis of cancer. Activated oncogenes have already been
    identified in many human cancers, and considerable progress has been
    made in elucidating the potential role of chemical carcinogens in
    the activation of oncogenes and the contribution of the latter to
    tumorigenesis in animal models (Balmain & Brown, 1988). Brandt-Rauf
    (1991) presented data from pilot studies that demonstrated the
    presence of the p21 protein product of the ras oncogene in the serum
    of 15 out of 18 lung cancer patients who were all current or former
    smokers. The protein was not found in the serum of any of the 18
    healthy non-smoking controls, but was present in 2 out of 8
    clinically healthy smoking controls. However, in another study, p21
    protein was not detected in 20 smokers in a normal population or in
    20 male healthy non-smokers (Brinkworth et al., 1992). The
    mutational spectrum for the tumour suppressor gene p53 in human
    tumours has been reviewed by Hollstein et al. (1991). Mutations of
    the p53 gene are the most common cancer-related genetic changes at
    the gene level and are widespread over the conserved codons of the
    p53 gene. Hence, mutational spectra could be compared for tumours at
    different sites and arising from different etiological backgrounds.
    The mutational spectrum appears to differ among cancers of the
    colon, lung, oesophagus, breast, liver, brain, reticuloendothelial
    tissues and haemopoietic tissues. In two populations where aflatoxin
    B1 exposure was implicated as one of the etiological factors in

    hepatocellular carcinomas, the same mutational hotspot (i.e. G-T
    transversion at codon 249) in the p53 gene has been identified (Hsu
    et al., 1991).

    7.3  Biomarkers for non-genotoxic carcinogenesis

         Although only a few non-genotoxic human carcinogens have been
    recognized (e.g., cyclosporin, diethylstilbestrol and estrogenic
    hormones), many non-genotoxic carcinogens have been identified in
    rodents. A compilation of NTP rodent data, designed to test the
    concordance between short-term tests and  in vivo carcinogenicity
    assays, showed that more than 30% of rodent carcinogens do not test
    positively for genotoxicity (Ashby & Tennant, 1991).

         The mechanisms of action for non-genotoxic carcinogens need to
    be considered in predicting human risk from chemical exposures.
    Although the modes of action of non-genotoxic carcinogens are poorly
    understood, several have been proposed, including immunosuppression,
    hormonal effects, promotion, inorganic carcinogenesis,
    co-carcinogenic effects and solid-state carcinogenesis (Weisburger &
    Williams, 1981). Recently some of these mechanisms were grouped
    under the headings of cytotoxicity and mitogenic growth stimulation
    (Butterworth et al., 1992). Non-genotoxic carcinogens are believed
    to exert their carcinogenic effects through mechanisms that do not
    involve direct binding of the chemical or its metabolites to DNA (UK
    DH, 1989). The key mechanism of non-genotoxic chemicals is to
    increase cell proliferation, either by mitogenesis of the target
    cells or by cytotoxicity, which is followed by regenerative cell
    proliferation (Ramel, 1992). Cohen & Ellwein (1990) suggested that
    non-genotoxic chemicals can be further categorized as to whether or
    not their main mechanism of action is mediated via receptor-binding
    (e.g., dioxin).

         Cell replication and proliferation are potential biomarkers of
    effect. Cell replication is the production of daughter cells by the
    process of replicative DNA synthesis, while cell proliferation is
    the enhanced replication of a selected cell population as observed
    in regenerating tissues. Cells undergoing replicative DNA synthesis
    (S-phase) are the most commonly used markers. The detection of cell
    proliferation involves the incorporation of DNA precursors like
    3H-thymidine or the base analogue 5-bromo-2'-deoxyuridine (BrdU)
    into cellular DNA during S-phase. This is accomplished by
    administering these precursors to animals by injection or through
    implanted osmotic pumps. These S-phase cells can be identified
    histoautotoradiographically or immunohistochemically (Goldsworthy et
    al., 1991). The invasiveness of these techniques currently limits
    their use to animal studies.

         The identification of biomarkers of effect for non-genotoxic
    carcinogens, whose major mechanism of action is via receptor

    occupancy, may be difficult. This is primarily because carcinogens
    of this type activate a variety of genes, some of which may not be
    involved in the carcinogenic process. However, a good example of a
    non-genotoxic carcinogen for which there is a good biomarker of
    effect is 2,3,7,8-tetrachlorinated dibenzo- p-dioxin (TCDD). TCDD
    interacts with a cytosolic receptor that is specific for it and its
    structural analogues (Poland et al., 1976). In addition to
    activating a number of growth factor and growth factor receptor
    genes, TCDD induces a number of enzymes, one of which is cytochrome
    P-450 1A1. Although the induction of this enzyme is probably not
    directly related to the biological mechanism leading to cancer from
    TCDD exposure, it is a sensitive marker for exposure (Tritscher et
    al., 1992).


         This chapter focuses on the genetic predisposition of an
    individual as it affects susceptibility to chemical materials. There
    are a number of external factors, such as age, diet and health
    status, that can also influence the susceptibility of an individual
    exposed to chemicals. Some discussion will be directed towards the
    effects of previous exposure on subsequent susceptibility, such as
    to sensitization and enzyme induction/inhibition by previous
    exposure. Table 3 lists some genetic and acquired factors affecting
    susceptibility (Calabrese, 1986).

         Although individuals may experience similar environmental
    exposures, genetic differences in metabolism may produce markedly
    different doses at the target site and thus a different level of
    response. Even when target doses are similar, markedly different
    responses may be noted in individuals due to varying degrees of
    inherent biological responsiveness. Biomarkers of susceptibility may
    reflect the acquired or genetic factors that influence the response
    to exposure. These are pre-existing factors and are independent of
    the exposure. They are predominantly genetic in origin, although
    disease, physiological changes, medication and exposure to other
    environmental agents may also alter individual susceptibility.
    Biomarkers of susceptibility identify those individuals in a
    population who have an acquired or genetic difference in
    susceptibility to the effects of chemical exposure.

         Biomarkers of susceptibility indicate which factors may
    increase or decrease an individual's risk of developing a toxic
    response following exposure to an environmental agent. Polymorphism
    is present for some metabolic activation/deactivation enzymes,
    including cytochrome P-450 isozymes (Nebert, 1988a, 1988b) and at
    least one form of glutathione transferase (Seidegard et al., 1990).
    Differing rates of enzyme activity controlling the activation or
    detoxification of xenobiotics lead to differences in susceptibility
    by increasing or decreasing the biologically effective dose of the
    environmental agent.

         The effect may vary between ethnic groups. For instance, there
    are approximately equal numbers of fast and slow acetylator
    phenotypes in a Caucasian population, whereas in a Japanese
    population the distribution is 10% slow acetylators and 90% fast.
    Genetic polymorphisms for drug metabolism have been widely studied
    using phenotypic assays which involves measuring drug clearance in
    individuals. Differential rates of metabolism will affect the
    distribution and persistence of metabolites, which may have
    implications for the site of toxicity. Epidemiological studies
    suggest that, with respect to aromatic amines, slow acetylators are
    more likely to contract bladder cancer but are at  decreased risk
    for colo-rectal cancer (Guengerich, 1991; Kadlubar et al., 1992).

        Table 3.  Some examples of established and suspected biomarkers of susceptibilitya
    Biomarker of susceptibility                       Environmental agent                             Disease


    Debrisoquine hydroxylation phenotype              cigarette smoke                                 lung cancer
    Acetylator phenotype                              aflatoxin,                                      liver cancer,
                                                      aromatic amines                                 bladder cancer
    Ataxia telangiectasia genotype                    bleomycin, epoxides                             cancer at various sites
    Xeroderma pigmentosum genotype                    agents that cause oxidative damage to           skin cancer,
                                                      DNA, PAH, aromatic amines and aflatoxin B1      other cancers
    Arylhydrocarbon hydroxylase inducibility          polycyclic aromatic hydrocarbons                lung cancer
    alpha-1-antitrypsin                               cigarette smoke                                 pulmonary emphysema
    Franconi's anaemia phenotype                      cross-linking agents                            acute leukaemia
    Glucose-6P-dehydrogenese deficiency phenotype     oxidative agents, aromatic amines,              poor resistance to oxidative 
                                                      nitro-aromatic compounds                        stress,
                                                                                                      aromatic amines
    Sickle cell phenotype                             aromatic amino and nitro compounds,             anaemia
                                                      carbon monoxide, cyanide
    Thalassemia phenotype                             lead, benzene                                   anaemia
    Erythrocyte porphyria                             chloroquine, hexachlorobenzene, lead,           anaemia
                                                      various drugs including barbituates,
                                                      sulfonamides, others

    Sulfite oxidase deficiency heterozygotes          sulfite, bisulfite, sulfur dioxide              pulmonary disease
    Alcohol dehydrogenase variant                     metabolize alcohols (e.g., ethanol)
                                                      more quickly than normal
    GSTµ phenotype                                    cigarette smoke                                 lung cancer
    Pseudocholinesterase variants                     organophosphate and carbamate insecticides,     neurotoxicity
                                                      muscle relaxant drugs
    IgA deficiency                                    respiratory irritants                           irritation of respiratory tract
    phenyl ketones in urine                           precursors of phenyl ketones                    phenylketonuria


    Table 3 (contd)
    Biomarker of susceptibility                       Environmental agent                             Disease


    Deficient diet                                    chemical                                        decreased resistance to
                                                                                                      effects of many chemicals 
    Induced P-45O IIE1                                alcohol consumption                             cancer at various sites
    Antigen-specific antibodies                       chemicals, dusts                                decreased pulmonary
                                                                                                      functions, skin rashes


    a  Modified from Calabrese (1986) 

    Polymorphism of N-oxidation has been linked to susceptibility to
    colonic cancer (Kadlubar et al., 1992) and polymorphism in
    glutathione  S-transferase to increased lung cancer, particularly
    adenocarcinoma (Seidegard et al., 1990).

         The methodology for determining the phenotypes of individuals
    for polymorphisms in metabolizing genes requires the administration
    of a relevant test drug to the person and the subsequent measurement
    of its clearance from the body. More recently, techniques based on
    polymerase chain reactions, using DNA isolated from lymphocytes and
    other cells, have been developed which allow the detection of
    genetypes of known polymorphisms involving a variety of
    xenobiotic-metabolizing enzymes, including GST1
    (gluthione- S-transferase µ) and NAT2( N-acetyl transferase), as
    well as two cytochrome P-450 isoenzymes: CYP1A1 and CYP2D6 (Bell,
    1991; Blum et al., 1991; Wolf et al., 1992; Hirvonen et al., 1992;
    Hollstein et al., 1992).

         Cigarette smoking provides another example that illustrates the
    effect of genetic polymorphisms on the response to chemicals.
    Cigarette smoking is associated with the development of lung cancer
    but not all smokers get lung cancer. This appears to be due in part
    to genetic variations in arylhydrocarbon hydroxylase activity, which
    results in a large variability in the binding of benz [a]pyrene to
    DNA in cigarette smokers. A genetically based low level of
    alpha-1-anti-trypsin activity greatly increases the risk of
    emphysema from cigarette smoking.

         In some situations a genetic trait may make an individual more
    susceptible to one environmental agent but less so to another. For
    example, the sickle cell trait predisposes to anaemia and altitude
    sickness but offers some protection to the individual from infection
    by the malaria parasite. Inherent differences in susceptibility
    depend upon variations in the function of genes controlling enzyme
    activity or the production of other proteins. Although a genotoxic
    agent may reach the target tissue, the significance of any
    chromosomal break will depend on the efficiency of the DNA repair
    mechanisms. In xeroderma pigmentosum the individual is at an
    increased risk of skin cancer after exposure to UV light because of
    an inherited defect in DNA repair proteins (Cleaver, 1969).
    Heterozygotes also have an increased risk of cancer and so the
    frequency of the gene may affect the incidence of this cancer. Other
    inherited diseases (e.g., ataxia-telangiectasia) that affect the
    efficiency of DNA replication or repair may affect susceptibility to
    carcinogenic agents (Swift et al., 1992). UV-DNA repair capacity has
    been found to be lower in the lymphocytes isolated from individuals
    with basal cell and squamous cell carcinoma than in the case of
    their age-matched controls, and this repair capacity has been found
    to decrease with increasing age in both groups (Wei et al., 1993).

         Another form of susceptibility has an immunological basis.
    Prior exposure to a chemical may induce an immune response that
    sensitizes the individual to subsequent exposures. Such responses
    occur in only a small fraction of the exposed population; an example
    is the development of pulmonary hypersensitivity to industrial
    agents such as toluene diisocyanate or cotton dust. The biomarkers
    of susceptibility are the antigen-specific antibodies developed
    against the chemical.

    9.  SUMMARY

         The Task Group considered biomarkers in three categories,
    biomarkers of exposure, of effect, and of susceptibility, while
    recognising that clear distinction of category is often not

         The Task Group agreed that the use of biomarkers can improve,
    and should be used in, the process for the assessment of human
    health risks caused by exposure to chemicals.  Biomarkers may be
    applied to the estimation of exposure and internal dose in
    individuals and in groups and may allow identification of those at
    greater or lesser risk than average.

         Biomarkers must be validated before application in the risk
    assessment process, i.e. the relationship between the biomarker, the
    exposure, and the health outcome must be established. The selection,
    validation and application of any biomarker is a complicated
    process, which will vary for different markers. Examples were
    selected to illustrate the concepts and principles. 

         Research and use of biomarkers involves complex ethical, social
    and legal issues, which may vary in different countries. These
    issues may impose constraints on research and use of biomarkers in
    risk assessment and risk management decisions. The ethical, social
    and legal aspects of biomarkers require careful consideration prior
    to any application.


         In making the following recommendations, the Task Group
    recognized the role given IPCS to facilitate and increase
    coordination of international activities in order to promote the
    further work needed to define human health effects associated with
    exposure to chemicals and to provide the basis for priority-setting
    actions in order to protect public health.

    10.1  General

    *    To promote the wider use of validated biomarkers in the
         risk-assessment process

    *    To promote interdisciplinary cooperation and communication in
         order to facilitate application of research findings

    *    To examine the feasibility of developing a data bank of
         information on biomarkers applied to the process of risk and
         their applications

    10.2  Research

    *    To develop, refine and validate models to relate biomarkers of
         exposure and of effect, qualitatively and quantitatively, to
         exposure and to health outcome, particularly for end-points
         other than cancer

    *    To identify and validate biomarkers of susceptibility in
         relation to the chemical, and to inter-individual variation in
         response, and investigate genetic polymorphism as a basis for
         individual hypersensitivity

    *    To assess the use of information on individual susceptibility
         in relation to protection of health with due respect to the
         ethical, social and legal issues

    *    To develop strategies to link exposure and internal dose with
         human health outcome by integration of mechanistically
         validated biomarkers of exposure, effect and susceptibility

    10.3  Applications

    *    To develop a practical protocol for use of biomarkers in human
         studies, taking into account scientific, emotional, ethical,
         legal and social aspects and including guidelines for risk
         communication, with emphasis on the right of participants to
         non-biased, intelligible information

    *    To encourage the production of certified reference materials
         for biomarker analyses and support the functioning of
         international quality assurance programmes

    *    To include consideration of biomarkers of exposure, effect, and
         susceptibility in future Environmental Health Criteria

    *    To consider the need to update this monograph on principles and
         concepts of biomarkers at an early date


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         Le groupe spécial a divisé les marqueurs biologiques en trois
    catégories, les marqueurs d'exposition, les marqueurs d'effet et les
    marqueurs de réceptivité, tout en admettant que bien souvent il
    n'était pas possible d'établir une distinction nette entre ces
    diverses catégories.

         Le groupe spécial a admis que le recours aux marqueurs
    biologiques pouvait améliorer l'évaluation des risques pour la santé
    humaine découlant d'une exposition à des produits chimiques et qu'il
    fallait donc en faire usage. Les marqueurs biologiques peuvent être
    utilisés pour évaluer l'exposition et la dose interne chez des
    individus et des groupes et peuvent faciliter l'identification de
    ceux de ces individus ou de ces groupes qui sont plus ou moins
    exposés aux risques que la moyenne.

         Avant d'utiliser les marqueurs biologiques pour l'évaluation du
    risque il faut les valider, c'est-à-dire établir la relation qui
    existe entre le marqueur, l'exposition et ses conséquences pour la
    santé. Le choix, la validation et l'utilisation de tout marqueur
    biologique constituent des processus complexes qui varient d'un
    marqueur à l'autre. Pour mettre en lumière ces notions et ces
    principes on a choisi un certain nombre d'exemples.

         La recherche et l'utilisation des marqueurs biologiques
    soulèvent des questions complexes sur le plan éthique, social et
    juridique, qui peuvent d'ailleurs varier d'un pays à l'autre. Il
    peut s'en suivre un certain nombre de contraintes imposées à la
    recherche et à l'utilisation des marqueurs biologiques dans
    l'évaluation des risques et dans les décisions relatives à la
    gestion de ces risques. Avant toute application il importe d'étudier
    avec soin les aspects éthiques, sociaux et juridiques des marqueurs


         En formulant les recommandations ci-après, le groupe de travail
    a pris en considération le rôle dévolu au PISC, à savoir de
    faciliter et de développer la coordination des activités
    internationales afin d'encourager les travaux à poursuivre pour
    définir les effets sur la santé humaine qu'entraîne l'exposition aux
    substances chimiques et de jeter les bases des actions prioritaires
    à entreprendre pour protéger la santé publique.

    1.  Généralités

    *    Encourager un plus large recours aux marqueurs biologiques dans
         l'évaluation des risques

    *    Favoriser la coopération et la communication
         inter-disciplinaires afin de faciliter l'application des
         résultats de la recherche

    *    Etudier la faisabilité d'une banque de données sur les
         marqueurs biologiques dans l'évaluation des risques et ses

    2.  Recherche

    *    Mettre au point, affiner et valider des modèles qui permettent
         de corréler les marqueurs biologiques de l'exposition et des
         effets tant qualitativement que quantitativement, à
         l'exposition et aux conséquences biologiques, en particulier
         aux conséquences autres que le cancer.

    *    Recenser et valider des marqueurs biologiques de réceptivité,
         par rapport à la réaction aux produits chimiques et aux
         variations interindividuelles à cette réaction et étudier le
         polymorphisme génétique en tant que base de l'hypersensibilité

    *    Voir dans quelle mesure il est possible d'utiliser certains
         renseignements sur la réceptivité individuelle pour la
         protection de la santé, dans le respect des impératifs
         éthiques, sociaux et juridiques.

    *    Mettre au point des stratégies afin de relier l'exposition et
         la dose interne aux conséquences biologiques pour l'homme
         grâceà une synthèse de biomarqueurs de l'exposition, de l'effet
         et de la réceptivité, mécanistiquement validés.

    3.  Applications

    *    Mettre au point un protocole pratique pour l'utilisation des
         marqueurs biologiques dans les études sur l'homme, qui prenne
         en considération les aspects scientifiques, émotionnels,
         éthiques, juridiques et sociaux et qui comporte des directives
         en matière de communication, en insistant sur le droit des
         participants à disposer d'informations intelligibles et non

    *    Encourager la production de substances de référence homologuées
         pour l'analyse des marqueurs biologiques et aider les
         programmes internationaux d'assurance de la qualité à

    *    Faire figurer des considérations sur les biomarqueurs
         d'exposition, d'effet et de réceptivité dans les futures
         monographies de la série Critères d'hygiène de l'environnement.

    *    Examiner s'il est nécessaire de mettre à jour à bref délai la
         présente monographie consacrée aux principes et conceptions en
         matière de marqueurs biologiques.


         El Grupo Especial distinguió tres clases de biomarcadores: de
    exposición, de efecto y de susceptibilidad, reconociendo sin embargo
    que a menudo resulta imposible establecer claramente la pertenencia
    a una de esas clases.

         El Grupo Especial coincidió en que los biomarcadores permiten
    mejorar, y deben emplearse a ese efecto, el proceso de evaluación de
    los riesgos que para la salud humana conlleva la exposición a
    productos químicos. Los biomarcadores se pueden emplear para
    calcular la exposición y la dosis interna recibida por individuos y
    grupos, con la consiguiente identificación de quienes sufren un
    mayor o menor riesgo que la media.

         Los biomarcadores deben ser validados antes de aplicarlos a la
    evaluación del riesgo, lo que significa que debe determinarse la
    relación entre el biomarcador, la exposición y el estado de salud.
    La selección, validación y empleo de cualquier biomarcador es un
    proceso complicado, distinto para cada marcador. Se eligieron
    algunos ejemplos para ilustrar los conceptos y principios

         La investigación y el empleo de los biomarcadores plantea
    complejas cuestiones éticas, sociales y jurídicas, que pueden
    diferir de un país a otro. Algunos de esos problemas limitan el
    alcance de las investigaciones sobre los biomarcadores y de su
    aplicación a la evaluación de riesgos y la adopción de decisiones
    relacionadas con la gestión de riesgos. Los problemas éticos,
    sociales y jurídicos que plantean los biomarcadores deben ser objeto
    de un detenido análisis antes de su eventual uso.


         Al formular las siguientes recomendaciones, el Grupo Especial
    reconoció la función asignada al IPCS de facilitar e intensificar la
    coordinación de las actividades internacionales con miras a fomentar
    los trabajos que aún será necesario realizar para determinar los
    efectos sobre la salud relacionados con la exposición a productos
    químicos, así como para establecer las bases que permitan señalar
    las prioridades a que haya que atenerse para proteger la salud

    1.  Recomendaciones generales

    *    Promover un mayor uso de los biomarcadores validados en el
         proceso de evaluación de riesgos.

    *    Fomentar la cooperación y la comunicación interdisciplinarias
         para facilitar la aplicación de los resultados de las

    *    Estudiar la posibilidad de crear un banco de datos sobre
         biomarcadores aplicados a la evaluación de riesgos y sus
         posibles usos.

    2.  Investigaciones

    *    Desarrollar, perfeccionar y validar modelos aptos para
         relacionar los biomarcadores de exposición y de efecto,
         cualitativa y cuantitativamente, con la exposición y con el
         estado de salud, sobre todo para puntos finales distintos del

    *    Identificar y validar biomarcadores de susceptibilidad en
         relación con el producto químico y con la variación
         interindividual de la respuesta, e investigar la influencia del
         polimorfismo genético en la hipersensibilidad individual.

    *    Evaluar el uso de la información referente a la susceptibilidad
         individual desde la perspectiva de una protección de la salud
         atenta a los aspectos éticos, sociales y jurídicos.

    *    Formular estrategias para relacionar la exposición y la dosis
         interna con el estado de salud mediante la integración de
         biomarcadores de exposición, efecto y susceptibilidad validados

    3.  Aplicaciones

    *    Elaborar un protocolo práctico para el uso de biomarcadores en
         los estudios realizados en el hombre, teniendo en cuenta los
         aspectos científicos, psicológicos, éticos, jurídicos y
         sociales, con inclusión de directrices para la comunicación del
         riesgo y haciendo hincapié en el derecho de los participantes a
         una información inteligible e imparcial.

    *    Fomentar la producción de material de referencia certificado
         para el análisis de biomarcadores y respaldar la aplicación de
         programas internacionales de garantía de la calidad.

    *    Incluir la consideración de los biomarcadores de exposición,
         efecto y susceptibilidad en las futuras monografías de la serie
         Criterios de Salud Ambiental.

    *    Tener presente la necesidad de actualizar con prontitud la
         presente monografía sobre los principios y nociones relativos a
         los biomarcadores.

    See Also:
       Toxicological Abbreviations