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1 Report of the Oversight Committee Reproduction and neurodevelopment are processes on which the continuation of any species depends. For humans, these processes carry a substantial emotional aura. We want our children to be born healthy with no impairment that would hin- der their structural or functional devel- opment, and we want reproduction to be successful at the appropriate time or times in life. In the United States, approximately 250,000 babies are born with birth defects each year. Twenty percent of these birth defects are attributed to multiple causes, 15% intrauterine infections, and 5% to a mutant gene. Environmental factors are identified as a cause with relative certainty in only 2-3% of the total number of cases. This leaves nearly 60% of birth defects that might involve unknown envi- ronmental factors. For every 3,000,000 U.S. births annually, at least 600,000 embryos or fetuses are aborted spontane- ously before the 20th week, and some 24,000 fetuses die before birth. Of live births, nearly 8% are premature and approx- imately 7% have low birthweight. Another 3-7% possess some type of malformation. Although the overall incidence of infer- tility remained stable between 1965 and 1982, infertility among married couples in which wives were ages 20 to 24 increased from 4% to 10%, and more than 2 million Amer- 15 lean couples who want to have a baby are unable to do so. This increase appears to be linked to several factors, including changes in the incidence of sexually transmitted diseases, but other factors, such as xenobiotic exposures, have not been well studied, and may contribute to reproductive impairment. The adverse effects on human reproduction of high doses of polychlorinated biphenyls, dibromo- chloropropane, and alcohol are well es- tablished, but the consequences of lower doses of these and other materials have not been well studied. The economic commitment to healthy children is difficult to measure, but Americans spent about $1 billion on medical care in 1987 to overcome infertility. Furthermore, the amount spent on remediat- ing developmental problems is large. In 1985, necrologic and communicative dis- orders alone were estimated to have af- fected 42 million Americans and cost $114 billion (Freeman, 1985~. Despite these expenditures, knowledge of basic repro- ductive and developmental processes and the environmental causes of adverse repro- ductive outcon'~s romaine ~t~sa~rinc~lv disappointing. _ 0 0 0 , . . %, Against this background desire for per- fect families with perfect children, concern is developing that environmental exposures might impair reproductive or

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16 developmental processes. Congress ex- pressed this concern in the recent Super- fund Amendments and Reauthorization Acts (SARA) by establishing the Agency for Toxic Substances and Disease Registry and recommending that epidemiologic stud- ies be conducted in populations having high risks of exposure to toxic substances from dumps. Also, beginning in the summer of 1988, the SARA requires federal, state, and local governments and industry to make publicly available an inventory of toxic chemical emissions from certain facili- ties. Through the Geographic Environmen- tal Monitoring system (GEMS), the public will have access to name, location, type of business, and quantity of chemicals entering each environmental medium an- nually. This type of information may cause the public to seek additional information on health effects and to request more local studies be performed. Such searches or studies undoubtedly will reveal that, in many cases, the impact of an environmental exposure on reproduction or development can be very difficult to determine. There is often little published information on the effect of an environmental chemical on reproduction or development in experi- mental animals and less on effects in hu- mans. By way of example, the committee reviewed evidence on an outbreak of prema- ture sexual maturity in children in Puerto Rico in the early 1980s, where no definitive cause could be identified. Where data are available on experimental animals, questions about experimental methods or about extrapolation of results across species or from high to low dose might hinder understanding of the poten- tial adverse human reproductive or devel- opmental effects. The adverse effects of high doses of polychlorinated biphenyls, dibromochlor- opropane, and alcohol for human reproduc- tion are well established, but the conse- quences of lower doses of these and other materials have not been well studied. Biologic markers, broadly defined, are indicators of variation in cellular or biochemical components or processes, structure, or function that are measurable in biologic systems or samples. For most . BIOLOGIC AL 4RKERS purposes in environmental health re- search, the reason for interest in bio- logic markers is a desire to identify the early stages of health impairment and to understand basic mechanisms of exposure and response in research and medical prac- tice. The growth of molecular biology and biochemical approaches to mane nas resulted in the rapid development of mark- ers for understanding disease, predicting outcome, and directing treatment. Many diseases are now defined, not by overt signs and symptoms, but by the detection of biologic markers at the subcellular or molecular level. For example, liver and kidney diseases are often diagnosed by measuring enzymes in blood or proteins in urine; lead poisoning can be diagnosed on the basis of blood lead concentrations and such biologic changes as increases in heme biosynthesis components in red cells and urine; and many inborn errors of metabolism, such as phenylketonuria, are diagnosed on the basis of cell biochem- istry, rather than expressed dysfunction. The identification, validation, and use of markers in medicine and biology depend fundamentally on increased understanding of mechanisms of action and the role of molecular and biochemical processes in cell biology. It is important to recognize that markers represent signals on a continuum between health and disease and that their defini- tions might shift as our knowledge of the fundamental processes of disease progres- sion increases. That is, today's markers of exposure may become tomorrow's markers of early biologic effect. What are perceived at first to be early signals of risk could come to be considered health impairments themselves because the pre- dictive relationship is so strong; i.e., the early signal could represent an effect at a stage in the progression at which, it is difficult to prevent a health impairment from occurring. Thus, biologic markers can be valuable in the prevention, early detection, and early treatment of disease. Figure 1-1 depicts the continuum involved. Recent advances in laboratory tech- niques in molecular biology have been ac- companied by increasing emphasis on the

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REPORT OF THE OVERSIGHT COM3fl7TEE EXPOSURE 1 1 hi_ ~ I NTERNAL Bl OLOG I CALLY EARLY ALTER ED CLI N I CAL -- L DISEASE | 1 1 _ _ ~ , J _ _ SUSCEPTIBILITY 1 1 . ~ _ ~ FIGURE 1-1 Sunplif~ed flow chart of classes of biological markers Vindicated by boxes). Solid lines indicate pros gression, if it occurs, to the not class of marker. Dashed lines indicate that ~ ridual susceptibility influences the rates of progression, as do other variables described In the text. Biological markers represent a continuum of changes, and the classification of change may not always be distinct. Source: Committee on Biological Markers of the National Research Council, 1987. use of markers in epidemiology. In 1977, Higginson described molecular epidemi- ology and its use of markers as the applica- tion of sophisticated techniques to the epidemiologic study of biologic material. Perera and Weinstein (1982) later defined molecular cancer epidemiology as an ap- proach combining analytic epidemiology and biochemical or molecular techniques to identify the role of exogenous agents or host factors in the causation of human cancer; these techniques included those for identifying carcinogens in human tis- sues, cells, or fluids and measuring early morphologic, biochemical, or fun- ctional responses to carcinogens. At the same time, Lower and Kanarek (1982) published an extensive discussion of the mechanisms of neoplastic disease and de- scribed molecular epidemiology as the measurement of molecular parameters re- lated to neoplastic disease. In June 19845 the National Institute of Environ- mental Health Sciences convened a major task force on research needs in environ- mental health. The report from that task force included recommendations for study of biochemical and cellular markers of chemical exposure and preclinical indica- tors of disease (NIEHS, 1985~. Other pub- lications that examined the use of biologic markers in environmental health research include those of Reff and Schneider (1982), Fowle ( 1984), IARC ( 1984), Silbergeld (1985), and Wogan and Gorelick (1985~. There is growing interest in the use of biologic markers to study the human health effects of exposure to environmen- tal toxicants in clinical medicine, epide- miology, toxicology, and related biomedi- cal fields. Clinical medicine uses markers to allow earlier detection and treatment of disease; epidemiology uses markers as indicators of internal dose or of health effects; toxicology uses markers to help determine underlying mechanisms of dis- eases, develop better estimates of dose- response relationships, and improve the technical bases for assessing risks at lower levels of exposure. This report focuses on the identifica- tion of indicators of differences between individuals or between cells that might be related to the reproductive potential of adults or the development of children. Few such biologic markers have been demon- strated to identify early stages of health impairment or toxicologically relevant internal doses. The detection of increased alpha-fetoprotein in a pregnant woman's serum and in amniotic fluid has been used to identify fetuses at risk of neural tube defects. Concentrations of lead in serum have been correlated with necrologic changes. Those two examples of biologic markers predict adverse health effects if measured concentrations are extreme. But for only few other biologic markers have particular values or ranges of values been demonstrated to be predictive of ad- verse health effects of specific toxic exposures. Therefore, this report dis-

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18 cusses a broad range of biologic markers and their use in studies of reproductive and developmental toxicology. The subcom- mittee does not discuss their utility for predicting adverse health effects of toxic exposures, because research results rele- vant to that interpretation are not avail- able. This introductory chapter presents concepts, definitions, and selected ap- plications of biologic markers, which reflects the efforts of the committee to create a framework for the overall project. The following four sections discuss bio- logic markers associated with male repro- ductive, female reproductive, pregnancy, and neurodevelopmental toxicology. Given the current status of research and development with respect to biologic markers in these areas, the approach to describing them varies. In each section, biologic markers are discussed in terms of their immediate and potential utility in environmental health research. The final chapter of each section is a summary of conclusions and recommendations. CONCEPTS AND DEFINITIONS As we use the term here, markers can be signals or indicators of normal physiology or forerunners of health impairment. A specific biologic marker can serve several purposes and is best defined by the use to which it is put in a particular context. Markers can indicate susceptibility, exposure to an exogenous agent, internal dose, biologically effective dose (dose at receptor site), early biologic effect, structural or functional alteration, physiologic status, or disease. Figure 1-1 shows the relationship among these and indicates that a biologically effec- tive dose can itself alter susceptibility. The choice of a marker and its interpreta- tion depend on the purpose of its use, and its intended use depends on characteris- tics specific to an exogenous agent in question, to the individual organism, and sometimes to a target organ or tissue (see Table 1-1~. When the goal is prevention, the major emphasis would be on markers that identify biologic changes that are predic- tive of health impairment or overt disease. BIOLOGIC MARS TABLE 1-1 Examples of Charactenstics of Exogenous Agents, Organic or Targets That Influence Choice of Biologic Marker Agent-specific characteristics Physicochemim1 properties Interactions Routes of exposure Exposure Exposure concentration Pattern of exposure Metabolism Activation Detonation Organism-specific characteristics Species Age Sex Physiologic state Pharmaco~netic characteristics Genetic factors Lifestyle factors Organ- or tissue-specific characteristics Location Blood flow Membrane permeability Transport Receptors Function Homeostasis Structure Physiologic state The committee has found it useful to define three general categories of biolog- ic markers: those of exposure to chemical or physical agents, those of effects of exposure, and those of susceptibility to the effects of exposure. A biologic marker of exposure is an exogenous substance or its metabolite~s) or the product of an interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organ- ism. A biologic marker of effect is a meas- urable alteration of an endogenous com- ponent within an organism that, depending on magnitude, can be recognized as a poten- tial or established health impairment or disease. A biologic marker of susceptibil- ity is an indicator of an inherent or ac- quired limitation of an organism to respond to the challenge of exposure to a specific xenobiotic substance.

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REPORT OF THE OVERSIGHT COMMITTEE Biologic Markers of Exposure External exposure is the amount or con- centration of xenobiotic material in the environment of an organism; internal dose is the amount of a xenobiotic material that is transferred or absorbed into the organism. Biologically effective dose, in general terms, is the internal dose that is quantitatively correlated with an iden- tifiable biologic effect; however, it is more precisely considered to be the amount of xenobiotic material that has interacted with a critical cellular or tissue receptor or target where the biolog- ic effect is initiated. Because such re- ceptor sites are often not known, or are not accessible for sampling, it is fre- quently necessary to use a surrogate site for which the dose has been correlated with the biologically effective dose or the identifiable biologic effect at the target. There is a continuing need for the devel- opment of more accurate markers of internal dose that reflect the biological- ly effective dose. The amount of a xeno- biotic that is actually absorbed is usually not known. Biologically effective dose might depend on individual characteris- tics, which account for a large part of observed differences in effect. Markers of exposure can be based on steady-state or pharmacokinetic measures, such as cir- culating peak concentration, cumulative dose, or plasma half-life. Individual variations in physiologic characteris- tics-such as sex, age, blood flow, mem- brane permeability, and respiratory rate- can significantly affect the absorption and distribution of a chemical and its metabolites (Table 1-2) (Doull, 1980~. For example, physiologic alterations in blood flow during pregnancy significantly alter distribution of drugs to the target tissue (Mattison, 1986~. Also, age and health status, such as disease, can alter respiratory rates and thus the pulmonary dose of a toxicant.. Exposure concentration (inhalation), size of delivered dose (ingestion), and dose rate also affect internal dose. When absorption capacities are exceeded, al- ternate pathways of clearance come into 19 TABLE 1-2 A Classification of Toxicity-Influencing Factors Factors related to the tome agent Chemical composition (pH, choice of anion, etc.) Physical characteristics (particle size, method of formulation, etc.) Presence of impurities or contaminants Stability and storage characteristics of the toxic agent Solubility of the toxic agent in biologic fluids Choice of the vehicle Presence of excipients: adjuvants, emulsifiers, surfactants, binding agents, coating agents, coloring agents, flavoring agents, preservatives, antioxidants, and other intentional and nonintentional additives Factors related to the exposure situation Dose, concentration, and volume of administration Route, rate, and site of administration Duration and frequency of exposure Time of administration (time of day, season of the year, etc.) Inherent factors related to the subject Species and strain (taxonomic classification) Genetic status (littermate, s~bl~g$ multigenerational effects, etc.) Immunologic status Nutritional status (diet factors, state of hydration, etc.) Hormonal status (pregnancy, etc.) Age, sex, body weight, and maturity Central nervous sytem status (activity, handling, presence of other species, etc.) Presence of disease or specific organ pathology Environmental factors related to the subject Temperature and humidity Barometnc pressure (hyper- and hypobanc effects) Ambient atmospheric composition Light and other forms of radiation Housing and caging effects Noise and other geographic influences Social factors Chemical factors play. High vapor concentrations may be "blown off," not absorbed. Species dif- ferences in metabolism can drastically alter internal doses of reactive metabol- ites. The internal dose of an xenobiotic can vary with route of exposure, chemical spe- cies, and physical form. To make qualita- tive or quantitative estimates of expo- sures with biologic markers, the concen- tration, duration and pattern of exposure,

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20 and physicochemical nature of a toxicant must be considered in the selection of an appropriate marker of exposure (Gibaldi and Perrier, 1982~. Other environmental factors, such as temperature, can affect exposure by changing amounts of water con- sumption and thus waterborne pollutants ingested. Diet alters intestinal motility and gastric emptying time, as well as the transport of specific substances, for example, diets low in iron appear to facil- itate intestinal uptake of lead (Silber- geldetal., 1988~. The presence of active mechanisms of transport into an organ or tissue and the density of receptor sites can all influence internal dose and biologically effective dose. Dependence on metabolic activation is critical; the tissue distribution of metabolizing enzymes is also an important determinant of biologically effective dose. Interpretation of dosimetric data also involves understanding the role of the receptor in overall cell-organ-or- ganism function, of coexisting or pre- existing stresses on the organism, and of the existence and availability of com- pensation during and after exposure (Doull, 1980~. The determination of the significance of a biologically effective dose depends on an understanding of how a predicted effect is induced. For exam- ple, prepubertal males and females appear to be less sensitive than sexually mature persons to the effects of alkylating agents on gonadal function. In the female, that is probably related to the greater number of oocytes in the ovary before the onset of ovulation (Mattison, 1985~; in the male, it appears to be due to the lower rates of cell proliferation and blood flow and lower capillary permeability that are characteristic of the sexually immature testis (Blatt et al., 1981~. Another important biologic marker of exposure is body burden, which is the total internal dose or that which has accumulated over time in the organism. Depending on the toxicokinetics of the agent, body bur- den might be a biologic marker of exposures that occurred recently or in the distant past. Body burden includes the dose at the target receptor sites, but it can also include amounts of xenobiotic material BIOLOGIC MARKERS stored in other, nontarget compartments. Although the body burden in remote com- partments might or might not be relatively inert biologically, it has the potential to be released under conditions of metabol- ic stress. Such release could result in a highly detrimental biologically effec- tive dose long after the original external exposure. Biologic Markers of Effect For purposes of environmental health research, biologic markers of effects in an organism after exposure to a toxicant are considered in the context of their relationship to health status-from normal health, through health impairment, to overt disease. In that context, an effect is defined as any of the following: An alteration in a tissue or organ. An early event in a biologic process that is predictive of development of a health impairment. A health impairment or clinically recognized disease. A response peripheral or parallel to a disease process, but correlated with it and thus usable in predicting develop- ment of a health impairment. Thus, a biologic marker of an effect can be any qualitative or quantitative change that is predictive of health impairment resulting from exposure to an exogenous agent. The same biologic marker might also be useful as an indicator of normal physi- ology, e.g., a particular range of blood glucose concentration. Markers of early biologic effects in- clude alterations in the functions of tar- get tissues after exposure. As early- warning signals, such markers can be useful dosimeters to guide intervention aimed at reducing or preventing further expo- sure. Such early-warning signals might also be observed in organs or tissues other than the sites that are critical for toxic action. A tissue affected by a toxicant might exhibit altered function even if the ex- posed person has no overt manifestations. Such altered function can in some cases

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REPORT OF THE OVERSIGHT COMMITTEE be determined by testing, particularly with biochemical methods. Biologic mark- ers of such altered functions are most useful if related to a specific organ or function-e."., ,B-microglobulin for kid- ney function and luteinizing hormone for ovarian function. If exposure to a toxicant and internal dose is great enough, disease will develop, because the biologically effective dose will be sufficient to affect some function irreversibly or for a substantial period. Disease that occurs soon after exposure might be directly linked to the toxicant. Disease that occurs long after exposure might be difficult to relate to a toxicant (e.g., ovarian or testicular failure or cancer), unless the findings are patho- gnomonic, i.e., are relatively specific to a particular type of exposure (such as mesothelioma) or are rare in unexposed persons (such as angiosarcoma or vaginal adenocarcinoma). The transition to overt disease can de- pend on properties of the toxicant, the nature of exposure, the disease process itself, or individual susceptibilities. Because people respond differently to toxicants, it is not surprising that only some members of a population similarly exposed to a given environmental agent will develop a given disease. Although scientists tend to divide bio- logic markers into groups, it seems evident that there is a continuum between health and disease, and advances in toxi- cology have demonstrated a continuum be- tween exposure and effect. Accordingly, what once appeared to be more or less dis- crete groups of biologic markers are now more difficult to discern. Biologic mark- ers are best divided operationally, de- pending on how they are assessed and how they will be used. but the divisions should not be interpreted to imply mechanistic distinctions. If a biologically effective dose is cor- related with an effect or concentration at a peripheral site, this can also func- tion usefully as a surrogate for the dose or effect that is occurring in the target tissue. Such surrogates can be used as markers of exposure and effect at the site of action. They include indicators 21 of the dose of indirectly acting toxi- cants-such as signals of altered hepa- tic metabolism of sex hormones, which can affect fertility (Mattison, 1985)and signals from surrogate compartments, such as measurements of red blood cell 6-amino- levulinic acid dehydrate (ALAD), an en- zymatic marker of a biologic effect of lead (Hernberg, 1980; Singhal and Thomas, 1980~. An example of a marker that is closely related to external dose, biolog- ically effective dose, and health status is the use of lymphocyte DNA adducts as markers of absorbed dose, dose at the mole- cular site of action, and likelihood of cancer (Perera and Weinstein, 1982~. Car- boxyhemoglobin (COHb) concentration after exposure to carbon monoxide has also bepen used to indicate internal dose and to predict effects. A major goal of biolog- ic marker research is to develop surrogates that link exposure and effect. It can be difficult to establish an as- sociation between biologic marker of ex- posure and a marker of effect. For example, blood lead content might appear to be a more direct indicator of internal lead dose than is a lead-induced increase in free erythrocyte protoporphyrin (FE P) , which is clearly an effect. In some clinical situations, however, FEP is a more valid marker of total lead body burden than is blood lead content, and it might also pro- vide more accurate information on the bio- logically effective dose of lead to target organs, such as the brain (Lauwerys, 1983~. Although the presence of hemoglobin and lymphocyte DNA adducts of mutagenic alkyl- ating xenobiotic compounds reflects a biochemical effect, they might also be considered markers of biologically effective doses of carcinogens that are uniformly distributed (Perera and Wein- stein, 1982; Osterman-Golkar and Ehren- berg, 1983; Shamsuddin et al., 1985; Wogan and Gorelick, 1985~. Unless the lymphocyte itself is the precursor of a tumor, the white-cell DNA adducts (and their surro- gates, the hemoglobin adducts) are appro- priately considered to be indirect markers of biologically effective dose in the target organ (NIEHS, 1985; NRC, in press).

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22 Biologic Markers of Susceptibility Some biologic markers indicate indivi- dual or population factors that can af- fect response to environmental agents. These factors are independent of whether exposure has occurred, although exposure sometimes increases susceptibility to the effects of later exposures (e.g., sen- sitization to formaldehyde). An intrinsic characteristic or pre-existing disease state that increases the internal dose or the biologically effective dose or that amplifies the effect at the target tissue can be a biologic marker of increased sus- ceptibility (NIEHS, 1985; Omenn, 1986~. Such markers can include inborn differ- ences in metabolism, variations in immuno- globulin concentrations, low organ re- serve capacity, or other identifiable genetically or environmentally induced factors that influence absorption, meta- bolism, detoxification, and effect of environmental agents. We do not discuss these types of biologic markers fully here, but cover them only to the extent that mark- ers of susceptibility also serve as mark- ers of exposure or effect. PRINCIPLES OF SELECTION OF MARKERS The selection of biologic markers of exposure or effect is based on a wide array of background data from in vitro and in viva experimental studies, from epidemiologic studies, and from meas- urements of exposure and on physico- chemical properties of the toxicant in question, as shown in Table 1-1. Biologic markers of exposure can be obtained by measuring the concentration of a particular toxicant or its metabolites alone or bound to DNA, RNA, proteins or receptors in body tissues or fluids, and in excretory products. The use of markers can be complemented by the use of question- naires that call for estimates of dura- tion and magnitude of exposure, such as work-history questionnaires or activity time-budget questionnaires. Markers of effect can be obtained by such procedures as biochemical analyses for organ-specific events. BIOLOGIC AWARDERS Biologic Considerations A mechanistic approach to the basic events that result in an adverse health effect must be taken in the selection of an appropriate biologic marker. The mech- anistic approach should yield biologic markers that identify the initial stages of disease. These markers are valuable tools for developing strategies to prevent progression of disease. Practical Limitations Ideally, the use of biologic markers to screen human populations involves mini- mally invasive techniques. Organ analy- sis, high-dose x irradiation, autoradio- graphy, or covalent binding assays can be used to identify sites of toxic action in laboratory animals, but cannot be readi- ly applied to human populations. Less invasive methods, such as nuclear magnetic resonance imaging, might eventually make it possible to estimate the concentrations of specific chemicals and specific types of effects (e.g., changes in cellular ener- getics and phosphorylation (Cohen et al., 1983) in remote target tissues in humans). In the meantime, detection must be done in surrogate compartments. The use of biologic markers also should involve test procedures that are readily acceptable by subjects. Unless a test is readily available, uncomplicated, and acceptable to the general public, partici- pation will be low. For example, fetal monitoring by ultrasonography, transab- dominal amniocentesis, and the karyotyp- ing of amniotic or chorionic villus cells for chromosomal abnormalities is relatively safe, but those procedures are not acceptable to the general population for routine biologic monitoring. Field studies-whether performed in the home, in the workplace, or elsewhere-often have the lowest refusal rate for human studies. In assessing the predictive value of biologic markers, it is necessary to ac- count for the heterogeneity of the human population, which is composed of persons who differ in age, genetic constitution, nutritional status, and general health. It is also necessary to identify persons

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REPORT OF THE: O~ERSIG~ COMMITTEE who are likely to exhibit the earliest and most severe effects of exposure to environ- mental agents. Animal models can sometimes be used to study the biologic mechanisms of hypersusceptibility and to examine in detail the effects of environmental toxi- cants on hypersusceptible groups. Batteries of Biologic Markers Limitations are associated with most biologic markers of exposure and of effect; therefore, it might be reasonable to use a battery of markers. And, it might be use- ful to develop markers of both exposure and effect for chemicals of concern. For example, to determine whether there is a potential or actual adverse effect of dimethylformamide on male reproductive function, a battery of functional tests of the male reproductive system might be tried, including concentration of for- mamide in urine or in seminal fluid (Kennedy, 1986~; sperm density in the eja- culate; sperm motility; sperm morphology (seminal cytology), including sperm- head shapes and Y-body test (Kapp and Jacobson, 1980~; interspecies sperm penetration assay (e.g., human sperm and Syrian golden hamster ovum) as an indicator of sperm chromosomal aberrations; and determination of plasma concentrations of hormones (e.g., testosterone). A1- though each of these tests has inherent limitations of specificity and sensitivi- ty and varying predictive value as a bio- logic marker of male reproductive function (e.g., sperm density fluctuates daily, and sperm motility is difficult to meas- ure), a battery of tests can be a powerful tool for indicating dysfunction and its association with exposure to a chemical. Ethical Issues The use of biologic markers has raised a number of important ethical issues (Ashford et al., 1984; Ashford, 1986; Samuels, 1986; Yodaiken, 1986~. Although the principal purpose of this project is to address scientific aspects of biologic markers, it is appropriate to draw atten- tion to the broader questions and issues that need to be addressed soon by society. 23 These are related particularly to markers of susceptibility. Does society have an obligation to pro- tect people beyond informing them of risks? Can an employee be forced to leave his or her job once a susceptibility marker has been detected or a biologically effec- tive dose has been received? There is a concern that focusing on the detection of susceptible persons could replace ef- forts to remove toxic chemicals from the workplace. Other ethical considerations arise from the degree to which susceptibil- ity markers are accurate predictors. For instance, it is important to distinguish between markers that are totally predic- tive of an adverse effect, reasonably pre- dictive, or only minimally predictive. Ethical issues are also pertinent in the consideration of using biologic mark- ers as a basis for making decisions about consumer products. For example, should an item of value or convenience to the gen- eral public be withdrawn from commerce because a few persons are susceptible to adverse effects of the item, or should those susceptible be responsible for avoiding contact with the item, given ade- quate labeling? Developments in science and technology have posed many ethical questions. As we move rapidly into an era of greater under- standing of the interactions between gene- tic material and exogenous chemicals and other biologic interactions, we must anticipate and be prepared to address the ethical issues that will certainly arise. VALIDATION OF BIOLOGIC MARKERS Sensitivity and Specificity To validate the use of a biologic meas- urement as a marker, it is necessary to understand the relationship between the marker and the event or condition of inter- est, e.g., potential for actual health impairment, health impairment, or suscep- tibility. Sensitivity and specificity are critical components in the process of validation (MacMahon and Pugh, 1970~. Sensitivity is the quality of an epidemio- logic test method that relates to the abil-

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24 ity to identify correctly those who have the disease or condition of interest. Specificity relates to identifying cor- rectly those who do not have the disease or condition of interest. Thus, markers of exposure or effect must be validated in terms of their ability to assess the true exposure or disease (sensitivity) and their ability to assess the lack of expo- sure or disease (specificity). Particularly critical for markers is the strength of biologic plausibility that allows an association between a change in a specified signal (designated as a marker) and the occurrence of a specific exposure or a change in probability of a specific outcome. A major purpose of mark- ers in environmental health research is to identify exposed persons, so that risk can be predicted and disease prevent- ed; therefore, validation involves both forward and backward processes of associa- tioni.e., from marker backward to exposure and from marker forward to effect. A complete understanding of the limita- tions of a given biologic marker is cru- cial for its appropriate application and interpretation. Uses Validation of a specific marker also depends on its expected use. Biologic markers observed well before the onset of disease might have low predictive value for the disease itself, nevertheless func- tion acceptably as criteria for defining exposed populations and thus be useful for long-term followup. For example, meas- urement of concentrations of pesticides, such as polychlorobiphenyls (PCBs) in human breast milk is clearly useful for exposure assessment and epidemiologic research, although its relationship to disease outcome might be difficult to de- termine. Conversely, an effect marker that is expressed long after exposure could be of relatively little use in exposure assessment, but be very important in pre- dicting progression of disease or calcu- lating risk. For example, a prenatal expo- sure that results in altered structure or function in the child or adult might be BIOLOGIC MARKERS difficult to identify on the basis of mark- ers associated with the altered structure or function. Animal Models In validating biologic markers, animal models are useful for understanding mech- anistic bases of the expression of markers and relationships among exposure, early effects, and disease. If a disease can be satisfactorily induced in experimental animals, then potential biologic markers for predicting eventual disease can be explored, and early indicators of the dis- ease might be identified for use in epide- miologic studies. Also, markers of expo- sure can be explored for utility as markers of effect by relating the concentrations in an accessible compartment (or surro- gate) to concentrations at the actual re- centor site. The goal is to develop markers that reliably indicate an early stage in the development of a disease in humans when effective intervention is still possible. A useful approach to the validation of marker data is to conduct experimental studies in animals and clinical studies in humans to develop information that per- mits interspecies comparisons. Markers of acute effects of short-term, low-dose, or high-dose exposures to a pollutant can be investigated in both animals and humans. Comparison of the results with information on markers of chronic effects of long- term low-dose exposure of animals to the same pollutant could lead to the develop- ment of markers that are predictive of health effects in chronically exposed humans. Quality Assurance Quality assurance and quality control are fundamental to the objective devel- opment and application of accurate and verifiable biologic markers. The objec- tive of laboratory quality-assurance practices is to ensure that findings re- ported by one laboratory are in fact veri- fiable and within acceptable limits of measurement error, that they accurately indicate the concentrations or presence of materials reported to have been found,

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REPORT OF THE OVERSIGHT COMMITTEE and that they are objective and free from sources of bias introduced through the analytic process. General issues of quality assurance and quality control have been addressed by documents produced by the Food and Drug Administration (FDA), the U.S. Environ- mental Protection Agency, the Organiza- tion for Economic Co-operation and Devel- opment, and other regulatory organiza- tions. FDA developed a set of guidelines known as Good Laboratory PracticesGLPs (U.S. FDA, 1988), which are now incorpor- ated into the standard procedures of most testing and analytic laboratories. GLPs are intended to reduce the chance of con- tamination (particularly important in the measurement of biologic markers of exposure) or of changes in biologic vari- ables introduced by sample storage, proc- essing, or measurement (Zeisler et al., 1983~. The application of GLPs to analysis of biologic samples, especially human tissue, has been reviewed by operational units of the Centers for Disease Control, the National Bureau of Standards, and vari- ous clinical laboratories (ACS, 1980; NCCLS, 1981, 1985~. Issues of quality assurance related to screening for muta- gens and reproductive toxins are discussed in Bloom ( 1981~. In establishing guidelines for quality assurance, the usual sequence is to develop methods of increasing authority based on accumulation of experience. Experience permits a method to be standardized, once it has been shown to be feasible, reprodu- cible, and accurate when used in various laboratories. In some cases, cost-effec- tiveness is also a factor, particularly for clinical measurements intended to be used for screening, rather than research. This approach to standardized guidelines has been followed for some biologic markers. Standardized reference methods, which have been well tested in the field, are available for measuring blood lead con- centration as a marker of exposure. In fact, a standardized procedure for inter- laboratory comparison of blood lead con- tent has been developed and used by the Centers for Disease Control (Annest et al., 1983~. Such a procedure, sometimes 25 known as round-robin testing, can be used to verify the performance of various test- ing laboratories. The preparation of biologic standards for measuring markers of effect is more complex. Cell-culture systems might pro- vide particular types of standards; in some cases, the chemicals that constitute the marker (metabolic product, intermedi- ate, or other material, such as protopor- phyrin) can be synthesized or derived from other biologic materials and incorporated into an appropriate biologic matrix to meet criteria of quality assurance. Sensitivity of Measurements Other general quality-assurance issues are related to sensitivity and specifici- ty. Estimations of sensitivity must in- clude considerations of the so-called background rate of events or concentra- tions likely to be found in persons without particular exposures, as well as consider- ations of the magnitude of external expo- sure or internal dose likely to be received by the population being sampled. In the presence of relative uncertainty as to the nature and extent of exposures in the reference or control population, deci- sions concerning sensitivity can be dif- ficult. Markers of Specific Exposures Ideally, we seek to correlate a biologic marker with a specific exposure. In order to measure concentrations of chemicals in body tissues and fluids, analytic tech- niques must be validated in the biologic media measured. Identification with atom- ic-absorption spectrophotometry, spec- trophotofluorometry, or electrochemistry can sometimes be confirmed by gas or liquid chromatography in conjunction with mass spectrometry or nuclear magnetic reso- nance Imaging. Biologic markers might be specific with respect to the system being investigated, but of unknown specificity with respect to the exposure or end point being studied. That is the case with markers of reproduc- tive function, such as plasma concentra- tion of human chorionic gonadotropin (a

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26 marker of early stages of gestation) or amniotic fluid concentration of alpha- fetoprotein (a marker of integrity of fetal development). These are highly specific markers of reproductive status, but of unknown and probably variable specificity with respect to exposure to xenobiotic agents. Other biologic markers are even less specific, such as some serum enzyme concentrations, which can reflect a wide variety of organ-level phenomena, includ- ing hepatic metabolism, renal clearance, turnover, release, and cytotoxicity. Determination of specificity involves consideration of variation, including age, sex, time of day, etc. When genetic markers are being measured, information on the possible impacts of heterozygous variation must be considered relevant. Age-matched and sex-matched cohorts should be established to control for age- and sex-related differences. Diurnal variation might be unknown for some mark- ers, but is clearly important for some reproductive, nervous-system, and immuno- logic markers. The handling of many specificity issues will improve as information is gained on aspects of human biology pertinent to un- derstanding the impact of xenobiotics. In the absence of such knowledge, it is critical to gather as much information on potentially confounding variables as possible through comprehensive history- taking and other methods. Whenever pos- sible, the collection of material for long-term storage should be encouraged, so that nested case-control studies can be conducted later, as new measures are developed, and hypotheses related to risk and outcome can be validated. ECOLOGIC MARKERS The biologic markers approach has great potential relevance to assessing and pre- dicting not only effects of exposure to xenobiotics, but effects of environmental modifications on ecologic systems and nonhuman target organisms. Biologic mark- ers of the status and function of an ecolog- ic system include morphologic and biochem- ical observations on individual members of the system, observations that are not BIOLOGIC MARKERS different from those in humans (although some tissues obtained from nonhuman or- ganisms might be unobtainable from humans because of ethical constraints). Markers might also be derived from functional groups, or communities, within ecosys- tems. Such markers would reflect the bio- logic consequences of exposure, such as shifts in the fixation of nitrogen or pho- tosynthesis by trees. They might involve control of rates of processes, such as Vitrification by a single species in the forest floor, or of the total photosynthe- sis of algal populations in a body of water. Physiologic effects in individuals in ecosystems can combine to affect a larger process, such as reproduction of field , mice in a grassland system. As a conse- quence of such effects, the relative abun- dance of species, the types of species, or the individuals within a species can change over time. The loss of a species is an ecologic mark- er of ecosystem damage, but it is unlikely to provide early warning of potential ef- fects before substantial damage has been sustained. Earlier, more subtle biologic markers in the sequence of events that leads to such a relatively drastic outcome should be sought. USE OF BIOLOGIC MARKERS IN RISK ASSESSMENT Cellular and molecular markers can be powerful new tools for the assessment of risks associated with exposure to environ- mental toxicants. Markers that indicate the receipt of an internal, biologically effective dose or the induction of a dis- ease process can be useful in hazard iden- tificationi.e., as the qualitative step by which an environmental agent is causally associated with an adverse effect. Biolog- ic markers can also be used to determine dose-response relationships, especially at the low doses relevant to exposure to most environmental chemicals. As Ehren- berg has demonstrated (1988), it is pos- sible to measure concentrations of ethyl- ene oxide-derived DNA adducts that cor- respond to an increased cancer risk of approximately one in a million, an in- crease often used to justify environmen-

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REPORT OF THE OVERSIGHT COMM17TEE tat regulation. The use of biologic mark- ers indicating exposure and dose in molecu- lar epidemiology studies provides the opportunity to determine the shape of the lower end of the dose-response curve in humans, an opportunity not available with standard epidemiologic or animal car- cinogenicity testing approaches. Another major role of biologic markers pertinent to risk assessment is in clarification of the extent and distribution of exposure and effect in human populations, as well as of the variability and susceptibility among individuals in a population (Fowle, 1984; Perera et al., 1986~. EXTRAPOLATION FROM ANIMALS TO HUMANS The validity of a specific biologic mark- er for the identification of an adverse health effect depends on the reliability of studies that provide the background data, particularly on mechanisms. In addi- tion, the direct relevance of studies in animals to humans needs to be assessed carefully. Laboratory animals and humans can differ in toxicokinetics. Thus, use of data from animals to determine health risks in humans might be inappropriate if the data are derived from monitoring of external exposure. The toxicity of some chemicals is mediat- ed either by activation or by detoxifica- tion biotransformation reactions. Inas- much as biotransformation differs among species, it is important to establish whether the routes and rates of human and animal metabolic pathways are similar. Health risks are often associated with combinations of effects in humans. For example, cardiovascular disease in humans can encompass both atherosclerosis and hypertension. Although swine are the most suitable animal model for studying spon- taneous atherosclerosis, young rats might be most appropriate for studying hypertension. For humans, estimating these diseases will necessarily entail some appropriate combination of the rele- vant animal test systems. A common source of uncertainty in risk assessment is the dose-response curve 27 relationship at low doses or for rare ef- fects (NRC, 1983, 1986~. It is often im- practical to conduct studies of effects at low doses, because large numbers of animals are required to detect a low in- cidence of effects (Wilkinson, 1987~. Demonstrable health effects in humans, given the limits of epidemiology, are often associated with high doses and hence high risk. Sensitive molecular markers being developed will permit study of the rela- tionships between exposure to chemicals at low ambient concentrations and the for- mation of a molecular marker predictive of human risk. The development of biologic markers might enable scientists to make better use of laboratory animal data in estimating the effects of chemicals in humans. QUALITY AND QUANTITY OF DATA Extrapolations to humans are to be based on the most sensitive animal species test- ed, barring clear evidence that the species is pharmacokinetically distinct from humans. Within the past 2 years, EPA has issued guidelines for evaluating reproductive studies (U.S. EPA, 1987~. These provide a means to estimate data quality and stip- ulate segment II developmental toxicity requirements of two species, three treat- ment groups, with 20 rodents or 10 non- rodent mammals. As this report went to press, EPA issued additional guidelines on animal reproductive studies. Quantity of data required can be deter- mined by statistical power considera- tions, and resource considerations. Sta- tistical power is related to the number of subjects in a group, to the rarity of the end point studied, and to the variabil- ity in the frequency of the end point's occurrence. The greater the expected rela- tive risk, the smaller the population that will need to be studied. The study of reproduction and develop- ment poses major resource and logistic problems for those working with laboratory animals. For instance, manageable sample populations do not reveal increases in toxic events of less than 5 to 10 percent. For some health effects, such as mutagene-

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28 sis and teratogenesis, incidences in a human population of 3 per 10,000 are sig- nificant. Obviously, these effects cannot be well studied in whole-body studies of thousands of experimental animals at a time. Classic toxicology studies of ro- dents involve the exposure and pathologic- al analyses of 200 animals for 2 years. In such assays, each animal is a surrogate for 1,000,000 people. Birth defects that would be undetectable in this rodent popu- lation, if occurring in humans, could pro- vide an epidemic within 10 generations. Approaches currently used for measuring human exposure can be divided into three major categories: modeling, ambient meas- urement, and biologic monitoring. The development of models for grossly estimat- ing human exposure is a major accomplish- ment of environmental scientists in the last few decades. Models, however, do not accurately take into account major sources of variation that affect dose received. e.g., variations in terrain or in human life styles. Ambient measurements, par- ticularly those related closely to actual human exposures (e.g., personal monitor- ing for air pollutants), can be useful techniques to estimate exposure. However, simplifying assumptions about respiration rates and other variables that affect in- ternal dose are necessary in deriving es- timates of human exposure from ambient measurements of air, water, food, or con- taminants in soil. Biologic monitoring is the measurement of exposures or effects of exposure direct- ly in receptor organisms, such as humans. The application of this approach has been limited principally by its expense and by the problems associated with sampling of humans. Furthermore, because the kine- tics and metabolism of most environmental chemicals are not known, it has been dif- ficult to develop strategies for measure- ment. Even when metabolic information is available, there might be no analytic methods to measure metabolites. Advances in analytic chemistry and molecular biolo- gy leading to such techniques as the use of monoclonal and polyclonal antibodies and the chemical analysis of DNA adducts have made it possible to detect biologic markers of exposure to a number of com- BIOLOGIC MARKERS pounds in humans. Those techniques should prove valuable for determining the extent of exposure to toxic chemicals and for establishing priorities for effort and resources; e.g., they might be directed at persons who have received the greatest exposure or who are at highest risk in con- nection with some environmental hazard. IMPLEMENTATION OF BIOLOGIC MARKERS IN POPULATION STUDIES The identification of biologic markers that indicate exposure, effect, or suscep- tibility is a complicated process involv- ing studies in animals, refinements in laboratory assays, and studies in special human populations. Moreover, even when a marker has been validated in such stud- ies, its use in larger populations is not straightforward. The framework for implementing an identified, potentially informative bio- logic marker in large population studies would include the following steps: Establish normal baseline values and distribution for the marker in laboratory animals and humans. Evaluate the sensitivity and specifi- city of the marker in predicting a health outcome (e.g., infertility or genetic damage). Understand in detail the time course of response of the marker to a toxic chemi- cal, with special attention to the reco- very process. Develop a strategy for and a consensus on the use of multiple species in toxico- logic studies. Develop human assays that use semen, saliva, or urine, rather than tissue or blood, whenever possible. Use noninvasive techniques, such as ultrasound or magnetic resonance imaging, whenever possible. Consider a battery of markers that reflect a wide array of physiologic func- tions and genetic damage and relate the marker in question to others in the bat- tery. Identify populations at high risk for reproductive or developmental health

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REPORT OF THE OVERSIGHT COMMI77EE impairment (perhaps populations exposed to drugs with reproductive or developmen- tal toxicity, aging populations, or off- spring of women exposed to diethylstil- bestrol), to serve as test subjects for the initial assessment and validation of biologic markers. Include among high-exposure popula- tions those with special or unique occupa- tional exposures (e.g., agricultural groups). Encourage and support institutions in the development of sample banks, to speed the identification and validation of markers. Establish a task force to develop and coordinate strategy. LONG-TERM TISSUE AND CELL STORAGE FOR RETROSPECTIVE ANALYSIS Long-term tissue and cell storage for retrospective analysis of exposures to previously unknown environmental toxi- cants has been used successfully by sev- eral organizations, e.g., the American Type Culture Collection and the Environ- mental Specimen Bank Program managed by the National Institutes of Standards and Technology. The oversight committee reviewed avail- able information on long-term tissue stor- age but found that national programs in this area are limited and inconsistent. The subcommittee on reproductive and de- velopmental markers also attempted to identify national storage programs for tissues from the reproductive tract; ex- cept for a few recently established sperm and ovary storage banks, no consistent programs were found. Some hospitals and medical research centers have attempted to store placenta; however, the protocols for storage are not developed sufficiently to make the tissue samples generally useful in reproductive markers research. The oversight committee and the subcom- mittee recognize that many difficulties are associated with long-term tissue ar- chiving. For example, establishing a standard protocol and training personnel to use it for collection and storage of samples is not easy to accomplish, nor can 29 adherence to a protocol be ensured easily. Collecting samples under sterile condi- tions; storing tissues in sterile, metal- free containers; and proper freezing (usu- ally at the temperature of liquid nitro- gen) upon collection and during storage are vital for preserving the cell and tis- sue integrity necessary for reliable fu- ture chemical analyses. In addition, space considerations and stable institu- tional commitment are necessary for the success of any banking program. The Na- tional Research Council's Committee on National Monitoring of Human Tissues, which is reviewing the EPA's National Hu- man Monitoring Program (an adipose tissue bank), is considering how many of these difficulties affect the collection, and ultimate analysis of archived tissues. Fixed and imbedded tissues generally are retained after toxicological studies are completed and might have some applica- tion in future biological markers re- search. Their use has been very limited because the processes of fixing and imbed- ding produce changes in the molecular con- stituents. However, recent developments in molecular biological techniques, in- cluding the polymerase chain reaction, show promise in detecting alterations in DNA and RNA in archival formaldehyde-fixed paraffin imbedded tissues (Burmer and Loeb, in press). In addition, antibodies to certain constituents also might work on aldehyde-fixed specimens. USE OF BIOLOGIC MARKERS IN REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY With the above general description of the concepts and definitions of biologic markers as background, the remainder of this report will focus on the use of biolog- ic markers in reproductive and neurodevel- opmental toxicology. Hypothetical exam- ples of the utility of biologic markers follow. These are meant to be instructive and do not represent the limits of utility of biologic markers in reproductive or developmental toxicology.

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30 Studies of Male Reproductive Toxicants Consider as a male reproductive toxicant a metal that' is used in an industrial proc- ess and emitted into the air (Fig. 1-2~. Assuming that uptake occurs by inhalation and ingestion (i.e., via the mucociliary elevator), biologic markers of internal dose can include sputum, blood, urine, sperm, and semen concentrations of the metal or its metabolites. For some metals, it might be possible, with noninvasive monitoring techniques, to measure the amount of metal in tissues, such as bone or testis. The biologic effects of the metal on male reproductive function might be measured on the basis of testicular volume (e.g., testicular swelling or shrinkage), circulating concentrations of protein or steroid hormones, altera- tions in sperm morphology or function, alterations in male reproductive behav- ior, changes in fertility and increased heritable mutations. Studies of Female Reproductive Toxicants Consider that a small-molecule organic compound is suspected as a female reproduc- tive toxicant (Fig. 1-3), and exposure is through ingestion (drinking water), skin (bathing and swimming), and inhala- tion (water droplets). Biologic markers of internal dose can include blood and urinary concentrations of the parent compound or metabolites. Measurements of exhaled parent compound or metabolites might also be of value. More invasive pro- cedures will be required to determine the concentrations of the xenobiotic or its metabolites in adipose tissue or fol- licular fluid. The biologic effects of the exposure could include alterations in the mean concentration or pulse fre- quency or amplitude of circulating steroid or protein hormones. Functional altera- tions in the female reproductive system might be reflected in menstrual irregular- ity, a change in ovulatory frequency, and a decrease in fecundity. BIOLOGIC MARKERS Studies of Pregnancy Toxicants Assume that an inorganic metal that is found in air, water, and some foods is a putative pregnancy toxicant (Fig. 1-4) External exposure occurs through air, water ingestion and contact, and dietary practices or preferences. The dose to a pregnant woman is the sum of inhaled, in- gested, and transdermal uptake. Individu- al variables that alter the dose include respiratory rate and volume, water and food ingestion, and contact with contami- nated water. Some of these factors can vary considerably In pregnancy. For example, minute volume increases by approximately 40% and blood flow to the skin increases by as much as a factor of 6 during pregnancy. Biologic markers of internal dose might include maternal and fetal blood concen- trations, amounts excreted by the mother in urine and feces, and concentrations in maternal or fetal tissues, including placenta. It might be possible to monitor for some metals in the target tissue with noninvasive techniques such as neutron activation or magnetic resonance imaging. If the metal is toxic to placental func- tion, the concentration of placental ster- oid or protein hormones in maternal serum or the rate of their excretion in maternal urine could change in response to placental concentration of the metal. The biologic effects of exposure either before or during pregnancy might include alterations in placental function, such as transport of gases, carbohydrates, amino acids, or other essential nutrients from maternal to fetal circulation. Alteration in pla- cental transport might impair fetal growth and later functional development or functional capacity of the child or adult. Exposure early in gestation might lead to fetal malformation, fetal death, or spontaneous abortion. Studies of Neurodevelopmental Toxicants Assume that an organometallic compound found as a contaminant in food is a putative developmental toxicant (Fig. 1-~. Ex- posure of an infant or child is determined by dietary practices, amounts of contami-

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REPORT OF THE OVERSIGHT COMMITTEE Xenobiotic Metal in Air CNS Hypothalamus Pituitary Testis Sertoli Cells Leydig Cells Germ Cells Epididymis Vas Deferens Accessory Sex Organs External Exposure Air in Workplace I ngestion I nhalation Dermal Exposure Environmental Monitoring Exfoli- >\ ation J 'kin Absorption Internal Dose I ndicators Blood Level Bone Level Urinary Excretion Fecal Excretion Testicular Level Semen Concentration Biologic Monitoring Inhalation Exhala- ~ tion | /` Tract | _ . (/ Sweat Semen \> ~ | Reproductive ~~ L Organs - 1 ~ ' 1 An/ ~ ( Blood ) ~ Hair ~ | 1 1 1 in/ 31 Ingestion It-,l-Tr~rt Liver Kidney At) Organs (a) Biologic Effects Testicular Swelling Decreased Testosterone Decreased Libido Altered Serum LH Sperm Concentration Changes in Sperm Quality and Function Reduced Fertility I ncreased Heritable Mutations Health Monitoring ( Feces) Media for biologic monitoring FIGURE 1-2 Hypothetical male reproductive toxicant used in an industrial process and emitted into air. Top potential monitoring media and markers. Bottom, metabolic model of hypothetical metal tomcant. Arrows indicate transfer of toxicant or metabolite. Source: Adapted from Committee on Biological Markers of the National Research Council, 1987.

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32 Xenobiotic Organic in Water Supply External Exposure Drinking Water Bathing I nhalation Water Droplets Environmental Monitoring Skin Absorption ~ Exfoli- '\ \: ation J ~ 1 ~~ 1 Skin :: Sweat ) CNS Hypothalamus Pituitary Ovary Stroma Follicle Oocyte Corpus Luteum Uterus Fallopian Tube Cervix Reproductive Organs it_ Internal Dose Indicators Blood Level Adipose Tissue Concentration Ovarian Concentration Granulosa Cell Concentration Oocyte Concentration Follicular Fluid Concentration Biologic Monitoring , Inhalation ~ Exhala-~ \ tion ] it\ 1 1 Ingestion Trac irato | ~ 3 ' 1 1 be, ( Blood / l l Kidney (,,l~ l Liver BIOLOGIC MARKERS Biologic Effects Decreased Serum E2 Increased Serum FSH Menstrual Irregularity Decreased Ovulatory Rate Decreased Fecundity Health Monitoring Organs | O Media for biologic monitoring FIGURE 1-3 Hypothetical female reproductive toxicant with exposure through ingestion, skin, and inhalation. Top, potential environmental monitoring media and markers. Bottom, metabolic model of hypothetical small- molecule organic toxicant. Arrows indicate transfer of toxicant or metabolite. Source: Adapted from Committee on Biological Markers of the National Research Council, 1987.

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REPORT OF THE OVERSIGHT COMMTITEE Xenobiotic Metal in Air Metal in Water Metal in Food External Exposure Concentration in Air Respiratory Rate Concentration in Water Water Exposure Concentration in Foods Dietary Practice Environmental Monitoring Skin Absorption Inhalation 1 /: Exfoli- ~ ~ Exhala-: ` \< tion J \; ( Blood \) \< t Sweat Fetus Placenta 1 Amniotic \ ~ ~ Hair Fluid \ 33 Internal Dose Indicators Blood Level Placental Level Fetal Level Maternal Excretion Placental Hormones Biologic Monitoring Respiratory Tract . . __ I ~ Blood Kidney - ( Urine Biologic Effects Placental Function Fetal Growth Fetal Malformation Fetal Death Health Monitoring Ingestion 1 ~ ~ I Liver I Organs Gl-Tract O Media for biologic monitoring FIGURE 1~ Hypothetical pregnancy to~ncant found in air, water, and some foods. Top, potential environmental monitoring media and markers. Bottom, metabolic model of hypothetical inorganic toxicant. Arrows indicate of toxicant or metabolite. Source: Adapted from Committee on Biological Markers of the National Research Council, 1987.

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34 Xenobiotic Organometallic in Food ~ 1 ~ ation J Ma wit ~ I BIOLOGIC AL4RKERS External Exposure Dietary Practice Food Consumption Food Preparation Environmental Monitoring Skin Absorption ~ Inhalation 1 ~1 Respiratory ~ , Tract Developing Organs _ Ingestion L Gl-Tract ] \-T ~ ~~ r Kidney | Kidney ( Urine ) a/ 3 0 Media tor biologic monitoring Internal Dose Indicators Blood Level Urine Level Fecal Level Enzyme Concentration Blood Urine Hormone Levels Biologic Monitoring Biologic Effects Cell Proliferation Cell-Cell Interaction Impaired Organ Growth Impaired Organ Function Health Monitoring FIGURE 1-5 Hypothetical developmental tomcant found as a contaminant In food. Top, potential environmental monitoring media and markers. Bottom, metabolic model of hypothetical organometallic tox~cant. Arrows indicate tom of topical or metabolite. Source: Adapted from Committee on Biological Markers of the National Research Council, 1987. nated foods ingested, and effects of pre- paration on the concentration of parent organometallic compound in the ingested foods. Markers of internal dose can in- clude blood, urinary, or fecal concentra- tions of the parent compound or metabo- lites. If the organometallic xenobiotic alters endocrine function, it might be possible to determine the biologically effective dose indirectly on the basis of alterations in the concentration or pulse frequency of hormones. The biologic effects of the organometallic compound could include altered rates of cell proli- feration or cell-cell interactions. Cel- lular effects might not be directly measur- able in some organs or systems. However, in the central nervous system, the effects may be discernable with sensitive diagnos- tic measures, including electroencepha- lography, computerized axial tomography, and magnetic resonance imaging. Later tests of organ or system function might also reflect disordered development, but latency could make identification of the etiologic xenobiotic difficult.

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REPORT OF THE OVERSIGHT COMMITTEE SUMMARY It is the job of the laboratory worker to develop tests that are as sensitive and specific as possible. It is the job of the clinician or public health worker to ensure that the benefits of using (invariably) imperfect tests outweigh the difficulties that arise from low predictive values when the tests are used in individuals or com- munities with low a priori probability of exposure or disease. Careful consideration must be given to how a test for a biologic marker of exposure will perform in the field. It is not generally appreciated that a key factor that will affect performance is the fre- quency of exposure in the population in 35 which the marker test is used. The use of even a good test in a population in which exposure is rare will result in a low pre- dictive value of a positive test result, that is, many false-positives results. Widespread application of such tests must be carefully considered to ensure that the benefits outweigh the risks. Few areas have changed in medicine as rapidly as those that are the subject of this report. The problems of reproductive and developmental toxicology remain com- plex because advances in our understanding of toxic chemicals have not kept pace with the introduction of new materials into the environment. A significant proportion of human reproductive wastage remains of unknown etiology.

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