The word biomarker is short for biological marker, a term that refers to a measurable event occurring in a biological system, such as the human body. This event is then interpreted as a reflection, or marker, of a more general state of the organism or of life expectancy. In occupational health, a biomarker is generally used as an indicator of health status or disease risk.

Biomarkers are used for in vitro as well as in vivo studies that may include humans. Usually, three specific types of biological markers are identified. Although a few biomarkers may be difficult to classify, usually they are separated into biomarkers of exposure, biomarkers of effect or biomarkers of susceptibility (see table 1).

Table 1. Examples of biomarkers of exposure or biomarkers of effect  that are used in toxicological studies in occupational health

Given an acceptable degree of validity, biomarkers may be employed for several purposes. On an individual basis, a biomarker may be used to support or refute a diagnosis of a particular type of poisoning or other chemically-induced adverse effect. In a healthy subject, a biomarker may also reflect individual hypersusceptibility to specific chemical exposures and may therefore serve as a basis for risk prediction and counselling. In groups of exposed workers, some exposure biomarkers can be applied to assess the extent of compliance with pollution abatement regulations or the effectiveness of preventive efforts in general.

Biomarkers of Exposure

An exposure biomarker may be an exogenous compound (or a metabolite) within the body, an interactive product between the compound (or metabolite) and an endogenous component, or another event related to the exposure. Most commonly, biomarkers of exposures to stable compounds, such as metals, comprise measurements of the metal concentrations in appropriate samples, such as blood, serum or urine. With volatile chemicals, their concentration in exhaled breath (after inhalation of contamination-free air) may be assessed. If the compound is metabolized in the body, one or more metabolites may be chosen as a biomarker of the exposure; metabolites are often determined in urine samples.

Modern methods of analysis may allow separation of isomers or congeners of organic compounds, and determination of the speciation of metal compounds or isotopic ratios of certain elements. Sophisticated analyses allow determination of changes in the structure of DNA or other macromolecules caused by binding with reactive chemicals. Such advanced techniques will no doubt gain considerably in importance for applications in biomarker studies, and lower detection limits and better analytical validity are likely to make these biomarkers even more useful.

Particularly promising developments have occurred with biomarkers of exposure to mutagenic chemicals. These compounds are reactive and may form adducts with macromolecules, such as proteins or DNA. DNA adducts may be detected in white blood cells or tissue biopsies, and specific DNA fragments may be excreted in the urine. For example, exposure to ethylene oxide results in reactions with DNA bases, and, after excision of the damaged base, N-7-(2-hydroxyethyl)guanine will be eliminated in the urine. Some adducts may not refer directly to a particular exposure. For example, 8-hydroxy-2´-deoxyguanosine reflects oxidative damage to DNA, and this reaction may be triggered by several chemical compounds, most of which also induce lipid peroxidation.

Other macromolecules may also be changed by adduct formation or oxidation. Of special interest, such reactive compounds may generate haemoglobin adducts that can be determined as biomarkers of exposure to the compounds. The advantage is that ample amounts of haemoglobin can be obtained from a blood sample, and, given the four-month lifetime of red blood cells, the adducts formed with the amino acids of the protein will indicate the total exposure during this period.

Adducts may be determined by sensitive techniques such as high-performance lipid chromatography, and some immunological methods are also available. In general, the analytical methods are new, expensive and need further development and validation. Better sensitivity can be obtained by using the ³²P post labelling assay, which is a nonspecific indication that DNA damage has taken place. All of these techniques are potentially useful for biological monitoring and have been applied in a growing number of studies. However, simpler and more sensitive analytical methods are needed. Given the limited specificity of some methods at low-level exposures, tobacco smoking or other factors may impact significantly on the measurement results, thus causing difficulties in interpretation.

Exposure to mutagenic compounds, or to compounds which are metabolized into mutagens, may also be determined by assessing the mutagenicity of the urine from an exposed individual. The urine sample is incubated with a strain of bacteria in which a specific point mutation is expressed in a way that can be easily measured. If mutagenic chemicals are present in the urine sample, then an increased rate of mutations will occur in the bacteria.

Exposure biomarkers must be evaluated with regard to temporal variation in exposure and the relation to different compartments. Thus, the time frame(s) represented by the biomarker, that is, the extent to which the biomarker measurement reflects past exposure(s) and/or accumulated body burden, must be determined from toxicokinetic data in order to interpret the result. In particular, the degree to which the biomarker indicates retention in specific target organs should be considered. Although blood samples are often used for biomarker studies, peripheral blood is generally not regarded as a compartment as such, although it acts as a transport medium between compartments. The degree to which the concentration in the blood reflects levels in different organs varies widely between different chemicals, and usually also depends upon the length of the exposure as well as time since exposure.

Sometimes this type of evidence is used to classify a biomarker as an indicator of (total) absorbed dose or an indicator of effective dose (i.e., the amount that has reached the target tissue). For example, exposure to a particular solvent may be evaluated from data on the actual concentration of the solvent in the blood at a particular time following the exposure. This measurement will reflect the amount of the solvent that has been absorbed into the body. Some of the absorbed amount will be exhaled due to the vapour pressure of the solvent. While circulating in the blood, the solvent will interact with various components of the body, and it will eventually become subject to breakdown by enzymes. The outcome of the metabolic processes can be assessed by determining specific mercapturic acids produced by conjugation with glutathione. The cumulative excretion of mercapturic acids may better reflect the effective dose than will the blood concentration.

Life events, such as reproduction and senescence, may affect the distribution of a chemical. The distribution of chemicals within the body is significantly affected by pregnancy, and many chemicals may pass the placental barrier, thus causing exposure of the foetus. Lactation may result in excretion of lipid-soluble chemicals, thus leading to a decreased retention in the mother along with an increased uptake by the infant. During weight loss or development of osteoporosis, stored chemicals may be released, which can then result in a renewed and protracted “endogenous” exposure of target organs. Other factors may affect individual absorption, metabolism, retention and distribution of chemical compounds, and some biomarkers of susceptibility are available (see below).

Biomarkers of Effect

A marker of effect may be an endogenous component, or a measure of the functional capacity, or some other indicator of the state or balance of the body or organ system, as affected by the exposure. Such effect markers are generally preclinical indicators of abnormalities.

These biomarkers may be specific or non-specific. The specific biomarkers are useful because they indicate a biological effect of a particular exposure, thus providing evidence that can potentially be used for preventive purposes. The non-specific biomarkers do not point to an individual cause of the effect, but they may reflect the total, integrated effect due to a mixed exposure. Both types of biomarkers may therefore be of considerable use in occupational health.

There is not a clear distinction between exposure biomarkers and effect biomarkers. For example, adduct formation could be said to reflect an effect rather than the exposure. However, effect biomarkers usually indicate changes in the functions of cells, tissues or the total body. Some researchers include gross changes, such as an increase in liver weight of exposed laboratory animals or decreased growth in children, as biomarkers of effect. For the purpose of occupational health, effect biomarkers should be restricted to those that indicate subclinical or reversible biochemical changes, such as inhibition of enzymes. The most frequently used effect biomarker is probably inhibition of cholinesterase caused by certain insecticides, that is, organophosphates and carbamates. In most cases, this effect is entirely reversible, and the enzyme inhibition reflects the total exposure to this particular group of insecticides.

Some exposures do not result in enzyme inhibition but rather in increased activity of an enzyme. This is the case with several enzymes that belong to the P450 family (see “Genetic determinants of toxic response”). They may be induced by exposures to certain solvents and polyaromatic hydrocarbons (PAHs). Since these enzymes are mainly expressed in tissues from which a biopsy may be difficult to obtain, the enzyme activity is determined indirectly in vivo by administering a compound that is metabolized by that particular enzyme, and then the breakdown product is measured in urine or plasma.

Other exposures may induce the synthesis of a protective protein in the body. The best example is probably metallothionein, which binds cadmium and promotes the excretion of this metal; cadmium exposure is one of the factors that result in increased expression of the metallothionein gene. Similar protective proteins may exist but have not yet been explored sufficiently to become accepted as biomarkers. Among the candidates for possible use as biomarkers are the so-called stress proteins, originally referred to as heat shock proteins. These proteins are generated by a range of different organisms in response to a variety of adverse exposures.

Oxidative damage may be assessed by determining the concentration of malondialdehyde in serum or the exhalation of ethane. Similarly, the urinary excretion of proteins with a small molecular weight, such as albumin, may be used as a biomarker of early kidney damage. Several parameters routinely used in clinical practice (for example, serum hormone or enzyme levels) may also be useful as biomarkers. However, many of these parameters may not be sufficiently sensitive to detect early impairment.

Another group of effect parameters relate to genotoxic effects (changes in the structure of chromosomes). Such effects may be detected by microscopy of white blood cells that undergo cell division. Serious damage to the chromosomes—chromosomal aberrations or formation of micronuclei—can be seen in a microscope. Damage may also be revealed by adding a dye to the cells during cell division. Exposure to a genotoxic agent can then be visualized as an increased exchange of the dye between the two chromatids of each chromosome (sister chromatid exchange). Chromosomal aberrations are related to an increased risk of developing cancer, but the significance of an increased rate of sister chromatid exchange is less clear.

More sophisticated assessment of genotoxicity is based on particular point mutations in somatic cells, that is, white blood cells or epithelial cells obtained from the oral mucosa. A mutation at a specific locus may make the cells capable of growing in a culture that contains a chemical that is otherwise toxic (such as 6-thioguanine). Alternatively, a specific gene product can be assessed (e.g., serum or tissue concentrations of oncoproteins encoded by particular oncogenes). Obviously, these mutations reflect the total genotoxic damage incurred and do not necessarily indicate anything about the causative exposure. These methods are not yet ready for practical use in occupational health, but rapid progress in this line of research would suggest that such methods will become available within a few years.

Biomarkers of Susceptibility

A marker of susceptibility, whether inherited or induced, is an indicator that the individual is particularly sensitive to the effect of a xenobiotic or to the effects of a group of such compounds. Most attention has been focused on genetic susceptibility, although other factors may be at least as important. Hypersusceptibility may be due to an inherited trait, the constitution of the individual, or environmental factors.

The ability to metabolize certain chemicals is variable and is genetically determined (see “Genetic determinants of toxic response”). Several relevant enzymes appear to be controlled by a single gene. For example, oxidation of foreign chemicals is mainly carried out be a family of enzymes belonging to the P450 family. Other enzymes make the metabolites more water soluble by conjugation (e.g., N-acetyltransferase and μ-glutathion-S-transferase). The activity of these enzymes is genetically controlled and varies considerably. As mentioned above, the activity can be determined by administering a small dose of a drug and then determining the amount of the metabolite in the urine. Some of the genes have now been characterized, and techniques are available to determine the genotype. Important studies suggest that a risk of developing certain cancer forms is related to the capability of metabolizing foreign compounds. Many questions still remain unanswered, thus at this time limiting the use of these potential susceptibility biomarkers in occupational health.

Other inherited traits, such as alpha₁-antitrypsin deficiency or glucose-6-phosphate dehydrogenase deficiency, also result in deficient defence mechanisms in the body, thereby causing hypersusceptibility to certain exposures.

Most research related to susceptibility has dealt with genetic predisposition. Other factors play a role as well and have been partly neglected. For example, individuals with a chronic disease may be more sensitive to an occupational exposure. Also, if a disease process or previous exposure to toxic chemicals has caused some subclinical organ damage, then the capacity to withstand a new toxic exposure is likely to be less. Biochemical indicators of organ function may in this case be used as susceptibility biomarkers. Perhaps the best example regarding hypersusceptibility relates to allergic responses. If an individual has become sensitized to a particular exposure, then specific antibodies can be detected in serum. Even if the individual has not become sensitized, other current or past exposures may add to the risk of developing an adverse effect related to an occupational exposure.

A major problem is to determine the joint effect of mixed exposures at work. In addition, personal habits and drug use may result in an increased susceptibility. For example, tobacco smoke usually contains a considerable amount of cadmium. Thus, with occupational exposure to cadmium, a heavy smoker who has accumulated substantial amounts of this metal in the body will be at increased risk of developing cadmium-related kidney disease.

Application in Occupational Health

Biomarkers are extremely useful in toxicological research, and many may be applicable in biological monitoring. Nonetheless, the limitations must also be recognized. Many biomarkers have so far been studied only in laboratory animals. Toxicokinetic patterns in other species may not necessarily reflect the situation in human beings, and extrapolation may require confirmatory studies in human volunteers. Also, account must be taken of individual variations due to genetic or constitutional factors.

In some cases, exposure biomarkers may not at all be feasible (e.g., for chemicals which are short-lived in vivo). Other chemicals may be stored in, or may affect, organs which cannot be accessed by routine procedures, such as the nervous system. The route of exposure may also affect the distribution pattern and therefore also the biomarker measurement and its interpretation. For example, direct exposure of the brain via the olfactory nerve is likely to escape detection by measurement of exposure biomarkers. As to effect biomarkers, many of them are not at all specific, and the change can be due to a variety of causes, including lifestyle factors. Perhaps in particular with the susceptibility biomarkers, interpretation must be very cautious at the moment, as many uncertainties remain about the overall health significance of individual genotypes.

In occupational health, the ideal biomarker should satisfy several requirements. First of all, sample collection and analysis must be simple and reliable. For optimal analytical quality, standardization is needed, but the specific requirements vary considerably. Major areas of concern include: preparation of the in- dividual, sampling procedure and sample handling, and measurement procedure; the latter encompasses technical factors, such as calibration and quality assurance procedures, and individual- related factors, such as education and training of operators.

For documentation of analytical validity and traceability, reference materials should be based on relevant matrices and with appropriate concentrations of toxic substances or relevant metabolites at appropriate levels. For biomarkers to be used for biological monitoring or for diagnostic purposes, the responsible laboratories must have well-documented analytical procedures with defined performance characteristics, and accessible records to allow verification of the results. At the same time, nonetheless, the economics of characterizing and using reference materials to supplement quality assurance procedures in general must be considered. Thus, the achievable quality of results, and the uses to which they are put, have to be balanced against the added costs of quality assurance, including reference materials, manpower and instrumentation.

Another requirement is that the biomarker should be specific, at least under the circumstances of the study, for a particular type of exposure, with a clear-cut relationship to the degree of exposure. Otherwise, the result of the biomarker measurement may be too difficult to interpret. For proper interpretation of the measurement result of an exposure biomarker, the diagnostic validity must be known (i.e., the translation of the biomarker value into the magnitude of possible health risks). In this area, metals serve as a paradigm for biomarker research. Recent research has demonstrated the complexity and subtlety of dose-response relationships, with considerable difficulty in identifying no-effect levels and therefore also in defining tolerable exposures. However, this kind of research has also illustrated the types of investigation and the refinement that are necessary to uncover the relevant information. For most organic compounds, quantitative associations between exposures and the corresponding adverse health effects are not yet available; in many cases, even the primary target organs are not known for sure. In addition, evaluation of toxicity data and biomarker concentrations is often complicated by exposure to mixtures of substances, rather than exposure to a single compound at the time.

Before the biomarker is applied for occupational health purposes, some additional considerations are necessary. First, the biomarker must reflect a subclinical and reversible change only. Second, given that the biomarker results can be interpreted with regard to health risks, then preventive efforts should be available and should be considered realistic in case the biomarker data suggests a need to reduce the exposure. Third, the practical use of the biomarker must be generally regarded as ethically acceptable.

Industrial hygiene measurements may be compared with applicable exposure limits. Likewise, results on exposure biomarkers or effect biomarkers may be compared to biological action limits, sometimes referred to as biological exposure indices. Such limits should be based on the best advice of clinicians and scientists from appropriate disciplines, and responsible administrators as “risk managers” should then take into account relevant ethical, social, cultural and economic factors. The scientific basis should, if possible, include dose-response relationships supplemented by information on variations in susceptibility within the population at risk. In some countries, workers and members of the general public are involved in the standard-setting process and provide important input, particularly when scientific uncertainty is considerable. One of the major uncertainties is how to define an adverse health effect that should be prevented—for example, whether adduct formation as an exposure biomarker by itself represents an adverse effect (i.e., effect biomarker) that should be prevented. Difficult questions are likely to arise when deciding whether it is ethically defensible, for the same compound, to have different limits for adventitious exposure, on the one hand, and occupational exposure, on the other.

The information generated by the use of biomarkers should generally be conveyed to the individuals examined within the physician-patient relationship. Ethical concerns must in particular be considered in connection with highly experimental biomarker analyses that cannot currently be interpreted in detail in terms of actual health risks. For the general population, for example, limited guidance exists at present with regard to interpretation of exposure biomarkers other than the blood-lead concentration. Also of importance is the confidence in the data generated (i.e., whether appropriate sampling has been done, and whether sound quality assurance procedures have been utilized in the laboratory involved). An additional area of special worry relates to individual hypersusceptibility. These issues must be taken into account when providing the feedback from the study.

All sectors of society affected by, or concerned with carrying out, a biomarker study need to be involved in the decision-making process on how to handle the information generated by the study. Specific procedures to prevent or overcome inevitable ethical conflicts should be developed within the legal and social frameworks of the region or country. However, each situation represents a different set of questions and pitfalls, and no single procedure for public involvement can be developed to cover all applications of exposure biomarkers.

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The study and characterization of chemicals and other agents for toxic properties is often undertaken on the basis of specific organs and organ systems. In this chapter, two targets have been selected for in-depth discussion: the immune system and the gene. These examples were chosen to represent a complex target organ system and a molecular target within cells. For more comprehensive discussion of the toxicology of target organs, the reader is referred to standard toxicology texts such as Casarett and Doull, and Hayes. The International Programme on Chemical Safety (IPCS) has also published several criteria documents on target organ toxicology, by organ system.

Target organ toxicology studies are usually undertaken on the basis of information indicating the potential for specific toxic effects of a substance, either from epidemiological data or from general acute or chronic toxicity studies, or on the basis of special concerns to protect certain organ functions, such as reproduction or foetal development. In some cases, specific target organ toxicity tests are expressly mandated by statutory authorities, such as neurotoxicity testing under the US pesticides law (see “The United States approach to risk assessment of reproductive toxicants and neurotoxic agents,” and mutagenicity testing under the Japanese Chemical Substance Control Law (see “Principles of hazard identification: The Japanese approach”).

As discussed in “Target organ and critical effects,” the identification of a critical organ is based upon the detection of the organ or organ system which first responds adversely or to the lowest doses or exposures. This information is then used to design specific toxicology investigations or more defined toxicity tests that are designed to elicit more sensitive indications of intoxication in the target organ. Target organ toxicology studies may also be used to determine mechanisms of action, of use in risk assessment (see “The United States approach to risk assessment of reproductive toxicants and neurotoxic agents”).

Methods of Target Organ Toxicity Studies

Target organs may be studied by exposure of intact organisms and detailed analysis of function and histopathology in the target organ, or by in vitro exposure of cells, tissue slices, or whole organs maintained for short or long term periods in culture (see “Mechanisms of toxicology: Introduction and concepts”). In some cases, tissues from human subjects may also be available for target organ toxicity studies, and these may provide opportunities to validate assumptions of cross-species extrapolation. However, it must be kept in mind that such studies do not provide information on relative toxicokinetics.

In general, target organ toxicity studies share the following common characteristics: detailed histopathological examination of the target organ, including post mortem examination, tissue weight, and examination of fixed tissues; biochemical studies of critical pathways in the target organ, such as important enzyme systems; functional studies of the ability of the organ and cellular constituents to perform expected metabolic and other functions; and analysis of biomarkers of exposure and early effects in target organ cells.

Detailed knowledge of target organ physiology, biochemistry and molecular biology may be incorporated in target organ studies. For instance, because the synthesis and secretion of small-molecular-weight proteins is an important aspect of renal function, nephrotoxicity studies often include special attention to these parameters (IPCS 1991). Because cell-to-cell communication is a fundamental process of nervous system function, target organ studies in neurotoxicity may include detailed neurochemical and biophysical measurements of neurotransmitter synthesis, uptake, storage, release and receptor binding, as well as electrophysiological measurement of changes in membrane potential associated with these events.

A high degree of emphasis is being placed upon the development of in vitro methods for target organ toxicity, to replace or reduce the use of whole animals. Substantial advances in these methods have been achieved for reproductive toxicants (Heindel and Chapin 1993).

In summary, target organ toxicity studies are generally undertaken as a higher order test for determining toxicity. The selection of specific target organs for further evaluation depends upon the results of screening level tests, such as the acute or subchronic tests used by OECD and the European Union; some target organs and organ systems may be a priori candidates for special investigation because of concerns to prevent certain types of adverse health effects.

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The functions of the immune system are to protect the body from invading infectious agents and to provide immune surveillance against arising tumour cells. It has a first line of defence that is non-specific and that can initiate effector reactions itself, and an acquired specific branch, in which lymphocytes and antibodies carry the specificity of recognition and subsequent reactivity towards the antigen.

Immunotoxicology has been defined as “the discipline concerned with the study of the events that can lead to undesired effects as a result of interaction of xenobiotics with the immune system. These undesired events may result as a consequence of (1) a direct and/or indirect effect of the xenobiotic (and/or its biotransformation product) on the immune system, or (2) an immunologically based host response to the compound and/or its metabolite(s), or host antigens modified by the compound or its metabolites” (Berlin et al. 1987).

When the immune system acts as a passive target of chemical insults, the result can be decreased resistance to infection and certain forms of neoplasia, or immune disregulation/stimulation that can exacerbate allergy or auto-immunity. In the case that the immune system responds to the antigenic specificity of the xenobiotic or host antigen modified by the compound, toxicity can become manifest as allergies or autoimmune diseases.

Animal models to investigate chemical-induced immune suppression have been developed, and a number of these methods are validated (Burleson, Munson, and Dean 1995; IPCS 1996). For testing purposes, a tiered approach is followed to make an adequate selection from the overwhelming number of assays available. Generally, the objective of the first tier is to identify potential immunotoxicants. If potential immunotoxicity is identified, a second tier of testing is performed to confirm and characterize further the changes observed. Third-tier investigations include special studies on the mechanism of action of the compound. Several xenobiotics have been identified as immunotoxicants causing immunosuppression in such studies with laboratory animals.

The database on immune function disturbances in humans by environmental chemicals is limited (Descotes 1986; NRC Subcommittee on Immunotoxicology 1992). The use of markers of immunotoxicity has received little attention in clinical and epidemiological studies to investigate the effect of these chemicals on human health. Such studies have not been performed frequently, and their interpretation often does not permit unequivocal conclusions to be drawn, due for instance to the uncontrolled nature of exposure. Therefore, at present, immunotoxicity assessment in rodents, with subsequent extrapolation to man, forms the basis of decisions regarding hazard and risk.

Hypersensitivity reactions, notably allergic asthma and contact dermatitis, are important occupational health problems in industrialized countries (Vos, Younes and Smith 1995). The phenomenon of contact sensitization was investigated first in the guinea pig (Andersen and Maibach 1985). Until recently this has been the species of choice for predictive testing. Many guinea pig test methods are available, the most frequently employed being the guinea pig maximization test and the occluded patch test of Buehler. Guinea pig tests and newer approaches developed in mice, such as ear swelling tests and the local lymph node assay, provide the toxicologist with the tools to assess skin sensitization hazard. The situation with respect to sensitization of the respiratory tract is very different. There are, as yet, no well-validated or widely accepted methods available for the identification of chemical respiratory allergens although progress in the development of animal models for the investigation of chemical respiratory allergy has been achieved in the guinea pig and mouse.

Human data show that chemical agents, in particular drugs, can cause autoimmune diseases (Kammüller, Bloksma and Seinen 1989). There are a number of experimental animal models of human autoimmune diseases. Such comprise both spontaneous pathology (for example systemic lupus erythematosus in New Zealand Black mice) and autoimmune phenomena induced by experimental immunization with a cross-reactive autoantigen (for example the H37Ra adjuvant induced arthritis in Lewis strain rats). These models are applied in the preclinical evaluation of immunosuppressive drugs. Very few studies have addressed the potential of these models for assessment of whether a xenobiotic exacerbates induced or congenital autoimmunity. Animal models that are suitable to investigate the ability of chemicals to induce autoimmune diseases are virtually lacking. One model that is used to a limited extent is the popliteal lymph node assay in mice. Like the situation in humans, genetic factors play a crucial role in the development of autoimmune disease (AD) in laboratory animals, which will limit the predictive value of such tests.

The Immune System

The major function of the immune system is defence against bacteria, viruses, parasites, fungi and neoplastic cells. This is achieved by the actions of various cell types and their soluble mediators in a finely tuned concert. The host defence can be roughly divided into non-specific or innate resistance and specific or acquired immunity mediated by lymphocytes (Roitt, Brostoff and Male 1989).

Components of the immune system are present throughout the body (Jones et al. 1990). The lymphocyte compartment is found within lymphoid organs (figure 1). The bone marrow and thymus are classified as primary or central lymphoid organs; the secondary or peripheral lymphoid organs include lymph nodes, spleen and lymphoid tissue along secretory surfaces such as the gastrointestinal and respiratory tracts, the so-called mucosa-associated lymphoid tissue (MALT). About half of the body’s lymphocytes are located at any one time in MALT. In addition the skin is an important organ for the induction of immune responses to antigens present on the skin. Important in this process are epidermal Langerhans cells that have an antigen-presenting function.

Figure 1. Primary and secondary lymphoid organs and tissues

Phagocytic cells of the monocyte/macrophage lineage, called the mononuclear phagocyte system (MPS), occur in lymphoid organs and also at extranodal sites; the extranodal phagocytes include Kupffer cells in the liver, alveolar macrophages in the lung, mesangial macrophages in the kidney and glial cells in the brain. Polymorphonuclear leukocytes (PMNs) are present mainly in blood and bone marrow, but accumulate at sites of inflammation.

Non-specific defence

A first line of defence to micro-organisms is executed by a physical and chemical barrier, such as at the skin, the respiratory tract and the alimentary tract. This barrier is helped by non-specific protective mechanisms including phagocytic cells, such as macrophages and polymorphonuclear leukocytes, which are able to kill pathogens, and natural killer cells, which can lyse tumour cells and virus-infected cells. The complement system and certain microbial inhibitors (e.g., lysozyme) also take part in the non-specific response.

Specific immunity

After initial contact of the host with the pathogen, specific immune responses are induced. The hallmark of this second line of defence is specific recognition of determinants, so-called antigens or epitopes, of the pathogens by receptors on the cell surface of B- and T-lymphocytes. Following interaction with the specific antigen, the receptor-bearing cell is stimulated to undergo proliferation and differentiation, producing a clone of progeny cells that are specific for the eliciting antigen. The specific immune responses help the non-specific defence presented to the pathogens by stimulating the efficacy of the non-specific responses. A fundamental characteristic of specific immunity is that memory develops. Secondary contact with the same antigen provokes a faster and more vigorous but well-regulated response.

The genome does not have the capacity to carry the codes of an array of antigen receptors sufficient to recognize the number of antigens that can be encountered. The repertoire of specificity develops by a process of gene rearrangements. This is a random process, during which various specificities are brought about. This includes specificities for self components, which are undesirable. A selection process that takes place in the thymus (T cells), or bone marrow (B cells) operates to delete these undesirable specificities.

Normal immune effector function and homeostatic regulation of the immune response is dependent upon a variety of soluble products, known collectively as cytokines, which are synthesized and secreted by lymphocytes and by other cell types. Cytokines have pleiotropic effects on immune and inflammatory responses. Cooperation between different cell populations is required for the immune response—the regulation of antibody responses, the accumulation of immune cells and molecules at inflammatory sites, the initiation of acute phase responses, the control of macrophage cytotoxic function and many other processes central to host resistance. These are influenced by, and in many cases are dependent upon, cytokines acting individually or in concert.

Two arms of specific immunity are recognized—humoral immunity and cell-mediated or cellular immunity:

Humoral immunity. In the humoral arm B-lymphocytes are stimulated following recognition of antigen by cell-surface receptors. Antigen receptors on B-lymphocytes are immunoglobulins (Ig). Mature B cells (plasma cells) start the production of antigen-specific immunoglobulins that act as antibodies in serum or along mucosal surfaces. There are five major classes of immunoglobulins: (1) IgM, pentameric Ig with optimal agglutinating capacity, which is first produced after antigenic stimulation; (2) IgG, the main Ig in circulation, which can pass the placenta; (3) IgA, secretory Ig for the protection of mucosal surfaces; (4) IgE, Ig fixing to mast cells or basophilic granulocytes involved in immediate hypersensitivity reactions and (5) IgD, whose major function is as a receptor on B-lymphocytes.

Cell-mediated immunity. The cellular arm of the specific immune system is mediated by T-lymphocytes. These cells also have antigen receptors on their membranes. They recognize antigen if presented by antigen presenting cells in the context of histocompatibility antigens. Hence, these cells have a restriction in addition to the antigen specificity. T cells function as helper cells for various (including humoral) immune responses, mediate recruitment of inflammatory cells, and can, as cytotoxic T cells, kill target cells after antigen-specific recognition.

Mechanisms of Immunotoxicity

Immunosuppression

Effective host resistance is dependent upon the functional integrity of the immune system, which in turn requires that the component cells and molecules which orchestrate immune responses are available in sufficient numbers and in an operational form. Congenital immunodeficiencies in humans are often characterized by defects in certain stem cell lines, resulting in impaired or absent production of immune cells. By analogy with congenital and acquired human immunodeficiency diseases, chemical-induced immunosuppression may result simply from a reduced number of functional cells (IPCS 1996). The absence, or reduced numbers, of lymphocytes may have more or less profound effects on immune status. Some immunodeficiency states and severe immunosuppression, as can occur in transplantation or cytostatic therapy, have been associated in particular with increased incidences of opportunistic infections and of certain neoplastic diseases. The infections can be bacterial, viral, fungal or protozoan, and the predominant type of infection depends on the associated immunodeficiency. Exposure to immunosuppressive environmental chemicals may be expected to result in more subtle forms of immunosuppression, which may be difficult to detect. These may lead, for example, to an increased incidence of infections such as influenza or the common cold.

In view of the complexity of the immune system, with the wide variety of cells, mediators and functions that form a complicated and interactive network, immunotoxic compounds have numerous opportunities to exert an effect. Although the nature of the initial lesions induced by many immunotoxic chemicals have not yet been elucidated, there is increasing information available, mostly derived from studies in laboratory animals, regarding the immunobiological changes which result in depression of immune function (Dean et al. 1994). Toxic effects might occur at the following critical functions (and some examples are given of immunotoxic compounds affecting these functions):

•  development and expansion of different stem cell populations (benzene exerts immunotoxic effects at the stem cell level, causing lymphocytopenia)
•  proliferation of various lymphoid and myeloid cells as well as supportive tissues in which these cells mature and function (immunotoxic organotin compounds suppress the proliferative activity of lymphocytes in the thymic cortex through direct cytotoxicity; the thymotoxic action of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) and related compounds is likely due to an impaired function of thymic epithelial cells, rather than to direct toxicity for thymocytes)
•  antigen uptake, processing and presentation by macrophages and other antigen-presenting cells (one of the targets of 7,12-dimethylbenz(a)anthracene (DMBA) and of lead is antigen presentation by macrophages; a target of ultraviolet radiation is the antigen-presenting Langerhans cell)
•  regulatory function of T-helper and T-suppressor cells (T-helper cell function is impaired by organotins, aldicarb, polychlorinated biphenyls (PCBs), TCDD and DMBA; T-suppressor cell function is reduced by low-dose cyclophosphamide treatment)
•  production of various cytokines or interleukins (benzo(a)pyrene (BP) suppresses interleukin-1 production; ultraviolet radiation alters production of cytokines by keratinocytes)
•  synthesis of various classes of immunoglobulins IgM and IgG is suppressed following PCB and tributyltin oxide (TBT) treatment, and increased after hexachlorobenzene (HCB) exposure).
•  complement regulation and activation (affected by TCDD)
•  cytotoxic T cell function (3-methylcholanthrene (3-MC), DMBA, and TCDD suppress cytotoxic T cell activity)
•  natural killer (NK) cell function (pulmonary NK activity is suppressed by ozone; splenic NK activity is impaired by nickel)
•  macrophage and polymorphonuclear leukocyte chemotaxis and cytotoxic functions (ozone and nitrogen dioxide impair the phagocytic activity of alveolar macrophages).

Allergy

Allergy may be defined as the adverse health effects which result from the induction and elicitation of specific immune responses. When hypersensitivity reactions occur without involvement of the immune system the term pseudo-allergy is used. In the context of immunotoxicology, allergy results from a specific immune response to chemicals and drugs that are of interest. The ability of a chemical to sensitize individuals is generally related to its ability to bind covalently to body proteins. Allergic reactions may take a variety of forms and these differ with respect to both the underlying immunological mechanisms and the speed of the reaction. Four major types of allergic reactions have been recognized: Type I hypersensitivity reactions, which are effectuated by IgE antibody and where symptoms are manifest within minutes of exposure of the sensitized individual. Type II hypersensitivity reactions result from the damage or destruction of host cells by antibody. In this case symptoms become apparent within hours. Type III hypersensitivity, or Arthus, reactions are also antibody mediated, but against soluble antigen, and result from the local or systemic action of immune complexes. Type IV, or delayed-type hypersensitivity, reactions are effected by T-lymphocytes and normally symptoms develop 24to 48hours following exposure of the sensitized individual.

The two types of chemical allergy of greatest relevance to occupational health are contact sensitivity or skin allergy and allergy of the respiratory tract.

Contact hypersensitivity. A large number of chemicals are able to cause skin sensitization. Following topical exposure of a susceptible individual to a chemical allergen, a T-lymphocyte response is induced in the draining lymph nodes. In the skin the allergen interacts directly or indirectly with epidermal Langerhans cells, which transport the chemical to the lymph nodes and present it in an immunogenic form to responsive T-lymphocytes. Allergen- activated T-lymphocytes proliferate, resulting in clonal expansion. The individual is now sensitized and will respond to a second dermal exposure to the same chemical with a more aggressive immune response, resulting in allergic contact dermatitis. The cutaneous inflammatory reaction which characterizes allergic contact dermatitis is secondary to the recognition of the allergen in the skin by specific T-lymphocytes. These lymphocytes become activated, release cytokines and cause the local accumulation of other mononuclear leukocytes. Symptoms develop some 24 to 48 hours following exposure of the sensitized individual, and allergic contact dermatitis therefore represents a form of delayed-type hypersensitivity. Common causes of allergic contact dermatitis include organic chemicals (such as 2,4-dinitrochlorobenzene), metals (such as nickel and chromium) and plant products (such as urushiol from poison ivy).

Respiratory hypersensitivity. Respiratory hypersensitivity is usually considered to be a Type I hypersensitivity reaction. However, late phase reactions and the more chronic symptoms associated with asthma may involve cell-mediated (Type IV) immune processes. The acute symptoms associated with respiratory allergy are effected by IgE antibody, the production of which is provoked following exposure of the susceptible individual to the inducing chemical allergen. The IgE antibody distributes systemically and binds, via membrane receptors, to mast cells which are found in vascularized tissues, including the respiratory tract. Following inhalation of the same chemical a respiratory hypersensitivity reaction will be elicited. Allergen associates with protein and binds to, and cross-links, IgE antibody bound to mast cells. This in turn causes the degranulation of mast cells and the release of inflammatory mediators such as histamine and leukotrienes. Such mediators cause bronchoconstriction and vasodilation, resulting in the symptoms of respiratory allergy; asthma and/or rhinitis. Chemicals known to cause respiratory hypersensitivity in man include acid anhydrides (such as trimellitic anhydride), some diisocyanates (such as toluene diisocyanate), platinum salts and some reactive dyes. Also, chronic exposure to beryllium is known to cause hypersensitivity lung disease.

Autoimmunity

Autoimmunity can be defined as the stimulation of specific immune responses directed against endogenous “self” antigens. Induced autoimmunity can result either from alterations in the balance of regulatory T-lymphocytes or from the association of a xenobiotic with normal tissue components such as to render them immunogenic (“altered self”). Drugs and chemicals known to incidentally induce or exacerbate effects like those of autoimmune disease (AD) in susceptible individuals are low molecular weight compounds (molecular weight 100 to 500) that are generally considered to be not immunogenic themselves. The mechanism of AD by chemical exposure is mostly unknown. Disease can be produced directly by means of circulating antibody, indirectly through the formation of immune complexes, or as a consequence of cell-mediated immunity, but likely occurs through a combination of mechanisms. The pathogenesis is best known in immune haemolytic disorders induced by drugs:

•  The drug can attach to the red-cell membrane and interact with a drug-specific antibody.
•  The drug can alter the red-cell membrane so that the immune system regards the cell as foreign.
•  The drug and its specific antibody form immune complexes that adhere to the red-cell membrane to produce injury.
•  Red-cell sensitization occurs due to the production of red-cell autoantibody.

A variety of chemicals and drugs, in particular the latter, have been found to induce autoimmune-like responses (Kamüller, Bloksma and Seinen 1989). Occupational exposure to chemicals may incidentally lead to AD-like syndromes. Exposure to monomeric vinyl chloride, trichloroethylene, perchloroethylene, epoxy resins and silica dust may induce scleroderma-like syndromes. A syndrome similar to systemic lupus erythematosus (SLE) has been described after exposure to hydrazine. Exposure to toluene diisocyanate has been associated with the induction of thrombocytopenic purpura. Heavy metals such as mercury have been implicated in some cases of immune complex glomerulonephritis.

Human Risk Assessment

The assessment of human immune status is performed mainly using peripheral blood for analysis of humoral substances like immunoglobulins and complement, and of blood leukocytes for subset composition and functionality of subpopulations. These methods are usually the same as those used to investigate humoral and cell-mediated immunity as well as nonspecific resistance of patients with suspected congenital immunodeficiency disease. For epidemiological studies (e.g., of occupationally exposed populations) parameters should be selected on the basis of their predictive value in human populations, validated animal models, and the underlying biology of the markers (see table 1). The strategy in screening for immunotoxic effects after (accidental) exposure to environmental pollutants or other toxicants is much dependent on circumstances, such as type of immunodeficiency to be expected, time between exposure and immune status assessment, degree of exposure and number of exposed individuals. The process of assessing the immunotoxic risk of a particular xenobiotic in humans is extremely difficult and often impossible, due largely to the presence of various confounding factors of endogenous or exogenous origin that influence the response of individuals to toxic damage. This is particularly true for studies which investigate the role of chemical exposure in autoimmune diseases, where genetic factors play a crucial role.

Table 1. Classification of tests for immune markers

As adequate human data are seldom available, the assessment of risk for chemical-induced immunosuppression in humans is in the majority of cases based upon animal studies. The identification of potential immunotoxic xenobiotics is undertaken primarily in controlled studies in rodents. In vivo exposure studies present, in this regard, the optimal approach to estimate the immunotoxic potential of a compound. This is due to the multifactoral and complex nature of the immune system and of immune responses. In vitro studies are of increasing value in the elucidation of mechanisms of immunotoxicity. In addition, by investigating the effects of the compound using cells of animal and human origin, data can be generated for species comparison, which can be used in the “parallelogram” approach to improve the risk assessment process. If data are available for three cornerstones of the parallelogram (in vivo animal, and in vitro animal and human) it may be easier to predict the outcome at the remaining cornerstone, that is, the risk in humans.

When assessment of risk for chemical-induced immunosuppression has to rely solely upon data from animal studies, an approach can be followed in the extrapolation to man by the application of uncertainty factors to the no observed adverse effect level (NOAEL). This level can be based on parameters determined in relevant models, such as host resistance assays and in vivo assessment of hypersensitivity reactions and antibody production. Ideally, the relevance of this approach to risk assessment requires confirmation by studies in humans. Such studies should combine the identification and measurement of the toxicant, epidemiological data and immune status assessments.

To predict contact hypersensitivity, guinea pig models are available and have been used in risk assessment since the 1970s. Although sensitive and reproducible, these tests have limitations as they depend on subjective evaluation; this can be overcome by newer and more quantitative methods developed in the mouse. Regarding chemical-induced hypersensitivity induced by inhalation or ingestion of allergens, tests should be developed and evaluated in terms of their predictive value in man. When it comes to setting safe occupational exposure levels of potential allergens, consideration has to be given to the biphasic nature of allergy: the sensitization phase and the elicitation phase. The concentration required to elicit an allergic reaction in a previously sensitized individual is considerably lower than the concentration necessary to induce sensitization in the immunologically naïve but susceptible individual.

As animal models to predict chemical-induced autoimmunity are virtually lacking, emphasis should be given to the development of such models. For the development of such models, our knowledge of chemical-induced autoimmunity in humans should be advanced, including the study of genetic and immune system markers to identify susceptible individuals. Humans that are exposed to drugs that induce autoimmunity offer such an opportunity.

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Genetic toxicology, by definition, is the study of how chemical or physical agents affect the intricate process of heredity. Genotoxic chemicals are defined as compounds that are capable of modifying the hereditary material of living cells. The probability that a particular chemical will cause genetic damage inevitably depends on several variables, including the organism’s level of exposure to the chemical, the distribution and retention of the chemical once it enters the body, the efficiency of metabolic activation and/or detoxification systems in target tissues, and the reactivity of the chemical or its metabolites with critical macromolecules within cells. The probability that genetic damage will cause disease ultimately depends on the nature of the damage, the cell’s ability to repair or amplify genetic damage, the opportunity for expressing whatever alteration has been induced, and the ability of the body to recognize and suppress the multiplication of aberrant cells.

In higher organisms, hereditary information is organized in chromosomes. Chromosomes consist of tightly condensed strands of protein-associated DNA. Within a single chromosome, each DNA molecule exists as a pair of long, unbranched chains of nucleotide subunits linked together by phosphodiester bonds that join the 5 carbon of one deoxyribose moiety to the 3 carbon of the next (figure 1). In addition, one of four different nucleotide bases (adenine, cytosine, guanine or thymine) is attached to each deoxyribose subunit like beads on a string. Three-dimensionally, each pair of DNA strands forms a double helix with all of the bases oriented toward the inside of the spiral. Within the helix, each base is associated with its complementary base on the opposite DNA strand; hydrogen bonding dictates strong, noncovalent pairing of adenine with thymine and guanine with cytosine (figure 1). Since the sequence of nucleotide bases is complementary throughout the entire length of the duplex DNA molecule, both strands carry essentially the same genetic information. In fact, during DNA replication each strand serves as a template for the production of a new partner strand.

Figure 1. The (a) primary, (b) secondary and (c) tertiary organization of human hereditary information

Using RNA and an array of different proteins, the cell ultimately deciphers the information encoded by the linear sequence of bases within specific regions of DNA (genes) and produces proteins that are essential for basic cell survival as well as normal growth and differentiation. In essence, the nucleotides function like a biological alphabet which is used to code for amino acids, the building blocks of proteins.

When incorrect nucleotides are inserted or nucleotides are lost, or when unnecessary nucleotides are added during DNA synthesis, the mistake is called a mutation. It has been estimated that less than one mutation occurs for every 10⁹ nucleotides incorporated during the normal replication of cells. Although mutations are not necessarily harmful, alterations causing inactivation or overexpression of important genes can result in a variety of disorders, including cancer, hereditary disease, developmental abnormalities, infertility and embryonic or perinatal death. Very rarely, a mutation can lead to enhanced survival; such occurrences are the basis of natural selection.

Although some chemicals react directly with DNA, most require metabolic activation. In the latter case, electrophilic intermediates such as epoxides or carbonium ions are ultimately responsible for inducing lesions at a variety of nucleophilic sites within the genetic material (figure 2). In other instances, genotoxicity is mediated by by-products of compound interaction with intracellular lipids, proteins, or oxygen.

Figure 2. Bioactivation of: a) benzo(a)pyrene; and b) N-nitrosodimethylamine

Because of their relative abundance in cells, proteins are the most frequent target of toxicant interaction. However, modification of DNA is of greater concern due to the central role of this molecule in regulating growth and differentiation through multiple generations of cells.

At the molecular level, electrophilic compounds tend to attack oxygen and nitrogen in DNA. The sites that are most prone to modification are illustrated in figure 3. Although oxygens within phosphate groups in the DNA backbone are also targets for chemical modification, damage to bases is thought to be biologically more relevant since these groups are considered to be the primary informational elements in the DNA molecule.

Figure 3. Primary sites of chemically-induced DNA damage

Compounds that contain one electrophilic moiety typically exert genotoxicity by producing mono-adducts in DNA. Similarly, compounds that contain two or more reactive moieties can react with two different nucleophilic centres and thereby produce intra- or inter-molecular crosslinks in genetic material (figure 4). Interstrand DNA-DNA and DNA-protein crosslinks can be particularly cytotoxic since they can form complete blocks to DNA replication. For obvious reasons, the death of a cell eliminates the possibility that it will be mutated or neoplastically transformed. Genotoxic agents can also act by inducing breaks in the phosphodiester backbone, or between bases and sugars (producing abasic sites) in DNA. Such breaks may be a direct result of chemical reactivity at the damage site, or may occur during the repair of one of the aforementioned types of DNA lesion.

Figure 4. Various types of damage to the protein-DNA complex

Over the past thirty to forty years, a variety of techniques have been developed to monitor the type of genetic damage induced by various chemicals. Such assays are described in detail elsewhere in this chapter and Encyclopaedia.

Misreplication of “microlesions” such as mono-adducts, abasic sites or single-strand breaks may ultimately result in nucleotide base-pair substitutions, or the insertion or deletion of short polynucleotide fragments in chromosomal DNA. In contrast, “macrolesions,” such as bulky adducts, crosslinks, or double-strand breaks may trigger the gain, loss or rearrangement of relatively large pieces of chromosomes. In any case, the consequences can be devastating to the organism since any one of these events can lead to cell death, loss of function or malignant transformation of cells. Exactly how DNA damage causes cancer is largely unknown. It is currently believed the process may involve inappropriate activation of proto-oncogenes such as myc and ras, and/or inactivation of recently identified tumour suppressor genes such as p53. Abnormal expression of either type of gene abrogates normal cellular mechanisms for controlling cell proliferation and/or differentiation.

The preponderance of experimental evidence indicates that the development of cancer following exposure to electrophilic compounds is a relatively rare event. This can be explained, in part, by the cell’s intrinsic ability to recognize and repair damaged DNA or the failure of cells with damaged DNA to survive. During repair, the damaged base, nucleotide or short stretch of nucleotides surrounding the damage site is removed and (using the opposite strand as a template) a new piece of DNA is synthesized and spliced into place. To be effective, DNA repair must occur with great accuracy prior to cell division, before opportunities for the propagation of mutation.

Clinical studies have shown that people with inherited defects in the ability to repair damaged DNA frequently develop cancer and/or developmental abnormalities at an early age (table 1). Such examples provide strong evidence linking accumulation of DNA damage to human disease. Similarly, agents that promote cell proliferation (such as tetradecanoylphorbol acetate) often enhance carcinogenesis. For these compounds, the increased likelihood of neoplastic transformation may be a direct consequence of a decrease in the time available for the cell to carry out adequate DNA repair.

Table 1. Hereditary, cancer-prone disorders that appear to involve defects in DNA repair

The earliest theories on how chemicals interact with DNA can be traced back to studies conducted during the development of mustard gas for use in warfare. Further understanding grew out of efforts to identify anticancer agents that would selectively arrest the replication of rapidly dividing tumour cells. Increased public concern over hazards in our environment has prompted additional research into the mechanisms and consequences of chemical interaction with the genetic material. Examples of various types of chemicals which exert genotoxicity are presented in table 2.

Table 2. Examples of chemicals that exhibit genotoxicity in human cells

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