human variability and susceptibility to trichloroethylene · 2017. 3. 23. · glutathione...

Human Variability and Susceptibility to TrichloroethyleneGina M. Pastino, Wendy Y. Yap, and Maria CarroquinoU.S. Environmental Protection Agency, National Center for Environmental Assessment, Washington, DC USA

Although humans vary in their response to chemicals, comprehensive measures of susceptibilityhave generally not been incorporated into human risk assessment. The U.S. EPA dose-response-based risk assessments for cancer and the RfD/RfC (reference dose-reference concentration)approach for noncancer risk assessments are assumed to protect vulnerable human subgroups.However, these approaches generally rely on default assumptions and do not consider the specificbiological basis for potential susceptibility to a given toxicant. In an effort to focus more explicitly onthis issue, this article addresses biological factors that may affect human variability and susceptibilityto trichloroethylene (TCE), a widely used halogenated industrial solvent. In response to ExecutiveOrder 13045, which requires federal agencies to make protection of children a high priority inimplementing their policies and to take special risks to children into account when developingstandards, this article examines factors that may affect risk of exposure to TCE in children. Theinfluence of genetics, sex, altered health state, coexposure to alcohol, and enzyme induction onTCE toxicity are also examined. Key words: children, gender differences, human variability,susceptibility, trichloroethylene. - Environ Health Perspect 1 08(suppi 2):201-214 (2000).http.//ehpnetl.niehs.nih.gov/docs/2000/suppl-2/201-214pastino/abstract.html

Throughout human history it has beenapparent that only some individuals in agiven population become sick or die afterexposure to a common environmental hazard.To what extent can we make predictions con-cerning the effects of environmental agentson individuals or groups? Determining therisk of an individual from a specific agentrequires studying the net influence of a largeset of variables on the known effects of thatagent. Some of these variables may enhancesusceptibility, while others may diminish it.

Risk assessment has been an importantenvironmental regulatory decision-makingtool in the United States and Canada sincethe 1980s. It is defined as a formalizedprocess for estimating the magnitude, likeli-hood, and uncertainty of environmentallyinduced health effects (1-3). While the U.S.Congress has tried to protect susceptiblegroups and workers since the 1970s throughlegislation [e.g., Clean Air Act (4)], bothresearch and risk assessment on potential tox-ins have lagged (5). Historically, U.S. govern-ment agencies have focused on the maximallyexposed individual in risk assessment. Forexample, the health of individual workers inan occupational setting is protected by settingstandards that restrict exposure beyond a per-missible limit of a substance (6).

Although humans vary in their responseto a given toxicant, measures of susceptibilityhave not been incorporated into human riskassessment methods (5). Since limited base-line information is available on exposure andhealth outcomes in humans, most humanhealth risk assessment is based on experimen-tal toxicity studies of homogenous animalpopulations; however, these usually do notevaluate differences in sensitivity, age, or

gender, particularly in human populations.While the U.S. Environmental ProtectionAgency (U.S. EPA) dose-response-based riskestimates are assumed to protect vulnerablehuman subgroups (7), the biological basis ofsusceptibility remains poorly understood. Inaddition, broadly based exposure baselinesand data on potentially susceptible groups,such as children or the elderly, are lacking.The U.S. EPA Guidelines for Risk Charac-terization (8) have emphasized the need toidentify, characterize, and include susceptiblepopulations in risk estimation and risk man-agement processes (9).

This article focuses on human variabilityand susceptibility in response to trichloro-ethylene (TCE) exposure. TCE and otherhalogenated hydrocarbons are widely usedindustrial solvents, and production of TCEincreased from approximately 260,000 poundsin 1982 to 320 million pounds in 1991.Heavy use of TCE has resulted in widespreadsoil and groundwater contaminants in theUnited States; TCE is present in as many as60% of the hazardous waste sites on the U.S.EPA National Priority List. The major envi-ronmental releases come from the air emissionsof metal degreasing plants. Thus, TCE hasbeen the subject of much toxicity testing andresearch, and an extensive database has beencompiled (10-13).

In an effort to address factors that affecthuman variability and susceptibility, this arti-cle examines the risks of TCE exposure tochildren. Executive Order 13045 requiresfederal agencies to make protection of chil-dren a high priority in implementing theirpolicies and to take special risks to childreninto account when developing standards. Theorder follows the recommendations of the

1993 National Research Council (NRC)report Pesticides in the Diets ofInfants andChildren (14), which suggested children areat disproportionate risk from environmentalhealth threats because they receive greaterexposures per unit of body weight, and theirdeveloping systems are immature.

The influence of genetics, altered state ofhealth, and the effect of enzyme induction onTCE toxicity are also examined in this article.Ethanol and TCE interactions are examinedin detail because of the availability of data.However, the studies on ethanol exposureand TCE interaction serve as an example ofthe potential toxic consequences of enzymeinduction and exposure to mixtures.

Health Effects of TCE Exposurein HumansMetabolism ofTCEThe metabolism of TCE is described exten-sively in another article in this issue (15).Briefly, TCE is rapidly absorbed followingacute exposure and distributes throughout thebody, preferentially to the fat. TCE under-goes metabolic activation primarily in theliver but also in the kidneys and lungs(Figure 1). The rate-limiting step in themetabolism of TCE is the P450-mediatedoxidation to chloral hydrate (CH). CH israpidly hydrolyzed to trichloracetic acid(TCA) and free trichloroethanol (TCOH) viaaldehyde and alcohol dehydrogenase (ADH),respectively. Free TCOH undergoes glu-curonidation and is excreted in the urine or isconverted back to TCA through CH. TCOHalso undergoes enterohepatic recirculation.That is, following excretion of TCOH fromthe liver into the bile and then the smallintestines, it is reabsorbed into the intestinal

This article is part of the monograph on TrichloroethyleneToxicity.

Address correspondence to G.M. Pastino, ScheringPlough Research Institute, 144 Route 94, PO Box 32,Lafayette, NJ 07848. Telephone: (973) 940-4554.E-mail: [email protected]

The authors thank the American Association for theAdvancement of Science for the support they receivedthrough the Environmental Science and EngineeringFellowship Program. The authors also thank C. Scott,D. Chen, and J. Cogliano for the editorial commentsthey carefully provided.

The views expressed in this paper are those of theauthor(s) and do not necessarily reflect the views or poli-cies of the U.S. Environmental Protection Agency or theAmerican Association for the Advancement of Science.

Received 20 October 1999; accepted 7 February2000.

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GSTTrichloroethylene > Dichlorovinyl Glutathione

CYP 450 1

Chloral Hydrate Dichlorovinyl Cysteine

ALDH

Trichloroacetic Acid

4,

ADH

Tnchloroethanol

f3-Lyase

Activation

NAT

Detoxification

Dichloroacetic Acid TrichloroethanolGlucuronide

Figure 1. Oxidative and conjugative pathways for the biotransformation of TCE.

circulation and passes back through the liver.TCA is metabolized to dichloracetic acid(DCA) or is excreted in the urine. TCA,TCOH, and DCA are thought to contributeto the toxicity ofTCE.

TCE also undergoes metabolism throughglutathione conjugation in the liver to formdichlorovinyl glutathione (DCVG) (15).DCVG in turn is metabolized to the cysteineconjugate in the kidney (DCVC), which ismetabolically activated to a thioacetylatingagent by P-lyase or detoxified by N-acetyltrans-ferase (NAT), which is excreted in the urine.

Acute EffectsAcute exposure to TCE results primarily incentral nervous system (CNS) effects.Neurotoxic effects, such as dizziness,headache, sleepiness, nausea, confusion,blurred vision, and weakness occur at concen-trations of approximately 100 ppm; anesthe-sia occurs at 2,000 ppm (10,12). Death hasoccurred at very high concentrations (10,000ppm) and was associated with cardiacarrhythmia and massive liver damage (12).

Reproductive/Developmental EffectsStudies on the various reproductive anddevelopmental effects of TCE have yieldedconflicting results. One study found anincrease in miscarriage among nurses exposedto TCE and other chemicals in the work-place, although no specific association withTCE was found. Another study found noincrease in malformations in the children of2,000 fathers and mothers exposed to TCEvia inhalation (12). An association, but nodirect cause-and-effect relationship, wasfound between elevated levels of chlorinatedhydrocarbons, including TCE, andchromium in drinking water and congenitalheart disease in children whose parents wereexposed to this contaminated drinking water(16,17); follow-up studies in rats showed thatTCE was a cardiac teratogen, but not a gen-eral teratogen during fetal organ development(18). An association between TCE exposure

and cardiac anomalies and eye malformationshas been found (18-20). Other studies, how-ever, found no adverse reproductive effects inhumans exposed to TCE in drinking water(12,21). An increase in abnormal sperm mor-phology in mice exposed to TCE by inhala-tion has also been found (12,21).

Cancer RiskLong-term exposure to TCE can result inhepatotoxicity, nephrotoxicity, and neurotox-icity, as reviewed by Bull (22), Lash (23), andBarton (20), respectively. Several studies inhumans have investigated the relationshipbetween TCE exposure and cancer. The U.S.EPA has for a number of years regulated TCEon the basis of carcinogenicity, although itsclassification has not been resolved betweengroup B2 (sufficient evidence in animals) andC (limited evidence) (24-26). Two largebodies in the past few years have reviewed theepidemiologic evidence on TCE. TheAmerican Council of GovernmentalIndustrial Hygienists (ACGIH) (27), lookingonly at the occupational studies, classifiedTCE as a compound not suspected as ahuman carcinogen, primarily on the basis ofobservations from large studies of degreasersat Hill Air Force Base in Utah (28,29).Several years later, the International Agencyfor Research on Cancer (IARC) (30)reviewed studies from both occupational anddrinking water exposures and noted consis-tent findings of liver/biliary tract cancer andnon-Hodgkin's lymphoma in the most infor-mative studies. Additionally, they noted ele-vated leukemia risks in the drinking waterstudies. However, these populations wereexposed to other solvents in addition to TCE.These findings led IARC to classify TCE as aprobable carcinogen in humans, or Group2A, based on limited human and sufficientanimal data (30). It should be noted that theACGIH and IARC used different methods toevaluate the epidemiologic database.

The evidence is increasing for an associa-tion between human kidney cancer and TCE

exposure; newer studies consistently reportelevated risk of cancer for this site (31-35).Additionally, nephrotoxicity and kidney dys-function following TCE exposure have beenreported (36,37). Several studies show ele-vated risks for breast and cervical canceramong women occupationally and environ-mentally exposed to TCE (33,38,39). Thisevidence is weaker than that for the afore-mentioned sites; however, it raises a questionregarding susceptibility ofwomen.

Although strict site concordance has notbeen established in animals and humans foreach specific tumor, increases in lung, liver,and kidney tumors in animals have been seen.Inhalation exposure results in the formation oflung, liver, and kidney tumors; oral gavageresults in increases in liver and kidney tumors(22,23). Male rats are more susceptible to theformation of kidney tumors, whereas mice aremore susceptible to the formation of liver andlung tumors. The toxicities resulting fromTCE exposure are likely due to the variousmetabolites formed from the oxidative andconjugative pathways. Qualitatively, the path-ways of biotransformation in humans and ani-mals are identical, with most metabolitesidentified in experimental animals also foundin humans. However, quantitative differencesexist that probably account for the observedspecies differences in the formation of TCE-induced tumors. Mice and rats metabolizeTCE more efficiently than humans. For exam-ple, the maximum rate of the in vitro metabo-lism ofTCE in humans is one-third that in therat and one-fourth that in the mouse (40).

While the tissue-specific tumors identifiedin rodents are due to metabolites rather thanTCE, the metabolite responsible for eachtissue-specific response is likely different.Animal studies suggest that the formation ofliver tumors is mediated through TCA andpossibly DCA, and species differences areprobably due to differences in the formationofTCA (22). Mice are more prone to the for-mation of liver tumors and metabolize TCEmore rapidly than rats or humans.

As reviewed by Lash (23), the formationof kidney tumors in rats is likely mediated bythe reactive thiol formed from the 3-lyasemetabolism of DCVC. DCVC has beenshown to be highly nephrotoxic and muta-genic in the Ames test (41). The kidneytumors formed following TCE exposure arevery rare and a single mode of action has notbeen identified, but studies in animals indi-cate that mutagenicity and cytotoxicity fromDCVC are involved (23).

The relevance of the formation of kidneytumors in rats to humans has not been estab-lished. A recent study reported blood levels ofDCVG, a precursor of DCVC, in humansexposed to occupationally relevant concentra-tions ofTCE (42). Sex-dependent differences

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were also found; peak blood levels in menwere 2-fold higher than in women and werereached sooner than in females. Since malerats are more susceptible to the nephrotoxicand nephrocarcinogenic effects of TCE andalso have a higher rate of glutathione (GSH)conjugation in the liver and kidney, thesefindings suggest that men may be at a greaterrisk than women of developing nephrotoxic-ity from TCE exposure.

The mechanism for the increased risk oflung tumors in mice is different from the liveror kidney and is thought to be mediatedthrough the formation and accumulation ofCH in the Clara cells (43). These cells lackthe capacity to metabolize CH to TCOH,and the subsequent accumulation ofCH leadsto marked vacuolization of the cells. The rele-vance to humans has not clearly been estab-lished because of differences in morphologyand metabolism in the lungs.

Analysis of Variabilityand SusceptibilityThere has been much debate about whetherenvironmentally related disease is attributablemore to toxicant exposure or to inherent fac-tors that affect a person's response to toxi-cants. Historically, many risk analyses havefocused on variations in responses followingdifferent levels of exposure (e.g., occupationalversus non-occupational exposure). However,the risk of developing an environmentallyrelated illness may also be influenced by aperson's genetic background, age, gender,nutritional status, behavior (e.g., exercise,alcohol, and smoking), past and currentexposure patterns, and interactions betweenthese factors and between genes.

It is important to note that susceptibilityseldom remains constant. A person's suscepti-bility to environmental hazards may vary con-siderably during his or her lifetime as he or shegrows and develops from infancy to adult-hood, changes jobs, adopts habits that affectoverall health, suffers from intermittent ill-ness, and develops chronic diseases in old age.Factors include fundamental physiologicalvariables such as the route of exposure, theportal of entry, and uptake route; for example,significant differences in physiology are foundbetween children and adults. Fundamentalvariables may in turn be affected by constitu-tive factors, such as a person's genetic back-ground (variations in metabolic proteins), sex(reproductive organs, endocrine systems), age(stage of life), and ethnicity (this combinesboth genetic and acquired factors such as diet,cultural practices). Acquired factors such ashealth status or diseases (e.g., diabetes, obe-sity), behavioral factors (e.g., diet, exercise,stress), and other exposures (e.g., alcohol con-sumption, exposure to other hazards) alsoaffect fundatmental physiological variables.

Fundamental Physiological VariablesGenetics of biotransformation reactions.Overview. Given equal exposure to a toxin,humans vary in their internal processingdepending on genetic background, acquiredcharacteristics, and other past or ongoingexposures (44). Inherited mutations thataffect all cells in a person (germline muta-tions) differ from those that arise in a singlecell and affect only the clonal descendants ofthat cell (somatic mutations) (45). Diseasesin the first category tend to be rare and resultfrom dominant single genes (e.g., Li-Fraumeni syndrome, familial, bilateralretinoblastoma, defective DNA repair andproofreading), and disease is usually indepen-dent of environmental exposure (44).

On the other hand, susceptibility genes aredefined as common polymorphic genes thatare found in over 1% of the population. Theyusually but not exclusively consist of genesinvolved in metabolic and repair function, orregulators of such genes, that affect activationor detoxification of environmental agents(46-49). In contrast to the rare germlinemutation diseases, specific environmentalexposures are required to induce diseases asso-ciated with susceptibility genes. While suscep-tibility genes confer only a modest increase inrisk to persons carrying them, larger numbersof people are affected, and these genes there-fore pose a greater public health threat thanthe rare, single-gene diseases (43). Unlike dis-eases resulting from rare germline mutations,those attributed to susceptibility genes do notshow strict familial aggregation. Indeed, thesame mutation may be found in a variety ofsporadic, nonhereditary diseases.

Historically, most observational studies ofpolymorphic metabolic genes have focused oncancer, particularly tobacco-linked lungcancer. Identifying specific metabolic genepolymorphisms in individuals may become apowerful tool in the future for identifyingthose at-risk persons and in targeting diseaseprevention. However, at present we are unableto predict environmental diseases based solelyon single metabolic gene mutations. The roleof each metabolic gene is complex, and maybe modulated by gene-gene interactions, envi-ronmental exposure, health, nutritional status,and other factors. Indeed, a trait that providesprotection against a disease resulting from onecompound may increase the risk of diseasefrom another (6).

There is considerable lack of data regard-ing genetic polymorphisms as they relate toTCE metabolism and toxicity. Most data onTCE toxicity have been obtained from studiesusing inbred animal strains. These studies areusually conducted on homogenous rodentstrains maintained on identical diets and liv-ing conditions, while humans are moreheterogeneous and vary widely in their

behavior and diet. As a further complication,TCE is metabolized differently between differ-ent rodent species. Since rodent toxicity dataare extrapolated as a basis for evaluatinghuman toxicity and form the basis for humanhealth risk assessment, a better understandingof the metabolic differences between species isneeded. In addition, further studies need to beconducted on genetic polymorphisms in TCEmetabolism in order to provide a completehealth assessment of susceptible populations.

The metabolism of xenobiotics includesvarious dasses of reactions, such as hydrolysis,reduction, and oxidation, which result in theformation ofcompounds that are slightly morehydrophiic. Examples of enzymes involved inthese types of reactions include cytochromeP450s (CYP), ADH, and aldehyde dehydroge-nase (ALDH). Additional biotransformationreactions include glucuronidation, sulfation,acetylation, methylation, and glutathione con-jugation, which typically lead to a significantincrease in the hydrophilicity of the com-pound, which provides for rapid urinary excre-tion. Each reaction as it pertains to TCEbiotransformation will be discussed in furtherdetail. Much TCE toxicity may result from itsmetabolites, and analyzing variations in TCEmetabolism is important in understandingvariable toxicity of this compound.

Metabolism by cytochrome P450. One ofthe major families of enzymes responsible forthe oxidation of xenobiotics is CYP. This fam-ily of enzymes contains many genetic variantsthat show different levels of metabolic activity;up to a 10-fold difference in CYP activity hasbeen seen in humans (48,50). Wide interindi-vidual variation is found in the CYP1A2,CYP2A, CYP2C, and CYP3 gene subfamilies(51), and associations between altered geno-types in CYPlAl, CYP2D6, and CYP2E1and tobacco smoke-induced lung cancer havebeen made (50).

In rats, several CYP450 enzymes meta-bolize TCE, including the CYP1A, 2B, 2C,and 2E subgroups (52,53); however, TCEmetabolism is most strongly dependent onCYP2E1 activity. CYP2E1 is well conservedin mammals, and also metabolizes ethanol anda wide range of other volatile organic solvents.At present, it is not clear what CYP450enzymes other than CYP2E1 are involved inTCE metabolism in humans (15). CYP2E1metabolizes a wide range of substances includ-ing nitrosamines and chlorinated solvents(54). As reviewed by Lieber (55), CYP2E1 isinvolved in the metabolism of over 80 exoge-nous substances. It is primarily expressed inliver but also in brain, kidney, and lung.

Interindividual variability in humans.CYP2E1 activity in vitro has been studied inliver microsomes from several human popula-tions. Peter et al. (56) found up to 10-foldrange in activity in 14 subjects, while


PASTINO ET AL.

Stephens et al. (57) and Yoo et al. (58) founda 50-fold variation among subjects.Pronounced differences have also beenobserved among Swedish, Japanese, andChinese subjects (54). According to Lieber(55), a 6- to 20-fold variability of CYP2E1protein or activity has been observed inhumans and cannot be attributed to liverdisease, cancer, alcohol, or smoking.

CYP2El-mediated TCE metabolism inhumans. In humans, CYP2E1 plays a crucialpart in TCE metabolism and is responsible forover 60% of TCE clearance (40). An 8-foldvariation in the in vitro CYP2E1-mediatedmetabolism of TCE was seen in 23 humanliver samples (40). There were no significantsex-dependent differences in overall TCEmetabolism or CYP2E1 activity, but the affin-ity (K,) was lower in females than in males.Greater variability was seen in women andmay have been due to the small sample size.

Although a better understanding ofCYP2E1 variation will allow persons moresusceptible to TCE toxicity to be more easilyidentified (54), CYP2E1 activity alone is nota sufficient indicator of risk. Genetic variationand diet both contribute to the wide variationin CYP2E1 activity, and the balance betweenactivation of TCE to toxic metabolites andtheir subsequent detoxification determinesthe risk of exposed persons (59). In addition,acquired factors that increase CYP2E1 activ-ity (e.g., alcohol exposure) may also increasesusceptibility to TCE-induced toxicity.

Interspecies variability in TCE metabo-lism. Several species-dependent differences inthe metabolism of TCE have been found.Lipscomb et al. (40) directly compared themetabolism ofTCE in hepatic microsomes ofmice, rats, and humans at occupationally rele-vant TCE concentrations. P450-dependentTCE metabolism was highest in mice, lower inrats, and substantially lower in humans. Themaximal rate ofTCE metabolism in humanswas one-fourth and one-third of that in themouse and rat, respectively. Clearance values(Vn,,j,/Km) also exhibited species dependence;human microsomes were the least efficient atmetabolizing TCE.

Species-dependent differences also existin the metabolism of CH, which occurs inboth the liver and blood. For example,metabolism of CH to TCOH was muchlower in human blood than in mice or rats,while TCA formation was significantlyhigher in humans and mice than in rats (60).Human liver showed only about a 60% con-version of CH to TCA and TCOH com-pared to rodent samples. In all species, theKm for TCOH formation in liver was at least10-fold lower than TCA formation. Km val-ues for both TCOH and TCA were higher inhumans than in rats and mice, and clearanceofTCOH was higher than TCA (60).

Nakajima et al. (52) examined differencesbetween rat and mouse liver microsomes inthe metabolism of benzene, toluene, andTCE by P450 and concluded the species dif-ferences observed during P450 inductionwere more relevant to TCE toxicity than thedifferences in basal enzyme levels. That is, theeffect of differences in CYP2E1 activity onthe toxicity of TCE became more apparentfollowing CYP2E1 induction. Differentisozyme distributions and substrate affinitieswere observed, with mice showing greaterTCE metabolism than rats (52).

Molecular markers and ethnic differences.Variability in the regulation of CYP2E1expression may play a role in susceptibility.Restriction fragment length polymorphisms(RFLP) have been identified in the CYP2E1gene using a variety of restriction enzymes(58). Variations in allelic distribution wereobserved when different ethnic groups, suchas European Americans, African Americans,and Taiwanese were compared, and particularalleles were positively correlated to protectionfrom lung cancer (61); however, this studydid not control for gender, age, or smoking.A rare allelic form of CYP2E1, c2, may becorrelated to an increased risk for lung cancer(62). There was an association betweenincreased incidence of a particular (DraI)allelic distribution in Japanese lung patients(62). This allele was less prevalent in Finnishand Swedish patients, and the correlationbetween the DraI marker and lung cancerwas not found in a Swedish study (63). Katoet al. (64) also found no correlation betweenRsaI markers and lung cancer risk in a studyof largely Caucasian lung cancer patients.Mutations in regions outside of the CYP2E1gene may also play a role. For example, a sig-nificant association was found for a RsaI poly-morphism in the gene promoter region ofCYP2E1 in Swedish lung cancer patients(64); however, no significant correlation wasfound in Finnish and Japanese patients withlung cancer (65,66). In another example, asignificantly higher distribution of the c2mutation was found upstream of CYP2E1 incontrols for lung cancer; this region containsa putative binding site for transcription factorHNF-4. CYP2E1 may be less efficientlyexpressed among carriers of the c2 mutation,making them less susceptible to xenobioticbioactivation (64). Examination of CYP2E1variants in nasopharyngeal cancer cases andcontrols showed an RFLP distribution similarto the Japanese population but higher than inthe Finnish or Swedish population (67). A 5-to 7.7-fold increase in cancer risk was foundin subjects homozygous for these CYP2E1variants.

Although molecular markers clearly indi-cate differences in metabolic genes in differentgroups, at present none of these sites can be

used as markers for predicting increaseddisease risk (10). In the absence of data directlyrelating polymorphisms to enzymatic activity,this information remains of limited use.

Alcohol dehydrogenase. ADH is a mul-tienzyme family found in the cytosol, mito-chondria, and endoplasmic reticulum ofmany tissues. Six human ADH genes havebeen characterized, and polymorphisms ofseveral ADH genes have been identified(68-73). The enzymes have been dividedinto three classes according to isozyme com-position and kinetic properties. Class I ADHconsists of three nonallelic loci, ADH 1,ADH2, and ADH3, which are involved inthe synthesis of three types of subunits, a, P,Iy, respectively (72). Genetic variants ofADH2 and ADH3 exist and encode for high-activity ADH (68). For example, the fre-quency of these variants is higher amongAsians (68,69). Atypical ADH contains avariant 0 subunit (32) instead of the usualp 1, and is found more frequently amongAsians than Caucasians (70). Class I ADHexhibits high catalytic activity toward short-chain aliphatic alcohols, including ethanol, aswell as biologically active amines (72). ClassII and Class III ADH exhibit catalytic activitytoward longer chain alcohols (71). Class IVADH consists of the enzyme that is primarilyresponsible for gastric metabolism of ethanol,a-ADH (74). ADH is involved in the metab-olism of CH to TCOH. Thus, ethnic vari-ability in ADH metabolism may result invariations in the formation ofTCOH, whichcan alter the hypnotic/CNS effects followingTCE exposure.

Aldehyde dehydrogenase. ALDH is agroup of enzymes that convert aldehydes tocorresponding acids. ALDH is inducible bycertain medications, such as barbiturates(75). Approximately 50% of East Asians havean inactive ALDH enzyme (mitochondrialALDH2), but active ALDH is present in vir-tually all Caucasian, African-American, andNorth and South American populations stud-ied (70). Persons lacking this enzyme developa flushing reaction following ethanol con-sumption due to the buildup of acetaldehyde(68).ALDH is involved in the metabolism of

CH to TCA. A decrease in the formation ofTCA, which could potentially occur in per-sons lacking this enzyme, could protectagainst the toxic effects of TCA. However, anincrease in flux through the conjugative path-way can occur secondary to a decrease in theformation of TCA from CH. Thus, geneticvariations in either enzyme will likely have aneffect on the metabolism and toxicity ofTCE.However, no studies have been conducted toexamine this.

Glutathione conjugation. Glutathione S-transferase (GST) conjugates'TCE with



glutathione to form DCVG; this is furthermetabolized in the kidney to the cysteinederivative DCVC, which is activated by 3-lyase or detoxified by NAT. While conjuga-tion products are generally less toxic than theparent compound, some TCE metabolites,such as the nephrotoxin DCVC, are morepotent (76). While variations in conjugativereactions of TCE biotransformation inhumans are not well documented, below aresummarized the known variants in GST.

Defective GST genes are associated withan increased risk of lung and bladder cancer(30). About 50% of Caucasians are homozy-gous for the null allele of GSTMI (51) andmay be at greater risk for certain cancers. Inaddition, different GST subfamilies showmarked tissue specificity and developmentalregulation. For example, placenta containsonly GST n activity, and 50% of fetal liverGST activity is from the n form, which is notexpressed in adult liver.

The GST family encodes multifunctionalenzymes that catalyze several reactionsbetween GST and electrophilic andhydrophobic compounds (51). The fourmultigene classes ofGST subunits are a, p, x,and 0. Approximately 60-70% of humans areconjugators who can conduct GSH-depen-dent detoxification of monohalomethanes,while nonconjugators cannot (51).

GST,u. GSTp1 detoxifies a number ofreactive electrophilic substances. About50-60% of Caucasians carry the GSTp1 nullphenotype (51). This is consistently associ-ated with a higher risk for lung and bladdercancer (77-79), and possibly skin (80) andcolon cancer (81,82). Indeed, 25% of allbladder cancers may be correlated to smokerscarrying the GST null phenotype (77). Geneand environmental interactions may also besignificant; nonsmokers carrying the GSTplnull phenotype do not show elevated bladdercancer risk, but risk increases 2- and 6-foldwith smoking (83).

GSTO. GSTO enzymes conjugate a numberof chlorinated low-molecular-weight com-pounds, and may be primarily responsible forconjugation ofTCE (15). The GSTO gene ispresent in 60-75% of humans, and at leasttwo classes of enzymes are found in liver andin erythrocytes (84). Distribution of theGSTO1 polymorphism varies among ethnicgroups (84-86), and is associated with anincrease in sister chromatid exchange rates,particularly with exposure to tobacco smoke ordiepoxybutane (87,88).

GSTp1 and GSTO1 polymorphisms maybe important in renal cell cancer developmentfollowing high occupational TCE exposure.Bruning et al. (89) found unequal distribu-tions of the GSTji1 and GSTO 1 genotypesbetween renal cell cancer patients and controls,suggesting that these polymorphisms indicate

predisposition toward this disease. Enzymepolymorphisms were found in renal cell cancerpatients who had high occupational TCEexposure, resulting in irreversible tubular dam-age. The authors concluded that glutathione-dependent formation of nephrotoxicmetabolites is responsible for induction ofrenal cell carcinoma by TCE, and that thisgenetic polymorphism may indicate a predis-position for TCE-induced renal cell cancer. Inaddition, persons with polymorphisms in boththe GST1i and GSTO genes show a synergisticrisk for lung cancer (90).

Ethnicity. Different ethnic groups vary intheir distribution of genotypes, and extrapola-tion of health outcomes among the groups isvery difficult (51). For example, genetic poly-morphisms in CYP450 were found indebrisoquin and (S)-mephenytoin hydroxyla-tion (catalyzed by CYP2D6 and CYP2CMPrespectively) when Japanese and Caucasianpopulations were compared. In a separatestudy, Shimada et al. (91) examined the dis-tribution and activity of P450 isoforms inliver microsomes from 30 Japanese and 30Caucasian patients. No significant gender-related differences were seen in either popula-tion, and the proportion of CYP2E1 inCYP450 was not affected by gender or age.While total CYP450 content and activitywere higher in Caucasians, the relativeCYP450 isoform levels were similar except forCYP2A6 and CYP2B6, which were higher inCaucasians. Differences in CYP4501A2, 2A6,2D6, 2E1, and 3A4 activity levels wereobserved, but no difference in CYP2CMPwas seen.

Biologically plausible mechanisms to linkspecific genotypes to specific outcomes arestill lacking for most environmental diseases;for example, many RFLP polymorphisms inClass I genes are the result of mutations inintrons or other silent areas of the humangenome (51). RFLP analysis is therefore oflimited use in predicting an individual's sus-ceptibility at this time but may provide someinsight into the mechanisms of susceptibilityamong different groups. DNA sequenceanalysis, either by direct sequencing-or byhybridization-based analysis of polymorphicsites via DNA chips, is likely to be of consid-erably greater use in the near future, however.

Sex. Susceptibility differences betweenmales and females have been studied only for afew environmental pollutants, most notablyinhaled agents such as cigarette smoke and car-bon monoxide (92,93). Several epidemiologicalstudies of cigarette smoke have shown that, atthe same exposure level, women differ in theirresistance to lung damage compared to men.Xu et al. (94) report that adverse effects ofsmoking on pulmonary function are greater inwomen than in men, and other studies indicatesex differences in lung dysfunction, and in

genetic and biochemical alterations in lungcancer (95-97).

In addition to differences in the internalprocessing of these toxins, sex affects manyaspects of lung growth and development aswell as structure and function. There are sex-related differences in how airborne pollutantsare deposited in the lung and in the pul-monary response. This may be importantbecause most TCE exposure comes frominhalation.

There are limited data on sex-dependentdifferences in the toxic response to TCE.Recent epidemiological studies show an excessrisk of breast and cervical cancer amongwomen exposed occupationally and environ-mentally to TCE (33,38,39). A recent analysisof the TCE and TCA National ExposureRegistry (NER) revealed significant sex-dependent differences in reported adversehealth outcomes (39). In this study, a com-parison between data from the registry andnational norms found significantly greaterincreases in diabetes, kidney problems, liverproblems, and urinary tract problems inwomen. Although this registry contains self-reported adverse health outcomes and thuscannot be used to draw definitive conclusions,it does provide a basis for further studies.

Recent studies have found significant sex-dependent differences in the toxicokinetics ofTCE. For example, higher blood concentra-tions of the glutathione conjugate DCVGwere found in men compared to women fol-lowing exposure to either 50 or 100 ppmTCE (42). Peak blood levels in men were 2-fold higher than in women at 100 ppm andwere obtained sooner (2 and 4 hr in men andwomen, respectively). Since male rats aremore susceptible to the nephrotoxic andnephrocarcinogenic effects of TCE and alsohave a higher rate ofGSH conjugation in theliver and kidney (98), these findings suggestthat men may be at a greater risk of develop-ing nephrotoxicity from TCE exposure.

Additional studies have also revealed sex-dependent differences in the toxicokinetics ofTCE. For example, in vitro studies usinghuman tissue found that males and femalesdiffered significantly in their affinities to oxi-dized TCE (40). An uncertainty and variabil-ity analysis of data obtained from subjectsexposed to 50 and 100 ppm TCE revealedsex-dependent differences in various simu-lated pharmacokinetic parameters (99). TCEalso distributes preferentially in fat, andwomen have a greater percentage of fat. Thus,these pharmacokinetic differences have thepotential to result in differential susceptibilitybetween men and women exposed to TCE.

Susceptibility of children to TCE. Themajority of research pertaining to environ-mental health risks in children relates toasthma, ozone, and environmental tobacco


PASTINO ET AL.

smoke. Research pertaining to the effects ofTCE in children has focused primarily onpotential teratogenic effects. Limited studiessuggest a link between exposure to TCE andcardiac malformations, but conflicting resultshave been found (100,101). The Agency forToxic Substance and Disease Registry(ATSDR) reported no increase in malforma-tions in children whose parents were exposedto TCE via inhalation (12). However, Swanet al. (16) and Goldberg et al. (17) found alink between the elevated levels of hydrocar-bons, including TCE, in the drinking waterand cardiac malformations. These resultswere consistent with studies in rats that foundan increase in cardiac malformations (100).Drinking water studies also found an increasein CNS defects, neural tube defects, and oralcleft defects (101). Although these effectswere attributed to TCE, there were other sol-vents present in the drinking water, making adistinct correlation with TCE difficult.

Attention is now being focused on thehealth effects of TCE in the child. Recently,in their comparison between self-reportedhealth outcomes from persons enlisted in theTCE and TCA NER and national norms,Burg and Gist (39) found an increase inreports of hearing and speech impairmentchildren 0-9 years of age, with girls reportinghigher rates. In addition, an increase in therate of urinary tract disorders was alsoreported for most age groups, induding chil-dren. As previously discussed, data obtainedfrom the registry are useful for identifyingareas of further research but do not provideconclusive evidence. However, ATSDR hasrecently undertaken studies to examine thelink between TCE exposure and hearing andspeech impairment in children.

The main exposure routes to TCE arethrough ingestion and inhalation. In chil-dren, oral exposures are most often associ-ated with consumption of water, whileinhalation may occur during use of water foractivities such as showering. Exposure canalso occur transdermally. In addition topotentially higher exposure in children,developmental differences in absorption, dis-tribution, metabolism, and excretion canalter susceptibility in children exposed toTCE. Specific factors that affect the forma-tion and excretion of toxic metabolitesdeserve consideration, as they may alter toxi-city. Data on the dosimetry of TCE in chil-dren are virtually nonexistent; therefore, thediscussion below focuses on potential differ-ences in response between adults, children,and the fetus. It should be noted that poten-tial differences do not necessarily reflect anincrease in susceptibility among children.The specific nature of each response must beconsidered and certain factors may actuallyprovide a protective role.

Age-dependent effects on absorption.The absorption of TCE occurs primarilythrough inhalation and through the gastroin-testinal tract but can also occur transdermally.Each of these processes is different in childrenversus adults and may affect the dosimetry ofTCE. Oral absorption of chemicals in theneonate can be affected by several factors. Forexample, the gastric pH will affect the absorp-tion of ionized drugs and chemicals. The gas-tric pH is neutral at birth as a result of thepresence of amniotic fluid in the stomach(102), decreases to approximately 1-3 withinhours after birth, reaches neutrality at 8 days,and slowly declines to 2-3 by 3 months(103). Although there are age-dependent dif-ferences in gastric pH, nonionized lipophiliccompounds such as TCE readily diffuseacross membranes. Thus, altered gastric pHwill likely not affect the absorption of TCE.Prolonged gastric emptying, which isobserved in the neonate (104), and increasedgastric and intestinal motility, which occursin young children (105), can potentiallyaffect the oral absorption of TCE. TCOH, ametabolite of TCE, undergoes glucuronida-tion and enterohepatic recirculation, which isaffected by a decrease in gastric acid secretion.

Transdermal absorption of chemicals issignificantly higher in the child than the adultbecause of the greater surface area relative tobody weight, which is approximately 2.7-foldgreater in children (103,105). In addition, inthe neonate and infant the epidermis andstratum corneum are thinner, facilitatingmuch greater dermal absorption (103,105).Because of its lipophilicity, TCE could betransdermally absorbed to a greater extent inchildren than in adults.

Inhalation also differs between childrenand adults because of physiological andanatomical differences, as reviewed inGuzelian et al. (106) and Snodgrass (107).The newborn has approximately 10 millionalveoli; adult levels of 300 million are notreached until 8 years of age. The alveolarsurface area increases from 3 m2 at birth to75 m2 in adulthood; as a result, the air-to-tissue gas exchange increases more than 20-fold. In addition, although the respiratoryvolume is the same in adults and children(10 mL/kg body weight/breath), the numberof breaths per minute is increased in theinfant [40 breaths/minute in the infant ver-sus 15 breaths per minute in the adult;(107)]. Thus, the respiratory minute venti-lation is greater in children [133 vs 2 mL/kgbody weight/m2 lung surface area/min;(107)]. One of the primary routes of expo-sure to TCE is via inhalation, and all ofthese factors may alter the dosimetry ofTCEin the child compared to the adult.However, no studies have been conducted toevaluate these factors.

Age-dependent factors in distribution.Factors such as blood flow, tissue volume,and protein binding affect the distribution ofchemicals and also vary with age. For exam-ple, protein binding is decreased in childrenprimarily as a result of an increase in the con-centration of nonesterified fatty acids(108,109). Plasma albumin is also decreasedin children (108,109). These factors willlikely not affect the distribution of TCE inchildren because TCE does not bind signifi-cantly to proteins.

However, the distribution of TCE willlikely be affected by factors that affect the vol-ume of distribution. Lipid-soluble chemicalssuch as TCE should have a smaller volume ofdistribution in infants because of children'slarger percentage of total body water. Totalbody water constitutes as much as 85% ofbody weight in preterm and 78% of bodyweight in full-term neonates (110,111),decreasing to 55% by 12 years of age (112).Because fat content also varies throughoutdevelopment, periods of fat reduction mayresult in increased dosage to the liver or othertarget organs. For example, fat contentincreases between 5 and 10 years of age, fol-lowed by a decrease in boys at age 17 (111).In girls there is a rapid increase at puberty;young females have approximately 2 timesgreater percentage body fat than boys, sug-gesting that females will accumulate TCE to agreater extent than males. The half-life ofTCE in the fat of adults is 3.5 hr, whereas inrapidly and slowly perfused tissue the half-lifeis 2-4 min (11). Therefore, TCE can bereleased from the fat for several hours, result-ing in prolonged delivery to various targetorgans.

TCE is a centrally acting chemical anddistributes to the brain. Thus, differences inbrain volume and blood flow to the brain canalter the toxicokinetics of TCE in childrencompared to adults. The brain weight in thenewborn is approximately 33% of adult val-ues, whereas the body weight of the newbornis 4%. After birth, the brain continues todevelop and is therefore susceptible to insult.Brain growth is very rapid during the first 2years and contains 75% of all cell types by age2, but further growth is due to myelination ofsubcortical white matter, elaboration of neu-ronal dendrites and axons, and an increase inthe number of glial cells. Thus, the newborn'sbrain is more lipophilic, which can affect thedistribution of chemicals such as TCE.

Age-dependent effects on metabolism. Ingeneral, children are believed to metabolizeand clear xenobiotics faster than adults.However, the metabolism of xenobiotics inthe infant and child is dependent on the spe-cific enzyme systems involved for each chemi-cal. Some enzymes present at birth do notreach adult activity levels for months or years.



Others are present in the fetus and reach adultcapacity at birth. It is therefore important toconsider chemical-specific metabolic pathwayswhen examining the potential risks in chil-dren, since the ontogenicity differs for eachenzyme system.

CYP2EL. As with adults, one of the mostimportant enzymes to consider in the evalua-tion of toxicity from exposure to environmen-tal chemicals in children is CYP2E1. Thisenzyme is responsible for the metabolism ofover 80 exogenous substances, including TCE(55). Expression of CYP2E1 in the placentaor fetus could potentially result in the forma-tion of high local concentrations of metabo-lites and lead to higher exposures in fetaltissues than would be expected from maternalexposure. Depending on the physiochemicalproperties, these metabolites may be unable toexit the placenta and accumulate in the fetalcompartment. Moreover, the possibility fortransplacental induction poses additionalthreats.

Recent studies have been conducted toexamine the expression and activity ofCYP2E1 in the human fetus and placenta.Many of these studies were motivated by anattempt to elucidate a mechanism for theeffects of ethanol on the fetus. In one study,CYP2E1 was detected in human fetal liverduring the second trimester, at about 16weeks of gestation (113). The molecularweight was slightly lower than in adults, butthe enzyme was able to metabolize ethanol at12 and 27% of adult capacity. However, at10 weeks of gestation, CYP2E1 mRNA levelswere undetectable in embryo liver samples.CYP2E1 was inducible in human fetal hepa-tocytes treated with ethanol and clofibrate,and a 4-fold induction in CYP3A1 was alsofound following treatment with rifampicin.CYP2E1 has also been detected in extrahep-atic tissues. For example, the expression ofCYP2E1 was detected in human placenta(114-116). CYP2E1 was also identified inprenatal human cephalic tissues (117).Northern blot analyses of mRNA levels fromtissues obtained between days 54 and 78 ofgestation showed that CYP2E1 levelsincreased as a function of age but were muchlower than hepatic levels.

Despite these findings, conflicting reportsexist regarding the presence of CYP2E1 infetal liver. Vieira et al. (118) found thatCYP2E1 was absent from fetal liver but roseimmediately after birth regardless of the gesta-tional age at birth, which ranged between 25and 40 weeks. This suggested CYP2E1 regula-tion was directly related to parturition eventsrather than temporal maturation itself. Thelevel of the protein and its catalytic activitysteadily increased during the first year to reachadult values in children age 1-10 years. Inaddition, the kinetic properties of CYP2E1

were investigated using cloroxazone as asubstrate in both neonatal and adult microso-mal preparation. The affinity was greater innewborns compared to adults; Km valuesobtained were 15.8 and 28.8 pM, respectively.However, the capacity was similar, with aVmax of 0.96 nmol/mg protein/min in theneonate and 1.0 nmol/mg protein/min in theadult. These findings are consistent with pre-vious studies examining the expression ofCYP2E1 in fetal tissues (119). In this study,CYP2E1 expression was absent in the placentaeither at 10 weeks or 18 weeks of gestation. Inaddition, CYP2E1 expression was notdetected in fetal liver, kidney, lung, and pla-centa at 18 weeks or in the liver at 6 weeksof gestation.

Differences between studies detecting thepresence of CYP2E1 and those that do notare due, at least in part, to timing of thestudies. The placenta and fetus are continu-ally growing and thus expression of CYP2E1is likely dependent upon the developmentalstage. While some human studies have shownthe presence of CYP2E1 in fetal hepatic andextrahepatic tissues of different gestationalages, in animals CYP2E1 is present only afterbirth. The rat CYP2E1 gene appears to betranscriptionally activated at birth, only afterwhich is it detected in rat hepatocytes (120).The expression of CYP2E1 in rabbits did notbegin until 2 weeks of age and reached twicethe adult level between weeks 3-5, the timeof weaning (121). In kidney, CYP2E1 wasexpressed at 1 week of age.

Clearly, the data indicate that develop-mental expression and activity of CYP2E1 inhumans must be explored more fully. This isparticularly important because this enzyme isresponsible for the biotransformation ofmany xenobiotics, including metabolicallyactivated compounds such as TCE. A com-plete assessment of the health effects of TCEin the fetus and child requires knowledge ofthe metabolism, particularly by CYP2E1, inthis population.

Additional metabolizing enzymes. As withthe CYP450 enzymes, the expression andactivity of several other enzyme systems inhumans vary depending on the developmen-tal stage. For example, glucuronidation in thechild reaches adult values at 3-6 months(122). Thus, the fetus and neonate probablylack adult capacity to form the glucuronide ofTCOH, which can potentially lead to anincrease in the formation of TCA and DCA.Since these metabolites are toxic, the fetusand neonate could potentially be more sus-ceptible to TCE-related toxicity.

Of additional concern to children is thedecrease in ADH activity. ADH activity ispresent in fetal liver at the second month ofgestation, but this activity is only 3% of adultlevels (123). The diminished ADH activity

appears to have an effect on the clearance ofseveral drugs and chemicals, including CH, ametabolite ofTCE. For example, the elimina-tion half-life of CH was much greater in thepreterm fetus (39.8 hr) than in the neonate(27.8) or child (9.7, age 1-13 years) (124).

Metabolism in Rodents. Studies in pregnantand immature rodents can lend insight intomechanisms of effects of exposure to environ-mental chemicals and potential toxicity tohumans, particularly with regard to the contri-bution of metabolic differences. Several studieshave been conducted examining the expressionof metabolic enzymes in the developingrodent. Hepatic microsomal monooxygenaseactivity is very low during fetal development inmost mammals but increases rapidly afterbirth. Sex-specific regulation of certain P450isozymes occurs during development. Forexample, CYP2C1 1 was found at significantlevels in immature (4-week-old) male rats, andliver levels increased 30-fold in male but notfemale rats at puberty (125,126). In addition,microsomal protein levels were higher inimmature male rats versus females; protein lev-els increased during development, but atpuberty no sex differences were found. No dif-ference in net P450 content was seen betweenimmature male and female rats, but markeddifferences were seen at puberty. Pregnancydecreased the P450 content but not the level ofmicrosomal protein.

Nakajima et al. (52) examined the effectsof sex, age, and pregnancy on toluene andTCE metabolism in rat liver. CYP2E1 levelswere higher in immature than in mature rats,especially at low substrate levels. At puberty,CYP2E1 levels were higher in females than inmales. In general, TCE metabolism washigher in immature than in mature rats, espe-cially at low substrate levels. No sex differ-ences in metabolism were seen with age orwith varied TCE concentration. However,pregnancy decreased the metabolism of bothtoluene and TCE.

Age-dependent effects on excretion.Several processes involved in the excretion ofchemicals may be affected by age. Polar com-pounds can be excreted unchanged, whereasmost lipophilic compounds are readilyexcreted following metabolism to more polarforms. Lipophilic chemicals are also elimi-nated through exhalation. Pulmonary excre-tion occurs primarily with gases and vapors,such as TCE. As described above, lung devel-opment is different in children and may affecthow TCE is excreted in breath.

Biliary and fecal excretion are additionalpathways. Many metabolites are excretedfrom the liver into the bile by specializedtransport systems similar to those in therenal tubule, and competition for transportcan occur between ions of like charges.These chemicals are either excreted in the


PASTINO ET AL

feces or reabsorbed into the blood andexcreted into the urine, a process known asenterohepatic recirculation (EHR). EHRoccurs primarily with organic ions, such asglucuronides or cations. In the child, biliaryacid secretion is decreased, which will alterEHR (127). The glucuronide conjugate,trichloroethanol glucuronide (TCOG), isexcreted in the bile, undergoes EHR, anddoes not get excreted into the feces (15).Thus, decreased biliary acid secretion in thechild will probably alter EHR ofTCOG andmay alter the toxicity ofTCE.

Elimination of chemicals can also beaffected by factors such as the glomerular fil-tration rate (GFR). The GFR in newborns islow but reaches adult levels by 3 months ofage (128). Since many chemicals are elimi-nated in the urine, including several TCEmetabolites, developmental differences in theGFR may affect overall elimination.A potential excretory pathway for TCE in

adult women that may be of particular con-cern for infants is lactation. TCE is lipophilicand is excreted in the breast milk. However,no studies have been conducted on thedosimetry of TCE in breast-fed infants.Recently, a physiologically based pharmacoki-netic (PBPK) model was developed todescribe the transfer of volatile chemicals inbreast milk, including TCE (129). An inter-mittent acute exposure to 50 ppm of TCEwas simulated using previously publishedphysiological and metabolic values for a lac-tating woman (2-3 months postpartum). Thesimulated nursing schedule consisted of eight12-min nursing bouts over a 24-hr period.The predicted amount of TCE ingested bythe infant over the 24-hr period was 0.496mg compared to the U.S. EPA HealthAdvisory Intake Limits of 0.6 mg/day for anadult consuming 2 L ofwater per day.

While this estimate would suggest thatbreast-fed infants are not at increased riskfrom exposure to TCE in breast milk, severalfactors need to be considered. The output,that is, the amount of TCE ingested by thelactating infant, is compared to drinkingwater limits for an adult and may or may notbe appropriate for the child. Moreover, themodel simulated the 2- to 3-month-oldinfant not the newborn or older infants. Themodel also simulates only parent compoundand does not consider the transfer of metabo-lites. Studies in rats showed that TCA reachesthe suckling pup at 30 and 50% of maternallevels following exposure of the mother toTCE in the drinking water and throughinhalation, respectively (130). Thus, giventhese factors and the small difference betweenPBPK model predictions and the U.S. EPAadvisory, conclusions regarding the safety ofexposure to TCE in breast milk need tobe reevaluated.

Clearly there are many factors that havethe potential to alter the dosimetry and toxic-ity of TCE in the child. Any one of these fac-tors alone may play a role in susceptibility.However, in order to provide a scientificallyjustifiable assessment of risk to children, thesefactors need to be considered simultaneously.The most useful tool for accomplishing thistask would be a PBPK model for TCE in thegrowing child. Despite potential limitations,the development of the PBPK modeldescribed above is a useful step in this process.

Acquired Factors That AfifctPhysiological VariablesA number of acquired factors affect metabolicgene expression and function, and includehealth status, prior and concurrent exposureto other substances, and behavioral patterns.Susceptibility to TCE can be altered by thesefactors because many of them have the poten-tial to alter toxicity secondary to CYP2E1induction. Factors that lead to an inductionof CYP2E1 include, but are not limited to,uncontrolled diabetes, obesity, and priorexposure to certain solvents (131). CYP2E1is also induced by certain medications, suchas barbiturates, and by excessive alcohol con-sumption (55,132). The chronic use of barbi-turates, as often occurs in epileptics, increasesprotein and lipid content of hepatic smoothendoplasmic reticulum, and also increases theactivity of glucuronyl transferase and variousCYPP450s, including CYP2E1 (133).Comprehensive reviews on the mechanismfor CYP2E1 induction in various popula-tions, and on the physiological and pathologi-cal role of CYP2E1, are provided by Raucy etal. (131) and Lieber (55), respectively.

It is important to consider these popula-tions in evaluating susceptibility, given thenumber of persons who fall into one ormore of these categories. For example, 14million people in the United States meet thediagnostic criteria for alcohol abuse or alco-hol dependence (68). The prevalence ofinsulin-dependent diabetes in the UnitedStates is 300,000-500,000, with 30,000new cases diagnosed each year (134). Theincidence of diabetes is affected by sex, race,and age (134,135).

Very few studies have been conducted ontoxicokinetic interactions between TCE andthese risk factors, with the exception ofethanol. Although the discussion below willfocus primarily on interactions betweenethanol and TCE, this serves as an example ofpotential consequences of TCE exposure inpersons with induced CYP2E1, as well as theinfluence of exposure to mixtures.

Ethanol Interactions. Concurrent expo-sure to TCE and ethanol is likely to occur in alarge number of people, and ethanol mayincrease susceptibility to the adverse health

effects of TCE. Alternatively, it can be viewedthat exposure to TCE can aggravate the effectsof ethanol. This interaction may be an impor-tant health problem because, as discussedabove, both alcohol and TCE exposure arewidespread in the United States. Ethanol andTCE are centrally acting chemicals and expo-sure to high levels can cause profound CNSdepression (75,136). Many enzymes responsi-ble for TCE metabolism are also involved inthe metabolism of ethanol.

Approximately 75-90% of ethanol ismetabolized through oxidation in the liver,primarily by ADH, although metabolism canalso occur in other tissues, such as the kidneys,gastric mucosa, and brain (Figure 2)(137-139). Ethanol is also metabolized byCYP2E1 (140). The contribution of CYP2E1to the metabolism of ethanol in vivo is unclearbut is thought to contribute at higher dosesand following chronic exposure to ethanol(55,132,138). Chronic ethanol consumptioncan result in up to a 10-fold induction inCYP2E1 (55). The catalase system also con-tributes to the metabolism of ethanol andrequires hydrogen peroxide. Thus, its relativerole in the metabolism of ethanol in vivo isthought to be minimal (141).

Several studies have been conducted onthe toxicokinetic interactions between ethanoland TCE in male rats. Exposure to ethanol inthe diet produced a 6-fold increase in the invitro metabolism of TCE and an increase incytochrome P450 content and activity (142).In vivo elimination was increased followinginhalation exposure to concentrations ofTCEranging from 500 to 8,000 ppm. Blood TCEconcentrations were decreased and the urinaryexcretion ofTCA and TCOH was enhancedat all exposure concentrations (142). Thesetoxicokinetic interactions between chronicethanol exposure and TCE in vivo are depen-dent upon TCE exposure concentration. For

NADPII

NADP

AcetaldehydeNAD+

.;, ALDHNADH

AcetateFigure 2. Biotransformation of ethanol. Metabolismoccurs primarily by hepatic ADH at low ethanol concen-trations and following acute exposure. At higher concen-trations and following chronic exposure to ethanolcytochromes, P450 (CYP450), particularly CYP2E1, con-tributes to the metabolism. Catalase plays a minor rolein ethanol metabolism. Each pathway results in the for-mation of acetaldehyde, which is metabolized to acetatevia ALDH.



example, an increase in the in vivo eliminationofTCE following chronic ethanol exposure inmale rats was found at TCE concentrationsgreater than 100 ppm; exposure to either 50or 100 ppm produced no difference in bloodTCE concentration or in the amounts ofTCAand TCOH in the urine (143). The durationof exposure (142,144) and the compositionof the ethanol-containing diet (55,145) canalso influence the metabolic interactions andtoxicity.

Data from human males also suggestpotential influences of chronic ethanol con-sumption on the toxicity of TCE. A study of188 factory workers exposed to TCE revealedincreased hepatotoxicity in subjects who con-sumed ethanol (146). On average, workerswere exposed for 7 hr/day for 7 years to TCEconcentrations ranging from 50 to 150 ppm.Heavy drinkers consumed 1.5 L of wine perday for at least 5 years. Of the 51 workersidentified as heavy drinkers, 41 showed clini-cal signs of liver impairment. This prevalencewas statistically significantly increased com-pared to the prevalence among workers whowere not heavy drinkers. However, becausechronic ethanol consumption causes liverimpairment, these observed effects may bedue to the presence of ethanol rather thanTCE. That is, TCE may be enhancing thetoxicity of ethanol.

Acute ethanol exposure also affects thetoxicokinetics of TCE. Concurrent exposureto ethanol and TCE in human males inhib-ited the metabolism of TCE at an exposureconcentration of 50 ppm for 6 hr on 5 con-secutive days (147). Plasma TCA andTCOH concentrations were decreased byone-half and one-third, respectively, com-pared to controls. The total amount of TCAand TCOH excreted in the urine alsodecreased 43 and 20%, respectively.Similarly, ethanol almost completely inhib-ited the metabolism of a single exposure to100 ppm TCE for 6 hr (147). Plasma TCAlevels were essentially unchanged, blood TCEconcentrations increased 2-fold, and breathTCE concentrations increased 3-fold whenethanol was consumed prior to and duringthe TCE exposure.

Studies in rodents confirm the acuteeffects of ethanol in adult men. Ethanol addeddirectly to microsomal incubations obtainedfrom rats inhibited the in vitro metabolism ofTCE (148). An acute ethanol administrationalso inhibited the in vivo elimination of TCEwhen ethanol was still present in the livers ofmale rats (149). Steady-state blood TCE con-centrations were elevated in female ratsexposed to 50 or 100 ppm TCE and 0.8mL/kg ethanol (149). These toxicokineticinteractions are likely affected by the balanceof cofactors necessary for TCE and ethanoloxidation, and inhibition of TCE following

acute exposure to ethanol is likely due to ashift in the NADH:NAD+ ratio (150-152).

Acutely, ethanol may also interact withthe metabolites of TCE, specifically CH andTCOH, since ethanol and CH are bothmetabolized by ADH and have similar phar-macological properties (75). For example,male subjects who were administered ethanolfollowing the administration of CH had sig-nificantly higher plasma TCOH levels com-pared to CH alone, and levels were prolongedin the ethanol-exposed subjects (153). Adecrease in plasma TCA was also found in theethanol-exposed subjects, as well as anincrease in urinary TCA and a decrease inurinary TCOH production. The authors sug-gest the ethanol-induced increase in plasmaTCOH concentration is due to an increase inthe metabolism of CH to TCOH and adecrease in the glucuronidation ofTCOH.

Although experimental studies in humansexposed to both TCE and ethanol are lim-ited, pharmacokinetic modeling has providedinsight regarding the effects of potentialinteractions. Simulation studies using aPBPK model for TCE and ethanol were con-ducted to examine the interactions betweenacute and chronic ethanol and TCE (154).Based on previous studies conducted in malerats, the metabolism ofTCE following acuteexposure to ethanol was assumed to be com-petitively inhibited and was described usingthe Michaelis-Menten expression for com-petitive inhibition. CYP2E1 induction fol-lowing chronic ethanol exposure wassimulated by an increase in the Vm,,, for themetabolism ofTCE.

Moderate doses of ethanol consumed 15min prior to a 6-hr exposure to 50 ppm TCEresulted in increases in blood TCE concentra-tions and corresponding decreases in the rateof excretion of urinary metabolites (152). Atthe highest ethanol dose (20 mmol/kg), bloodTCE concentrations increased 70% and theurinary excretion rate of total urinarymetabolites decreased 40%. This dose ofethanol corresponds to approximately 6-9standard drinks in a 70-kg man and 5-8drinks in a 60-kg female.

The induction of CYP2E1, simulated bya 5-fold increase in the Vmax, had a slighteffect on TCE metabolism (154). BloodTCE concentrations were decreased 10% andthe excretions of urinary TCA and TCOHwas increased by less than 10%. Simulationsat TCE concentrations higher than 500 ppmshowed a much greater effect on the decreasein blood TCE and the increase in urinaryTCA and TCOH formation.

The minimal effect of CYP2E1 inductionon the metabolism of low concentrations ofTCE is due, at least in part, to the intrinsicclearance of TCE. When concentrations ofTCE are low, the metabolism is blood-flow

limited and the effect of CYP2E1 induction isminimal. At higher concentrations whenmetabolism is saturated, CYP2E1 inductionresults in an increase in the capacity to metab-olize TCE, which increases the in vivo elimi-nation and formation of toxic metabolites.

Predictions based on in vitro studies ofTCE metabolism (40) and PBPK model sim-ulations in human males (155) suggest themetabolism ofTCE in humans at occupation-ally relevant concentrations (50 ppm) is notsaturated. Accordingly, a significant increasein the formation of toxic metabolites formedfrom the oxidative pathway would not beexpected. However, a 10% increase in the for-mation of toxic metabolites, as predicted bythe PBPK model, might be enough toenhance the toxicity ofTCE, particularly overlong-term TCE exposure. The simulationsaccounted for a 5-fold induction in CYP2E1,and chronic ethanol consumption can resultin up to a 10-fold induction of CYP2E1(132). Moreover, up to a 20-fold variation inCYP2E1 activity can occur (138).

The extent of TCE metabolism inhumans was found to be correlated withcytochrome 450 activity and content (40).Although Lipscomb et al. found thatCYP2E1 activity varied less than 10-fold,these studies were conducted on a limitednumber of subjects and cannot necessarily begeneralized to all populations. For example,subjects who consumed alcohol were notincluded in this study. Furthermore, theshort-term exposure limit for TCE is 200ppm and was set to protect against the anes-thetic effects ofTCE (29). The minimal risklevel (MRL), which is the estimated dailyhuman exposure to a hazardous substancethat is likely to be without appreciable risk ofadverse noncancer health effects over a speci-fied duration of time and is set by ATSDR, iswell below 200 ppm for TCE (12). TheMRLs for acute and intermediate inhalationexposure to TCE are 2 and 0.1 ppm, respec-tively. Thus, when CYP2E1 is induced, anincrease in the formation of toxic metaboliteswill likely occur at TCE concentrations at ornear 200 ppm. The anesthetic effects of acuteethanol and TCE will be enhanced whenexposure to both occurs at this concentration.

Another possible interaction that has notbeen studied is the effect of ethanol-inducedglutathione depletion on the metabolism ofTCE to DCVG. Ethanol decreases hepaticmitochondrial glutathione levels (156),which in turn impairs mitochondrial function(157). The formation of DCVG from glu-tathione conjugation has been implicated as apossible mechanism for the nephrotoxicityand nephrocarcinogencity of TCE (15).Thus, ethanol exposure has the potential toaffect susceptibility to TCE via interactionwith this pathway.


PASTINO ET AL.

Most studies on the interaction betweenTCE and ethanol were conducted in malerats, as well as a limited number of studies inhuman males. Caution should be used whenextrapolating these findings to females, partic-ularly since significant sex-dependent differ-ences in the affinity to metabolize TCE wereobserved in humans (40). As discussed above,sex-dependent difference in the toxicity ofTCE and TCA also exists. In addition, thereare sex-dependent differences in the pharma-cokinetics and toxicity of ethanol; women aremore susceptible to the adverse effects ofethanol (158-163).

While studies have not been conductedon the interactions between ethanol and TCEin pregnancy, studies in rats showed an eleva-tion in cardiac malformation in rat pupsexposed to DCA, TCA, or TCE in utero(18,100,164). An increase in cardiac anom-alies was found in children born to mothersexposed to drinking water containing, amongother contaminants, TCE and DCA (17,18).Exposure to ethanol in utero also results incardiac malformations in humans (165).

The induction of CYP2E1 by ethanol inthe fetus and developing infant may alsoincrease the likelihood of developmentaleffects ofTCE and/or ethanol. Recent studiesfound elevated CYP2E1 levels and an increasein activity in rat pups exposed to ethanol inutero (166) as well as pups exposed translac-tationally (167). Ethanol has been measuredin the breast milk of lactating women (168).Recent studies in women with a history ofheavy drinking found an increase in theexpression ofCYP2E1 in third-term placentas(169). A 2-fold induction in CYP2E1 levelswas found in fetal hepatocyte cultures treatedwith ethanol (170). The estimated incidenceof fetal alcohol syndrome (FAS), a syndromeof anomalies resulting from exposure toethanol in utero, is 9.7 per 10,000 live births;among heavy drinkers, 4.3% of children areborn with FAS (171). Therefore, the possibil-ity of induction of CYP2E1 in the placentaand/or human fetus exists and this may leadto a subsequent increase in susceptibility tothe developmental effects ofTCE.

The data presented thus far illustrate thatinteractions between ethanol and TCE occurin both rodents and humans followirng acuteand chronic ethanol exposure. There are lim-ited data in rodents and humans, suggestingthat chronic ethanol consumption leads togreater hepatotoxicity of TCE and that acuteethanol consumption increases the CNSeffects of TCE. Alcoholics are often warnedabout a potential increase in the adversehealth effects of many drugs. For example,the U.S. Food and Drug Administration willbe requiring an alcohol warning on all over-the-counter medications containing aceta-minophen because induction of CYP2E1

causes an increase in the hepatotoxicity ofacetaminophen (172). Given that there areover 80 exogenous chemicals metabolized byCYP2E1, physicians should probably warnalcoholics of the potential increase in adversehealth effects of environmental contaminants.

Prior and concurrent exposures. Suscepti-bility may be altered simply by increasing thebody burden of a given environmental agentor its metabolites. In some cases a thresholdmay be reached. Previous exposures mayenhance susceptibility by sensitizing the indi-vidual to the compound, either directly or bycross-sensitization. Prior exposure to variouschemicals may also interfere with metabolismthrough inhibition or induction, which inturn can alter susceptibility.

For example, Lipscomb et al. (40) studiedthe effect of TCE on specific cytochromeP450 isozyme activities using mouse, rat, andhuman hepatic microsomes. TCE was a com-petitive inhibitor of CYP2E1 activity, and anoncompetitive inhibitor of CYP3A andCYP2B. Addition of TCE at 1,000 ppminhibited CYP2E1 activity in all three species,and CYP3A activity in mice and rats. Priorexposure to TCE had no effect on CYP2Aactivity but did increase CYPlA1/1A2 activ-ity. In addition, animal studies show DCAinhibits its own metabolism (173). Thus,DCA levels may accumulate with prior orprolonged exposure.

Nakajima et al. (52) compared the rela-tive contribution of the P450 isozymes in themetabolism of TCE in rats. Hepatic micro-somes were obtained from control rats, andrats previously exposed to phenobarbital,ethanol, or 3-methylcholanthrene were used.CYP2E1, CYP2E11/6, CYPIIB1/2, andCYPIA1/2 were involved in conversion ofTCE to CH, with some variation in isozymelevels at different TCE concentrations. At lowTCE levels, adding anti-CYP2E1 antibodiesinhibited TCE metabolism in ethanol-treatedrats more greatly than in controls. In contrast,CH formation was inhibited by anti-CYP2C1 1/6 in control and PB-treated rats athigh but not low TCE concentrations, with alower net inhibition than from using anti-CYP4502E1. Anti-CYP3B1/2 and anti-CYPA1/2 inhibited CH formation fromphenobarbital and 3-methylcholantherene-treated rats, particularly at high TCE concen-trations. CYP2B1 contributed more to TCEoxidation than CYPIC1/6, but the reversewas seen for toluene metabolism. In conclu-sion, CYP2E1, 1A1/2, 2C11/6, and 2B1/2are all involved in metabolism of benzene,toluene, and TCE, indicating broad andoverlapping substrate specificity of thesevolatile compounds.

Prior exposure to solvents such as toluenecan induce CYP450s, which in turn canaffect TCE metabolism (174). Both sex and

age influence CYP450 induction after tolueneexposure; in general, induction is greater inyounger animals and higher in males (55).Neonatal exposure of rats to toluene greatlyaffected liver microsomal CYP450 activity atbirth, whereas minimal effects were seen inrats exposed at 3 weeks of age (174).

Altered health state. An additional popula-tion that deserves consideration in the evalua-tion of susceptibility to environmental toxinsis persons whose health state is altered. This isimportant because of the effects of certain dis-eases on the ability of the body to processchemicals. For example, the ability to renallyexcrete chemicals can be diminished in peoplewith kidney impairment, leading to anincrease in toxicity. The phenomenon is read-ily apparent with antibiotics. The majority ofantibiotics and their metabolites, such as van-comycin and aminoglycosides, are eliminatedby the kidneys. In patients with renal insuffi-ciency, dosages of these drugs are adjustedaccordingly to avoid overt excessive toxicity(175). Liver disease also affects the ability tometabolize and eliminate chemicals. Forexample, rifampin and isoniazid have pro-longed half-lives in people with cirrhosis(175). These same principles apply followingexposure to environmental chemicals and maycontribute to differences in susceptibility.

As previously discussed, uncontrolleddiabetes results in an induction of CYP2E1,which can lead to an increase in the forma-tion of toxic metabolites. Diabetics are also atgreater risk for liver cancer. Recent studies inthe Swedish population show a 4-foldincrease in the risk for development of pri-mary liver cancer in patients with diabetesmellitus versus the general population (176).Similar results were reported in studies fromItaly, Denmark, and Los Angeles (177-179).Diabetics may be a population susceptible tothe potential hepatotoxic effects ofTCE.

The effect of DCA on blood glucose andserum insulin levels is of additional concernfor diabetics. An increase in serum insulinlevels in mice exposed to < 0.5 mg/L DCA inthe drinking water was found (22). This hasnot been found in humans. But DCA reduceshyperglycemia in insulin and non-insulin-dependent diabetics by stimulatingblood glucose oxidation in peripheral tissues,stimulating glycogensis from glucose inadipocytes, and depleting hepatic and muscleglycogen levels (180). These effects have beenobserved in both animals and humans. DCAalso decreases hyperglycemia through stimu-lation of pyruvate dehydrogenase (PDH)(181). DCA inhibits pyruvate dehydrogenasekinase, which maintains PDH in its unphos-phorylated, catalytically active state (181).PDH controls the rate of glucose and pyru-vate oxidation, and the net effect is a decreasein blood glucose levels, as well as a dramatic



and prolonged decrease in lactate and anilinelevels (181).

Several studies have been conducted inhumans to evaluate the potential use of DCAin the treatment of hyperglycemia in diabetics,as well as in the treatment of lactic acidosis,hyperlipidemia, ischemic heart disease, andheart failure. The treatment regimen used inlactic acidosis consists of a 30-min intravenousinfusion of 50 mg/kg followed by a secondinfusion of the same dose 2 hr after initiationof the first dose (182). This dosing regimen,which results in a rapid decrease in plasma lac-tate and glucose concentrations, producedmaximum plasma DCA concentrations of 144± 92 mg/L and 180 ± 92 mg/L following thefirst and second infusion, respectively, in adultpatients with lactic acidosis (182). These lev-els are much greater than those measured inhealthy human subjects exposed to occupa-tionally relevant concentrations of TCE(183). In this study, subjects were exposed to100 ppm TCE for 4 hr, and TCE andmetabolite levels were measured in blood andurine. DCA was detected intermittently, withas high as 0.014 mg/L measured in one sub-ject. It is unlikely that exposure to occupation-ally relevant TCE concentrations will produceblood DCA levels that would result in adecrease in hyperglycemia in diabetics.

However, DCA administration in humanshas been limited to 2 weeks and chronicadministration has not been examined.Because DCA inhibits its own metabolism,prolonged and/or repeated administration maylead to sustained concentrations that mayresult in an irreversible inactivation of thePDH (180). Thus, an increase in susceptibility.of diabetics to the effects of the TCE metabo-lite DCA on blood glucose levels cannot beruled out until further studies are conducted.

DiscussionAs discussed earlier, for most environmentalchemicals data on susceptible subgroups arerarely available. The use of linear low-doseextrapolation in cancer assessments has beenassumed to be conservative enough not tounderestimate risk for susceptible members ofthe population (9). Noncancer assessmentsacknowledge response variability within thepopulation by an uncertainty factor (currentpractice uses a default value of 10)(183).However, the scientific basis for defaultassumptions remains questionable, particu-larly in light of the myriad factors that canpotentially alter toxicity. A person's responseto environmental hazards is affected by physi-ological variables including exposure route(respiratory rates, dermal penetration/irrita-tion, etc.), pharmacokinetics (distribution,absorption, metabolism, excretion), and phar-macodynamics (receptor density, organ speci-ficity). These variables are in turn affected by

constitutive factors (genetics, gender, age,ethnicity) and acquired factors (disease state,diet, exercise, stress, effects of previous orconcurrent exposures).

While a small number of human carcino-gens show variable effects in outcome follow-ing exposure, our understanding of theinherent variability in subpopulations is lim-ited, and epidemiological and laboratorystudies on this subject are in their infancy(184). While heredity clearly has an impacton exposure-driven cancer, not enough isknown about the correlation to harness it inimproving public health (184).

Ethnic groups vary in their distributionsof genotypes, and extrapolation of health out-comes between the different groups is verydifficult (51). In the United States, overalldisease rates are higher in African-Americansthan in Caucasians. Molecular epidemiologysuggests different patterns of exposure and/orinternal handling of exogenous or endoge-nous carcinogens along racial or ethnic lines,and different patterns of susceptibility mark-ers are seen (184).

Despite the observational correlations,biologically plausible mechanisms to linkspecific genotypes to specific outcomes arestill lacking for most diseases; for example,many RFLP polymorphisms in Class I genesare the result of mutations in introns or othersilent areas of the human genome (51), andmolecular methods are still of limited utilityin predicting susceptibility at this time.Furthermore, it is difficult to disentangle out-comes that are the result of genetic traits, par-ticularly when associated with ethnicity, fromsocioeconomic and behavioral factors thatalso may affect environmental exposureor susceptibility.

In some cases, risk reduction can beachieved by going beyond risk regulation andtaking a public health approach promotinghealthy activities in conjunction with anincreased understanding of the biologicalmechanisms of toxicity, which may allowdevelopment of more effective interventions.Examples of successful interventions includecontrolling smoking and radon exposure,smoking and asbestos exposure, and CYP450induction and alcohol/smoking.

While environmental prevention strate-gies focus on reducing exposure in the work-place and environment, interventionstrategies are geared toward the population asa whole and do not inherently addressgenetic differences among individuals (185).Some contend that current environmentalrisk assessment methods do not provide suffi-cient protection, as they assume similar bio-logical responses from all individuals for aspecified dose of a toxic agent (185). Thisresults in regulatory and health policiesaimed at protecting the "average American,"

ignoring a sizable, more vulnerable fractionof the population (51,184). Conversely, oth-ers contend that the default safety factorsused in human risk assessment are overpro-tective of the population, resulting in highregulatory costs at the expense of industry.

The new U.S. EPA cancer guidelines pro-posed in 1996 discuss the importance ofincorporating information on susceptibility.The NRC and the Presidential Commissionon Risk Assessment and Risk Managementhave recommended that the U.S. EPAdevelop explicit methods for assessing suscep-tibility, using molecular epidemiology andother population-based information to deter-mine the magnitude ofvariability (5).

In order to better protect vulnerablegroups and the population as a whole, accu-rate data on susceptible groups are neededand should be directly incorporated into riskassessment processes to allow development ofbetter health-based policies (3,5). Moremechanistic information is needed to allowbetter understanding of how different factorsinfluence toxicity, and how these vary accord-ing to genetic profile, behavior, and age. Forexample, molecular biomarkers of exposureand effect are increasingly being used in epi-demiological analyses to improve the resolu-tion of risk factors and to better understandthe basic underlying mechanisms responsiblefor disease (185). Biomarkers can serve as anearly indicator of exposure or susceptibilitybefore clinical signs manifest, allowing earlyinterventions. The distribution of biomarkersin a population can potentially be used toimprove the quantitative estimates of riskfrom a given exposure, and to identify suscep-tible groups or even individuals who may beat risk (51,185).A more comprehensive documentation

and understanding of sex- and species-depen-dent differences in metabolism is essential forextrapolating animal data to humans, forrefining risk assessment models and methods,and for better determining safe exposure levelsfor humans.

REFERENCES AND NOTES

1. National Research Council. Risk Assessment in the FederalGovernment: Managing the Process. Washington, DC:NationalAcademy Press, 1983.

2. National Research Council. Science and Judgment in RiskAssessment. Washington, DC:National Academy Press, 1994.

3. Sexton K, Callahan M, Bryan EF. Estimating exposure and dose tocharacterize health risks: the role of human tissue monitoring inexposure assessment. Environ Health Perspect 103:13-29 (1995).

4. Clean Air Act, 33 United States Code Sec. 1241 Et Esq, 1970.5. Presidential/Congressional Commission on Risk Assessment

and Risk Management. Risk Assessment and Risk Managementin Regulatory Decision-Making. Final Report, Vol. 2. U.S.Government Printing Office: 1997-519348/90015.

6. Vainio H, Husgafvel-Pursiainen K. Elimination of environmentalfactors or elimination of individuals: biomarkers and prevention.J Occup Environ Med 37:12-13 (1995).

7. Renwick AG, Lazarus NR. Human variahility and noncancer riskassessment -an analysis of the default uncertainty factor.Regul Toxicol Pharmacol 27:3-201(1998).


PASTINO ET AL.

8. U.S. Environmental Protection Agency. Guidelines for RiskCharacterization. Washington, DC:Science Policy Council, 1995.

9. U.S. EPA. Proposed Guidelines for Carcinogen Risk Assessment.EPA/600/P-92/003C. Washington, DC:U.S. EnvironmentalProtection Agency, 1996.

10. Barton HA, Flemming, CD, lipscomb, JC. Evaluating human vari-ability in chemical risk assessment: hazard identification anddose-response assessment for noncancer oral toxicity oftrichloroethylene. Toxicology 111:271-287 (1996).

11. Davidson IWF, Beliles RP. Consideration of the target organ tox-icity of trichloroethylene in terms of metabolite toxicity andpharmacokinetics. Drug Metab Rev 23:493-499 (1991).

12. ATSDR. Toxicological Profile for Trichloroethylene. Atlanta,GA:Agency for Toxic Substances and Disease Registry, 1993.

13. Maltoni C, Lefemine G, Cotti G, Perino G. long-term carcino-genicity bioassays on trichloroethylene administered by inhala-tion to Sprague-Dawley rats and Swiss and B6C3F1 mice. AnnNY Acad Sci 534:316-342 (1988).

14. National Research Council. Pesticides in the Diets of Infantsand Children. Washington, DC:National Academy Press, 1993.

15. Lash LH. Metabolism of trichloroethylene. Environ HealthPerspect 108(suppl 2):177-200 (2000).

16. Swan S, Deane M, Harris J, Neutra R. Pregnancy outcomes inrelation to water contamination, 1980-81, San Jose, CA. In:Pregnancy Outcomes in Santa Clara County 1980-82: Reports ofTwo Epidemiological Studies. Berkeley, CA:CaliforniaDepartment of Health Services, 1985.

17. Goldberg SJ, Lebowitz D, Graver EJ, Hicks S. An association ofhuman congenital cardiac malformations and drinking watercontaminants. J Am Coil Cardiol 16:155-164(1990).

18. Dawson B, Johnson P, Goldberg S, Ulreich J. Cardiac teratogen-esis of halogenated hydrocarbons-contaminated drinking water.J Am Coil Cardiol 21:1466-1472(1993).

19. Nartosky MG, Weller EA, Chinchilli VM, Kaviock RJ.Nonadditive developmental toxicity in mixtures of trichloroeth-yiene, di-(2-ethylhexyl)phthalate and heptachlor in a 5 X 5 X 5design. Fundam Appi Toxicol 27:203-216(1995).

20. Barton HA, Ciewell HC. Non-cancer effects due to trichloroeth-yiene: pharmacokinetics and risk assessment. Environ HealthPerspect 108(suppi 2:323-334 (2000).

21. U.S. EPA. Health Assessment Document for Trichloro-ethylene-Final Report. EPA/600/8-82/006F. Washington,DC:U.S. Environmental Protection Agency, 1985.

22. Bull RJ. Mode of action of liver tumor induction by trichloro-ethylene and its metabolites, trichloroacetate and dichloroac-etate. Environ Health Perspect 108(suppi 2):241-259 (2000).

23. Lash LH. Modes of action for kidney tumorigenisis. EnvironHealth Perspect 108(suppi 2):225-240 (2000).

24. U.S. EPA. Addendum to the Health Assessment Document forTrichloroethylene: Updated Carcinogenicity Assessment forTrichloroethylene. EPA/600/8-82-006FA. Washington, DC:U.S.Environmental Protection Agency, 1987.

25. U.S. EPA. Addendum to the Health Assessment Document forTrichloroethylene. Prepared by the Office of Health andEnvironmental Assessment, Environmental Criteria andAssessment Office. EPA/600/8-82/006FA. External ReviewDraft. Research Triangle Park, NC:U.S. Environmental ProtectionAgency, 1998.

26. U.S. EPA. Letter from Science Advisory Board, HalogenatedOrganics Subcommittee, to Hon. Lee Thomas, Administrator,March 9, 1988.

27. American Conference on Governmental Industrial Hygienists.Notice of intended change - trichloroethylene. Appi OccupEnviron Hyg 7:786-791 (1992).

28. Spirtas R, Stewart RA, Lee JS, Marano DE, Forbes CD, GraumanDJ, Pettigrew HM, Blair A, Hoover RN, Cohen JL. Retrospectivecohort mortality study of workers at an aircraft maintenance faci-ity. I: Epidemiological results. Br J Ind Med 48:515-530 (1991).

29. Stewart PA, Lee JS, Marano DE, Spiritas R, Forbes CD, Blair A.Retrospective cohort mortality study of workers at an aircraftmaintenance facility. I: Exposures and their assessment. Br JInd Med 48:531-537 (1991).

30. IARC. IARC Monographs on the Evaluation of CarcinogenicRisks to Humans. Vol 63: Dry Cleaning, Some ChlorinatedSolvents and Other Industrial Chemicals. Lyon:lnternationalAgency for Research on Cancer, 1995.

31. Henschier D, Vamvakas S, Lammert M, Dekant W, Kraus B,Thomas B, Ulm K. Increased incidence of renal cell tumors in acohort of cardboard workers exposed to trichloroethene. ArchToxicol 69(5):291-299 (1995).

32. Axelson A, Sledon A, Anderson K, Hogstedt C. Updated andexpanded Swedish cohort study on trichloroethylene and cancerrisk. J Dccup Med 36(5):556-562 (1994).

33. Blair A, Hartge P. Stewart PA, McAdams M, Lubin J. Mortality

and cancer incidence of aircraft maintenance workers exposedto trichloroethylene and other organic solvents and chemicals:extended follow up. Occup Environ Med. 55(3):161-71 (1998)

34. Morgan RW, Kelsh MA, Zhao K, Heringer S. Mortality of aero-space workers exposed to trichloroethylene. Epidemiology9(4):424-431 (1998).

35. Vamvakas S, Bruening T, Thomasson B, Lammert M, BaumuellerA, Bolt HM, Dekant W, Birner G, Henschler D, Ulm K. Renal cellcancer correlated with occupational exposure to trichloroethyl-ene. J Cancer Res Clin Oncol 1247(7:374-382 (1998).

36. David NJ, Wolman R, Milne FJ, Nierkerk, I. Acute renal failuredue to trichloroethylene poisoning. Br J Ind Med 46:347-349(1989).

37. Rasmussen K, Brogen CH, Sabroe S. Subclinical affection ofliver and kidney function and solvent exposure. Int Arch OccupEnviron Health 64(6):445-448 (1993).

38. Anttila A, Pukkala E, Sallmen M, Hernberg S, Hemminki K.Cancer incidence among Finnish workers exposed to halo-genated hydrocarbons. J Environ Occup Med 37:797-806 (1995).

39. Burg J, Gist Gl. The potential impact on women from environ-mental exposures. J Women's Health 6(2):159-161 (1997).

40. Lipscomb JC, Garrett CM, Snawder JE. Use of kinetic andmechanistic data in species extrapolation of bioactivation:cytochrome P-450 dependent trichloroethylene metabolism atoccupationally relevant concentrations. J Occup Health40:110-117 (1998)

41. Vamvakas S, Elfarra AA, Dekant W, Henschler D, Anders MW.Mutagenicity of amino acid and glutathione S-conjugates in theAmes test. Mutat Res 206(1):83-90 (1988).

42. Lash, LH, Putt DA, Fisher JW, Brashear WT, Parker JC.Identification of S-(1,2-dichlorovinyl) glutathione in the blood ofhuman volunteers exposed to trichloroethylene. J ToxicolEnviron Health 56(1(:1-21 (1999).

43. Green T. Pulmonary toxicity and carcinogenicity of trichloro-ethylene: species differences and modes of action. EnvironHealth Perspect 108(suppl 2):261-264 (2000).

44. Venitt S. Mechanisms of carcinogenesis and individual suscep-tibility to cancer. Clin Chem 40:1421-1425 (1994).

45. Ommen GS. Comment: Genetics and public health. Am J PublicHealth 86:1701-1704 (1997).

46. Nebert OW. Polymorphism of human CYP2D genes involved indrug metabolism: possible relationship to individual cancer risk.Cancer Cells 3:93-96 (1991).

47. Gonzalez FJ, Idle JR. Pharmacogenetic phenotyping and geno-typing. Present status and future potential. Clin Pharmacokinet26:59-70 (1994).

48. Guengerich FP, Shimada T. Oxidation of toxic and carcinogenicchemicals by human cytochrome P-450 enzymes. Chem ResToxicol 4:391-407 (1991).

49. Poulsen E. Risk assessment for people exposed from differentenvironments. Pharmacol Toxicol 72(S1):51-54 (1993).

50. Wrighton SA, Stevens JC. The human hepatic cytochromesP450 involved in drug metabolism. Crit Rev Toxicol 22:1-21(1992).

51. Raunio H, Husgafvel-Pursiainen K, Anttila A, Hietanen E,Hirvonen A, Pelkonen 0. Diagnosis of polymorphisms in carcino-gen-activating and inactivating enzymes and cancer susceptibil-ity-a review. Gene 159:113-121 (1995).

52. Nakajima T, Wang R, Elovaara E, Park S, Harry G, Vainio H. Acomparative study on the contribution of cytochrome P450isozymes to metabolism of benzene, toluene, and trichloroethyl-ene in rat liver. Biochem Pharmacol 43:251-257 (1992).

53. Nakajima T, Wang RS, Elovaara E, Park S, Gelboin H, Vainio H.Cytochrome P450-related differences between rats and mice inthe metabolism of benzene, toluene, and trichloroethylene inliver microsomes. Biochem Pharmacol 45:1079-1085 (1993).

54. Raucy J. Risk assessment: toxicity from chemical exposureresulting from enhanced expression of CYP2E1. Toxicology105:217-223 (1995).

55. Lieber CS. Cytochrome P-4502E1: its physiological and patho-logical role. Physiol Rev 77:517-543 (1997).

56. Peter R, Bocker R, Beaune PH, Iwasaki M, Guengerich FP, YangCS. Hydroxylation of chlorzoxazone as a specific probe for humanliver cytochrome P-45011E1. Chem Res Toxicol 3:566-573 (1990).

57. Stephens EL, Taylor JA, Kaplan N, Yang CH, Hsieh IL, LucierGW, Bell DA. Ethnic variation in the CYP2E1 gene: polymor-phism analysis of 695 African-Americans, European-Americansand Taiwanese. Pharmacogenesis 4:185-192 (1994).

58. Yoo JS, Guengerich FP, Yang CS. Metabolism of -nitrosodi-alkyl-amines in human liver microsomes. Cancer Res88:1499-1505 (1988).

59. Medinsky MA, Asgharian B, Farris GM, Gonzalez FJ, SchlosserPM, Seaton MJ, Valentine JL Benzene risk assessment trans-genic animals and human variability. CIIT Activ 17:1-5)1997).

60. Lipscomb JC, Mahle DA, Brashear WT, Garrett CM. A speciescomparison of chloral hydrate metabolism in blood and liver.Biochem Biophys Res Commun 227:340-350 (1996).

61. Uematsu F, Kikuchi H, Motomiya M, Abe T, Sagami I, OhmachiT, Wakui A, Kanamura R, Wantanabe M. Association betweenRFLP of the human cytochrome P45011E1 gene and susceptibilityto lung cancer. Jpn J Cancer Res 82:254-256 (1991).

62. lngelman-Sundberg M, Alexandrie AK, Persson I, Rannug A.Genetic polymorphism of cytochromes P450 lA1, 2D6 and 2E1.Regulation and toxicological implications. Pharmacol ToxicolSuppl 64 (2):S10 (1994).

63. Persson I, Johansson I, Bergling H, Dahl ML, Seidegard J,Rylander R, Rannug A, Hogberg J, Sundberg Ml. Genetic poly-morphism of cytochrome P4502E1 in a Swedish population.Relationship to incidence of lung cancer. FEBS Lett319:207-211 (1993).

64. Kato S, Shields PG, Caporaso NE, Hoover RN, Trump BF,Sugimura H, Weston A, Harris CC. Cytochrome P4511E1 geneticpolymorphisms, racial variation, and lung cancer risk. CancerRes 52:6712-6715 (1992).

65. Hirvonen A, Husgafvel-Pursiainen K, Anttila S, Karjalainen A,Vanio H. The human CYP2E1 gene and lung cancer: Dral andRsal restriction fragment length polymorphisms in a Finnishstudy population. Carcinogenesis 14 (1):85-88 (1992).

66. Wantanabe J, Yang JP, Eguchi H, Hayashi S, lmai K, Nakachi K,Kawajiri K. An RSA/ polymorphism in the CYP2E1 gene does notaffect lung cancer risk in a Japanese population. Jpn J CancerRes 86:245-248(1995).

67. Hilidesheim A, Chen CJ, Caporaso NE. Cytochrome P4502E1genetic polymorphisms and risk of nasopharyngeal carcinoma:Results from a case-control study conducted in Taiwan. CancerEpidemiol Biomarker Prev 4(6):607-610 (1995).

68. U.S. Department of Health and Human Services. Ninth SpecialReport to the Congress: Alcohol and Health, 1997. NIHPublication No. 97-4017. Bethesda MD: National Institute ofAlcohol Abuse and Alcoholism, 1997.

69. Thomasson HR, Edenberg HJ, Crabb DW, Li XL, Jerome RE, LiTK, Wang SP, Lin YT, Lu RB, Yin SJ. Alcohol and aldehyde dehy-drogenase genotypes and alcoholism in Chinese men. Am JHum Genet48:677-681 (1991).

70. Agarwal OP, Goedde HW. Pharmacogenetics of alcohol metab-olism and alcoholism. Pharmacogenetics 2(2):48-62 (1992).

71. Edman K, Maret W. Alcohol dehydrogenase genes: restrictionfragment length polymorphisms for ADH4 (p-ADH) and ADH5 (c-ADH) and construction of haplo types among different ADHclasses. Hum Genet 90:395-401 (1992).

72. Yoshida A. Molecular genetics of human aldehyde dehydroge-nase. Pharmacology 2:139-147 (1992).

73. Yoshida A, Hsu LC, Yasunami M. Genetics of human alcohol-metabolizing enzymes. Prog Nucleic Acid Res 40:255-287(1991).

74. Morena A, Pares A, Ortiz J, Enriquez J, Pares X. Alcohol dehy-drogenase from human stomach: variability in normal mucosaand effect of age, gender, ADH3 phenotype and gastric region.Alcohol Alcohol 29(6):663-671 (1994).

75. Hobbs WR, Rail TW, Verdoorn TA. Hypnotics and sedatives:ethanol. In: The Pharmacological Basis of Therapeutics(Hardman JG, ed). New York:McGraw Hill, 1996;390.

76. Jaffe DR, Gandolfi AJ, Nagle RB. Chronic toxicity S-(trans-1,2-dichlorovinyl)-i-cysteine in mice. J AppI Toxicol 4(6):315-319(1984).

77. Bell DA, Taylor JA, Paulson DF, Robertson CN, Mohler JL,Lucier GW. Genetic risk and carcinogen exposure: a commoninherited defect of the carcinogen-metabolism gene glutathioneS-transferase Ml (GSTM1) that increases susceptibility to blad-der cancer. J NatI Cancer Inst 85(14):1159-1164 (1993).

78. Daly AK, Thomsa DJ, Cooper J, Pearson WR, Neal DE, Idle JR.Homozygous deletion of a gene for glutathione S-transferaseMl in bladder cancer. Br Med J 307:481-482 (1993).

79. Nazar-Stewart V, Motulsky AG, Eaton DL, White E, Homung SK,Leng ZT, Stapleton P, Weiss NS. The glutathione S-transferasemu polymorphism as a marker for susceptibility to lung carci-noma. Cancer Res 53(suppl 10):2313-2318 (1993).

80. Heagerty AH, Fitzgerald D, Smith A, Bowers B, Jones P, FryerAA, Zhao L, Alidersea J, Strange RC. Glutathione S-transferaseGSTM1 phenotypes and protection against cutaneous tumors.Lancet 343:266-268(1994).

81. Zhong S, Wyllie AH, Bames D, Wolf CR, Spurr NK. Relationshipbetween the GSTM1 genetic polymorphism and susceptibilityto bladder, breast and colon cancer. Carcinogenesis14(9):1821-1824 (1993).

82. Peters WH, Nagengast FM, Wobbes T. Glutathione S-trans-ferases in normal and cancerous human colon tissue.Carcinogenesis 101121:2371-2374 (1989).

212 Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000

SUSCEPTIBIUTY TO TCE

83. Lin HJ, Han CY, Bernstein DA, Hsiao W, Lin BK, Hardy S. Ethnicdistribution of the glutathione transferase Mu 1-1 (GSTM1) nullgenotype in 1473 individuals and application to bladder cancersusceptibility. Carcinogenesis 15(5):1077-1081 (1994).

84. Pemble S, Schroeder KR, Spencer SR, Meyer DJ, Hallier E, BoltHM, Ketterer B, Taylor JB. Human glutathione Stransferase 0(GSTT1): cDNA cloning and the characterization of a geneticpolymorphism. Biochem J 300(1):271-276 (1994).

85. Lee EJ, Wong JY, Yeoh PN, Gong NH. Glutathione S transferase-o (GSTT1) genetic polymorphism among Chinese, Malays andIndians in Singapore. Pharmacogenesis 5(51:332-334 (1995).

86. Nelson HH, Wiencke JK, Christiani DC, Chieng TJ, Zuo ZF,Schwartz BS, Lee BK, Spitz M, Wang M, Xu XP, et al. Ethnicdifferences in the prevalence of the homozygous deleted geno-type of glutathione S-transferase 0. Carcinogenesis16(5):1243-1245 (1995).

87. Schroder KR, Wiebel FA, Reich S, Dannappel D, Bolt HM, HallierE. Glutathione-S-transferase (GST) 0 polymorphism influencesbackground SCE rate. Arch Toxicol 69(7):505-507 (1995).

88. Wiencke JK, Pemble S, Ketterer B, Kelsey KT. Gene deletion ofglutathione-S-transferase 8: correlation with induced geneticdamage and potential role in endogenous mutagenesis. CancerEpidemiol Biomark Prev 4(3):253-259 (1995).

89. Bruning T, Lammert M, Kempkes M, Thier R, Golka K, Bolt HM.Influence of polymorphisms of GSTM1 and GSTT1 for risk ofrenal cell cancer in workers with long-term high occupationalexposure to trichloroethene. Arch Toxicol. 71(9):596-599 (1997).

90. Kelsey KT, Spitz MR, Zuo ZF, Wiencke JK. Polymorphisms inthe glutathione S-transferase class p and 0 genes interactand increase susceptibility to lung cancer in minority popula-tions (Texas, United States). Cancer Causes Control8(4):554-559 (1997).

91. Shimada T, Yamazaki H, Mumura M, lnui U, Guengerich FP.Interindividual variation in human liver cytochrome P-450enzymes involved in the oxidation of drugs, carcinogens andtoxic chemicals: studies with liver microsomes of 30 Japaneseand 30 Caucasians. J Pharmacol Exp Ther 270:414-423 (1994).

92. Zang EA, Wynder EL. Differences in lung cancer risk betweenmen and women: examination of the evidence. J NatI CancerInst 88(3-4):183-192 (1996).

93. Long W, Tate RB, Neuman M, Manfreda J, Becker AB, AnthonisenNR. Respiratory symptoms in a susceptible population due toburning of agricultural residue. Chest 113(2):351-357 (1998).

94. Xu X, Li B, Wang L. Gender difference in smoking effects onadult pulmonary function. Eur Respir J 7(3):477-483 (1994).

95. Guinee DG Jr, Travis WD, Trivers GE, De Benedetti VM, CawleyH, Welsh JA, Bennett WP, Jett J, Colby TV, Tazaelaar H, et al.Gender comparisons in human lung cancer: analysis of p53mutations, anti-p53 serum antibodies and C-ergB-2 expression.Carcinogenesis 16(5):993-1002 (1995).

96. Chen Y, Horne SL, Dosman JA. Increased susceptibility to lungdysfunction in female smokers. Am Rev Respir Dis143:1224-1230(1991).

97. Britt EJ, Shelhamer J, Menkes H, Cohen B, Meyer M, PermuttS. Sex differences in the decline of pulmonary function withage. Chest 80(1):79-80 (1981).

98. Lash LH, Xu Y, Elfarra AA, Duescher RJ, Parker JC. Glutathione-dependent metabolism of trichloroethylene in isolated liver andkidney cells of rats and its role in mitochondrial and cellulartoxicity. Drug Metab Dispos 23(8(:846-853 (1995).

99. Bois FY. Statistical analysis of Fisher et al. PBPK model oftrichloroethylene kinetics. Environ Health Perspect 108(suppi2):275-282 (2000).

100. Dawson B, Johnson P, Goldberg S, Ulreich J. Cardiac teratogen-esis of trichloroethylene and dichloroethylene in mammalianmodel. J Am Coil Cardiol 16:1304-1309 (1990).

101. Bove FJ, Fulcomer MC, Klotz JB, Esmart J, Dufficy EM, SavrinJE. Public drinking water contamination and birth outcomes.Am J Epidemiol 141:850-862 (1995).

102. Avery GB, Randolf JG, Weaver T. Gastric acidity during the firstday of life. Pediatrics 37:1005-1007 (1966).

103. Morselli PL. Clinical pharmacokinetics in neonates. ClinPharmacokinet 1:81-89 (1976).

104. Seigner E, Fridrich R. Gastric emptying in newborns and younginfants. Acta Ped Scand 64:525-530 (1975).

105. Crom WR. Pharmacokinetics in the child. Environ HealthPerspect 102:111-118(1994).

106. Guzelian PS, Henry CJ, Olin SS, eds. Similarities and DifferencesBetween Children: Implications for Risk Assessment.Washington, DC:ILSI Press, 1992;32.

107. Snodgrass WR. Physiological and biochemical differencesbetween children and adults as determinants of toxic responseto environmental pollutants. In: Similarities and DifferencesBetween Children and Adults: Implications for Risk Assessment

(Guzelian PS, Henry CJ, Olin SS, eds). Washington, DC:1lSIPress, 1992;35-42.

108. Ehrnebo M, Agurell S, Jailing B, Boreus L. Age differences indrug binding by plasma proteins: studies on human foetuses,neonates and adults. Eur J Clin Pharmacol 3:189-193 (1971).

109. Herngren L, Ehrnebo M, Borehus L0. Drug binding to plasmaproteins during human pregnancy and in the perinatal period.Dev Pharmacol Ther 6:110-124 (1983).

110. Friis-Hansen B. Body composition during growth: biochemicaldata and in vivo measurements. Pediatrics 47:264-274(1971).

111. Friis-Hansen B. Body water compartment changes in children:changes during growth and related changes in body composi-tion. Pediatrics 28:169-181 (1961).

112. Milsap RL, Hill MP, Szefler SJ. Special pharmacokinetic consid-erations in children. In: Applied Pharmacokinetics: Principles ofTherapeutics, 3rd ed. (Evans WE, Schentag JJ, Jusko WJ, eds).Spokane, WA:Applied Therapeutics, 1992.

113. Carpenter SP, Lasker, JM, Raucy, JL. Expression, induction andcatalytic activity of the ethanol-inducible cytochrome P450(CYP2E1) in the human fetal and liver hepatocytes. MolPharmacol 49(2):260-268 (1996).

114. Hakkola J, Pasanen M, Hukkanen J, Pelkonen 0, Maenpaa J,Edwards RJ, Boobis AR, Raunio H. Expression of xenobiotic-metabolizing cytochrome P450 forms in human full-term pla-centa. Biochem Pharmacol 51:403-411 (1996).

115. Hakkola J, Raunio H, Purkunen R, Pelkonen 0, Saarikoski S,Cresteil T, Pasanen M. Detection of cytochrome P450 geneexpression in human placenta in first trimester pregnancy.Biochem Pharmacol 52:379-383 (1996).

116. Hakkola J, Pasanen M, Pelkonen 0, Evisalmi S, Anttila S, RaneA, Mantyla M, Hukkanen J, Purkunen R, Saarikoski S, et al.Expression of CYP1 B1 in human adult and fetal tissues and dif-ferential inducibility of CYP1B1 and CYPlA1 by Ah receptor lig-ands in human placenta and cultured cells. Carcinogenesis18:391-397 (1997).

117. Boutelet-Bochan, Huang Y, Juchau MR. Expression of CYP2E1during embryogenesis and fetogenesis in human cephalic tis-sues: implications for the fetal alcohol syndrome. BiochemBiophys Res Commun 238:443-447 (1997).

118. Vieira I, Sonnier M, Cresteil T. Developmental expression ofCYP2E1 in the human liver. Hypermethylation control of geneexpression during neonatal period. Eur J Biochem238:476-483 (1996).

119. Jones SM, Boobis AR, Moore GE, Stanier PM. Expression ofCYP2E1 during human fetal development: methylation of theCYP2E1 gene in human fetal and adult liver. BiochemPharmacol 43:1876-1879 (1992).

120. Song B-J, Gelboin HV, Park S-S, Yang CS, Gonzales FJ.Complementary DNA and protein sequences of ethanol-inducible rat and human cytochrome P450a: transcriptional andpost-transcriptional regulation of the rat enzyme. J Biol Chem261:16689-16697 (1986).

121. Peng HM, Porter TD, Ding XX, Coon MJ. Differences in thedevelopmental expression of rabbit cytochromes P450 2E1 and2E2. Mol Pharmacol 40:58-62 (1991).

122. Routiedge PA. Pharmacokinetics in children. J AntimicrobChemother 24:19-24 (1994).

123. Pikkarainen P, Raiha N. Development of ADH activity in thehuman liver. Pediatr Res 1:165-168 (1967).

124. Reimche LD, Sankaran K, Hindmarsh KW, Kasian GF, GoreckiDK, Tan l. Chloral hydrate sedation in neonates and infants-clinical and pharmacologic considerations. Dev PharmacolTher.1 2(2):57-64 (1989).

125. Waxman DJ, Dannan GA, Guengerich FP. Regulation of rathepatic cytochrome P-450: age-dependent expression, hor-monal imprinting, and xenobiotic inducibility of sex-specificisoenzymes. Biochemistry 24(16):4409-4417 (1985).

126. Kamataki T. Purification and regulation of forms of cytochromeP-450 present specifically in liver microsomes of male andfemale rats. Yakugaku Zasshi 105(7):607-618 (1985).

127. Murphy GM, Signer E. Progress report: bile acid metabolism ininfants and children. Gut 15:151-163 (1974).

128. Schwartz GJ, Haycock GB, Edelman, CM, Spitzer A. A simpleestimate of glomerular filtration rate in children derived frombody length and plasma creatinine clearance. Pediatrics58:259-263 (1976).

129. Fisher JW, Mahle D, Bankston L, Greene R, Gearheart J.Lactational transfer of chemicals in breast milk. Am Ind HygieneAssoc J 58:425-431 (1997).

130. Fisher JW, Whittaker TA, Taylor DH, Clewell HJ, Andersen ME.Physiologically based pharmacokinetic modeling of the lactat-ing rat and nursing pup: a multiroute exposure model fortrichloroethylene and its metabolite, trichloracetic acid. ToxicolAppl Pharmacol 102:497-513 (1989).

131. Raucy J, Kraner J, Lasker J. Bioactivation of halogenatedhydrocarbons by cytochrome P4502E1. Crit Rev Toxicol23:1-20 (1993).

132. Lieber CS. Ethanol metabolism and cirrhosis and alcoholism.Clin Chem Acta 257:59-B4 (1997).

133. Hobbs WR, Rail TW, Verdoon TA. Hypnotics and sedatives;ethanol. In: The Pharmacological Basis of Therapeutics, Ninthed. New York:MCGraw Hill, 1996;377.

134. LaPort RE, Matsushima M, Chang Y. Diabetes in America. In:Diabetes in America (Harris Ml, ed). Bethesda, MD:NationalInstitutes of Diabetes and Digestive and Kidney Diseases,1995;37-46.

135. Kenny SJ, Aubert RE, Geiss LS. Diabetes in America. In:Diabetes in America (Harris Ml, ed). Bethesda, MD:NationalInstitute of Diabetes and Digestive and Kidney Diseases,1995;47-67.

136. Gist GL, Burg JR. Trichloroethylene: a review of the literaturefrom a health effects perspective. Toxicol Ind Health11:253-306(1995).

137. vonWartburg JP. The metabolism of alcohol dehydrogenase innormal and alcoholics: enzymes. In: The Biology of Alcoholism,Vol 1 (Kissin B, Begleiter, H, eds). New York:Plenum Press,1971;1-29.

138. Lieber CS. The metabolism of ethanol. In: Metabolic Aspects ofAlcoholism (Lieber CS, ed). Baltimore, MD:Baltimore UniversityPark Press, 1977;1-29.

139. Crabb DW, Bosron W, Li T. Ethanol metabolism. Pharmacol Ther34:59-73 (1987).

140. Lasker JM, Raucy J, Kubota S, Bloswick BP, Black M, Lieber CS.Purification and characterization of human liver cytochromeP450. Biochem Biophys Res Commun 148(1):232-238 (1987).

141. Inatomi N, Kato S, Ito D, Lieber CS. Role of peroxisomal fattyacid betaoxidation in ethanol metabolism. Biochem Biophys ResCommun 163:418-423(1989).

142. Nakajima T, Okino T, Okuyama S, Kaneko T, Yonekura I, Sato A.Ethanol-induced enhancement of trichloroethylene metabolismand hepatotoxicity: difference from the effect of phenobarbital.Toxicol AppI Pharmacol 94:227-237 (1988).

143. Kaneko T, Yang PY, Sato A. Enzymes induced by ethanol differ-ently affect the pharmacokinetics of trichloroethylene and1,1 ,1-trichloroethane. Occup Environ Med 51:113-119 (1994).

144. Okino T, Nakajima T, Nakano M. Morphological and biochemi-cal analyses of trichloroethylene hepatotoxicity: differences inethanol- and phenobarbital-pretreated rats. Toxicol AppIPharmacol 108:379-389 (1991).

145. Sato A, Nakajima T, Koyama Y. Interaction between ethanol andcarbohydrate on the metabolism in rat liver of aromatic and chlo-rinated hydrocarbons. Toxicol AppI Pharmacol 68:242-249 (1983).

146. Barret L, Faure J, Guilland B, Chomat D, Didier B, Debru JL.Trichloroethylene occupational exposure: elements for betterprevention. Int Arch Occup Environ Health 53(4):283-289 (1984).

147. Muller G, Spassowski M, Henschler D. Metabolism oftrichloroethylene in man. Ill: Interactions with ethanol. ArchToxicol 33:173-189 (1975).

148. Sato A, Nakajima T, Koyama Y. Dose-related effects of a singledose of ethanol on the metabolism in rat liver of some aromatic andchlorinated hydrocarbons. Toxicol AppI Pharmacol 60:8-15(1981).

149. Jakobson I, Holmberg B, Ekner A. Venous blood levels ofinhaled trichloroethylene in female rats and changes induced byinteracting agents. Acta Pharmacol Toxicol 59:135-143 (1986).

150. Kawamoto T, Hobara T, Kobayashi H, Iwamoto S, Sakai T,Takano T, Miyazaki Y. The metabolite ratio as a function ofchloral hydrate dose and intracellular redox state in the per-fused rat liver. Pharmacol Toxicol 60(5):325-329 (1987).

151. Larson J, Bull R. Effect of ethanol on the metabolism oftrichloroethylene. J Toxicol Environ Health 28:395-406 (1989).

152. Yuki T, Thurman RG. The swift increase in alcohol metabolism.Time course for the increase in hepatic oxygen uptake and theinvolvement of glycolysis. Biochem J 1869(1):119-126 (1980).

153. Sellers EM, Lang M, Koch-Weser J, LeBlanc E, Kalant H.Interaction of chloral hydrate and ethanol in man: I. Metabolism.Clin Pharmacol Ther 13:37-49 (1972).

154. Sato A, Endoch K, Kaneko T, Johanson G. Effects of consump-tion of ethanol on the biological monitoring of exposure toorganic solvent vapors: a simulation study with trichloroethyl-ene. Br J Ind Med 48:548-556(1991).

155. Fisher JW, Allen BC. Evaluating the risk of liver cancer inhumans exposed to trichloroethylene using physiological mod-els. Risk Anal 13(1):87-95 (1993).

156. Fernandez-Checa JC, Garcia-Ruiz C, Ookhtens M, Kaplowitz N.Impaired uptake of glutathione by hepatic mitochondria fromchronic ethanol-fed rats. Tracer kinetic studies in vitro and invivo and susceptibility to oxidant stress. J Clin Invest87(21:397-405 (1991).


PASTINO ET AL.

157. Garcia-Ruiz C, Morales A, Ballesta A, Rodes J, Kaplowitz N,Fernandez-Checa JC. Effect of chronic ethanol feeding on glu-tathione and functional integrity of mitochondria in periportal andperivenous rat hepatocytes. J Clin Invest 94(1):193-201 (1994).

158. Norton R, Batey R, Dwyer T, MacMahon S. Alcohol consump-tion and the risk of alcohol related cirrhosis in women. Br JMed 295:80-82 (1987).

159. Rabinowitz M, Van Theil DH, Dindzans V, Gavaler JS.Endoscopic findings in alcoholic liver disease: does gendermake a difference? Alcohol 6:465-468 (1989).

160. Urbano-Marques A, Estruch R, Fernandez-Sola J, Nichols JM,Pare, JC, Rubin MD. The greater risk of cardiomyopathy andmyopathy in women compared to men. J Am Med Assoc274:149-154 (1995).

161. Bowlin SJ, Leske MC, Varma A, Nasca P, Weinstein A, CaplanL. Breast cancer risk and alcohol consumption. Int J Epidemiol26:915-923 (1997).

162. Jones BM, Jones MR. Male and female intoxication levels forthree alcohol doses, or, do women really get higher than men?Alcohol Tech Rep 5:11-14 (1976).

163. Frezza A, DiPadova C, Pozzato G, Terpin M, Baraona E, LieberCS. High blood alcohol levels in women: role of decreased gas-tric alcohol dehydrogenase activity and first pass metabolism ofethanol. N EngI J Med 322:95-99 (1990).

164. Smith MK, Randall JL, Read EJ, Stober JA. Teratogenic activityof trichloracetic acid in the rat. Teratology 40:445-451 (1989).

165. Scheneker S. Fetal alcohol syndrome: current status of patho-genesis. Alcohol Clin Exper Res 14:635-643 (1990).

166. Wu D, Cederbaum A. Ethanol consumption by the nursingmother induces cytochrome P-4502E1 in neonatal rat liver. JPharmacol Exp Ther 267:560-567 (1993).

167. Chhabra SK, Perella C, Anderson LM. Induction of hepatic andrenal P4502E1 of neonatal rats exposed translactationally toethanol. Food Chem Toxicol 34(5):469-476 (1996).

168. Binkiewicz AR, Senior B. Pseudo-Cushing syndrome caused byalcohol in breast milk. J Pediat 93:965-967 (1978).

169. Rasheed A, Hines R, McCarver-May D. Variation in induction ofhuman placental CYP2E1: possible role in susceptibility to fetalalcohol syndrome? Toxicol AppI Pharmacol 144:396-400 (1997).

170. Carpenter SP, Lasker JM, Rauch JL. Expression, induction andcatalytic activity of ethanol-inducible cytochrome P450(CYP2E1) in human fetal liver and hepatocytes. Mol Pharmacol49: 260-268 (1996).

171. Abel EL. An update on incidence of FAS: FAS is not an equal oppor-tunity birth defect. Neurotoxicol Teratol 17(4):437-443 (1995).

172. Department of Health and Human Services. Over-the-counterdrug products containing analgesic/antipyretic active ingredi-ents for internal use. Required alcohol warning. (Food and DrugAdministration, ed). Fed Reg 62:61041-61057 (1997).

173. Gonzalez-Leon A, Schultz IR, Bull RJ. Pharmacokinetics andmetabolism of dichloroacetic acid in the F344 rat after pro-longed administration in drinking water. Toxicol Appi Pharmacol146:189-195 (1997).

174. Nakajima T, Wang RS. Induction of cytochrome P450 bytoluene. lnt J Biochem 26(12):1333-1340 (1994).

175. Chambers HF, Sande MA. Antimicrobial agents. In: Goodmanand Gilman's the Pharmacological Basis of Therapeutics, Ninthed. New York:McGraw-Hill, 1996;1029-1056.

176. Adami HO, Chow WH, Nyrene 0, Berne C, Linet MS, Ekbom A,Wolk A, McLaughlin JK, Fraumeni JF. Excess risk of primaryliver cancer in patients with diabetes melilitus. J Nati CancerInst 88:1472-1477 (1996).

177. LaVecchia C, Negri E, Franceschi S, D'Avanzo B, Boyle P. A case-control study of diabetes mellitus and cancer risk. Br J Cancer70:950-953(1994).

178. Wideroff L, Gridley G, Mellemkjaer 1, Chow HH, Linet M, KeehnS, Borch-Johnson K, Olsen JH. Cancer incidence in a popula-tion-based cohort of patients hospitalized with diabetes melli-tus in Denmark. J NatI Cancer Inst 89:1360-1365 (1997).

179. Yu MC, Tong MJ, Govindarajan S, Henderson BE. Nonviral riskfactors for hepatocellular carcinoma in a low-risk population,the non-Asians of Los Angeles County, California. J NatI CancerInst 83:1820-1826 (1991).

180. Stacpoole PW. The pharmacology of dichloroacetate.Metabolism 38(1 1):1124-1144 (1989).

181. Whitehouse S, Cooper RH, Randle PJ. Mechanism of activationof pyruvate dehydrogenase by dichloroacetate and other halo-genated carboxylic acids. Biochem J 141(3):761-774 (1974).

182. Henderson GN, Curry SH, Derendorf H, Wright EC, StacpoolePW. Pharmacokinetics of dichloroacetate in adult patients withlactic acidosis. J Clin Pharmacol 37(5):416-425 (1997).

183. Fisher JW, Mahle D, Abbas R. A human physiologically basedpharmacokinetic model for trichloroethylene and its metabo-lites, trichloroacetic acid and free trichloroethanol. Toxicol AppIPharmacol 152:339-359 (1998).

184. Barnes DG, Dourson M. Reference dose (RfD): description anduse in health risk assessment. Regul Toxicol Pharmacol8(4):471-486 (1988).

185. Khoury MJ, Genetics Working Group. From genes to publichealth: the applications of genetic technology in disease pre-vention. Am J Public Health 86:1717-1722 (1997).

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