environmental health perspectives

Trichloroethylene (TCE), a common metaldegreasing solvent, is considered to be themajor component in more than half the U.S.Environmental Protection Agency hazardouswaste sites (Fay and Mumtaz 1996). Althoughrodent bioassay studies have established thatexposure to TCE is associated with develop-ment of neoplasia in a variety of organ systems(Davidson and Beliles 1991), epidemiologicstudies have not provided support for analo-gous susceptibility of humans (Lewandowskiand Rhomber 2005; Wartenburg et al. 2000).Efforts to redefine the risk of TCE-inducedcarcinogenicity have been driven by the abilityof this solvent to induce hepatocellular carci-noma in the B6C3F1 mouse strain (Herren-Freund et al. 1987). It is now well acceptedthat hepatocarcinogenicity is caused by ametabolite, trichloroacetate (TCA), although acontribution by its dichloroacetate (DCA) ana-log cannot be discounted at this time (Bull2000; Bull et al. 2002).

TCE is metabolized in the liver (Figure 1),predominately by cytochrome P450 enzymeCYP2E1 (Lipscomb et al. 1997; Nakajimaet al. 1992), to chloral hydrate (CH). In turn,CH is metabolized by either of two pathways:oxidation to TCA catalyzed by aldehyde

dehydrogenase (ALDH), or reduction to anoncarcinogenic metabolite, trichloroethanol(TCOH), mediated by alcohol dehydrogenase(ADH). Although the conversion of CH toTCOH is considered to be reversible, theextent to which this back-reaction contributesto TCA pools in vivo is unclear. In the main,the distribution of CH into its carcinogenic(TCA) and noncarcinogenic (TCOH) metabo-lites appears to depend on the relative activityof the two pathways.

Animals and humans appear to handleTCE in regard to absorption, distribution,metabolism, and elimination in a qualitativelysimilar fashion. It is thus attractive to usephysiologically based pharmacokinetic(PBPK) models for dose and species extrapola-tion. Several models have been developed forthis purpose using kinetic parameters (e.g., Kmand Vmax) derived from both human and ani-mal studies (Clewell and Andersen 2004;Fisher 2000).

It is well known that both ADH andALDH are polymorphic in humans and thatthe isoforms may differ markedly in theirsteady-state kinetic constants (Li et al. 2001).The human hepatic ADH concerned with themetabolism of ethanol is designated as class I

and is encoded by three genes, ADH1, ADH2,and ADH3, which code for peptides α, β, andγ (Ehrig et al. 1990; Xu et al. 1988; Yin et al.1984). Because the active enzyme is a dimer,human hepatic ADH may occur as any of 21possible forms depending on the individual’sgenotype. Among Caucasians, β1 predomi-nates (80–95%) over β2 (4–20%). Asians showa reverse pattern (β1 ~ 30% and β2 ~ 70%).About 25% of African Americans express theβ3 form of ADH in their livers. Of importancefor the present study, the kinetic parametersVmax and Km toward ethanol vary considerablyamong the isomeric forms, with a resultantrange of about a 50-fold difference in theirfirst-order rate constants (Vmax/Km). At ethanolblood levels associated with intoxication(10–20 mM), the rate of elimination ofethanol in the liver appears to be most closelyassociated with the Vmax of the individual’sALDH isoform and with the rate of regenera-tion of the essential cofactor NAD+ from itsreaction product, NADH. Regarding CH, thesituation is less clear. At the low levels of TCEin drinking water, the steady-state productionof CH may be very low, with correspondinglow levels of CH in the liver. Theoretically,the first-order rate constant for CH metabo-lism would be the prime determinant ofelimination by this pathway.

ALDH is also polymorphic (Ehrig et al.1990). For acetaldehyde metabolism, ALDH1(cytosolic) and ALDH2 (mitochondrial)appear to be the most important, with themitochondrial form contributing most of theclearance. An aberrant (inactive) form ofALDH2 occurs in many individuals of Asiandescent and has been considered to be respon-sible for their lesser ability to metabolizeacetaldehyde generated from ethanol by ADH.ALDH is active as a tetramer, and the presenceof even one inactive monomer in the tetramer

Environmental Health Perspectives • VOLUME 114 | NUMBER 8 | August 2006 1237

Research

Address correspondence to D.G. Hoel, Department ofBiostatistics, Bioinformatics and Epidemiology,Medical University of South Carolina, 135 CannonSt., Suite 303, P.O. Box 250835, Charleston, SC29425 USA. Telephone: (843) 876-1109. Fax: (843)876-1126. E-mail: [email protected]

We thank J. Schulte for her technical assistance in thepreparation of the manuscript.

This work was supported by a U.S. Department ofEnergy cooperative agreement (DE-FC02-98CH1092).

The authors declare they have no competing financialinterests.

Received 27 January 2006; accepted 30 May 2006.

Application of Cryopreserved Human Hepatocytes in TrichloroethyleneRisk Assessment: Relative Disposition of Chloral Hydrate to Trichloroacetateand Trichloroethanol

Apryl Bronley-DeLancey,1 David C. McMillan,2 JoEllyn M. McMillan,2 David J. Jollow,2 Lawrence C. Mohr,1,3

and David G. Hoel1

1Department of Biostatistics, Bioinformatics and Epidemiology, 2Department of Cell and Molecular Pharmacology, and 3Department of Medicine, Medical University of South Carolina, Charleston, South Carolina, USA

BACKGROUND: Trichloroethylene (TCE) is a suspected human carcinogen and a common ground-water contaminant. Chloral hydrate (CH) is the major metabolite of TCE formed in the liver bycytochrome P450 2E1. CH is metabolized to the hepatocarcinogen trichloroacetate (TCA) by alde-hyde dehydrogenase (ALDH) and to the noncarcinogenic metabolite trichloroethanol (TCOH) byalcohol dehydrogenase (ADH). ALDH and ADH are polymorphic in humans, and these polymor-phisms are known to affect the elimination of ethanol. It is therefore possible that polymorphismsin CH metabolism will yield subpopulations with greater than expected TCA formation with asso-ciated enhanced risk of liver tumors after TCE exposure.

METHODS: The present studies were undertaken to determine the feasibility of using commerciallyavailable, cryogenically preserved human hepatocytes to determine simultaneously the kinetics ofCH metabolism and ALDH/ADH genotype. Thirteen human hepatocyte samples were examined.Linear reciprocal plots were obtained for 11 ADH and 12 ALDH determinations.

RESULTS: There was large interindividual variation in the Vmax values for both TCOH and TCA for-mation. Within this limited sample size, no correlation with ADH/ALDH genotype was apparent.Despite the large variation in Vmax values among individuals, disposition of CH into the two compet-ing pathways was relatively constant.

CONCLUSIONS: These data support the use of cryopreserved human hepatocytes as an experimentalsystem to generate metabolic and genomic information for incorporation into TCE cancer riskassessment models. The data are discussed with regard to cellular factors, other than genotype, thatmay contribute to the observed variability in metabolism of CH in human liver.

KEY WORDS: alcohol dehydrogenase, aldehyde dehydrogenase, chloral hydrate, genetic variability,human hepatocytes, metabolism, risk assessment, trichloroacetate, trichloroethylene. EnvironHealth Perspect 114:1237–1242 (2006). doi:10.1289/ehp.9047 available via http://dx.doi.org/[Online 30 May 2006]

significantly impairs acetaldehyde eliminationand results in a “flushing” reaction and nauseain these individuals after ethanol ingestion.

Collectively, consideration of the knownpolymorphisms of ADH and ALDH inhumans raises the possibility of significant vari-ation in the contribution of the two pathways(conversion to TCOH by ADH or to TCA byALDH) in the elimination of CH formed inthe liver from TCE ingested in drinking water.Because hepatocarcinogenicity is considered tobe related to TCA levels in the liver, this varia-tion could contribute significantly to relativesusceptibility among exposed humans.

The question of relative distribution of CHto TCOH and TCA was examined byLipscomb et al. (1996) using 700 × g super-natant fractions of homogenized livers fromhumans, mice, and rats. They reported that inall three species, TCOH was the majormetabolite when concentrations of CH werebelow 1 mM. However, these studies were per-formed by incubating CH/liver homogenateseparately with either NAD+ (for TCA forma-tion) or NADH (for TCOH formation) atoptimal concentrations (0.9 mM) of nucleo-tide. As discussed below, these experimentalconditions may not reflect the environment ofthe intact hepatocyte, so it is unclear whetherkinetic constants obtained in vitro are predictiveof the in vivo situation.

Cryopreserved human hepatocytes are nowreadily available from commercial sources andhence offer the possibility of rapid assessment ofa large number of individuals with varyinggenotypes. The present studies were undertakento determine whether cryopreserved hepato-cytes could be used to examine the distributionof CH into its carcinogenic and noncarcino-genic metabolites and, if so, whether the activi-ties of each pathway could be correlated withADH and ALDH genotypes. The data indicatethat cryopreserved hepatocytes readily metabo-lized CH and that the data were amenable toLineweaver-Burke kinetic analysis. Although theindividual samples showed major differences inactivity for both pathways, the ratio of oxida-tion to reduction was relatively constant. Inview of the relatively small number of humansamples (i.e., 13) in these initial studies, no cor-relation could be made between enzymaticactivities and ADH/ALDH genotype. We alsodiscuss the possibility that factor(s) other thanADH/ALDH genotype may influence activityat low substrate concentrations.

Materials and Methods

Chemicals and materials. Cryopreserved humanhepatocytes were purchased from InVitroTechnologies (Baltimore, MD) and ZenBio(Research Triangle, NC). The cells were storedin liquid N2 until use. InVitroGRO HI incuba-tion medium and Torpedo antibiotic mix werepurchased from InVitro Technologies. CH,

TCA, DCA, TCOH, RNase A, and ethidiumbromide were obtained from Sigma ChemicalCo. (St. Louis, MO). The DNeasy tissue kitwas obtained from Qiagen Inc. (Valencia, CA).Eppendorf hot master mix was obtained fromFisher Scientific (Freehold, NJ). Bovine serumalbumin (BSA) was purchased from PromegaScientific (Madison, WI). ALDH and ADHprimers and restriction enzymes were obtainedfrom Integrated DNA Technologies (Coralville,IA). Ten percent TBE (Tris-borate-EDTA)Novex gels and TBE-running buffer were pur-chased from Invitrogen (Carlsbad, CA). Allother reagents were analytical grade and pur-chased from commercial sources.

Metabolism studies. Just before use thehepatocytes were thawed according to the sup-pliers’ instructions and counted; cell viabilitywas determined by trypan-blue exclusion.Hepatocytes were diluted to a concentration of1 × 106 cells/mL with InVitro Technologies HIincubation medium containing Torpedoantibiotic mix and transferred to reaction vialsin 0.5-mL aliquots. CH (0.06–2.5 mM) wasadded in a small volume of incubation mediumto each vial. The cells were incubated with CHfor 10 min at 37°C with gentle shaking. Afterthe incubation period, three 10-µL aliquotswere withdrawn from each vial and placed in a20-mL gas chromatography (GC) vial contain-ing 200 µL esterizer (H2O/H2SO4/methanol,6:5:1) to enable volatilization of the acetates.The esterized samples were analyzed by gaschromatography (GC) with electron-capturedetection for the presence of CH metabolites.

The reproducibility of this experimentalmethod was assessed in liver homogenates(700 × g supernatant) prepared from anuntreated male Sprague-Dawley rat (five sepa-rate suspensions of the same liver) anduntreated male B6C3F1 mice (four suspensionsfrom four individual male mice of the sameage). Excision of livers from anesthetized ani-mals was carried out in accordance with the

Guide for the Care and Use of LaboratoryAnimals (Institute of Laboratory AnimalResources 1996) guidelines as adopted by theNational Institutes of Health. Frozen (–80°C)pieces of these livers were thawed and homoge-nized in 4 vol of homogenization buffer(250 mM sucrose, 25 mM KCl, 1 mM dithio-threitol, 0.5 mM EDTA, 10 mM HEPES,20% glycerol) and centrifuged at 700 × g for10 min at 4°C. The supernatant was removedand kept on ice until use. Protein content ofthe supernatant was determined as describedpreviously (Smith et al. 1985). NAD+, NADP+

(0.9 mM), and various concentrations of CHwere added to 0.5 mL of the supernatant. Thereactions were carried out at 37°C for 10 min.Aliquots of each reaction mixture (10 µL) wereprepared for GC analysis as described above forhuman hepatocytes. In each case, the experi-mental variability did not exceed 10%.

GC analysis of CH metabolites was per-formed essentially as described previously(Muralidhara and Bruckner 1999). Sampleswere analyzed on a PerkinElmer AutosystemXL gas chromatograph fitted with a headspaceautosampler and an electron-capture detector(PerkinElmer, Inc., Wellesley, MA). Metabo-lites were resolved using a 10 inch × 1/8 inchouter-diameter stainless-steel column packedwith 10% OV-17 on Supelcoport (Supelco,Bellefonte, PA). The temperatures for analyseswere as follows: the column was run isothermalat 150°C, injector at 200°C, transfer line at120°C, and detector at 360°C. Nitrogen wasused as the carrier gas at 60 mL/min with aheadspace pressure of 20 psi. The samples wereheated at 110°C in the autosampler chamberfor 30 min before injection into the GC; runtime was 8 min. The metabolites were quanti-fied against a standard curve using authenticDCA, TCOH, and TCA (10–1,000 ng).

ADH and ALDH genotyping. Determina-tion of ADH and ALDH genotype was per-formed on the same cryopreserved hepatocytes

Bronley-DeLancey et al.

1238 VOLUME 114 | NUMBER 8 | August 2006 • Environmental Health Perspectives

Figure 1. Metabolism of TCE to CH and subsequent disposition into its carcinogenic (TCA) and noncarcinogenic(TCOH) pathways.

CI CI

CI

CI

CI

CI

CI

CI

CI

CI

CI

CIC C C C

C C

C C

HH

OH

OH

OH

OH

H

H

O

TCECH

TCA

TCOH

ADH

ALDH

NADH

NADH

NAD+

NAD+

CYP2E1 TCE–CYPintermediate

that were used for the metabolism studies.Detection of ADH2, ADH3, and ALDH2 wasperformed using previously described methods[Nakamura et al. 1993; human alcohol dehy-drogenase (ADH; alcohol:NAD+ oxidoreduc-tase, EC 1.1.1.1), UniGene accession no.P07327 (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=unigene);human (mitochondrial) aldehyde dehydroge-nase (ALDH2), EC 1.2.1.3, UniGene accessionno. P05091; and human (cytosolic) aldehydedehydrogenase (ALDH1), EC 1.2.1.3, UniGeneaccession no. P00352]. DNA was isolated fromapproximately 5 × 105 cells using a QiagenDNeasy Tissue Kit with the RNase A step.Polymerase chain reaction (PCR) amplificationof the ADH and ALDH alleles was performedusing the primers and restriction enzymes listedin Table 1. Primers were constructed basedupon previously reported sequences (Burnellet al. 1989; Ehrig et al. 1990; Mulligan et al.2003; Osier et al. 1999; Yin et al. 1984). Eachreaction mixture contained 20 µL Eppendorfhot master mix, 1 µL of 5 µM forward primer,1 µL of 5 µM reverse primer, 17 µL sterilewater, and 10 µL isolated DNA template. PCRwas carried out using the following conditions:4 min at 95°C, 1 min at 95°C, 1 min at 55°C,1 min at 72°C, second and fourth steps cycled34 times, 30 min at 72°C, and hold at 4°C.

The PCR products were isolated from thewhole DNA by restriction enzyme digest.Briefly, to the 20 µL PCR reaction mixture weadded 5.5 µL H2O (RNase, DNase free), 3 µLbuffer, 1 µL restriction enzyme, and 0.5 µLBSA. The reaction was carried out at 37°Cfor 2–3 hr. The digest product (10 µL) was

separated by gel electrophoresis using a 10%TBE Novex gel and visualized with ethidiumbromide. The gel was run for 20 min at 200 Vto obtain optimal PCR fragment separation. A100-bp DNA ladder was run concurrently withthe samples for determination of productmolecular weight. The gels were photographedfor analysis using Biorad Quantity OneChemDoc software (BioRad, Hercules, CA).

Statistical analysis. Data were analyzedusing Stata software (version 8.2; StataCorp LP,College Station, TX), and robust regressionswere performed to remove outliers. The “rreg”command was used to fit the regression model;the final kinetic values were determined usingthis method. SAS software (version 9.0; SASInstitute Inc., Cary, NC) was used to determinethe reproducibility of the assay procedure thatemployed the mouse and rat liver homogenates.

Results

CH metabolism by liver homogenates: speciescomparison. Appreciable species-related dif-ferences have been reported for the kineticconstants of CH metabolism into TCOH andTCA by liver homogenates (Lipscomb et al.1996). To validate our experimental assay pro-cedure, we examined the capacity of rat andmouse liver homogenates to metabolize CH toTCOH and TCA. The kinetic constants (Kmand Vmax) obtained for the conversion of CHto TCOH (Table 2) were in the 100- to400-µM range and were similar to valuesreported previously.

The Km values obtained for TCA formationwere in the 100- to 200-µM range and compa-rable to those seen for TCOH formation by the

same liver fraction. Although these values arenotably lower than those observed by Lipscombet al. (1996), it is of interest that the calculatedfirst-order rate constants (Vmax/Km) for TCAformation showed the same species rank order:mouse > human > rat.

CH metabolism in isolated human hepa-tocytes. Because metabolism of CH to TCAand TCOH by liver homogenates requiresaddition of NAD+ and NADH, respectively,the activity of each pathway must be examinedin separate incubations. To avoid the compli-cation introduced by the different reactionconditions in the assessment of the distribu-tion of CH formed in the liver after ingestionof TCE, we examined the suitability of iso-lated human hepatocyte preparations for thispurpose. Cryogenically preserved humanhepatocytes (~ 10 × 106 cells/sample) wereobtained commercially, thawed, and equili-brated in the recovery buffer as recommendedby the supplier. Cell viability was then deter-mined, and the cells were resuspended to givea final density of 1 × 106 viable cells/mL ofincubation mixture.

Preliminary studies established that themetabolism of CH in the hepatocyte suspen-sions was linear with time for 10 min at 37°Cover a concentration range of 0.05–2.0 mM.The double-reciprocal plot of the simultaneousmetabolism of CH to TCOH and TCA by onehuman hepatocyte sample (CHD) is illustratedin Figure 2.

Table 3 lists the demographic and lifestylecharacteristics of the donors of the 13 humanhepatocyte samples supplied by the vendors.Nonlinear kinetics was observed in the forma-tion of TCOH in two individuals (HL7 andHL8), and for TCA formation in one individ-ual (HL6). The reason for nonlinear kinetics inthese samples is unknown, although this phe-nomenon has been described previously usingethanol as a substrate (Dalziel and Dickinson1966). The apparent kinetic constants for theother individual hepatocyte samples and theirgenotypic forms of ADH and ALDH areshown in Table 4. The means of these values

Human hepatocytes in TCE risk assessment


Table 1. Primer sequences and restriction enzymes used in ALDH and ADH genotyping.

Isoform (primer ID) Primer sequence Restriction enzyme

ALDH2 (YC3) 5´-TTG GTG GCT AGA AGA TGT C-3´ MboIIALDH2 (YC4) 5´-CCA CAC TCA CAG TTT TCT CTT-3´ MboIIADH2 (A2F) 5´-ATT CTA AAT TGT TTA ATT CAA GAA G-3´ MsIIADH2 (A2R) 5´-ACT AAC ACA GAA TTA CTG GAC-3´ MsIIADH2 (424) 5´-TGG ACT CTC ACA ACA AGC ATG GT-3´ AluIADH2 (290) 5´-TTT CTT TGG AAA GCC CCC AT-3´ AluIADH2 (352) 5´-TCT TTC CTA TTG CAG TAG C-3´ AluIADH3 (321) 5´-GCT TTA AGA GTA AAT ATT CTG TCC CC-3´ SspIADH3 (351) 5´-AAT CTA CCT CTT TCC GAA GC-3´ SspI

Table 2. Kinetics of TCOH and TCA formation from CH in 700 × g supernatants obtained from present datacompared with data in the literature.

Present data Lipscomb et al. (1996) dataVmax Km Vmax Km

TCOHRat 9.1 0.37 24.3 0.52Mouse

High affinity 8.9 0.11 6.3a 0.12Low affinity 6.1 0.51

Human ND ND 34.7 1.34TCA

Rat 2.5 0.20 4.0 16.41Mouse 8.9 0.10 10.6 3.50Human 3.9 0.17 65.2 23.90

ND, not determined. Vmax is expressed as nmol/min/mg 700 × g supernatant protein. Km is expressed as mM CH. aInhibited above 0.57 mM CH.

Figure 2. Lineweaver-Burke plot of the raw datapoints (n = 3) for TCOH and TCA formation in a sus-pension of one human hepatocyte sample (CHD).

–20 –15 –10 –5 50 10 15 201/S (mM)

4.5

4.0

3.5

3.0

2.5

2.0

1.5

1.0

1/V

(nm

ol/m

in/1

06 cel

ls) TCOH

TCA

(Km of 0.06 ± 0.18 and 0.12 ± 0.03 mM, andVmax of 75.3 ± 38.4 and 53.9 ± 26.2nmol/min/mg, respectively, for TCOH andTCA formation) are generally similar to thosepreviously reported for the 700 × g fraction ofhuman liver homogenates (Lipscomb et al.1996). Of note, however, is the wide variationin the derived Km and Vmax values among indi-viduals for both TCOH and TCA formation.

Because the two pathways may be regardedas competing for CH, it was of interest to com-pare the relative activity of the two pathways inthe various individuals. Figure 3 shows a plotof the Vmax for TCOH formation against theVmax for TCA formation for each of the hepa-tocyte preparations. The values appear to clus-ter along the diagonal of the graph, indicatingthat increase in one activity is matched byincrease in the other. This relationship suggeststhat, although the activity of each pathwayvaries significantly among individuals, the dis-position of CH into the two metabolites isconstant. This relationship was further exam-ined by calculation of the first-order rate con-stants (Vmax/Km) for the two enzymaticpathways. Figure 4 shows the plot of the first-order rate constants for TCOH versus TCA.Although less striking than those for the Vmaxplots, there appears to be a similar tendency tocluster along the diagonal, again indicating that

the disposition of CH into the two pathwaysamong the individual samples is relatively con-stant regardless of the large variation in activityof the two individual pathways.

Discussion

It is now well appreciated that humans varyconsiderably in susceptibility to environmentaltoxicants and that this variability is of majorconcern in assessing the risk posed by hazardouschemicals for human health (Aldridge et al.2003). The factors determining variabilityinclude genetics, age, sex and disease state, bothalone and in combination. The complexity ofthe situation is further compounded by theneed to extrapolate from high-dose levels usedin rodent bioassays to chronic low-dose levelstypical of drinking water exposure in humans.Currently, uncertainty factors may be used toaccount for human variability, and “average”kinetic values are used in PBPK models fordose extrapolation. Clearly, our ability toaccount for human variability in response tohazardous chemicals would be greatly improvedif arbitrary uncertainty factors could be replacedby quantitative measurement and the “average”kinetic constants by values appropriate to thevarious populations at risk (Gentry et al. 2002).

Obtaining kinetic data from prospectiveclinical studies to encompass the range of

human genetic variability clearly presentsmajor difficulties. The growing availability ofcryogenically preserved human hepatocytescould partially solve this dilemma in that itmay be practical to perform kinetic and geneticanalyses on a large number of donors in a rapidand cost-effective manner. The present studieswere undertaken to determine the feasibility ofusing cryopreserved cells as a sampling sourceto probe the relationship between genotypeand metabolic disposition into toxic versusnontoxic pathways of metabolism. CH waschosen for this purpose because a) it is aknown intermediate in the pathway that gener-ates a carcinogenic metabolite (TCA) from anenvironmental contaminant (TCE); b) it iscleared competitively by two pathways, oneleading to TCA and the other to the non-carcinogenic metabolite TCOH; and c) theenzymes concerned with its clearance (ALDH/ADH) are polymorphic in humans. Becausethe objective was to determine if the cells couldbe used immediately after thawing, no recoveryor passage procedures were used beyond thesuppliers’ instructions.

Experimentally, 13 samples of cryopre-served human hepatocytes were obtained com-mercially, thawed and reconstituted as directed,and incubated with various concentrations ofCH. Metabolite formation was quantitated andused to generate the kinetic constants Km andVmax (Figure 2). These cells were also used todetermine ADH and ALDH genotypes.

As shown in Table 4, nine of the sampleshad the ADH2β1β1 genotype for ADH, andfour were ADH2 β1β2. Nine samples wereADH3γ1γ1 and four were ADH3γ1γ2. Inregard to metabolism, two samples (HL7 andHL8) yielded nonlinear kinetics for TCOHformation and were excluded from calcula-tion. The basis for the aberrant kinetic behav-ior is unknown. The remaining values gavemean Km and Vmax values of 0.06 ± 0.018mM and 75.3 ± 38.4 nmol/min/106 cells,



Figure 3. Plot of Vmax versus Vmax for ADH andALDH activities in human hepatocyte suspensionsgiven CH. Vmax values were determined from linearregression analysis of the raw data points.

0.1 1 10

Vmax TCOH

100 1,000

EJR

KTG

AOK

IOE

HL10

CHD

CEC

ZAG

DAD

HL12

1,000

100

10

1

0.1

V max

TCA

Table 3. Donor characteristics of cryopreserved human hepatocytes.

Cellsa Donor age (years) Sex Race Tobacco use Alcohol use Substance abuse

AOK 47 M C Y N NCEC 48 M C Y Y YDAD 62 M C Y Y NIOE 79 M C Y Y NKTG 59 M C Y Y NZAG 59 M C Y Y NCHD 72 F AA N N NEJR 56 F C N N NHL10 42 M AA Y Y YHL6 48 F C N N NHL7 55 F C N N NHL8 56 M C Y Y NHL12 0.03 F C N N N

Abbreviations: AA, African American; C, Caucasian; F, female; M, male; N, no; Y, yes. aViability of hepatocytes when thawed was 84.6 ± 2.6% (mean ± SE).

Table 4. Kinetics of TCOH and TCA formation in relation to ADH and ALDH genotypes.

TCOH TCADonor Km Vmax ALDH2 ALDH3 Km Vmax ALDH2

HL12 0.043 0.590 β1β1 γ2γ2 0.005 1.200 2*1CHD 0.829 2.360 β1β1 γ1γ2 0.040 1.220 2*1KTG 0.700 33.330 β1β1 γ1γ1 1.130 4.150 2*1HL10 2.440 12.070 β1β1 γ1γ1 0.240 4.350 2*1CEC 0.093 4.420 β1β1 γ1γ1 0.294 4.590 2*1IOE 0.270 6.990 β1β1 γ1γ1 0.410 6.760 2*1HL6 0.001 0.540 β1β1 γ1γ2 ND ND 2*1HL7 ND ND β1β1 γ1γ1 0.013 0.140 2*1AOK 0.160 3.340 β1β1 γ1γ1 1.270 16.940 NonreactingHL8 ND ND β1β2 γ1γ1 0.329 0.410 2*1DAD 0.130 1.450 β1β2 γ1γ1 0.220 0.710 2*1ZAG 0.022 2.220 β1β2 γ1γ1 0.142 2.280 2*1EJR 0.027 222.220 β1β2 γ1γ1 0.009 158.730 NonreactingMean ± SE 0.060 ± 0.018 75.30 ± 38.36 0.124 ± 0.031 53.91 ± 26.21

ND, not determined. Km is expressed as mM CH; Vmax is expressed as nmol/min/mg cell protein.

Human hepatocytes in TCE risk assessment


respectively. In view of the limited representa-tion of β2 and γ2 forms of the isoforms withinthe samples, no conclusions could be drawn asto the influence of genotype on susceptibilityto TCE hepatocarcinogenicity.

A similar situation was seen with theALDH kinetic parameters. All but two of thesamples had the same genotype (ALDH2 *1 ).The two samples that were not ALDH2 *1 weretermed “nonreacting”; that is, the phenotypewas not that of the inactive form associatedwith acetaldehyde intolerance. An additionalsample showed nonlinear kinetics. Within the series, the mean values for Km and Vmaxwere 0.12 ± 0.03 mM and 53.9 ± 26.2nmol/min/106 cells, respectively.

The most striking aspect of these results isthe large variation among the individual sam-ples regardless of genotype. For both ADHand ALDH, the Vmax values showed morethan a 10-fold variation, raising the possibilitythat the uncertainty factor of 10 commonlyused to assess human variability may be anunderestimation.

However, as illustrated in Figure 1, pro-duction of TCA from CH depends on theratio of the activities of both ADH and ALDHrather than on ALDH alone. Accordingly, theVmax values for each pathway were plottedagainst each other for each individual sample(Figure 3). Although there was a significantdifference in the activities of the pathwaysamong the samples, it is noteworthy that theirratios fall reasonably close to the diagonal. Ofinterest, the sample EJR, which was an outlierfor both pathways, also showed a ratio thatapproximated unity. Although not as striking,a similar relationship could be observed whenthe first-order rate constants (Vmax/Km) for thetwo pathways were plotted against each other(Figure 4). Collectively, these data suggest thatin spite of large differences in the activities ofthe individual pathways, the distribution ofCH into its noncarcinogenic and carcinogenicmetabolites may be relatively constant.

The reason for the large variability in ADHand ALDH activity among these samples is notknown. However, a plausible explanation maylie in the steady-state kinetics of CH elimina-tion. It has long been known that ADH-catalyzed oxidation of ethanol/reduction ofacetaldehyde is an equilibrium reaction inwhich the direction and rate depend on boththe concentration of substrate (alcohol or alde-hyde) and the concentration and form of thepyridine cofactor (i.e., NAD+ or NADH). Thereaction has been described in terms of eightkinetic constants (Dalziel and Dickinson1966). Although the reaction yields linear dou-ble-reciprocal plots and is hence amenable toLineweaver-Burke analysis, the kinetic parame-ters derived are simplifications of the overallTheorell-Chance mechanism (Theorell andChance 1951). Special cases exist where the rec-iprocal plots are not linear. Of importance forthe present studies, the reaction appears to beordered with addition of pyridine nucleotidepreceding ethanol and the release of NADHbeing the rate-limiting step (Ehrig et al. 1990).Thus, the availability and form of the cofactorcan explain differences in the Vmax of the reac-tion. Differences in Km values are less wellunderstood but may reflect inclusion of acatalytic constant (Kcat) of ethanol oxidation/acetaldehyde reduction in experimentallyobserved values.

For the present studies, a simplified schemaappears adequate to explain most of theobserved activities and the apparent constantratio between the two pathways. As shown inFigure 5, reduction of CH by the NADH/ADH complex yields TCOH with subsequentrelease of NAD+. The NAD+ would then beavailable to react with ALDH, and the complexwould oxidize CH to TCA with subsequentrelease of NADH. Thus, the reduction and oxi-dation of CH by ADH and ALDH would beindependent of the initial NAD+:NADH ratio.This interdependence may normalize the ratioof oxidation and reduction, thus yielding theobserved approximate linear correlationbetween the two pathways. This proposedmechanism implies that there is no kinetic bar-rier for pyridine nucleotide exchange between

the two enzymatic pathways, suggesting thatboth enzymes are located in the same cellularcompartment. This situation would be satisfiedif the ALDH used for CH oxidation was thecytosolic isoform (ALDH1) rather than themitochondrial isoform (ALDH2), which isbelieved to be more important for the elimina-tion of acetaldehyde generated from ethanol.Future studies are needed to probe the role ofcellular pyridine nucleotide levels in the appar-ent Km and Vmax values and to distinguish thecontribution of the two ALDH isoforms toCH oxidation by their sensitivity to disulfiram(Ehrig et al. 1990).

Previous studies on the relative dispositionof CH into its oxidative and reductive pathwaysused liver homogenates (Lipscomb et al. 1996).Each pathway was measured in separate incu-bations with optimal pyridine nucleotide con-centrations and a concentration range of CHof up to 20 mM. These investigators con-cluded that TCOH formation predominated atsubmillimolar concentrations of CH. Theunequal disposition of CH with preponderanceof TCOH formation has also been observed inexperimental animals. Abbas et al. (1996)administered CH to B6C3F1 mice at doses of10–300 mg/kg and reported an approximate3:1 ratio for the first-order rate constants forTCOH versus TCA formation. The presentstudies relied on endogenous levels of NAD+

and NADH within the hepatocytes and used aCH substrate concentration range of 0.060–2.0mM. Additional studies are warranted to deter-mine whether the approximate 1:1 ratio seen inthese studies reflects the low level of CH sub-strate or a low level of pyridine nucleotide inthe cryopreserved hepatocytes. It is also possiblethat hepatocellular pyridine nucleotide levels inhumans are under genetic control and that atvery low levels of CH generated from drinkingwater levels of TCE, the genetically determinedconcentration of pyridine nucleotides may bemore important than the ADH/ALDH geno-types of the exposed individual.

Collectively, the present studies show thatcryogenically preserved hepatocytes can providea cost-effective approach to the simultaneousdetermination of the kinetics of elimination of

Figure 5. Proposed interdependence of oxidation and reduction of CH by ADH and ALDH (see “Discussion”for description).

TCOH

CH

ADH

TCA

ADH–NAD+

ADH–NADH

ADH–NADH

ALDH–NAD+

ALDH

CH

NADH

NAD+

Figure 4. Plot of first-order rate constants (Vmax/Km)for ADH versus ALDH activities in human hepato-cyte suspensions given CH.

0.1 1 10

Vmax /Km TCOH100 1,000

EJR

KTGAOK

IOEHL10

CHD

CEC

ZAG

DAD

HL12

100,000

10,000

1,000

100

10

1

0.1

V max

/Km

TCA

10,000

CH by ADH and ALDH and the determina-tion of ADH and ALDH genotypes of individ-ual donors. Both metabolism and genotypingcan be done on as few as 107 cells. Applicationof this approach to a larger number of ethni-cally diverse donor samples would allow fordefinition of the relationship between genotypeand susceptibility to TCE-induced livertumors. However, the studies also raise a num-ber of fundamental questions, such as whichgenes are important at very low (i.e., drinkingwater) levels of exposure to environmentalchemicals. Is it the isoform of the metabolizingenzyme or of a “housekeeping” gene that setsthe physiologic homeostasis of the cell? Genesthat control nucleotide formation and removalwould fall into this category. It is also impor-tant, however, to characterize the cryogenicallypreserved cells in greater detail. Although therecovered cells in this study all showed highviability, and their metabolic activity isexpressed in terms of viable cell number, it ispossible that their physiologic state may vary inways that affect the kinetic interpretation ofthe data for PBPK modeling and subsequentrisk assessment. Additional studies directed atnormalizing the cellular physiologic homeosta-sis of individual donor samples before experi-mentation would allow distinction betweengenetically endowed differences and treatment/handling effects.

The 11 individuals in the present studywho showed the 2*1 polymorphism forALDH2 had a Vmax variability for the pathwaygenerating TCA of between one and two ordersof magnitude (Table 4). When the two non-2*1 genotypic individuals were included, therange of Vmax values was increased by an addi-tional order of magnitude. This observationmay have implications for quantitative risk esti-mation for environmental exposures to TCE inthe general population. In risk assessment adefault value of 10 is used as an uncertainty fac-tor for genetic variability in the population.The results of this study indicate that if Vmax isused as the appropriate correlate to risk, then a10-fold uncertainty factor is an underestimate.For TCE, however, what is relevant in risk

assessment is the amount of TCA, whichdepends on the Vmax values for TCA metabo-lism as well as the Vmax for the competitivepathway to TCOH. Thus, Figure 3 is the rele-vant comparison and the Vmax values for TCAand TCOH are about equal, at least within anorder of magnitude for all the samples. At lowerexposures, a comparison of the rate constants(Vmax/Km) for the two competing pathwayswould be relevant. Figure 4 shows a good corre-lation with the two rate constants being approxi-mately within an order of magnitude of eachother. This suggests that a 10-fold uncertaintyfactor for genetic susceptibility is reasonable forTCA-driven TCE carcinogenesis. Furtherresearch is warranted to quantify the geneticvariation in liver cancer risk following environ-mental TCE exposures due to the polymorphicgenes involved in its metabolism.

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