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4 Use of Biological Markers in Assessing Human Exposure to Airbome Contaminants INTRODUCTION As used in this report, biological markers are indicators of changes or events in human biological systems. For example, a metabolite of some exog- enous substance found in a person's blood or urine might be considered a marker of the person's exposure to that substance in the environment. If a tissue response to the person's exposure to the substance parallels a disease process, but does not itself constitute a disease process, the tissue response might be considered a marker of effect. The use of biological indicators of exposure to toxic substances is not a new concept it has been used in occupa- tional health monitoring for many decades. However, as the emphasis on conducting accurate exposure assessments increases, biological markers of exposure will be used more frequently A dramatic indicator, particularly in occupational epidemiology studies, is death. Less drastic and more manipula- ble biological measures of health outcome do exist and continue to be devel- oped. Unfortunately, terminology is confusing, the array of technological subtle- ties is bewildering, and there is a potential for ethical dilemmas. By virtue of their collaborative and interdisciplinary nature, investigations involvingbiologi- ca] markers are highly demanding in terms of personnel, cost, and effort to develop mutual understanding between researchers in different fields. Incor- poration of biological markers into exposure assessment engenders new and significant methodological problems in the design of studies that use human subjects and in analysis of data. It also introduces important ethical questions into health effects research, particularly when information on biological mark- ers of uncertain relation to adverse health effects is given to a possibly ex- posed person. Successful use of biological markers will require an under- standing of the fate and effects of a contaminant within a person to permit the 115

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116 ASSESSING HUMAN EXPOSURE relation of marker data to the exposure and the establishment of relationships among biological markers of effect and exposure. This chapter discusses how biological markers might be used as surrogates for or in combination with other measures of exposure, including comments on their limitations. FROM EXPOSURE TO HEALTH EFFECTS: KINDS OF MARKERS As defined in Chapter 1' exposure to an airborne contaminant is the prod- uct of the concentration of the contaminant and the period during which exposure is at that concentration. "Dose" is defined as the actual amount of a given contaminant that is absorbed or deposited in the body of an exposed organism in ~ given period (usually with reference to a single medium or route of exposure). "Potential dose" assumes total absorption of a contaminant. The term dose can be subdivided into internal dose and biologically effective dose~oth are discussed in this chapter with regard to biological markers. ~Effect" is defined as an adverse health outcome or nuisance resulting from exposure. An exposure might or might not lead to a dose, and a dose to a health or nuisance effect. Each of these compartments may also be subdivid- ed into stages representing a progression from exposure to adverse health effect, as shown in Figure 1.2 in Chapter 1. These stages are not discrete. The progression from exposure to effect is a result of the physiological chang- es that can occur within the organism. The progression can be affected at any point by internal biological factors or by external interventions. The classes of biological markers are depicted in Figure 4.1, an elaboration of Figure 1.2 in Chapter 1. Biological markers can be used for each stage in the progression and can be used either to determine the exposure to the causative agent or to predict adverse health effects (Committee on Biological Markers of the National Research Council, 1987~. Markers can be divided into three broad classes: markers of exposure, markers of health effects, and markers of susceptibility (NRC, 1989~. The committee focuses in this chapter on the application of biological markers to assessment of exposure to contami- nants rather than their use to predict health effects. However, the use of markers to predict health effects should not be disregarded. As discussed in Chapter 1, biological markers of exposure integrate all routes of exposure to a particular contaminant. For example, lead concentra- tions as measured in a human tissue, such as blood, can reflect exposure via any or all of such routes, including inhalation of lead in ambient air, ingestion of vegetables contaminated by deposition of airborne lead and ingestion of

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BIOLOGICAL MARKERS 117 Exposure Markers of exposure Markers of health effects Internal dose Biologically effective dose Early biological effect Altered function or structure Clinical disease ~1 13 Markers of susceptibility FIGURE 4.1 Kinds of biological markers. Source: Adapted from Committee on Biological Markers, 1987. lead in drinking water or dust, especially by children with pica. This integrat- ing process of biological markers of exposure can be important for risk assess- ment and risk management. In the case of lead, for example, the amount inhaled might be assessed with procedures described in Chapter 3. If only a very small portion of the observed dose of lead could be accounted for via inhalation, other exposure routes and sources might then be sought. Biological markers provide information on dose that can be related to exposure by using pharmacokinetic or pharmacodynamic models (e.g., blood lead or carboxyhemoglobin). Internal dose is the amount of contaminant that is absorbed into the body in a given period. Biological markers of internal

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118 ASSESSING HURON EXPOSURE dose directly measure the exposure substance or its metabolites in cells, tis- sues, or body fluids. The biologically effective dose is the amount of contami- nant or its metabolites that has interacted with a target site over a given peri- od so as to alter a physiological function. Such an interaction can lead to disease or be subjected to repair. Biological markers of health effects (referred to in this chapter as biological markers of effect) represent a further step away from exposure and a step closer to the clinical manifestation of disease. A marker of early biological effect provides information on an event resulting from a toxic interaction, either at a target or at an analogous site. An early effect is considered an irreversible step in pathogenesis or a qualitative or quantitative correlate of a disease process. Biological markers of effect also include preclinical alter- ations in organs, tissue structure, or function directly associated with disease. To be useful in disease prevention, these markers of effect should be measur- able at preclinical stages. Biological markers of susceptibility span the entire range of markers show n in Figure 4.1. They indicate an organism's inherent or acquired limitations that affect its response to an exposure to a particular contaminant. These markers can be useful in understanding the relationships that exist between markers of exposure and effect. Relating a biological marker directly to an exposure becomes more uncer- ta~n as one obtains information on the stages within the progression shown in Figure 4.1. Accordingly, the Committee on Biological Markers of the Nation- al Research Council (1987) has stated that "markers of health effects are often less readily related to environmental exposures than are the markers of expo- sure.- Therefore, greater emphasis is given to markers of exposure. Biological markers of effect can be used for exposure analysis if and only if information concerning contaminant identity and route of exposure is incorporated into the exposure assessment. An attempt to use biological markers as surrogates for measurements of exposure might raise ethical questions. When humans are directly involved in biological-marker studies, caution must be exercised in reporting results to study subjects, to avoid undue psychological stress when the significance of the marker for future adverse health effects is uncertain (Ashby, 1988~. Pharmacokinetics is the quantitative description of the rates of absorption, distribution, metabolism, and elimination of a contaminant taken into a bio- logical system (Leung and Pastenbach, 1988~. Pharmacodynamics is the de- scription of the processes that relate biologically effective dose to health ef- fects. Pharmacokinetic modeling techniques are used to aid in the extrapola- tion of test results from animals to humans; pharmacodynamic models are just being developed (Menzel, 1987~. The pha~acokinetic models attempt to

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BIOLOGICAL ~4RKER5 119 compensate In part for the physiological and biochemical differences between humans and test animals, which can cause markedly different responses to the same substance. The models use mechanistic, biochemical, and physiological information to describe the disposition of a contaminant once taken in. Mechanistic studies provide information on the target tissues, metabolic path- ways, and nature of a contaminant's chemically stable metabolites or reactive intermediates. Pertinent biochemical data include partition coefficients and permeation coefficients. Pertinent physiological factors include tissue volumes, blood flow rates, and ventilation rates. Much of this information can be obtained from published sources or through the use of in vitro techniques. A physiologically based pharmacokinetic model is constructed as a set of biologically defined compartments-tissues and target organs are grouped into well-perfused, moderately perfused, or poorly perfused compartments for describing the process of metabolic transformation in the lungs, kidneys, or liver. Each compartment has a defined volume, blood flow, partition coeffi- cient, and metabolic constants (Travis et al., 1990~. The growing availability of sophisticated simulation packages that can solve large numbers of mass- balance differential equations makes the construction and testing of models ncreasmgly easier. An example of use of pharmacokinetic techniques to relate various biologi- cal markers to each other and to an exposure is the study of halothane con- centrations in the breathing zone and blood of operating-room personnel (Fiserova-Bergerova, 1987~. Anesthesiologists had the highest breathing-zone concentrations but lower blood concentrations than nurses, who had substan- tially lower breathing-zone concentrations. Pharmacokinetics accounted for the difference: the anesthesiologists had a primarily sedentary job, whereas the nurses were actively moving about the room and therefore had higher ventilation rates and cardiac output. Pharmacokinetics and pharmacodynamics can also be used to indicate which tissues are to be sampled and when samples should be taken, as dis- cussed in Chapter 2. For example, the pharmacokinetics of cadmium indicate that, if one is interested in recent cadmium exposure, one should analyze blood; if accumulation is of interest, one should analyze urine (ACGIH, 1986~. Another study involved the effect of ethylene glycol ethers on sperm count. Pharmacodynamic studies did not show.a relationship between sperm count and metho~yacetic acid, the primary metabolite of ethylene glycol ethers (Smith, 1988~. However, because the sperm production cycle takes 80 days, there is a lag of approximately 80 days between toxic exposure and reduced sperm count. Therefore, exposure and effect measurements had to be offset by approximately 80 days. Art understanding of the pharmacokinetics of a contaminant is important

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120 ASSESSING HUMAN EXPOSURE In relating biological markers to the original exposure. As pharmacodynamic modeling advances, relationships will then be established between biological markers of exposure and biological markers of effect (Menzel, 1987~. APPLICATIONS OF HUMAN BIOLOGICAL MARKERS Markers of Exposure Sensitive physicochemical methods can detect and measure very low con- cer~trations of xenobiotic substances in the body (Sheldon et al., 1986~. They are being used in industrial hygiene with biologic exposure indexes as refer- ence values for workplace exposure monitoring (ACGIH, 1986, 1988b; Fiser- ova-Bergerova, 1987~. Concentrations of a contaminant are usually measured in exhaled air, blood, or urine, but breast milk and semen have also been used. Each biological medium has a unique relevance to exposure and health outcome, and interpretation of the resulting data requires an understanding of the pharmacokinetics of the substance in question. For example, concen- trations in exhaled air generally reflect only inhalation, whereas concentrations in blood, other tissues, and other fluids might reflect recent exposures from several sources and stored body burdens. Measures of internal dose can be characterized according to chemical specificity and selectivity. Highly selective markers of exposure typically repre- sent measures of unchanged contaminants in biological media and thus pro- vide the most clear-cut evidence of a specific environmental exposure. Studies of volatile airborne contaminants usually involve the measurement of a specific substance in exhaled air. The unchanged compound or its metabolite in urine may also be analyzed where variations in urinary volume can be accounted for by normalizing the concentration of the analyte to the creatinine levels. If it is necessary to analyze for a metabolite, rather than its parent substance, a procedure can lose some specificity in relating the biological marker to expo- sure. For example, exposure to styrene and exposure to ethyl benzene each give rise to mandelic acid in urine, so a finding of mandelic acid in urine would have to be supplemented with another assay (e.g., of breath) to deter- mine which compound the subject was exposed to. Measures of biologically effective dose include DNA and hemoglobin ad- ducts in peripheral blood and other cells and tissues c.g., lung macrophages, sputum, bronchial washes or bronchoalveolar ravage fluid, buccal mucosa, bone marrow, placental tissue, and lung tissue. Such measures can be used as markers of exposure only when the analyzed cells or tissues are the target of the exposure contaminant or its metabolites. Many carcinogens and repro

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BIOLOGICAL MARKERS 121 ductive toxicants are metabolically activated to clectrophilic metabolites that covalently bind to DNA. AdJucts on DNA, if they occur at critical sites and are not repaired, can cause gene mutation, which has been shown to be an initiating step in the multistage carcinogenic process. Several methods to detect DNA-chemical abducts In lymphocytes and target tissues are available, including radio- or enzyme-linked immunoassays that use polyclonal or mono- clonal antibodies, 32P-postlabeling, and synchronous fluorescence spectropho- tometry (Watson, 1987; Santella, 1988~. The first use of antibodies to detect polycyclic aromatic hydrocarbon (PAH)-DNA adducts involved lung tissue and peripheral white blood cells from lung-cancer patients and controls (Perera et al., 1982~. They have since been used to analyze white blood cells and other tissues from persons exposed to PAHs in cigarette smoke and in occupational settings (e.g., foundries and coke ovens) (Harris et al., 1985; Shamsuddin et al., 1985; Haugen et al., 1986; Perera et al., 1988~. Antibodies are available to assess formation of DNA abducts in humans with several carcinogenic substances: aflatoxin Be, benzoa- pyrene (BaP) and other PAHs, cisplatinum, and metho~ypsoralen. Immuno- assays can detect frequencies as low as one adduct per 108 nucleotides. As- says that use monoclonal antibodies are highly specific for a given substance, but those with polyclonal antibodies, such as PAM-DNA antibodies, can react with multiple structurally related compounds and thus lose specificity. The value of immunoassays depends on the development of appropriate antibodies, and the highly specific monoclonal assays can be time-consuming and techni- cally difficult. In contrast with the monoclonal assays, 32P-postlabeling can be used to recognize various adducts without characterizing their chemical compositions. It can be more sensitive than monoclonal assays; it can detect one abduct per 10~ nucleotides. The method produces an image that constitutes an idiosyn- cratic ~fingerprint" of the exposure. However, researchers developing this assay have faced difficulties in identifying the abducts formed. If the adducts can be isolated and identified, further analyses can be carried out with immunoas- say techniques, once the appropriate antibody has been developed. The post- labeling method generally is limited to the measurement of bulky adJucts that might limit the usefulness of the technique to smaller airborne contaminants, and the results of the method are only semiquantifiable. Effective use of the postlabeling method requires the synthesis of an internal standard since the efficiency of labeling can vary according to the substance. The technique of 32P-postlabeling has been applied to the identification of various alkylating and methylating agents (Ready and Randerath, 1987~. It is possible to characterize methylating agents using HPLC (high performance liquid chromatography); however, this is not a routine technique. Application

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122 ASSESSING HUMAN EXPOSURE to populations exposed to airborne pollutants has been limited to assessment of BaP and other PAH exposure of foundry workers and roofers (Hemminki et al., 1988; Phillips et al., 1988~. Some investigators have related placenta and lung adJuct measurements to cigarette smoking with postlabeling tech- niques (Emerson et al., 1986~; others have not seen smoker-nonsmoker differ- ences in bone marrow, white blood cells, or buccal mucosa (Dunn and Stich, 1986; Phillips et al., 1986; Jahnke et al., 1990~. A third approach uses synchronous fluorescence spectrophotometry, which has recently been applied to coke-oven workers with a reported sensitivity of one BaP adduct per lo? nucleotides (Vahakangas et al., 1985~. The method has been used to confirm the presence of BaP-DNA abducts In placental tissue from smokers (Weston et al., 1988~. HPLC and fluorescence spectros- copy have been used to detect excised carcinogen-DNA adJucts in urine (Autrup et al., 1983~. The technique is limited in that it is useful only for detecting compounds that fluoresce, such as PAHs. Assays that measure protein abducts, including adducts of direct binding agents and metabolites with hemoglobin, can in some cases be a good surro- gate for DNA-adduct measurements. That use is supported by correlations in animal studies between protein and DNA binding by BaP, vinyl chloride, ethylene oxide, methyl methanesulfonate, and transit dimethylaminostilbene (LARC, 1984; Neumann, 1984; Bartsch, 1988~. Methods available for measur- ing those adJucts include immunoassays, amino acid analysis by ion-exchange LC, and the combination of gas chromatography and mass spectrometry (GC-MS) with both conventional ionization and negative chemical ionization techniques. GC-MS has been successfully applied to the quantitation of aminobipheny} (lABP) hemoglobin adJucts in smokers (Bryant et al., 1987; Perera et al., 1987~. Sensitive GC-MS methods can measure protein adJucts in persons exposed to ethylene oxide from cigarette smoke and workplace sources (Osterman-Golkar et al., 1984; Farmer et al., 1986; Tornqvist et al., 1986~. Because of the 3-month life span of hemoglobin, those assays reflect relatively recent exposures; DNA adducts in lymphocyte subpopulations reflect exposure integrated over a longer period. Protein adducts are more abundant than their DNA counterparts and therefore provide a more sensitive measure of exposure. Markers of Effect Markers of biological effect can be useful for exposure assessment, provid- ed that they can be related to the exposure responsible for an effect. A num- ber of markers might signal a preclinical or presymptomatic stage in disease

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BIOLOGICAL MARKERS 123 development, some of which are specific.to a chemical; others might signal adaptive changes that are not themselves pathological. For example, the presence of carboxyhemoglobin in the blood signals that damage related to carbon monoxide exposure is occurring, but the source could be the inhalation of carbon monoxide or the metabolism of methylene chloride. Red blood cell delta-aminolevulinic acid dehydratase (deIta-ALAD) has been used as an indicator of early toxic effects of lead exposure (Friberg, 1985), but the Ameri- can Conference of Governmental Industrial Hygienists (ACGIH) has not rec- ommended its use as a biological exposure index, because of Interpretative diff~culties~ (ACGIH, 1986~. Reduction in plasma acetylcholinesterase activity has been specifically linked to organophosphate insecticides, but thiocarba- mates can induce the same effect (Fiserova-Bergerova, 1987~. Nonspecific markers of reproductive impairment include plasma ollicle-stimulating hor- mone, plasma luteinizing hormone, salivary progesterone, and urinary hydro~y- proline or hydroxylysine (which could reflect tissue remodeling (collagen turnover) after inhalation exposure to environmental pollutants) (NRC, 1988~. Cytogenetic techniques provide another direct, although nonspecific, meth- od of identifying changes that occur on the chromosomal level after exposure to environmental contaminants. Cytogenetic changes include alterations in chromosome number, such structural chromosomal changes as breakage and rearrangement, and exchanges between reciprocal portions of a single chromo- some referred to as sister-chromatic exchanges (SCE). The mechanism re- sponsible for inducing SCEs is not well understood. Many classes of carcino- gens and mutagens are known to increase SCE frequency, and that could limit the usefulness of SCE frequency for exposure assessment. For example, increased SCE frequency has been found in workers exposed to ethylene once, styrene, benzene, arsenic, chloromethyl ether, chloroprene, organophos- phates, or ionizing radiation (Evans, 1982~. Chromosomal aberrations have been used successfully as a biological do- simeter to measure absorbed radiation in humans (LAEA, 1986~; however, ionizing radiation acts through mechanisms that are different from those of most other atmospheric contaminants. Micronuclei, fragments of nuclear material left in the cytoplasm after replication, are considered an indication of the prior existence of chromosomal aberrations. Cytogenetic markers can be identified in lymphocytes and sometimes in other tissue with stimulated cell culture and staining techniques in conjunction with light microscopy (Liv~ng- ston et al., 1983; Carrano and Natarajan, 1988~. Many types of human cancers are associated with specific alterations (e.g., adult leukemia) or nonspecific aberrations, and there is increasing evidence that chromosomal aberrations might be linked to the carcinogenic process related to exposure to some chemicals (Yunis, 1983~.

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124 ASSESSING HUMAN EXPOSURE New techniques have been based on the fact that a specific mutagen (such as a chemical agent) induces a specific pattern of gene mutation (Benzer and Freese, 1958~. Techniques have been developed to screen for patterns In genes from human peripheral blood lymphocytes that contain mutations in the non-essential enzyme hypoxanthine guan~ne phosphoribosyl transferase (HGPRT) (Cariello and Thilly, 1986~. One technique involves the use of denaturing gradient gel electrophoresis, which can separate short DNA mole- cules according to their melting (uncoiling) properties. The melting behavior of the DNA fragments is extremely sequence-dependent; a single base-pair substitution can change migration on the gel. That type of tool provides a convenient means of examining HGPRT for genetic alterations (according to the generation of mutation-related spectra on the gel) and then relating the alterations to specific causative agents (Cariello et al., 1988~. The technique depends on the use of a high-fidelity polymerase chain reaction to provide in vitro amplification of a given region of the genome and thus improve sensitivi- ty and provide enough material for sequencing. The application of the tech- nique to exposure assessment holds the potential for being specific for a sub- stance causing mutation and so would be useful in epidemiological studies, risk assessment, and risk management. In addition, it leads to a measure of a biological effect and thus could be more directly related to potential adverse health outcomes than biological markers of exposure. Extensive validation of the technique is necessary. Another new approach to assessing genetic effects involves measurement of single-strand breaks in lymphocyte DNA. A linear dose-response relation- ship was found in mice exposed to styrene (Welles and Orsen, 1983), and increased frequencies of breaks were found in workers exposed to styrene (Welles et al., 1988~. The assay was also used to demonstrate differential styrene metabolism and clearance by various organ systems (Welles and Or- sen, 1983~. Alkylating agents, such as N-nitroso-N-methylnitroguanidine and ethyl methylsulfonate, which are negative in standard cell transformation assays, have also been shown to be strong inducers of single-strand breaks (Lubes et al., 1983~. DNA hyperploidy measured in e~ol~ated bladder and lung cells has been shown to be a biological marker of response to exposure to carcinogens. It has been detected with flow cytometry of stained cells (Melamed et al., 1977), but this approach requires large samples and cannot be used to evaluate individual cells. The more recently developed technique of quantitative fluo- rescence image analysis involves the scanning of the fluorescence of individual stained cells in a microscopic field (Hemstreet et al., 1986~. This technique has show n DNA hyperploidy to be highly correlated with magnitude of expo- sure in workers exposed to aromatic amines (Hemstreet et al., 1988~. Further

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BIOLOGICAL MARKERS 125 validation is needed to determine the specificity of DNA hyperploidy markers for causative agents and for actual magnitudes of exposure in the environment. Activated oncogenes and their protein products can be used as markers of effect. During oncogenesis, a normal segment of DNA (a proto-oncogene) is activated to a form that causes cells to become malignant. Activation can occur through several mechanisms, including gene mutation and chromosomal breaks and rearrangements. Of particular interest in assessment of ambient environmental exposures is the ras oncogene, first identified in rat sarcoma, later in human bladder, colon, and lung cancers (Slamon et al., 1984; Span- didos and Kerr, 1984~. Activated ras oncogenes containing a point mutation have been produced in vitro by a number of ambient pollutants, including BaP, dimethylbenzanthracene, and N-nitroso compounds (Sukumar et al., 1984~. Activation of the ras oncogene can be measured with complex im- munoblotting techniques. A more convenient and relatively simple method involves measurement of the gene's abnormal protein product, designated P21. P21 can be measured in tissue or sputum with monoclonal antibodies, as well as in blood and urine with immunoprecipitation techniques. The assay is still in the early validation stage (Brandt-Rauf, 1988~. It is likely that more than one oncogene needs to be activated to convert a normal cell to a cancerous one (Stowers et al., 1987~. More studies will be needed to reveal the sequen- tial requirements for oncogene activation. Assays that detect changes in the function of target or analogous tissue, such as decreased sperm counts after dibromochloropropane exposure, pro- vide markers that are closely linked to disease end points. An increased concentration of erythrocyte zinc protoporphyrin (EPP) indicates a later stage in lead toxicity than does delta-ALAD deficiency (Friberg, 1985) and Is a better masticator of past chronic exposure to lead then is blood lead concentra- tion (Blumberg et al., 1977; Franco et al., 1984~. ACGIH recommends the use of EPP as a measure of lead exposure (ACGIH, 1986), although it points out that iron deficiency can also increase EPP (Lamola and Yamane, 1974~. Some markers can indicate the presence of disease at preclinical or early clinical stages. For example, serum alpha-fetoprotein, although not specific to liver cancer, has been successfully used in China to indicate preneoplasia of the liver (Committee on Biological Markers of the National Research Council, 1987~. However, the lack of specificity of tumor markers (such as alpha-fetoprotein) severely restricts their usefulness (Hulka and Wilcosky, 1988~.

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132 ASSESSING HUMAN EXPOSURE from the same worker population (Hemminki et al., 1988; Phillips et al., 1988~. However, despite the correlation between DNA adJucts and exposure at the group level, there was significant variation among individuals within the exposed groups. Adducts measured with immunoassay ranged from nondetec- table to 2.8 fmol/~g. On the basis of the work of Lioy et al. (1988), it is possible that workers were receiving exposures by other routes, mainly food. Harris et al. (1985) did not find BaP-DNA abducts In 10 of 41 coke-oven workers evaluated. They suggested that that was probably due to variation in exposure, ~ individual metabolic balance between activation and deactivation, and in DNA repair capacity. Because DNA repair is both inducible and saturable, differences related to dose are likely (Swenberg et al., 1987; Dan- heiser et al., 1989~. The manner and timing of sample collection can produce ambiguous or meaningless results. For example, in a study to determine exposure to envi- ronmental tobacco smoke with nicotine or its metabolites as markers, differ- ent results were obtained when different sampling protocols were used. Physi- ological pharmacokinetic modeling has demonstrated that the single-time point sampling protocol is of little use, unless a marker has a long half-life, which is rare for most exposures (Schwartz and Baiter, 1988~. Therefore, a good understanding of the pharmacokinetics is essential for proper timing of sample collection. Difficulty of Establishing Links Between Exposure and Markers of Effect Biological markers of effect are usually not exposure-specific. For example, pathophysiological changes, such as alterations in sperm structure or function and hormonal changes indicative of early reproductive disease, are not specific to chemicals and routes of entry. The same is true of most markers of pre- clinical and clinical immunological, cardiovascular, neurological, and renal dysfunction (EPA, 1988a). With respect to tumorigenesis, most preclinical effect markers- such as fetal proteins, oncogene protein products, structural changes in oncogenes, and chromosomal deletions not reveal the identity of the environmental exposure responsible. Increased specificity can be achieved if future studies, such as those suggested by Cariello and Thilly (1986), show that mutagens are specific with regard to the kind and position of mutation, and this specificity can be considered as a chemical fingerprint left on the DNA. The fingerprint would be a powerful tool in establishing the cause-effect relationship. However, Sukumar et al. (1984) observed the same

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BIOLOGICAL MARKERS 133 ras oncogene is activated in neoplastic cells derived from exposure to several different chemicals, so this lesion cannot act as a fingerprint. The search for effect markers for pulmonary to~ncants is also frustrated by the lack of specificity of pulmonary responses to etiologic agents (NRC, 1988~. Those responses include altered breathing patterns and airway constriction, cell injury leading to inflammation, persistent alteration of lung structure (e.g., fibrosis, chronic obstructive pulmonary disease, and granulomatous disease), and neoplasia. There is a need for research to identify biochemical correlates of structural changes, as well as to distinguish effects caused by a chemical from effects that are responses to cell injury (Smith et al., 1986~. It is therefore desirable to incorporate chemical-specific markers of exposure and early biological effect into a study design to complement the nonspecific biological markers. As discussed later, however, relating even a chemical-specific marker to a defined cxposurc period is not clear-cut. Confounding Influences on Biological Markers Biological markers of exposure reflect diverse behavioral, biological, and methodological processes. Hence, they can be more subject to confounding influences than are measurements of ambient airborne contaminants. Droz (1985) evaluated effects of confounding factors on dose with simulation mod- els. He identified six confounding factors that could influence the dose of organic solvents: intraday and interday fluctuation of exposure, repetition of exposure, physical workload, body build, and metabolism. The factors were applied to four solvents (benzene, styrene, methyl chloroform, and tetrachloro- ethylene) that differ in blood solubility and metabolism. Droz suggested that such models provide a means for assessing the role of confounding factors in internal dose. Complexity and Resource Intensiveness By virtue of their collaborative and interdisciplinary nature, biological marker investigations are highly demanding in terms of personnel, cost, and effort to develop understanding among researchers in different fields. Most also require clinical interaction with subjects. Incorporation of biological markers engenders new and important methodological problems in study design and in analysis of data (Schulte, 1987, 1989~. It also introduces impor- tant ethical questions into health-effects research, particularly if information

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134 ASSESSING lIUMAN EXPOSURE is given to a person concerning a biological marker of effect when the rela- tionship to an adverse health effect is unclear. Use of Exposure Markers in Conjunction with Traditional Measures The most effective method of characterizing exposure is to combine meth- ods for assessing ambient exposure with various biological markers (Friberg, 1985~. Used together, they enhance the precision and accuracy of exposure information, as exemplified in a study of hospital sterilizer operators occupa- tionally exposed to ethylene oxide (Yager et al., 1983~. Ambient and breathing-zone ethylene oxide concentrations were measured, as well as SCEs. Workers were classified into two groups according to estimates of amounts potentially absorbed: low, a mean of 13 mg of ethylene oxide over 6 months; and high, a mean of 500 mg over 6 months. A nonexposed control group was also evaluated. The high-exposure group had a mean of 10.7 SCEs/cell, which was significantly different ~ = 0.002) from both that in the low-exposure group (7.8 SCEs/cell) and that in the control group (7.6 SCEs/cell). Without the use of ambient measurements to distinguish the two exposure groups, the difference in mean SCE frequency between exposed and control groups would have been only marginally significant (p < 0.05~. CRITERLt GOVERNING THE VALIDATION AND USE OF BIOLOGICAL MARKERS Biological markers can be used to identify discrete components in the e~osure-health-effects relationship (Figure 4.1~. For a given exposure and disease, it is possible to identify most of the components of the relationship. Whether the relationship is linear or follows some other form, such as a branched network, is uncertain, but the linear concept of a relationship should suffice for research planning. Use of this model, however, should not obscure the need for efforts to explore the relationship between markers that might be represented by more accurate, nonlinear models (see Schulte, 1989, for review). The current use of biological markers for exposure assessment is not with- out its pitfalls, and in some cases it cannot occur without adjustments and a stipulation of an independent determination of confounding variables. For example, there will be requirements to account for exposure to background concentrations of contaminants from the same and other media and to adjust

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BIOLOGICAL MARKERS 135 for seasonal or regional variation in some markers. These adjustments not- withstanding, the conventional techniques for assessing e~osure-disease asso- ciations, screening for exposure of individuals in populations and handing multiple variables can be practiced for any two components in the relationship (e.g., exposure and internal dose). The major assumption that permits this approach is the causal or positive association between components of the relationship. For investigations involving markers of components from the central regions of the relationship (e.g., biologically effective dose), there is an increased need to test assumptions and hypotheses, evaluate misclassification, and identify and control confounding factors. That is because it is difficult to define the central markers in terms of either exposure or health effect when so many confounding factors (e.g., kinetics, medical history, and rates of tran- sition) can affect the pathognomonic relationships. Validation and Selection of Biological Markers The validity of a biological marker for environmental studies should be determined on the basis of the fundamental criteria reviewed by Perera (1987) and discussed below. Exposure Assessment There must be a clear hypothesis or model of exposure-response relation- ships defining the role of the specific marker in relation to the possible expo- sure scenario. The relevance of markers of exposure is generally easier to establish than that of effect markers when assessing exposure. Relating mark- ers of early biological effect to an original causative agent and routes of expo- sure is much more difficult, requiring extensive validation studies with careful linkage of an exposure to a marker of effect. Understanding of Pharmacokinetics and Temporal Relevance The proper selection and use of biological markers depend on an under- standing of the underlying pharmacokinetics and pharmacodynamics of sus- pected contaminants (WHO, 1986; Andersen, 1987; Smith, 1987; Yager, 1988). Knowledge of pharmacokinetics is important in determining the frequency and timing of sampling and the tissues or fluids most appropriate for study. It also

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136 ASSESSING HUMAN EXPOSURE guides the interpretation of dose and effect data obtained in a target tissue or a surrogate. The temporal relationship of markers to external exposure or to disease end points must be clear. Whether an exposure marker reflects recent or cumulative exposures, or peaks or averages, depends on the pharmacokinetics of the contaminant, the persistence of the marker In the biological sample being assayed (which is in large part a function of the turnover rate or half-life of the sample), and repair rates. Understanding of temporal relevance is essential for developing monitoring strategies and interpreting results. Most measures of internal dose, for exam- ple, reflect recent exposures. An exception would be a substance that is fat- soluble and is stored in adipose tissue. Hemoglobin is a good integrating dosimeter over the Month half-life of erythrocytes, which, unlike white blood cells, lack repair systems. Human serum albumin has a half-life of 20-25 days. Albumin is synthesized in the liver, where many carcinogens are metabolically activated, so it might reveal adducts not detected in hemoglobin. The period reflected by white blood cells or lymphocytes is considerably more complex (Perera, 1987; Carrano and Natarajan, 1988~. For example, adducts on DNA from white cells can reflect both past and current exposures, because a subset (T cells) is very long-lived. A review of white-cell subpopu- lations shows that measurements of DNA adducts in these cells will be influ- enced by the longer-lived T cells and will therefore reflect exposures that occurred both recently and several decades in the past. T cells make up approximately 60-90% of lymphocytes, which in turn constitute about 22-28% of peripheral white cells in circulating blood. Thus, T cells constitute a ma~n- mum of 25% of white cells. The estimated half-life of T ceils is 3 years In contrast, B cells and monocytes constitute roughly 1-2% and 1-7%, respective- ly, of circulating white cells and have lifetimes ranging from days to weeks. Granulocytes make up the remaining 66-85% of white cells and are short-lived (hours to days). Thus, one should consider DNA adducts only in cells dam- aged while in circulation and not white-cell adducts in circulating white cells, which might result from damage to stem or precursor cells in the bone mar- row. When all DNA from a sample of peripheral blood is assayed for DNA adduces, one sees that the long-lived T cells are the major contributor in cases of past discontinued exposure, largely because of their lifetime, which is 100- 1,000 times longer. In addition, lymphocytes have lower repair activity than do circling cells, so DNA lesions are likely to be more persistent in lympho- cytes In cases of current, recent, or long-term uninterrupted exposure to carcinogens, T cells will contribute less importantly to total abducts. The preponderance of adducts will be measured in the shorter-lived granulocytes, B cells, and monocytes.

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BIOLOGICAL MARKERS 137 In retrospective case-control studies, for example, one would want a perma- nent marker left decades earlier by an initiating carcinogen. In the case of discontinued exposures, even the longest-lived markers wiD be diluted by cell turnover, thereby leading to underestimation of past or cumulative dose. Only if exposure had been continuous and had not changed substantially over the decades (and only if the disease had not altered metabolism) would current measurements of the marker be directly representative of critical prior e~o- sure. At the very least, however, such markers as adducts reflect a person's responsiveness to carcinogenic exposures, provided that metabolism was un- changed by disease. Many other exposure patterns are relevant to case-con- trol and cohort studies (current but interrupted, continuous but of varying magnitude, etc.~. Each pattern would lead to a different distribution of ad- ducts among white-cell populations and hence to a varied pattern of persis- tence. Understanding of Background Variability and Confounding Variables It is essential to know the range of values of a given biological marker in a "normal" population. Care must be taken not to be deceived by the extensive variation in biochemical individuality; what is considered Healthy in some might indicate a health risk in others (Schulte, 1987~. The range of ~normal" can be large. For example, it is well known that cholinesterase activity in people not exposed to organophosphorus insecticides covers a wide range (WHO, 1975~. Interindividual variation and intraindividual variation are important contri- butors to "noise. or background in monitoring or epidemiological studies and should be characterized before large-scale application of a particular biological marker. Such data, however, can be generated only by large-scale surveys with repeated sampling and efforts to control for confounding variables. Thus, a background study is an important exposure-assessment exercise in itself. With respect to carcinogen-DNA and carcinogen-protein adducts, substantial interindividual variation and intraindividual variation have been observed with PAHs, ABE, and nitrosamines (Harris et al., 1985; Umbenhauer et al., 1985; Bryant et al., 1987; Perera, 1987) SCEs also vary widely within and between subjects (Carrano and Natarajan, 1988~. As discussed earlier, biological markers are subject to greater variability than conventional exposure-assessment techniques, because the body actively participates in the collection, distribution, and elimination of absorbed con- taminants. Confounding variables that must be accounted for in studies that

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138 ASSESSING HUMAN EXPOSURE use biological markers include age, sex, race, cigarette smoking, alcohol con- sumption, diet, drugs or other environmental exposures, genetic factors, and pre-e~nsting health impairment. In fact, it is known that alcohol intake is the most common cause of reduced metabolism of industrial chemicals (Fiserova- Bergerova, 1987~. Thus, in a study of those chemicals in which the chemical or a metabolite is used as a biological marker of dose, one would have to account for alcohol consumption if one were to understand and interpret the data properly. Most reports of cytogenetic studies have given no information on the ~m- pact of smoking and other exposures to carcinogens or mutagens on their results. Life-style factors (e.g., smoking and diet) and other chemical eypo- sures (e.g., environmental, recreational, and therapeutic) are also potential confounding factors with respect to SCEs and most other markers. Additional potential confounders are host factors (eeg., health and immune status) and exposures that influence the marker of interest. In a study of PAM-DNA adJucts in workers, for example, it is necessary to account for all other back- ground exposures to PAHs and, ideally, for factors that could induce or inhibit metabolism and binding. Those inducers include cigarette smoke, char-broiled meat, alcohol, sedatives, and PCBs; the inhibitors include methylxanthines in foods, steroids, solvents, and spray paints. Thus, even pilot studies attempting to evaluate biological markers can become complex epidemiological exercises that involve careful interviewing. Reproducibility, Sensitivity, and Specificity Chemical specificity is a prime criterion for marker selection. Markers should be chemical-specific or at least highly correlated with the contaminant exposure of concern. That is important if effective mitigating steps are to be taken to prcvcat later exposures. In addition to being specific, markers should also be "sensitive" to the agents involved in the exposures, detecting a high percentage of persons in the ex- posed group. Given interindividual variation, not all exposed persons would be expected to be positive; but it is important that the method for assaying a marker be sensitive enough to determine background, so that a true compari- son of the exposed and unexposed can be made. Another criterion for sensi- tivity might be biological relevance. Some postlabeling techniques are able to detect progressively lower numbers of adJucts, so a point might well be reached where adducts are of no phenotypic significance. That point could be detennined in animal carcinogenicity studies (Ashby, 1988~. Assays should be reproducible, with limited variability ascribable to labora

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BIOLOGICAL MARCHERS 139 tory personnel or the assay method itself (Gann et al., 1985~. Reproducibility includes interlaboratory and intralaboratory reproducibility. When it is possi- ble, different methods for assaying a given marker are useful for detecting artifacts In a method. An example of such a process is the cross-validation of various DNA-adduct marker techniques with immunoassays, 32P-postlabeling and GC-MS, such as that carried out on PAH adducts of Finnish foundry workers. Feasibility The biological sample to be assayed must be readily available in humans so that procedures cause minimal discomfort to the participant. Often, under- stand~ng of the pharmacokinetics can assist In determining which tissue is most appropriate for sampling. Assays must be cost-effective. Most assays are still in the experimental stage and cost about $300-500/sample. If multiple assays are performed in tandem on the same biological samples, costs of a study are greatly reduced. That is also the case if stored samples from a prospective study or biological specimen banks are available. Storage of samples must not allow degradation of the relevant marker. Preparation and assay of samples are generally time-consuming, because repeat assays are needed to ensure reproducibility, but an assay should not require unusually complicated processing or storage efforts. Study Design Adequate Sample Size Sample size is determined by the elected differences in marker frequency or concentration between comparison groups, by the anticipated background in the exposed group, and by variability in measurement data. Considerable groundwork is required to establish those characteristics. Appropriate Control Populations Ideally, depending on the type of study (e.g., cohort vs. case-control), con- trols must be selected from persons expected to be nonexposed. However, in most studies of environmental exposures, there are numerous and often im

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140 ASSESSING HUMAN EXPOSURE portent background sources. For example, when one is evaluating inhalation exposure of PAH, ethylene ounce, or styrene from industrial sources, back- ground exposures could include cigarette smoke, diet, and indoor-air pollution. It is necessary to control and analyze for potentially confounding sources of exposure. The definition of the none~osed group will be the results of analy- sis of the chemical and biological markers. In many instances, assays have detected a substantial background of industrial compounds in nonindustrial persons, owing to the domestic use or misuse of the compounds. Hence, it may not always be necessary to be marker-free at the beginning of a longitudi- nal cohort study; however, they should have similar frequencies in each group. Control of Potential Confounding Variables This issue has been discussed above. Adequate control necessitates elicit- ing detailed environmental histories from study subjects. Where it is possible, an array of biological markers should be used to quantify their relationship to the exposure of interest, confirm the validity of the markers, and attempt to detect artifacts. Biological markers of susceptibility are useful to document the presence of confounding conditions. Use of Batteries of Biological Markers As noted previously, combination of markers is necessary to account for confounding factors. Multiple markers should also be used to distinguish between recent and earlier exposures. A battery of markers designed to assess the role of occupational exposure to aromatic amines in bladder cancer, for example, might include breathing-zone monitoring for the amines, im- munoassays of specific aromatic amine-DNA adducts in white cells (lifetimes of days to years), and cytogenetic effects in T cells (half-life, 3 years). Coti- nine in plasma could be used to learn about cigarette-smoke exposure, a potential confounding variable for bladder cancer. Analysis For a detailed discussion of the analytical considerations in biological-mark- er studies, the reader is referred to Thompson (1983), Sheldon et al. (1986), Schulte (1987, 1989), and Margolin (1988~.

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BIOLOGICAL MARKERS 141 ETHICAL ISSUES The availability of highly sensitive assays that can identify dose and effects resulting from the interplay between low-level exposures and genetic or ac- quired susceptibility raises several thorny ethical and moral questions. The reader is referred to Ashford (1986), Committee on Biological Markers of the National Research Council (1987), Schulte (1987), and Weiss (1989) for dis- cussion of those issues. Primary among them is the use that is to be made of biological-marker information. Take, for example, the theoretical case of a biological marker known to reflect susceptibility. Should a worker who tests positive or has an increased measurement be removed from the workplace? If so, should he or she be offered an equivalent job in the same industry? Or should the workplace be cleaned up to protect the most sensitive worker? In reality, few, if any, biological markers are established as predictors of individual risk associated with inborn traits, exposure, or a combination of the two. Therefore, it is important to inform research-study participants in ad- vance that the results are interpretable only on the group level. Participants in such studies should be provided test results that are presented and dis- cussed in context with available information (or lack thereof) on the variability within and between people in the normal (nonexposed) population, as well as that observed in the research-study group. SUMMARY Biological markers can be divided into three broad classes: markers of exposure, markers of effects, and markers of susceptibility. The committee focused on the application of biological markers to assessment of exposure to contaminants rather than their use to predict health effects. Markers provide information on dose that can be related to exposure using pharmacokinetic or pharmacodynamic models. However, these markers integrate all routes of exposure, and the actual routes cannot be detected without personal or micro- env~ronmental exposure information. Biological markers, therefore, are best used in conjunction with conventional measures of exposure. Biological markers range from measures of the intact original contaminant to measures of adverse health effects caused by that contaminant. Relating a marker directly to an exposure becomes more uncertain as measurements are obtained within the progression toward an effect, because the variability associated with the human "receptor" increases along that progression. Properly measured, biological markers can reduce misclassification errors of degree of exposure and can increase the power of epidemiological studies

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142 ASSESSING lIUM4N EXPOSURE to identify associations between exposure and disease. Markers can, in theory, be useful in developing and validating pharmacokinetic models aimed at relat- ing exposure to dose. They have the potential to improve risk extrapolation between species and between populations and to allow better predictions of human risk. Markers can provide an early warning of hazard or potential risk of disease. Markers measured in stored specimen banks can allow retrospec- tive determination of whether a particular contaminant was elevated in the general population at an earlier time. Use of biological markers for assessing exposure has several disadvantages and limitations. A major limitation is that most markers are in the develop- ment stage and have not been fully validated or field tested. A key question regarding markers of exposure is what compounds and exposures could have produced a marker of a measured amount in a specific tissue. The variability of markers can present problems in establishing quantitative relationships between exposure and response at low-exposure concentrations. Markers reflect diverse behavioral, biological, and methodological processes. Hence, they can be subject to more confounding influences than are measurements of ambient airborne contaminants. By virtue of their collaborative and inter- disc~plinary nature, investigations of biological markers are demanding in terms of personnel, cost, and effort to develop understanding among research ers. Analytical techniques with improved chemical specificity and sensitivity for biologically significant markers are needed to apply to exposure assessments, especially flexible assays that can analyze a number of markers simultaneously or be readily adapted to analyze numerous markers sequentially. For such techniques, validation studies are needed to link conclusively biological mark- ers to putative causative agents. Better pharmacokinetic data are needed for an increasing range of chemi- cals. These data are needed to further the development and validation of more sophisticated biological-marker models and to further understanding of how to model multiple metabolism pathways as a function of exposure level.