Environmental Health Perspectives Vol 90, pp. 261-269, 1991 The Potential of Exposure Biomarkers in Epidemiologic Studies of Reproductive Health by Carol J. Rowland Hogue* and Marge A. Brewstert 1l urthr tbe delopment and ap a of exposure marker in ffid igons in reproductive epidemiooy, we have synthesizd recent min s of the iums sn dng expoe _ in rt ep oductivee The specific godds Of this pper are to die e e d e le their pkuiel u, ar took The tests for glucaric ad, m idty, and po n p meet the genal criteria for usel ex- posure screens. For certain enobodc agents, the tests y diferentateposure k , as demonstrated in oc- cuptional and niromental studies. As urinary screens, they are noninvasve and apble on a large scale with current labo techniques For short-term esue, glucaric acid, thloetbers, and mug tests are usefl. Fpyn pattrs may measr cumulative effects asswell acurrent eqxu klveh The ness of these tests in epidemiologic udesof ei ental effects on repducthealth ha yet to be studied. 'lb do so, the battery must be _ed for pregant wemen, and ts reults must be correlaed with measured adverse ep u comes such as g I nllgh and l ht. Tis a i p il y Im at bece m a e e rater tIu feti exposure isbeingm d Theex eUttowhichIntohtic c hemiaiscrooathep barriermqvary greadyt de-pendng on the type of epsues, tming in p and materal Wi better eqre measures, ep studies of halt probably Wm not ci ienobiotic fegotoc* agents in the environment. However, with an adeq battery of necf expsre blmarkers, prospective studies of en- vironmental effects on pregnancy outcomes might be possible. lb narrow the lst of potentil exposures, these prospec- tive studies could be foLowed by case-cntrol studies of more specific blomarks dired at upect exposures. Introduction The research of Hatch (1) has greatly increased our understand- ing of the use and potential limitations of biological markers for adverse reproductive effects. Likewise, there are great potentials and pitfalls in the emerging field of exposure biomarkers for reproductive health. This area of research has lagged con- siderably behind the field of developing new effect markers, such as semen analysis and early pregnancy loss. However, its impor- tance in quantitative risk assessment cannot be overemphasized. As Hulka has so clearly stated, "the most important current limitation in many epidemiological studies is the relative inac- curacy of methods for measuring the exposure variable" (2). Exposure to a potential fetal health hazard can be estimated through ecological assessment (e.g., testing the community water supply), questionnaires (e.g., classifying residents accord- ing to whether they drink bottled or tap water), or biological markers (e.g., testing for exposures to chemicals or solvents found in tap water). In a community with an environmental factr suspected of adversely affecting reproductive outcome, *Division of Reproductive Health, Centers for Disease Control, Atlanta, GA 30333. tDepartments of Pathology and Pediatrics, University of Arkansas for Medical Sciences, and Arkansas Children's Hospital, Little Rock, AR 72201. Address reprnt requests to M. A. Brewster, Metabolic Labomaory, Children's Hospital, 804 Wblfe Street, Little Rock, AR 72201. measuring environmental contaminants provides a gross, ecological esimate of the exposure incurred by pregnant women and their fetuses. However, ecological estimates can lead to significant misclassification of individual exposure (3). Such misclassification, if nondifferential, will underestimate the true effect of the exposure. If misclassification is differential, misleading results in either direction can occur. Using questionnaires to assess the extent of an individual's potential for exposure may help to reduce misclassification bias. Yet reports of individual exposures can be erroneous in either direction (4). In addition, people are often unaware of their potential for exposure, and researchers may not know or be able to account for all the pathways of exposure. For example, certain mothers in a Yugoslav community with a lead smelter had elevated blood-lead levels. Questionnaire data detennined which of these women were wives of men employed in the lead industry. However, these data could not distinguish between women with low blood-lead levels and women with elevated levels (4). In this example, a biological exposure marker (blood lead-level) was available for classifying mothers according to their exposure to lead. To date, such biological markers have not been widely available nor have they been widely used when they are available. Several years ago, the Environmental Protection Agency co- sponsored a National Research Council study on The Role of Biomarkers in Reproductive and Developmental Toxicology (5). 2HOGUE AND BREWSYER After reviewing the situation, Longo described a "paradox" (6). Although several techniques for identifying individual exposures have been developed and tested, and although more and more xenobiotics have been recognized to have teratogenic and mutagenic potential, "essentially no specific biomarkers are cur- rently available to indicate that exposure to a given xenobiotic is directly associated with a cellular, subcellular, or phar- macodynamic event" (6). The paradox continues, despite con- tinuing advancements in laboratory science and the growing recognition of the need for biological markers to improve ex- posure measurement in the field of environmental epidemiology (3,4,7-9). To further the development and application of exposure markers in studying the environmental hazards to reproductive health, we have attempted to synthesize recent examinations of the issues surrounding exposure measurements in reproductive epidemiology. The specific goals of this paper are to explore the potential uses of biomarkers as measures of exposure, particular- ly as they may be used in an environmental setting as screening tools. Biomarkers As Measures of Exposure Hulka defines biological markers in environmental epidemiology as "cellular, biochemical or molecular alterations which are measurable in biological media such as human tissues, cells or fluids and are indicative of exposure to environmental chemicals" (2). Biomarkers are not environmental measures in air, soil, water, or food; nor reports from research subjects; nor results of physical, anthropometric, or mental examinations. Rather, they are material measures obtained from physical samples (4). Biomarkers used to estimate environmental exposues must be distinguished from those used to estimate the effects of those ex- posures. Biological markers of effects can be subclassified into biologically effective doses (such as DNA adducts) and biological responses (2). Examples of the biological response in- clude sensitive tests of early pregnancy loss and serum alphafetoprotein to detect etal neurl tube defect. The National Research Council study prinarily dealt with effect markers (5). Although both exposure and outcome measures are necessary for an epidemiologic investigation, we will focus on measuring ex- posures. In Hulka's classification, these are "internal dose markers" (2). Internal dose-exposure markers may be useful to improve the quality of exposure measurement in an epidemiologic investiga- tion of a known environmental hazard; to serve as the "gold stan- dard" for other infrmation sources; to provide a screening tool for environmental exposures to a target tissue (in this case, the fetus); and to provide quantification of the biological load from a known exposure (4). To be useful in epidemiologic investiga- tions of reproductive health an exposure marker should be bet- ter than the woman's ability to recall an exposure; allow for dif- ferentiation between exposure levels, at least qualitatively; allow the use of noninvasive procedures that are applicable on a large scale; and provide interprable data for short-term or cumulative exposure regarding time, dose, and duration (4). For the environmental Sherlock Holmes, internal dose markers offer strong circumstantial evidence that the perpetator xenobiotic has invaded the human victim. This evidence is very specific if the chemical is retrieved unaltered. However, sub- stantial circumstantial evidence can be gleaned from metabolically altered chemicals. The metabolic outcome can be very specific (e.g., urinary cotinine for nicotine in cigarette smoke) or nonspecific (e.g., thioethers for cigarette smoking). Nonspecific markers measure a biochemical pathway affected by a variety of xenobiotic agents. Exposure Markers As Screening Tools In addition to possessing the characteristics of all useful ex- posure markers, biomarkers used as exposure screens should be able to detect subtoxic exposures and be nonspecific (8). Nonspecificity of the marker is important because the environ- ment commonly includes complex and unknown chemical mix- tures, such as those found in drinking water, that could be misclassified by selecting a few specific markers for a screening battery. For epidemiologic research, nonspecific markers tend to be held in lower esteem than specific markers, since it is impossi- ble, without further evidence, to identify which chemical has triggered the metabolic response being measured. However, as screening tools, nonspecific markers hold some promise. A biomarker that can be used to detect that one or more of a class of xenobiotic agents to which the pregnant woman has been exposed and may have exposed her fetus could be useful for targeting a subset of women for further investigation and follow-up. First, however, the fiat that the metabolic pathway has been altered must be correlated with adverse human reproduc- tive outcomes so that such alteration can be shown to reflect fetotoxicity. We have previously proposed three nonspecific urinary biomarkers as potential screening tools for reproductive epidemiology (8): glucaric acid, thioethers, and porphyrin patterns. Vainio et al. (10) proposed mutagenic activity in bacteria as another nonspecific urinary screening tool, and Brewster (9) has added that biomarker to her proposed battery forpreantm. Let us briefly review the usefulness of tiese biomarkers. Bacterial Urinary Assay for Mutagenic Activity Vainio et al. (10 critically reviewed the bacterial bioassay pro- cedu as a test of mutagenic activity in urine. Like the oher tests proposed, it has the advantage of demonstrating biological activity rather than the mere presence of xenobiotic substances. However, this procedure poses problems that lead the reviewers t recommend that it be used in conjunction with other screening measures. We agree. For example, a bacterial bioassay cannot detect cumulative exposures and may react to substances normally present in urine (such as amino acids). Ikble 1 gives examples of the numerous studies of reported alterations in muagenicity by extrnal agents (11-59). Occupational exposures have been associated with alterations in mutagenicity; however, not all studies are positive. For example, oncology nurses handling cytotoxic drugs have been studied by several investigators. i00 studies were positive for altered mutagenicity (13,17), but two later reports were negative (25,42). This dif- ference may reflect changes in routines for handling these drugs. 262 EXPOSURE BIOMAR SU RN PR EGNANCY 'Ibbe 1. Reported ons in Imtencity by xenobotics. Human studies Anesthesiologists (halogenated anesthetic gases) Epichlorhydrin-exposed workers Oncology nurses (cytotoxic drugs) Foundry workers (PAHs) Refinery workers (coal tar and pitch) Chemical workers (various) Oncology nurses (cytotoxic drugs) Pharmacists (cytotoxic drugs) Pharmacists (ctotoxic drugs) Carbon electmde industry workers Chemical and coke oven workers Cold-rolling steel plant workers (mineral oils); synergistic with smoking Vegan diet Cancer patients on chemotherapy Workers at a coal tar distillation plant Workers at sewage treatment plants Tool and die workers (N-nitroso diethanolamine, diethylnitosamine) Cancer patients receiving cyclophosphamide (or doxorubicin) Fried salmon diet to nonsmokers (reversed by co-intake of parsley) Tannery workers Steel mill workers, coal processing Explosives manufacturing workers (trinitrotoluene) Smoking, passive Smoking >25 years Snuff users Pharmacists handing cytostatic drugs Coke oven emissions exposure Tire plant workers Tire plant workers (possible smoking synergism); tetramethyl (thiuram disulfide, poly-p-dinitrosobenzene, diaryl-p-phenylendiamies) Oncology nurses handling cytotic drugs Anode plant workers exposed to coal tar and pitch Oncology nurses handling cytostatic drugs Autopsy serviceworkers (formaldehyde) Nonsmokers on low PAH diet exposed passively to tobacco smoke Hospital employees handling cancer che_mo py agents Macrobiotic versus typical western diet Workers exposed to 2,4,7-tnnitro- 9-fluorenone (low dose) Operating room personnel References McCoy et al. (11) Kilian et al. (12) Falck et al. (13) Schimberg et al. (14) Inamasu et al. (15) Dolera et al. (16) Bos et al. (17) Anderson (18) Nguyan et al. (19) Theiss (20) Pasquinin et al. (21) Kriebel et al. (22) Pasquini et al. (23) Sasson et al. (24) Benhamou et al. (25) Jongeneelen et al. (26) Scarlett-Krans et al. (27) Garry et al. (28) Tuffnall et al. (29) Ohyama et al. (30) Gostantini et al. (3) DeMeo et al. (32) Ahlborg et al. (33) Mohtashamipur et al. (34) Kriebel et al. (35) Curval et al. (36) Curvall et al. (36) Kohmodin-Hedmanetal. (37) Moller and Dybing (38) Falck et al. (39,40) Grebeili et al. (41) Barale et al. (42) Venier et al. (43) Benhamou et al. (25) Connor et al. (44) Scherer et al. (45) Everson et al. (46) Staiano et al. (47) Sasson et al. (24) Grebeili et al. (48) Baden et al. (49) Animal studies + + + + + Amniotic fluid + Direct chemical mutagenicity Acrylonitrile Food mutagens (qurAetin and mtin) Coal tar, dermal Hazardous industrial waste samples 2,4,7-Trinitro-9-fluorenone Benzo(a)pyrene Cyclophoshamide Azo dye tartrazin Mori et al. (50) Grebelli et al. (51) Jongeneelen et al. (26) DeMarini et al. (52) Crebelli et al. (48) Jongeneelen et al. (53) Duverger-van Bogaert et al. (54) Henschler and Wild (55) Smoking (heavy) versus nonsmoking, at term Smokin versus nonsmoking, 2nd trimester Rivrud et al. (56) Everson et al. (58) Rivrud et al. (57) Compilation of results of >5000 chemicals N(+) Indicates a slight increase of questionable significance. EMIC Idex (58) Urea + + + + + + + + + + + + - 263 _ .. . . _ _ _ . . HOGUE AND BREWSTER Exrin 2. ReporRrd rinn ed e by me Excretion' Study t Pesticide production workers (aldrin, dieldrin, endrin, DlYD) Hunter et al. (65) Endrin manuficturing plant workers Nottenand Henderson (60) Ottervanger and Van Sitter (66) Viij-Stanhardt et al. (67) t (In some) Metal and chemical factory employees Nottenand Henderson (60) t Pesticide packaging workers Seutter-Berlage et al. (68) t Electrical workers exposed to PCBs Seutter-Berlage et al. (69) -b Workers in polyester industry (styrene) Hotz et al. (70 -~ Resident of Tmies Beach, Missouri, in high-exposure risk group for dioxin Steinberg et al. (71) -~ Steel plant employees (low-risk exposure to minerl oils) Pasquini et al. (23) t Diuroin, hexachlorobenzene, heptaclor, dieldrin, akdrin, rhotane, disulfiram, 2-phenylphenol Notenand Henderson (61) in guinea pigS it Toluene, terethyl lead, Aroclor 1260, ethnl, n-hexane, dodin, atrazine in guinea pigs NoUenand Henderson (61) -. Captan, dimethoate, nitubenzene, aniline, naphthylacetic acid, benzene, rotenone, Nottenand Henderson (61) binapacryl in guinea pigs a() Unchanged; (?) slight elevation of questionable significance. Glucaric Acid NottenandHenderson (60) firstproposedusingurinaryglucaric acidtoscreenforxenobioticexposures. Glucuronidationisamajor transformation route for a number of xenobiotics, including azides, nitrites, alkylamines, and alkyl and aryl alcohols (61). Stimulation of the glucuronidation pathway results in increased ecretion ofglucaric acid, primarily thugh reduced production ofglycogenandpossiblythroughdirectinductionofpathway en- zymes (9,62). Although pregnancy (63) and estrogen therapy (64) produce a nonsignificant increase in glucaric acid eretion, a number ofex- ogenouschemicalshave produced significant changes in glucaric acid excretion (Table 2) (65-71). Intraindividual variation, which can be as high as 50%, may reflect daily variations in environ- mental exposures (72). Such variations would suggest that daily measures during vulnerable points in pregnancy would be re- quired to assure accurate classification of exposure through the pathways that increase glucaric acid excretion. Laboratory methods for measuring urinary glucaric acid have been dev- eloped and stadardized (9). However, standards for pregnant women do not yet exist. Thioethers Alkylating agents can be detoxified by reaction with gluta- thione or other sulfhyryl compounds. These conjugates fre- quently appear in urine as mercapturic acids or other thioether (R-S-R) products. Xenobiotics known to be detoxified through this sequence include aromatic hydrocarbons, arylamines, and many other chemical agents (9). Seutter-Berlage et al. (73) first posed using urine thioedhers as a screening tool for xenobiotic exposures. Sincethen, numeros studieshavedocumentedeleva- tions in urinary excretions related to occupational, enironmen- tal, and behavioral (i.e., smoking) exposures (Table 3) (73-104). Henderson et al. (105) and Van Doorn et al. (76) have critically reviewed the literature on the urinary thioether assay as an ex- posure screening tool. They note that positive results reflect true exposures, but negative results may not reflect lackof exposure. This false negativity occurs because thioethers measure short- term exposure and thus may miss past exposures that could have a future biological effect. This limitation, although prhaps not as great for exposures in pregnancy (because short-term expo- sures may be the most valid for measuring fetal exposure), sug- gests prospective urine collection at several points during pregnancy. Porphyrins Urinary porphyrin patterns are assessable through automated laboratory methods using high-pressure liquid chromatography (105). Brewster (9) has reviewed the usefulness of measuring total urine porphyrins to detect xenobiotic exposures to heavy metals, hormones, drugs, and halogenated aromatic hydrocarbons. These chemicals induce chronic disturbances in hepatic syn- thesis of porphyrins (Tible 4) and thus lead to excess porphyrin excretion and skin symptoms in the final stage (107-124). Presumably, all xenobiotics dtat produce chronic changes would also show the urinary patten at early stages prior to overt toxici- ty, but this assumption has not been tested in all circumstances. Strengths and Limitations of Proposed Screening Battery These four tests discussed previously generally meet the criteria for useful exposure screens. For certain xenobiotic agents, they accurately differentiate exposure levels, as demonstrated in occupational and environmental epidemiologic studies. As urinary screens, they are noninvasive and applicable on a large scale with current laboratory techniques. For short- term exposure, glucaric acid, thioethers, and mutagenicity tests are useful. Pbrphyrin patterns may measure cumulative effects as well as current exposure levels. The potential for this battery to identify groups of pregnant women at risk ofeavironenal insults to their ftuses can be il- lustraedby cite smoking. Glucaric acid may (23) or may not (125) be elevated by cigaette smoke. However, thioethers are elevated by cigarette smoke (73,80,90,91). Also, mutagenicity tests are very sensitive to cigarette smoking (10). Although cotinine is a specific marker for cigaette smoking (and thus would be the biomarker of choice if cigarette smoking is the specific exposure of interest), the fact that this battery is respon- sive to a known feltotoxic agent lends credence to its potential value for detecting other, as yet unknown, fetotoxins. However, as we have previously coented (8), to be effective screening 264 EXPOSURE BIOMARKERS lN PREGNANCY Table 3 Reported alterations in urinary excretion of thioethers by xenobiotics. Effecta t Group mean t Group mean t Group mean t Across shift t Across shift t From pre-employment t Group mean T Group mean T Group mean t Group mean and across shift t Across shift t Group mean t Group mean t Across work week t Across wvrk shift t Across work shift t Post therapy t Across work shift t Group mean t Group mean - Group mean - Group mean Group mean Group mean Group mean - Across shift - Across shift - Across shift - Across shift t T I T Study Chemical workers (possible exposure: acrylonitrile and biphenyl) Rubber and tire workers Pesticide packaging workers Operators of chemical waste incinerators Spinners in viscose-rayon Rubber industry (carbon disulfide) women Nurses handling cytotoxic drugs (cyclophosphamide, vincristin, cytoxan) Explosives manufacturers (trinitrotoluene) Oncology nurses (cyclophosphamide, adriamycin) Petroleum retailers and garage mechanics Dry cleaning workers Urban school chldren in smoke-polluted areas (polcyclic aromatic hydrocarbons) Oncology nurses handling cytotoxic drugs Road and asphalt plant workers (asphalt) Chemical plant workers (3,3 -dichlorobenzidine) Hospital employees handling cytotoxic drugs Cancer patients receiving cytotoxic drugs Chemical plant workers (ethylene oxide, epichlorohydrine, formaldehyde, organic solvents including toluene) Workers producing polyurethane foams (isocyanates and tertiary amines) Cigarette smokers Chemical manufacturing workers (methylchloride) Pharmaceutical manufacturing workers Wrkers at waste water treatment plant (chlorinated cyclodiene pesticides and flame reardants) Crews of roll-on, roll-off ships and car ferries; bus garage staff (particulates, benzene, formaldehyde, NO2, benzo(a)pyrene) Road asphalt plant workers (asphalt) Roadmen (PAHs) Asphalt production workers (PAHs) Petrochemical plant workers (benzene) Coke plant workers (PAHs) Toluene and xylenes in rats 1,3-Dibromopropane in rats 2-cl-and 3-cl-benzylidene malonitrile and benzaldehyde metabolites (chloro BMNs) in rats Ethylene dichloride (1,2-dichloroethane) in rats trans- and cis-epoxy cinnamates in rats Trichloroethylene in rats Benzene, naphthalene, anthracene, phenanthrene, benzanthracene, styrene, aniline, 2-naphthylamine, acetophenetidine, halobenzenes, l-chloronaphthalene, halonitrobenzenes, benzyl chloride, phenethyl bromide, 1-menaphthyl chloride, benzyl acetate, l-menaphthyl acetate, 3,5-di-tert-butyl-4-hydroxytoluene, iodomethane, bromoethane, allyl chloride, 1-nitropropane, cyclopentene, bromocyclohexane, maleic acid, isolvaleric acid, ethyl methanesulfonate, urethane, benzothiazole-2-sulfonamide, J6iv_R.xhInrethvI aidlfifh- -.thad-rwnir skgiil sirnrnlino- salivl :nrotsito in vskrimtic s:nimsil-c Reference Seutter-Berlage et al. (73) Salvolainen and Vainio (74) Seutter-Berlage et al. (i5) Van Doorn et al. (76) Van Doom et al. (77) Kilpikari and Salvoainen (78) Jagun et al. (79) Ahlberg et al. (80) Que Hee et al. (81) Stock et al. (82) Lafuente and Mallol (83) Lafuente and Mallol (84) Bayhan et al. (85) Lafuente and Mallol (86) Triebig et al. (87) Triebig et al. (87) Triebig et al. (87) Hagmar et al. (88) Holmen et al. (89) Seutter-Berlage et al. (73) Ahlborg et al. (80) Van Doom et al. (90) Buffoni et al. (91) Heinonen et al. (92) Lafuente and Mallol (93) Van Doom et al. (94) Ahlborg et al. (80) Que Hee et al. (95) Ulfvarson et al. (96) Burgaz et al. (97) Triebig et al. (87) Triebog et al. (87) Triebig et al. (87) Triebig et al. (87) Van Doom et al. (98) Onkenhout et al. (99) Reitveld et al. (100) Igwe et al. (101) Rietveld et al. (102) Rouisse and Chakrabarti (103) Boyland (104) a(-+) Unchanged. 265 266 HOGUE AND BREWSTER TIble 4. Reported alterations in porphyrin excretion by xenobiotics Effecta Study Referenced Abnormal pattern Polyvinylchloride workers (vinylchloride) Lange et al. (107) Abnormal pattern Michigan farm families exposed to polybrominated biphenyls (PBBs), 2 years prior Strik et al. (108) Abnormal pattern Seveso explosion 2 years prior, dioxin exposure Strik et al. (109) Abnormal pattern Occupational exposure to 2,4,5-T Strik et al. (110) t All porphyrins Transformer/condenser production workers exposed to polychlorinated biphenyls (PCBs) Colombi et al. (111) t Uro Turkish residents ingesting hexachlorobenzene in wheat, 20-30 years prior Cripps et al. (112) t Mean uro Residents of Times Beach, Missouri, in dioxin exposure risk group Hoffman et al. (113) t Uro Persons exposed to smoke from a PCB transformer fire Octerloh et al. (114) t Copro III Lead-exposed workers Tohiba and Tomokuni (115) t Copro Alcohol ingestion Doss (116) t Total porphyrins Children exposed transplacentally to polyhalogenated aromatic hydrocarbons (PCBs, PCDFs, Gladen et al. (117) PC quaterphenyls) Normal pattern PCBs exposure via contaminated rice oils, 10 years prior (Yusho) Strik et al. (118) Normal pattern Possible occupational exposure to hexachlorocyclopentadiene, allyl chloride, Nagelsmit et al. (119) epichlorohydrin, endrin t Uro and copro Hexachlorobenzene in rats Kondo and Shimizu (120) t Uro PCBs, PBBs (20 different congeners) at least 2 Cl or 2 Br at lateral adjacent positions each Sassa et al. (121) phenyl ring; negative in < 2 or >3 halogens each ring, in CEL cellsb t Uro 3,4,5,3 ,4 ,5 - Hexachlorobiphenyl, lindane, parathion, nifedipine, verapimil in CEL cells Sinclair et al. (122) t Uro and Hepta 3,4,3 ,4 - Tetrachlorobiphenyl in CEL cells Sinclair et al. (122) t Uro Chlorobenzenes (4 chlorines) in CEL cells Sinclair et al. (123) t Uro Aroclor 1254, oxidized 3,5-diethoxycarbonyl collidine, sodium phenobarbitone in CEL cells Ferioli et al. (124) and embryos in ovo t Copro III Lead in rats Tohiba and Tomokumi (115) a(t) Increased concentration. Uro, uroporphyrin; hepta, heptacarboxygloporphyrin; copro, coproporphyrin; Copro III, coproporphyrin m. bCEL, chick embryo liver cells in culture. tools for adverse reproductive health exposures, several steps have yet to be taken. First, tests must be standardized for pregnant women. Although there is little evidence to suggest that pregnancy itself can alter these test outcomes, it is important to establish standard levels for pregnant women with normal pregnancy outcomes. Se- cond, tests should be administered to women with known ex- posures, such as maternal smoking, so that patterns of alterations can be correlated with reported exposures. Third, the tests must be associated with adverse pregnancy outcomes, such as reduced birthweight or gestational length. This last element in the valida- tion research is particularly important since maternal exposure rather than fetal exposure is being measured. The extent to which xenobiotic chemicals cross the placental barrier may vary great- ly, depending on the type of exposures, timing in pregnancy, and maternal detoxification capability. If the battery of screening tests proves useful, further field investigations would be warranted to determine the tests' ability to measure environmental exposures that adversely affect fetal development. In reproductive epidemiology, we may be at a unique point for implementing this validation process. Because a number of studies of early pregnancy loss are collecting serial urines dur- ing pregnancy, the moment may be opportune to begin examin- ing these urines for metabolic alterations, as tests of the poten- tial usefulness of these nonspecific biomarkers to predict adverse pregnancy outcomes. Progress is being made in learning about these tests' response to specific environmental chemicals, but more research needs to focus on the quantitative relationship of these agents to body burdens. It would be helpful if this battery of tests were routinely applied to pregnant women in known ex- posure situations. Also, if pregnant wvmen with abnormal tests (with and without adverse outcomes) were investigated further, much could be learned about the metabolic functions that are af- fected and the specific chemicals that are creating the effect. Conclusion Without better exposure measures, epidemiologic studies of reproduction will probably fail to identify xenobiotic fetotoxic agents in the environment. However, with an adequate battery of nonspecific exposure biomarkers, prospective studies of en- vironmental effects on pregnancy outcomes might be possible. A proposed battery of nonspecific biomarkers should be tested to determine their usefulness for predicting adverse pregnancy outcomes. We acknowledge the extensive and excellent editorial work and typing of Patricia Huckaby. REFERENCES 1. Hatch, M., and Friedman-Jiminez, G. Using reproductive effect markers to observe subclinical events, reduce misclassification, and explore mechanism. Environ. Health Perspect. 90: 255-259 (1991). EXPOSURE BIOMARKERS IN PREGNANCY 267 2. Hulka, B. S. Biological markers in epidemiologic research. Arch. Environ. Health 42(2): 83-89 (1988). 3. Brunekreef, B., Noy, D., and Clausing, P. 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