Trichloroethylene biotransformation in human and rat primary hepatocytes

Toxic. in Vitro Vol. 4, No. 4/5, pp. 537-541, 1990 Printed in Great Britain.All rights reserved

0887-2333/90 $3.00+ 0.00 Copyright © 1990 Pergamon Press plc

TRICHLOROETHYLENE BIOTRANSFORMATION IN H U M A N A N D RAT PRIMARY HEPATOCYTES S. A. KNADLE*, C. E. GREEN, M. BAUGH, M. VIDENSEK, S. M. SHORT, X. PARTOSt and C. A. TYSON SRI International, Menlo Park, CA 94025, USA Abstract--The biotransformation of trichloroethylene (TCY) was studied in male Sprague-Dawley rats

and in human hepatocyte suspensions to aid in estimating the potential for hepatocarcinogenesis in humans. The major metabolites were qualitatively identical in both species, but rat hepatocytes metabolized about four times more TCY than did human hepatocytes under the same experimental conditions. The quantities of chloral hydrate, trichloroethanol (free plus conjugated) and trichloroacetic acid (TCA) were 15, 5 and 20 times greater, respectively, in rat hepatocyte suspensions. Since the TCA metabolite has been implicated in TCY-induced peroxisomal proliferation and hepatocarcinogenesis and rats form less TCA than do mice, which are susceptible to these effects, the results suggest that humans are at low risk from TCY exposure.

controlled respiratory exposures (Ertle et al., 1972; Fernandez et al., 1977; Monster et al., 1976; Nomiyama and Nomiyama, 1979; Ogata et al., 1971; Soucek and Vlachova, 1960). Although these studies provide data on the percentage of the retained TCY dose metabolized, data on the comparative rates of TCY biotransformation in humans and rodents are needed to predict human susceptibility to the peroxisome-inducing and carcinogenic effects of this chemical. Since in vitro hepatocyte incubations simulate in vivo metabolism (Billings et al., 1977; Fry and Bridges, 1979) and replicate interspecies differences in predominant pathways (e.g. Gee et al., 1983; Green et al., 1986), experiments were conducted using hepatocyte suspensions to compare the metabolism of TCY in human and rat liver cells.

Introduction

Trichloroethylene (TCY) is hepatocarcinogenic in B6C3F~ mice, but not in F344/N or Osborne-Mendel rats (NCI, 1976; NTP, 1983). Since hepatocarcinogenicity and peroxisomal proliferation appear to be associated (Elcombe et al., 1985; Nemali et al., 1989; Reddy et aL, 1980), and equal doses of TCY cause peroxisome proliferation in mouse but not rat liver (Elcombe et al., 1986; Goldsworthy and Popp, 1987), pharmacokinetic studies have focused on the relationship between susceptibility to TCY-induced hepatocarcinogenicity and species differences in TCY metabolism (Bolt et al., 1985; Green and Prout, 1985; Mitoma et al., 1985; Stott et al., 1982). The major pathway for TCY disposition is oxidation by cytochrome P-450 to chloral hydrate (CH), followed by either subsequent reduction to trichloroethanol (TCEt) or oxidation to trichloroacetic acid (TCA) (Miller and Guengerich, 1982). In vivo doses of TCA increase peroxisome content in mouse and rat liver in a dose-dependent manner (Elcombe, 1985; Goldsworthy and Popp, 1987). In contrast, in vivo doses of TCY that increase the peroxisome density in mouse liver by 1000% do not significantly increase peroxisome content in rat liver (Elcombe et al., 1985). Thus a quantitative difference in formation of TCA from TCY was hypothesized to be the basis for the species difference in peroxisomal proliferation and hepatocarcinogenicity (Elcombe, 1985; Elcombe et al., 1985). In humans, the extent of TCY metabolism has been estimated by measuring total urinary metabolites or the expired unchanged TCY during and after

Materials and Methods

*Present address: Environmental Epidemiology and Toxicology Section, Department of Health Sciences, Sacramento, CA 95818, USA. tPresent address: Barnes-Hind, Inc., Sunnyvale, CA 94086, USA. Abbreviations: BSA = bovine serum albumin; CH = chloral

hydrate; EGTA=ethylene glycol (bis(fl-aminoethyl ether)-N,N-tetraacetic acid; TCA = trichloroethanol glucuronide; TCY = trichloroethylene. TW4J4/~--R

Liver sources. Human liver specimens were obtained from organ transplant donors through the California Donor Transplant Network, San Francisco, (~A, USA. The liver was perfused intact, immediately after surgical removal, with ice-cold Collins Kidney Preservation Solution or perfusate containing EGTA (Green et al., 1983), or dissected into large pieces, immersed in EGTA-containing perfusate, and stored on ice for transport to the laboratory. The cell isolation procedure commenced within 2 hr of cessation of life-support systems. Table 1 presents donor characteristics. Adult male Sprague-Dawley rats (300-350 g) were obtained from Simonsen Laboratories (Gilroy, CA, USA). They were maintained for at least 1 wk before the experiments on hardwood bedding and allowed feed (Purina) and water ad lib. Hepatocyte isolation and culture. Human liver lobes were cut into wedge-shaped biopsy sections (10-25 g each) and hepatocytes were isolated by a two-step perfusion with EGTA-containing buffered salt solution followed by collagenase (Green et al., 1986; Reese and Byard, 1981). Rats were anaesthetized with 65 mg pentobarbital/kg body weight ip, and hepatocytes were isolated from whole liver or biopsy

537

538

S.A. KNADLEet al. Table 1. Donor information Specimen No. Sex H-7 M H-9 M H-10

F

Age 43 28

Race C C

18

C

Drug history NA Cocaine user. Past history of intravenous drug use and Xanax overdose None NA = not available

sections using the same perfusion method (Green et al., 1983). For human liver, digestion with col-

lagenase (250 to 300 U/ml) required 45-60 min; rat liver required 25min of collagenase (90U/ml) digestion. Hepatocytes were washed in Hunk's balanced salt solution buffer containing 2% bovine serum albumin (BSA). Cell viability (trypan blue exclusion) was 90 + 6.5% for human hepatocytes and 91 ___3.6% for rat hepatocytes. Chemicals a n d reagents. Collagenase (Type I), Waymouth's 752/1 culture medium, TCY, CH, TCEt, TCA and other analytical-grade biochemicals and reagents were obtained from Sigma Chemical Company (St Louis, MO, USA). [IaC]TCY (15.5 mCi/mmol), from Amersham (Arlington Heights, IL, USA), was a gift from C. Mitoma of SRI International. H e p a t o c y t e incubations. Hepatocytes were suspended at 1 x 106 (viable) cells/ml in 4.0 ml of supplemented Waymouth's 752/1 culture medium (Green et al., 1983), prepared as described by Salocks et al. (1981), except 2% fatty-acid-poor BSA was substituted for 0.2%. [14C]TCY (0.5, 1 or 3#1) was added to the watchglass-covered centerwell of 25-ml gas-tight flasks having a side-arm fitted with a Mininert valve (Pierce Chemical Co., Rockford, IL, USA) for serial sampling (Tyson et al., 1983). The flasks were gassed for 30 sec with air: CO 2 (95 : 5) and stoppered. The watchglass was dislodged from the centerwell to initiate volatilization of TCY, and the flasks were placed in a water-bath at 37°C, 60-70 osc/min. Equilibration between airspace and culture medium was complete within 20min. The actual concentration of TCY and its partition between airspace and culture medium was determined after equilibration of t4C-radiolabel at 30min by sampling of air and media through the Mininert valves. TCY concentration in culture medium exhibited a steep temperature-dependence curve with 84% being dissolved at 4°C, 76% at 21°C and 57% at 37°C. Therefore, flasks were always maintained at 37°C when sampling. Measured TCY contents ranged from 1.4 to 2.3 #mol/flask for 0.5 #1, 6.5 to 8.7#mol/flask for l / d and 14.1 to 29.4/~mol/flask for 3/~1 added to the centerwell. M e t a b o l i t e analyses. To quantify metabolites, aliquots of incubation medium were removed with a gas-tight syringe at predesignated intervals through the side-arm valve. For CH and TCEt quantitation, 100/~1 were injected immediately into 1900/~1 of methanol in a 0.5 dram vial sealed with a Tuf-bond silicone septum (Pierce Chemical Co., Rockford, IL, USA). A 100-/ll aliquot for TCEt-glucuronide (TCEt-G) and a third (200 #1) for TCA were quickfrozen, using acetone:dry ice, for later analysis. CH and TCEt were quantified on a Varian 3700 gas chromatograph with a 1%o SP-1000 60/80 mesh

Cause of death Intracranial haemorrhage Gunshot wound in head Intracerebral haemorrhage

Carbopak B column (2 mm I.D. x ]" O.D. × 6', glass) using a temperature gradient of 4°C/min from 100-160°C. Detection was by electron capture at 250°C. The retention times were: CH, 4,4 min; TCEt, 11.4 min; 1,1,2-trichloroethane (internal standard), 6.0 min. TCEt-G was quantified identically to TCEt after adjusting to pH 5.0 with 20 pl of 1.0 M-sodium acetate buffer and digesting with 1000 U Glusulase (fl-glucuronidase/sulphatase, from Sigma Chemical Co., St Louis, MO, USA) for 24hr. TCA was separated from cellular constituents by acidifying the medium with 10% sulphosalicylate solution containing chlorobutanol (7.2 #g/ml) as an internal standard (Garrett and Lambert, 1973). After extraction into diethylether and esterification with diazomethane, TCA was quantified by gas chromatography at 150°C as above. Retention time for TCA methylester was 6.7 min; for chlorobutanol, 12.5 min. Results

Both human and rat hepatocytes oxidized TCY to CH, TCEt, TCEt-G and TCA. The amounts of each metabolite varied between individuals, especially among rat hepatocyte preparations. Despite this variation, rat hepatocytes clearly produced greater quantities of each metabolite than did human hepatocytes (compare data, Tables 2 and 3). The percentage of TCY present as CH by 4 h r was about 15 times greater and the percentages of TCEt and TCEt-G about 4 - 6 times greater in rat hepatocyte incubations than in human hepatocytes. Likewise, the percentage of TCA averaged 20 times greater in rat than in human hepatocytes. Comparison of the percentage metabolites formed at 1.4 to 2.3/~mol TCY/flask with those formed at 14.1 to 29.4/~mol TCY/flask indicates that the formation of each metabolite was saturated in both rat and human hepatocytes in this concentration range (Table 4). In rat hepatocytes, free and conjugated TCEt accounted for 57-76% of the TCY metabolites, while TCA comprised 20-28% of the metabolites at the lowest TCY concentration and 8% at the highest concentration (Table 5). In human hepatocytes, total TCEt comprised 81-93%o of the metabolites and TCA comprised only 2-5%0 at both concentrations. The total T C E t : T C A ratio varied from 3:1 to 12:1 with TCY concentration in rat hepatocyte suspensions, whereas it was approximately 25 at both TCY concentrations ill human hepatocytes (Table 5). Discussion In vivo studies in several laboratories have demonstrated that the mouse, which is susceptible to TCYinduced hepatocarcinogenicity, metabolizes TCY much more actively than does the rat, in which TCY is not hepatocarcinogenic. Prout et al. (1985)

Comparative metabolism of trichloroethylene

539

Table 2. Metabolite formation from TCY in human hepatocytes Exp. H-7

TCY (#mol/fl~k)* 2.3 6.5 18.5

H-9

2.3 8.7 29.4

H-10

2.3 6.5 16.3

Time (hr) 2 4 2 4 2 4 2 4 2 4 2 4 2 4 2 4 2 4

CH (gM)t 1.1 _+0.1 0.6±0 1.8±0.7 n.a.~ n.a. 1.3±0.5 1.4±0.2 1.4±0.2 1.8_+0.2 1.6±0.1 2.1_+0.3 2.1_+0.6 2.0_+0.2 1.9_+0.4 1.5_+0.2 1.6_+0.1 1.7±0.3 1.7±0.1

TCEt (#M) 3.9±2.6 9.2±0.4 5.1_+0.5 14.4±4.6 6.4+_1.2 19.6±12.0 3.5_+0.2 4.1±2.4 3.6±0.3 6.5±0.3 5.0_+0.6 8.1_+0.8 3.2_+0.2 6.1±1.4 2.8_+0.2 4.7_+0.6 3.1±0.6 4.8+_1.1

TCEt-G (#M) n.a. 5.2_+1.8 n.a. n.a. n.a. 5.2±1.8 2.1_+0.7 7.5±2.1 2.4_+0.3 2.6_+0.8 3.2_+1.5 3.2_+1.2 5.4_+1.7 9.0±1.1 4.9±0.7 9.1+_0.4 5.6±1.3 9.3±1.6

TCA (#M) n.a. 0.5±0.~ n.a. n.a. n.a. 0.5_+0.01 0.2_+0.03 0.4_+0.03 0.4_+0.08 0.4±0.01 1.0±0.~ 0.5±0.02 0.4_+0.02 0.8±0.3 0.4_+0.1 0.6_+0.2 1.0±0.9 0.8±0.0

*[14C]TCY concentration measured by liquid scintillationcounting of airspace and media samples from 25-ml flasks at 30 rain incubation. tConeentration of individual metabolites produced by 4 x 106 hepatocytes in 4.0ml of culture medium as determined by gas chromatography and described in Materials and Methods. :~Sample not available for chromatography.

reported that the mouse metabolized four times more T C Y / k g b o d y weight t h a n the rat at a dose o f 2000 mg/kg body weight, and accordingly was exposed to a four-fold greater concentration o f TCY metabolites. Similarly, our results indicate that rat hepatocytes metabolized T C Y a b o u t four times faster than did h u m a n hepatocytes. This finding is consistent with in vivo studies o f N o m i y a m a and N o m i y a m a (1979), w h o estimated a six-fold difference between the two species. Therefore the rate o f T C Y biot r a n s f o r m a t i o n by species, in decreasing order, is: mouse > rat > human. The in vivo metabolism o f T C Y was saturable in the rat (Prout et al., 1985; Stott et al., 1982), in contrast to the mouse, in which metabolism was linear even at a dose o f 2000 mg/kg body weight. The in vitro data in Table 3 c o r r o b o r a t e these observations in the rat, d e m o n s t r a t i n g that T C Y biotransformation is saturable in rat hepatocytes, and show

additionally that T C Y b i o t r a n s f o r m a t i o n is saturable in h u m a n hepatocytes. The data in Table 4 d e m o n s t r a t e that reduction o f C H to free and conjugated T C E t p r e d o m i n a t e d over oxidation to T C A in b o t h rat and h u m a n hepatocytes, which has also been f o u n d to o c c u r / n vivo in both species (Green and Prout, 1985; M o n s t e r et al,, 1976 and 1979; N o m i y a m a and N o m i y a m a , 1979). However, the rate o f f o r m a t i o n o f T C E t was greater in h u m a n hepatocyte suspensions. H u m a n hepatocytes p r o d u c e d 87 _+ 5% free and conjugated T C E t at all T C Y concentrations, whereas rat hepatocytes produced 59 _+ 13% T C E t from similar T C Y concentrations. Greater variation (20-fold) was observed in the T C E t : T C A ratio in rat t h a n in h u m a n hepatocytes. A c o m p a r i s o n o f reported T C E t : T C A ratios suggests that inter-individual variation is c o m m o n ( M o n s t e r et al., 1976 and 1979; N o m i y a m a and N o m i y a m a ,

Table 3. Metabolite formation from TCY in rat hepatocytes TCY Time CH TCEt ,TCEt-G TCA (tzmol/flask)* (hr) (#M)t (/~M) (/~z) (/tM) 1.5 2 n.a.~: 10.6 + 5.8 n.a. n.a. 4 9.6+4.6 1 8 . 5 + 2 . 7 11.8+4.3 10.0 + 0.0 17.4 2 31.9-t-11.6 28.4+7.1 n.a. n.a. 4 23.6+ 1.9 59.9+9.7 25.7+ 15.5 4.0+ 1.3 n.a. R-3 1.6 2 25.9_+9.1 20.9_+4.1 n.a. 4 22.2 + 5.7 28.8 _+1.8 n.a. 8.7 + 2.0 14.1 2 82.5 _ 16.2 56.2 + 8.8 n.a. n.a. 4 76.7_+10.1 74.2_+15.0 37.4_+0.14 6.9_+4.4 R-4 1.4 2 5.5-1-4.8 5.2_+ I.I 1 8 . 8 + 2 . 1 7.4+2.1 4 18.5_+4.6 4.7+2.0 25.8_+7.5 29.8_+12.0 5.4 2 4.0+1.0 7.1_+2.5 17.1+_9.5 6.9-+0.2 4 12.1 +5.1 8.0+_2.7 28.3-+4.6 20.9_+8.3 17.4 2 12.6+3.9 8.8+3.1 18.0_+4.9 3.9+2.3 4 14.7+_1.5 15.0_+4.1 27.8+8.5 11.4_+5.9 *Concentrationof ['4C]TCY measuredby liquid scintillationcountingof airspaceand media samples from 25-ml flasks at 30 rain incubation. tConcentration of individual metabolites produced by 4 x 106 hepatocytes in 4.0ml of culture medium as determined by gas chromatography and described in Materials and Methods. ~:Sample not available for chromatography. Exp. R-2

540

S.A. KNADLE et al.

Table 4. Percentage metabolite formation ~om TCY in 4-hr incubations TCY Percentageof TCY metabolized to:? (#mol/flask)* CH TCEt TCEt-G TCA 2.3 0.3 4.2 2.4 0.20 6.5 n.a.~ 2.4 n.a. n.a. 18.5 0.1 1.2 0.3 0.03 H-9 2.3 0.6 1.9 3.4 0.20 8.7 0.2 0.8 0.3 0.05 29.4 0.1 0.3 0.1 0.02 H-10 2.3 0.9 2.8 4.1 0.40 6.5 0.3 0.8 1.5 0.10 16.3 0.1 0.3 0.5 0.04 R-2 1.5 6.9 13.2 8.4 7.1 17.4 1.5 3.7 1.6 0.6 R-3 1.6 14.8 19.2 n.a. 5.8 14.1 5.9 5.7 2.9 0.5 R-4 1.4 14.4 3.6 19.9 22.9 5.4 2.4 1.6 5.7 4.2 17.4 0.9 0.9 1.7 0.7 *[14C]TCY concentration measured as described in Table 2. ?Percentage of [IaC]TCY concentration from airspace and media samples converted to individual metabolites after 4-hr incubation with 4 x 106 hepatocytes/4ml. Metabolite concentrations were determined by gas chromatography as described in Materials and Methods. ~Sample not available for chromatography. Exp. H-7

1979), b u t the v a r i a t i o n o f the d a t a in the three h u m a n specimens studied here was r e m a r k a b l y small. K a w a m o t o et al. (1987) f o u n d t h a t in isolated rat liver infused with C H the T C E t : T C A ratio was inversely related to the C H c o n c e n t r a t i o n a n d thus to the redox state o f the liver cells, since oxidation o f C H to T C A utilizes oxidized N A D as a cofactor, whereas reduction to T C E t requires reducing equivalents. However, in the present studies, the T C E t : T C A ratio was n o t correlated with the C H c o n c e n t r a t i o n .

Table 5. Percentage of metabolites converted to TCE or TCA in 4-hr incubations Total Percentage metabolite of metabolites as:t TCY* concn Exp. (,umol/flask) (pM) TCE+TCE-G TCA H-7 2.3 15.5 92.9 3.2 18.5 26.6 93.2 1.9 H-9

2.3 29.4

13.4 13.9

86.6 81.3

3.0 3.6

H-10

2.3 16.3

17.8 16.5

84.8 85.5

4.5 4.8

1.5

17.4

49.9 112.8

60.7 75.5

20.0 3.5

1.6 14.1

n.a.:~ 195.2

n.a. 57.2

n.a. 3.5

R-2 R-3 R-4

1.4 79.0 38.6 37.7 17.4 69.0 62.1 16.5 *[14C]TCY concentration measured as described in Table 2. tPercentage of the sum of CH, TCEt, TCEt-G and TCA concentrations produced by incubating TCY with 4 x 106 bepatocytes/4ml for 4 hr at 37°C. Metabolite concentrations were determined by gas chromatography as described in Materials and Methods. :~Sample not available for chromatography.

T h e T C E t : T C A ratio a n d the q u a n t i t y o f T C A p r o d u c e d are i m p o r t a n t considerations for estimating the susceptibility o f h u m a n s to the peroxisomeproliferating a n d carcinogenic effects o f T C Y observed in mice (Elcombe, 1985; Elcombe a n d Mitchell, 1986; Elcombe et al., 1985; H e r r e n - F r e u n d et al. 1986). E l c o m b e (1985) provides evidence t h a t T C A is responsible for the increase in hepatic peroxisomal fl-oxidation elicited by T C Y in mice. The in vitro studies here suggest t h a t h u m a n s are at less risk from peroxisomal proliferation a n d hepatocarcinogenicity from TCY, since (1) h u m a n hepatocytes were less active in metabolizing T C Y t h a n rat hepatocytes a n d (2) the T C E t : T C A ratio in h u m a n hepatocytes was greater t h a n t h a t in rat hepatocytes. In addition, E l c o m b e (1985) a n d Elcombe a n d Mitchell (1986) reported t h a t neither T C A n o r mono(2-ethylhexyl) phthalate, a n o t h e r inducer of peroxisomal proliferation, increased peroxisome n u m b e r s or volume density in h u m a n hepatoctye cultures. Several other p o t e n t inducers in rats a n d rat hepatocyte assays were also relatively ineffective in increasing peroxisomal E - o x i d a t i o n activity in h u m a n hepatocytes. These results in toto suggest t h a t h u m a n s are inherently less susceptible to peroxisomal induction (Allen et al., 1987; B u t t e r w o r t h et al., 1989). In conclusion, the relatively low rate of T C Y metabo!ism in h u m a n s , the saturability o f h u m a n T C Y metabolism, a n d the relatively high T C E t : T C A ratio p r o d u c e d by h u m a n hepatocytes suggest t h a t T C Y produces neither peroxisomal proliferation n o r carcinogenicity in h u m a n liver. In addition, the resuits d e m o n s t r a t e t h a t d a t a o n species c o m p a r i s o n s o f b i o t r a n s f o r m a t i o n profiles a n d rates o b t a i n e d from hepatocyte i n c u b a t i o n s c o m p a r e f a v o u r a b l y with data o b t a i n e d in vivo, which provides further s u p p o r t for using hepatocyte systems to assess species similarities a n d differences in the m e t a b o l i s m o f environm e n t a l chemicals. Addendum

Recently Klaunig et al. (1989) reported a correlation between species differences in inhibition of gap junctionmediated intercellular communication and hepatocarcinogenic sensitivity that was also attributed to the TCA metabolite as well as to intrinsic factors that render mouse hepatocytes more susceptible than rat hepatocytes. Acknowledgements--This work was supported in part by NIEHS Contract ES-55109. Technical discussions on analytical methodology with Mr V. Hanko, Mr G. Ross Gordon and Dr Ronald J. Spanggord were greatly appreciated. REFERENCES

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