The role of glutathione conjugation in the development of kidney tumours in rats exposed to trichloroethylene

ELSEVIER

Chemico-Biological

Interactions

105 (1997) 99- 117

The role of glutathione conjugation in the development of kidney tumours in rats exposed to trichloroethylene T. Green *, J. Dow, M.K. Ellis, J.R. Foster, J. Odum Zeneca

Received

Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire, SK10 4TJ, 27 December

1996; received

in revised

form 21 April

1997; accepted

23 April

UK 1997

Abstract

Trichloroethylene is metabolised to a very minor extent ( < 0.01% of the dose) by conjugation with glutathione, a metabolic pathway which leads to the formation of S-(1,2dichlorovinyl)-L-cysteine (DCVC), a bacterial mutagen and nephrotoxin activated by the renal enzyme /J-lyase. The role of this metabolic pathway in the development of the nephrotoxicity and subsequent tumour formation seen in rats exposed to trichloroethylene has been evaluated. The pathway has been assessed quantitatively in vivo in rats, and in rats, mice and humans in vitro. Trichloroethylene was found to be a very weak nephrotoxin. There was no evidence of morphological change in the kidneys and only small increases in biochemical markers of kidney damage in rats dosed with 2000 mg/kg trichloroethylene by gavage for 42 days. N-acetyl-S-( 1,2-dichlorovinyl)-r.-cysteine was detected in the urine of rats dosed with 500 and 2000 mg/kg trichloroethylene for up to 10 days at levels equivalent to O.OOl-0.008% of the dose. In vitro, the rate of conjugation of trichloroethylene with glutathione in the liver was higher in the mouse, 2.5 pmol/min per mg protein, than the rat, 1.6 pmol/min per mg protein, and in human liver the rates were extremely low, 0.02-0.37 pmol/min per mg protein. Comparisons of the metabolism of DCVC by renal #I-lyase and N-acetyl transferase showed that metabolism by N-acetyl transferase was two orders of magnitude greater than that by P-lyase and that /I-lyase activity in rat kidney was ll-fold greater than that in human kidney. When the nephrotoxicity of DCVC was compared in rats and mice, the mouse was found to be 5-10 fold more sensitive than the rat. The no effect level in the rat was 10 mg/kg, a dose which is three orders of magnitude higher than the * Corresponding

0009-2797/97/$17.00

author.

Tel: 01625-515458;

0 1997 Elsevier

PII SOOOS-2797(97)00040-9

Science

fax: 01625-586396.

Ireland

Ltd. All rights

reserved.

loo

T. Green et (11./ Chemico-Biologicul

Interactions

IO5 (1997) 99-l 17

amount of DCVC formed from trichloroethylene in vivo. The lack of correlation between metabolism by this pathway and the rat specific tumours, together with questions concerning the potency of DCVC at the levels formed from trichloroethylene, suggests that DCVC may not be involved in the renal toxicity and subsequent tumour development seen in rats and that further evaluation of the mechanism(s) involved in the nephrotoxic response is warranted. 0 1997 Elsevier Science Ireland Ltd. Keywords: Trichloroethylene;

Kidney tumours; Glutathione

conjugation

1. Introduction

Trichloroethylene has caused low incidences of renal tumours in several lifetime studies in which rats were exposed either by gavage or by inhalation [l-5]. Several of the bioassays were judged inadequate because of poor survival as a result of nephrotoxicity [l-3]. In yet further studies, kidney tumours were not seen in rats [6-81, nor have kidney tumours been reported in mice in any of the studies. Nephrotoxicity has been a common feature of all of the rat lifetime studies. kidney tumours were observed in rats exposed to Thus, although trichloroethylene in a number of studies, the increases were either not statistically significant or the studies were compromised by poor survival. Nevertheless, the rarity of renal adenocarcinomas in the rat has prompted investigations into the mechanisms involved in their formation. Most toxicological and carcinogenic responses seen in animals exposed to trichloroethylene are known to arise from metabolites of the cytochrome P-450 pathway, however, the renal tumours and nephrotoxicity seen in rats have previously been attributed to a second minor pathway involving glutathione conjugation of trichloroethylene (Fig. 1) [9-121. This pathway produces S-( 1,2-dichlorovinyl)-L-cysteine (DCVC) and its isomer S-(2,2dichlorovinyl)-L-cysteine, the former being the major metabolite [ 131.These cysteine conjugates may undergo sulphur oxidation and/or be detoxified by N-acetylation to form the mercapturic acids which are excreted in urine. Both the cysteine conjugates and their sulphoxides may also be cleaved by the renal enzyme p-lyase leading to a variety of reactive metabolites (see Commandeur et al. [14] for review). As a result of cleavage by this enzyme the isomers of DCVC are mutagenic in bacteria and nephrotoxic in animals [15]. Of the two isomers, S-(1,2-dichlorovinyl)-~-cysteine is significantly more toxic than the 2,2-isomer [16]. The pathway has been quantified in rats and mice in vivo [9,10,12,13,17,18], and in rat liver and kidney fractions in vitro [19]. In vivo, metabolism of trichloroethylene by this pathway occurs at very low levels in all species studied; typically the levels of the mercapturate, N-acetyl-S-( 1,2-dichlorovinyl)-L-cysteine (N-acetyl DCVC), found in the urine of rats and mice exposed to trichloroethylene account for less than 0.005% of the administered dose 113,171.Where the 2,2-isomer of DCVC has been quantified, the amounts found (0.0013% of the dose) were half that of the 1,2-isomer [13]. No other products of this pathway have been identified in animals or humans exposed to trichloroethylene.

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Where species comparisons have been made, the mouse is significantly more susceptible to the acute nephrotoxic effects of both trichloroethylene and DCVC than the rat [20,21]. Similarly, the amounts of N-acetyl DCVC found in the urine of rats and mice dosed with trichloroethylene [17] are not consistent with the observed species difference in carcinogenicity, the levels in mouse urine being several fold higher than those in the rat. These conclusions have been based solely on measurements of the 1,2-isomer of N-acetyl DCVC in urine, one of only two detectable products of glutathione conjugation of trichloroethylene, the other being the 2,2-isomer. Whilst both isomers are known, the 1,Zisomer is both the major metabolite and is by far the most toxic and mutagenic isomer [16]. Thus, based on these observations it would appear that there is no direct correlation between either the nephrotoxic potency of trichloroethylene and DCVC, nor the amount of trichloroethylene metabolised by this pathway, and the rat specific renal tumours. The nephrotoxicity of DCVC has been extensively studied in a number of species including rats and mice [22,23]. Of these studies, only that by Terracini and Parker [23] in which rats were dosed with 10 mg/kg DCVC for 46 weeks and killed at 78 weeks could be considered a test for carcinogenicity. At 78 weeks, kidney damage was evident but there were no increases in the incidences of kidney tumours. The dose levels used in this study were several orders of magnitude higher than the levels of DCVC found in rats dosed with trichloroethylene. Thus, in addition to the lack of species specificity, there is a question about the toxicological significance of the very low levels of DCVC formed from trichloroethylene.

Cytwhrome P-450 - Majorpathway

ccl,cHo

Chloral

Dcvc

---c

CCf3CH20H(G) Tfichlom&hanol (ghrcuronide)

\ N-aoetvl

N-aceiylDCVC

Fig. I. The metabolism of trichloroethylene by the major and minor pathways. A number of additional minor metabolites (not shown) are known from the cytochrome P-450 pathway. DCVG and DCVC are shown as the 1,24somers; S-(1,2-dichlorovinyl) glutathione and S-(1,2-dichlorovinyl)-L-cysteine, respectively.

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N-acetyl DCVC has been detected in human urine, albeit at levels even lower than those seen in the rat [17,18]. Although there is no evidence from a number of large well conducted epidemiology studies of human populations exposed to trichloroethylene [24] that the presence of this metabolite is toxicologically significant, a study of a small population believed to be exposed to very high dose levels of trichloroethylene has reported a small cluster of kidney tumours [ll]. This observation has led some authors to conclude that both the kidney tumours seen in rats, and those in humans, are caused by a common mechanism, namely that involving metabolism of trichloroethylene to DCVC [ll]. Others have questioned this mechanism based on the lack of a correlation between the nephrotoxicity of trichloroethylene and DCVC in rats and mice and the observed carcinogenicity [20,21], and on the very minor nature of this pathway 1121.Thus, while it is facile to attribute these tumours to this pathway in both rats and humans, there is currently a lack of mechanistic data to support this assumption. The present study is an attempt to further clarify the role of glutathione conjugation of trichloroethylene and the formation of DCVC in the development of renal toxicity in the rat and human. The renal toxicity of trichloroethylene has been assessed following repeated dosing, and the metabolism of trichloroethylene by this pathway has been compared in rat, mouse and human tissues.

2. Material and methods

2.1. Chemicals and radiochemicals

Trichloroethylene (TCE), with a minimum purity of > 99% (w/w), was supplied by BDH Ltd, Poole, Dorset, UK. [1,2-‘4C]-l,1,2-trichloroethylene ([‘4C]trichloroethylene) was supplied in sealed glass ampoules by Sigma, Gillingham, Dorset (specific activity 5.4 mCi/mmol; 98%) and by Cambridge Research Biochemicals, Northwich, Cheshire (19.3 mCi/nmol; 97%). Each batch received was diluted with dimethylformamide (DMF) and the radiochemical purity was assessed by high performance liquid chromatography with radiochemical detection. [14C]trichloroethylene was purified by overnight extraction with aqueous glutathione (5 mM) at room temperature followed by vacuum distillation to achieve a minimum radiochemical purity of 99O/o. Radiolabelled S-( 1,2-dichlorovinyl) glutathione (DCVG; 2.26 mCi/mmol) and unlabelled S-( 1,2-dichlorovinyl) cysteine (DCVC) were synthesised as described previously [25]. [l-‘4C]-Acetyl coenzyme A (50-60 mCi/mmol) was purchased from Amersham International, Amersham, UK. Acetyl coenzyme A (CoASAc) and cyclohexanone were purchased from Sigma, Poole Dorset, UK. All other chemicals were of analytical grade or the highest grade available.

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2.2. Animals Male Fischer 344 rats (180-220 g bodyweight) and male B6C3Fl mice (20-35 g bodyweight) were supplied by Harlan Olac, UK and Charles River, UK. The animals were group housed in stainless steel cages in temperature controlled rooms equipped with a 12-h light-dark cycle. Food (CTR diet, Special Diet Services Ltd, Witham, Essex, UK) and water were provided ad libitum. 2.3. In vivo studies 2.3.1. Trichloroethylene metabolism Rats (n = 5) were dosed daily by gavage with either 500 or 2000 mg/kg trichloroethylene in corn oil (10 ml/kg) for either 1 or 10 days. [‘4C]trichloroethylene (250 pCi/kg) was incorporated into the dose on days 1 and 10. Urine was collected for 24 h following these doses. Urine samples were combined according to dose and duration of dose and acidified (pH 1) with sulphuric acid. Fifteen ml aliquots were extracted with diethyl ether (30 ml), the ether extract evaporated to 1 ml, and methylated with an ethereal solution of diazomethane. The derivatized extracts were analysed for the presence of trichloroacetic acid and N-acetyl DCVC by gas chromatography and by gas chromatography-mass spectrometry. The methyl ester of trichloroacetic acid was analysed using a Hewlett Packard 5890 gas chromatograph fitted with an electron capture detector and a 530 pm x 10 m methyl silicone column operated from 60 to 75°C at S”/min. The methyl ester of N-acetyl DCVC was analysed using the same instrument fitted with a 5-m column operated at 180 to 210°C (5”/min) or with the same column fitted to a VG 7070E mass spectrometer. The extraction efficiency for the compounds of interest was shown to be quantitative using the above procedure. Retention times and spectra were compared with authentic standards. 2.3.2. Trichloroethylene toxicity Male rats (n = 10) received a single gavage dose of trichloroethylene (2000 mg/kg in corn oil, 10 ml/kg) daily for 42 days. Urine was collected for 24 h after dosing on days 1, 9, 17, 28 and 42. Blood was collected by tail bleeding at each time point, 24 h after the dose was given. At the end of the study, the rats were killed by exsanguination under terminal anaesthesia (Fluothane, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) and the livers and kidneys removed, weighed and taken for histopathological examination. Portions of liver from left, median and right lobes and portions of left and right kidneys were fixed in 10% (w/v) neutral buffered form01 saline, dehydrated through an ascending ethanol series and embedded in paraffin wax. Sections (5 pm) were cut and stained with haematoxylin and eosin. Blood samples were centrifuged to separate plasma. Plasma alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and urea were determined by standard automated methods. Urinary creatinine, protein, glucose, ALP, N-acetyl glucosaminidase (NAG) and gamma-glutamyl transpeptidase (GGT) were also determined.

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T. Green et al. / Chemico-Biological Interactions 105 (1997) 99-l 17

2.3.3. S-(1,2-dichlorovinyl)-L-cysteine

toxicity

Rats and mice (n = 5) received either 10 daily gavage doses of DCVC (0.1, 0.5, 1.0 or 5.0 mg/kg; in saline, 10 ml/kg) or a single dose of 10 or 50 mg/kg. Control animals received saline (10 ml/kg). Urine was collected in containers cooled in dry ice for the 24 h period prior to dosing, for 24 h following the single doses, and on days 1, 5 and 10 of the lo-day study. At termination, animals were killed by exsanguination under terminal anaesthesia (Fluothane, Zeneca Pharmaceuticals, Macclesfield, Cheshire, UK) and blood was collected into tubes containing lithium heparin. Blood and urine samples were analysed for biochemical markers of liver and kidney damage as described above. Slices of liver and kidney were also processed as above for histological examination. 2.4. In vitro studies 2.4.1. Prepuration of’ subcellular fractions Rats or mice were killed with a slowly rising concentration of CO, followed by cardiac puncture. Following removal, livers and kidneys were weighed and 25% (w/v) homogenates made in ice cold SET (250 mM sucrose, 5 mM EDTA, 20.0 mM Tris) buffer, pH 7.4 using a Teflon/glass homogeniser (5 passes). The homogenates were centrifuged at 9000 x g (Beckman 52.21 centrifuge) for 20 min at 4°C and the supernatent (S9 fraction) further centrifuged at 105 000 x g (Beckman L8-70 M Ultracentrifuge) for 70 min at 4°C. The supernatents (cytosol fraction: rat liver lo-20 mg protein/ml; mouse liver, 14-26 mg protein/ml; rat kidney, 4-12 mg protein/ml; mouse kidney, lo- 14 mg protein/ml) were stored at - 70°C prior to use. The microsomal pellets were resuspended in 20 mM Tris, 1.15% KC1 pH 7.4 (Tris/KCl buffer) and centrifuged at 105000 g at 4°C. The washed microsomal pellets (rat liver, 9-26 mg protein/ml; mouse liver, 4-26 mg protein/mg; rat kidney, 2-7 mg prqtein/ml; mouse kidney, 5- 10 mg protein/ml) were finally resuspended in 0.1 M phosphate buffer pH 7.4 (1 ml/2 g of original tissue weight) and stored at - 70°C. Human liver samples (n = 5) were obtained from the Queen Elizabeth Hospital, Birmingham, UK. and were from fresh liver excess to transplantation requirements. Samples were provided in compliance with local ethical guidelines. Microsomal (15.3 mg protein/ml) and cytosolic (19.5 mg protein/ml) fractions were prepared as described above. Human male kidney cytosol (16.3 and 18.1 mg protein/ml) and microsomes (13.6 and 7.8 mg protein/ml) were prepared from kidneys obtained from the International Institute for the Advancement of Medicine, Exton, PA, USA. The protein content of the liver and kidney fractions were measured using the method of Bradford [26] with reagents obtained from Bio-Rad Laboratories. 2.4.2. Trichloroethylene metabolism to S-(1,2-dichlorovinyl) glutathione (DCVG) in vitro

[r4C]Trichloroethylene (l-3 mM; 2-12 ,&i in 2 ~1 DMF) was incubated for up to 2.5 h at 37°C with either liver or kidney fractions (final protein concentration 7

T. Green et al. / Chemico-Biological Interactions 105 (1997) 99-I I7

105

mg/l) in 0.1 M potassium phosphate buffer pH 7.4 supplemented with GSH (5 mM). Controls were incubated in the absence of tissue fraction, both with and without GSH. The reaction was started by the addition of trichloroethylene and 250 ~1 aliquots were removed for analysis after 0, 15, 30, 45 and 60 min or after 2.5 h. The reaction was terminated by addition of 12.5 ~1 of ice-cold trichloroacetic acid (50% w/v). Protein was removed by centrifugation and the supernatents neutralised with sodium hydroxide solution (2.5% w/v) before analysis by hplc. A Hypersil HSODS column eluted at 1 ml/min with a gradient consisting of 25 mM sodium acetate (pH 6.1) with increasing concentrations (5-90%) of acetonitrile over a period of 20 min. was used for the analysis. The eluate was passed through an ultraviolet detector (220 nm) and fractions were collected at 30 s intervals. Radioactivity in the fractions was assayed by scintillation counting. Typical retention times of authentic samples of DCVC, DCVG and trichloroethylene were 9, 11 and 23 min respectively. The limit of detection of DCVG was 0.9 pmol injected on the column. Metabolite structures were confirmed by lc-ms using a Finnigan MAT TSQ7000 (Finnigan MAT, Hemel Hempstead, Herts) fitted with an electrospray source. The HPLC conditions described above were used, except that sodium acetate was replaced with 0.1 M ammonium acetate and the initial condition for the gradient was 2%. The eluent was split between the mass spectrometer and the UV detector in the ratio of 1:5. Negative ion spectra of samples and standards were recorded. Glutathione-S-transferase activity was determined in all of the tissue fractions using I-chloro-2,4_dinitrobenzene as the substrate [27]. The conjugation of tetrachloroethylene was also measured in rat liver cytosol using the method described by Green et al. [28]. 2.4.3. Cysteine Conjugate N-acetykransferase The N-acetylation of DCVC by N-acetyl transferase was measured in rat, mouse and human kidney microsomes using the method described by Duffel and Jakoby [29]. Enzyme activity was measured with 1.0 mM DCVC, 0.8 mM [l14C]CoASAc (0.25 pCi/mmol) and microsomes (rat, 0.1 mg protein/ml; mouse, 0.1 mg protein/ml; human, 0.6 mg protein/ml) in a final volume of 0.25 ml of 0.1 M phosphate buffer, pH 7.0 at 37°C. The mixture was preincubated for 2 min at 37°C after which time the microsomes were added and the incubation continued for 4 min. The reaction was terminated by the addition of 0.75 ml of 1.33 M acetic acid and 3 ml cyclohexanone. The mixture was subject to vortex mixing for approximately 10 set and the phases separated by centrifugation at 1000 rpm for 3 min (extraction efficiency into cyclohexanone 103 f 4.3%). Aliquots of the cyclohexanone layer (2 x 1 ml) were mixed with Ultra Gold liquid scintillation fluid (Packard) and radioactivity was determined with a Packard 2500TR Liquid Scintillation Analyser. Control incubations containing no DCVC were conducted in parallel. Under the conditions of the assay, product formation was linear up to 6 min with 0.1 mg microsomal protein and 1.0 mM DCVC.

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Table I N-acetyl-S-(1,2-dichlorvinyl)-L-cysteine trichloroethylene

Dose (w/W

500

2000

a Pooled bN=3.

samples

Interactions

and trichloroacetic

No. of daily doses

N-acetyl

I 5 IO

0.0015

I

0.0046 0.0169

5 IO

0.0328

DCVC”

10.5 (1997) 99-l

17

acid levels in urine from

(mg/24

h)

Trichloroacetic

2.69 6.39 8.36 4.97 27.48 30.77

rats dosed

acidb (mg/24

with

h)

+ 0.93 + I .22 (n = 2) k 0.86 + 4.88 + 6.21

from five rats.

2.4.4. Cysteine conjugate /I’-lyuse Cysteine conjugate p-lyase activity was measured in rat, mouse and human kidney cytosol using the method of Stevens and Jakoby [30]. Incubation mixtures contained 100 ,uM NADH, 0.1 units of lactate dehydrogenase, DCVC and kidney cytosol (rat, 1.0 mg protein/ml; mouse, 1.0 mg protein/ml; human, 1.0 and 0.5 mg protein/ml) in a final volume of 1 ml of 50 mM potassium phosphase buffer, pH 7.4 at 37°C. The mixture was incubated for 2 min at 37°C prior to initiation of the enzyme reaction by addition of DCVC. Pyruvate production over a 4-min period, monitored spectrophotometrically at 340 nm, was used as a measure of enzyme activity. Pyruvate formation from DCVC was linear with respect to time.

3. Results 3.1. Trichloroethylene metabolism in vivo Preliminary experiments dosing rats with non-radiolabelled trichloroethylene failed to detect N-acetyl DCVC in urine. Consequently, radiolabelled trichloroethylene was incorporated into the dose to increase the sensitivity of the assay for N-acetyl DCVC and the urine samples were pooled before analysis. N-Acetyl DCVC was found in these samples at levels ranging from 1.5 to 33 pg/24 h urine sample. The amounts found increased with both dose and duration of dosing (Table 1). However, they only accounted for between 0.001 and 0.008% of the administered dose of trichloroethylene and were present at levels at least three orders of magnitude lower than those of trichloroacetic acid. 3.2. The toxicity of trichloroethylene Rats dosed with 2000 mg/kg trichloroethylene for 42 days showed no significant clinical abnormalities throughout the study. Body weights were unaffected, but

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both liver to body weight (1.6 x ) and kidney to body weight (1.3 x ) ratios were increased after 42 days. The effects of trichloroethylene on plasma and urinary markers of liver and kidney function at day 42 are shown in Table 2. The plasma markers for nephrotoxicity were virtually unchanged throughout the study. Increases were seen in all of the urinary parameters with the exception of creatinine. Increases in urine volume and protein (both 1.8 x ) and NAG (1.6 x ) were maximal at 42 days; glucose (2.2 x ) and ALP (2.0 x ) showed maximum increases after 9 and 28 days respectively. These increases were only slightly higher than those seen at 42 days (Table 2). Livers taken at the end of the study showed marked hypertrophy in the centrilobular region. There were no morphological changes in the kidneys of any of the animals. 3.3. The toxicity

of DCVC

A marked difference in the sensitivity of rats and mice to the toxicity of DCVC was observed following either a single dose (10 or 50 mg/kg) or repeated dosing (0.1-5.0 mg/kg for 10 days). In the rat, following a single dose, there was no evidence of kidney damage, assessed either morphologically or biochemically, at the 10 mg/kg dose level. At 50 mg/kg there was evidence of slight degeneration and necrosis of the straight portion of the proximal tubule accompanied by increases in urinary volume (2.0 x ; P < O.Ol), NAG (2.5 x ; P < 0.05), glucose (11.9 x ; P < 0.05) and protein (1.9 x ; P < 0.05). Blood urea and creatinine were unaffected throughout the study but markers of liver damage, ALT (6.7 x ; P < O.Ol), AST (5.0 x ; P < 0.01) and ALP (1.9 x ; P < O.OOl), were increased following a single dose of 50 mg/kg DCVC. Overall, DCVC was more hepatotoxic than nephrotoxic in the rat. Following repeated dosing for 5 or 10 days, urinary volume, creatinine, Table 2 Plasma and urinary parameters of liver and kidney toxicity in male rats dosed with trichloroethylene for 42 days Parameter Plasma Urea (mg%) ALP (r/ml) ALT @/ml) Creatinine (mg%) Urine Volume (ml/24 h) Protein (mg/24 h) Glucose (mg/24 h) Creatinine (mg/24 h) ALP (bmol/h/24 h) NAG @mol/h/24 h)

Control (corn oil 10 ml/kg)

Trichloroethylene

32.28 & 2.39 417.75 f 27.70 40.25 k 4.24 0.55 * 0.05

32.33 k 468.33 + 49.83 k 0.52 f

5.74 + 1.77 31.96 f 9.96 4.79 f I .47 7.38 + 1.13 3.76 f 1.18 1.05 kO.19

Values are mean f SD. Statistically significant: *P
3.20 47.7@ 13.28 0.07

10.43 + 2.41** 57.99 & 14.75** 8.51 & 3.41** 7.50 f 0.64 6.57 k 1.38** 1.70 + 0.20**

(2000 mg/kg)

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105 (1997) 99- 117

glucose and NAG levels were unchanged at all dose levels. Protein (1.2 x ; P < 0.01 at both 5 and 10 days) and GGT (1.9 x ; P < 0.001 at 5 days and 2.2 x ; P < 0.05 at 10 days) were both increased at the 5 mg/kg dose level. In contrast to the rat, the mouse was found to be more sensitive to the nephrotoxic effects of DCVC. Following a single dose of DCVC, GGT levels were increased at 1 mg/kg and above (3-10 fold) and at doses of 5 mg/kg and above, urinary volume (2.0-3.3 fold) and protein levels (1.9-5.1 fold) were increased in a dose dependant manner. Urinary creatinine (2.5 fold), NAG (3.0 fold) and glucose levels (5.9 fold) were increased at 50 mg/kg. Blood urea (2.0-4.4 fold) and creatinine levels (1.8-3.3 fold) were also elevated following single doses of 10 and 50 mg/kg respectively. The no-effect level for morphological change in the 10 day study was 0.5 mg/kg DCVC. Following 1.0 mg/kg there was minimal karyomegaly, increasing to moderate tubular basophilia at the 5 mg/kg dose level. Marked necrosis of the straight portion of the proximal tubule was seen following a single dose of 10 mg/kg, and at 50 mg/kg necrotic tubules were observed throughout the medulla with only a few viable tubules remaining in the cortex. There was no evidence of liver damage in mice. 3.4. Trichloroethylene metabolism to DCVG HPLC analysis following the incubation of [14C]trichloroethylene (1.4 mM) and GSH (5 mM) with rat liver cytosol(7 mg protein/ml) for 2.5 h at 37°C showed that at least four minor metabolites had been produced (Fig. 2). In the absence of cytosol, these metabolites were not formed. Metabolites A, B, C and D eluted at 3.1, 6.4, 12.8 and 17.6 min respectively and accounted for O.SO%,0.lo%, 0.09% and 0.14% of the added trichloroethylene. Attempts to identify metabolites A, B and D using LC-MS were unsuccessful, however, metabolite C (1.79 PM) was isolated and identified as DCVG by co-chromatography with an authentic standard using 3 different HPLC systems. The identity of DCVG was also confirmed by LC-MS. Similar metabolite profiles were seen with mouse cytosol but not human cytosol, where there was no evidence for the formation of these additional metabolites. Using highly purified [‘4C]trichloroethylene, glutathione conjugation could be detected in liver cytosol fractions from all three species, but not in hepatic microsomal fractions, nor in fractions prepared from rat kidney (Fig. 3, Table 3). The rate of formation of DCVG in incubations containing only buffer, glutathione and substrate was assumed to be due to residual levels of dichlorovinylcysteine in the added trichloroethylene. Consequently, only rates in excess of this background rate were considered to be due to the enzymic conjugation of trichloroethylene with glutathione. The rates of formation of DCVG from [‘4C]trichloroethylene in rat, mouse and human liver cytosol (7 mg protein/ml) are shown in Figs. 3 and 4. The highest rate, 2.5 pmol/min per mg protein, was found in mouse liver cytosol followed by that in rat liver cytosol, 1.62 f 0.02 pmol/min per mg protein. In human liver the rates were extremely low (0.19 f 0.14; range 0.02-0.37 pmol/min per mg; Fig. 4), approximately g-fold lower than that in the equivalent rat tissue fraction. All of the rates were linear from O-60 min (Fig. 3), and dependant upon

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T. Green et al. / Chemico-Biological Interactions 105 (1997) 99-I 17

A 1.40

=psx100

1.20

3L

'A

1.00 0.00 0.60

B

C

0.20

0.00,y 0.40

00”W

B

14C

05’00

10’00

D

IS'00

20’00

25’00

lc?s mm

14c

1.20 1.00 0.80 0.60 0.40 0.20 1.40 _;__; 0.00 00"00

05'98

10'88

15'00

20'00

25'00.

Fig. 2. The in vitro metabolism of trichloroethylene in rat liver cytosol. [%]Trichloroethylene (1.4 mM; 5.4 mCi/mmol) was incubated with glutathione (5 mM) in the presence (A) and absence (B) of rat liver cytosol fractions (7 mg protein/ml) for 2.5 h. The samples were analysed by HPLC as described in Section 2.

the presence of cytosol fraction. Full metabolic rate constants (I&, V,,,) were not obtained for these reactions due to the very low rates which were at the limit of detection of the assay. The tissue fractions used in these experiments were active in metabolising chlorodinitrobenzene, a broad spectrum substrate for the glutathione S-transferases (e.g. liver cytosol, rat, 985; mouse, 2520; human, 685-1337 nmol/ min per mg protein). Conjugation of tetrachloroethylene could also be readily detected in rat liver cytosol fractions (17.75 pmol/min per mg protein). 3.5. p-lyase and N-acetyl transferase activities DCVC may be metabolised by two competing pathways, the enzyme /3-lyase leading to toxic and potentially mutagenic metabolites, N-acetyltransferase leading to N-acetyl DCVC which is excreted in urine (Fig. 1). The maximal rates (V,,,) for

110

T. Green et al. / Chemico-Biological

0

10

20

Interactions

105 (1997) 99-I 17

30 40 Time (min)

50

Fig. 3. The comparative metabolism of [‘%Jtrichloroethylene to S-( I ,2-dichlorovinyl) glutathione in rat, mouse and human liver cytosol fractions. [“‘ClTrichloroethylene (1.9 mM; 19.3 mCi/mmol) was incubated with GSH (5 mM) and cytosol fractions (7 mg protein/ml) for 1 h.

the /3-lyase pathway were similar in kidney fractions from rat, mouse and human (Table 4). However, K,,, varied significantly with the value for rat being lo-fold lower than that for mouse and 17-fold lower than that for human kidney. Overall, the metabolic clearance (V,,,/K,) through this pathway was 11-fold greater in rat kidney than in human kidney. The metabolic rate constants for the N-acetyl transferase pathway were virtually identical for rat and mouse kidney, but V,,, for human kidney was an order of magnitude lower than that for rodents, and Km was 5-fold higher (Table 4). Comparisons of the relative clearance of DCVC by the two pathways show the flux Table 3 The in vitro metabolism of trichloroethylene to S-( I ,2-dichlorovinyl) glutathione (DCVG) in rat liver and kidney cytosolic and microsomal fractions Incubation

DCVG (nM)

Control Control with GSH Liver cytosol Liver microsomes Kidney cytosol Kidney microsomes

25 638 1179 631 642 495

[‘4C]Trichloroethylene (1.4 mM; 5.4 mCi/mmol) was incubated with GSH (5 mM) and cytosol or microsomal fractions (7 mg protein/ml) for 2.5 h. Control samples consisted of [‘4C]trichloroethylene and buffer or [“‘Cltrichloroethylene and buffer plus GSH (5 my).

T. Green et al. /Chemico-Biological

Interactions 105 (1997) 99-l I7

111

20

0

20

40

30 Tie

50

60

(miu)

Fig. 4. The metabolism of [“‘C]trichloroethylene to S-(1,2-dichlorovinyl) glutathione in 4 individual human liver cytosol fractions. [‘4C]Trichloroethylene (1.9-2.5 mM; 19.3 mCi/mmol) was incubated with GSH (5 mM) and cytosol fractions (7 mg protein/ml) for I h.

through the N-acetyl transferase pathway in rodents to be two orders of magnitude higher than that via p-lyase, but only 27-fold higher in humans.

4. Discussion Lifetime administration of trichloroethylene to rats has in a number of studies caused a small increase in kidney adenocarcinomas, a relatively rare tumour in control rats. In some studies the incidence was not statistically significant; in others it was considered that the maximum tolerated dose had been exceeded, and in one study the increases were neither dose, strain nor sex dependant. It is arguable therefore whether trichloroethylene has ever been demonstrated unequivocally to be a rat kidney carcinogen. These tumours are, however, rare in control rats and

Table 4 The in vitro metabolism of S-(1,2-dichlorovinyl)-L-cysteine Species

Rat Mouse Human

by renal B-lyase and N-acetyl transferase

N-acetyl transferase

/?-Lyase

&I

VEX+,

(mM)

(nmol/min per mg)

0.58 5.6 10.1

2.04 2.10 2.95

VmaxIKm Km 3.5 0.4 0.3

Vmax

(mM)

(nmol/min per mg)

0.25 0.27 1.44

118.4 93 11.5

V,,xlIL

474 344 8.0

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trichloroethylene has been shown consistently to be nephrotoxic in all of the chronic studies. Thus, it is clear that the rat kidney is a target organ for trichloroethylene toxicity and that in some instances the kidney damage is accompanied by a low incidence of renal tumours. A number of in vivo studies, including the present one, have shown that trichloroethylene is conjugated with glutathione in the liver, albeit to a very minor extent. Isomers of N-acetyl DCVC, the ultimate metabolites of this pathway, have been detected in rat, mouse and human urine at concentrations corresponding to approximately 0.005% of the administered dose of trichloroethylene [9,10,13,17,18]. Since the immediate precursors to these metabolites, isomers of S-dichlorovinylcysteine (DCVC), are known bacterial mutagens and nephrotoxins [15], it has been postulated that the renal tumours seen in rats exposed to trichloroethylene are a result of metabolism by this pathway [9]. A similar argument has been used to explain an excess of kidney tumours observed in a group of humans reportedly exposed to high levels of trichloroethylene [l I]. Clearly a quantitative assessment of this pathway in rodents and humans is important not only to assess the potential risks to humans exposed to trichloroethylene but initially to seek confirmation that a metabolite produced in such small amounts is indeed responsible for these tumours. Previous studies [20,21] have failed to find the expected correlation between metabolism to DCVC and renal cancer in rats and mice. Glutathione conjugation and processing through the mercapturic acid pathway is complex, involving hepatic glutathione conjugation, biliary excretion, enterohepatic recirculation and renal processing. In the kidney, the cysteine conjugate may be N-acetylated and excreted or in the case of DCVC, cleaved by /?-lyase. C-S bond cleavage by this enzyme leads to a number of products, including some which are reactive electrophiles [14]. Acylases are also present in the kidney which can de-acetylate the mercapturic acid to reform DCVC. In vivo, only the mercapturates are detectable in urine following either dosing of cysteine conjugates or chemicals metabolised by these pathways. The study of this pathway for trichloroethylene is extremely difficult because of its very minor nature. Consequently, all species comparisons have been based on either in vitro comparison of glutathione conjugation in the liver or on the levels of N-acetyl DCVC excreted in urine, predominantly as the 1,2-isomer. The rate of glutathione conjugation in the liver is clearly important since it determines the amount of substrate available for all of these subsequent reactions and the 1,Zisomer of N-acetyl DCVC is both the major urinary metabolite and is derived from the most toxic form of DCVC itself. Measurement of these two end-points would therefore seem to be an appropriate way of making comparisons between species. The extremely low rate of glutathione conjugation in all three species could only be detected using radiolabelled trichloroethylene. Even this method was not without problems since all of the samples of [i4C]trichloroethylene were found to contain trace amounts of [14C]dichloroacetylene (DCA), an impurity formed readily from trichloroethylene which reacts rapidly with glutathione to form DCVG [31]. Because of the very low rate of metabolism of trichloroethylene even trace amounts of

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DCA are sufficient to interfere with the assay for DCVG. Consequently, the [14C]trichloroethylene had to be further purified before use by washing with a solution of glutathione followed by vacuum distillation. Even after this procedure a background rate was still present in incubations containing buffer, glutathione and trichloroethylene (Table 3). The need for extensive purification of the trichloroethylene used for the in vitro studies is a reflection of the potential problems that may be encountered when measuring such a minor metabolic pathway. Trichloroethylene is not available with a purity of > 99% and consequently the possibility of interference from impurities, even at the 1% level, is considerable when the metabolic conversion represents < 0.01% of the administered material. This is particularly so when one of those impurities may be DCA. The rates of glutathione conjugation of trichloroethylene found in this study, 2.5, 1.6 and 0.19 pmol/min/mg protein for mouse, rat and human liver respectively, are in marked contrast to the rate of approximately 400 pmol/min/mg protein reported by Lash et al. [19] for rat liver cytosol fractions. Both studies used liver fractions prepared from the same strain of rat (male F344) and identical substrate concentrations, cofactor concentrations and incubation times. Only the method of analysis of the glutathione conjugate differed. Previous attempts to measure this reaction in rat liver cytosol in this laboratory had failed to detect a rate even though the limit of detection of the assay used was almost two orders of magnitude lower than the rate found by Lash et al. [19]. Further, the remarkably high rates found by Lash et al. [19] would appear not to be consistent with the reported rates for tetrachloroethylene conjugation with glutathione. In vivo, tetrachloroethylene is conjugated with glutathione to a significantly greater extent, l-2% of the dose [32] than trichloroethylene ( < 0.005% of the dose). It seems highly improbable therefore that the in vitro rate reported by Lash et al. [19] for trichloroethylene should be up to one order of magnitude higher than the in vitro rate reported for tetrachloroethylene [28,33]. The lower in vitro rate for the conjugation of trichloroethylene with glutathione found in this study is consistent with the previous observations with tetrachloroethylene and the relative metabolism of the two chemicals by this pathway in vivo. Attempts to reproduce the methodology used by Lash et al. [ 191 were unsuccessful due to interference from endogenous components present in the reaction mixture suggesting that the basis of these descrepancies may lie in the differing methodologies used. Alternatively, interference from low levels of impurities such as DCA may have been a factor. The rate of glutathione conjugation of trichloroethylene in human liver was an order of magnitude lower than that in rats and is therefore compatible with the relative in vivo excretion of N-acetyl DCVC in the two species [18]. Similarly, the higher rate seen in mouse liver is also consistent with the higher levels of N-acetyl DCVC found in the urine of this species [17]. The rates of conjugation in the rat and mouse also reflect the relative in vivo nephrotoxicity of trichloroethylene [21]. In contrast to the correlation between glutathione conjugation, N-acetyl DCVC excretion and the nephrotoxicity (but not carcinogenicity) of trichloroethylene, in vitro /?-lyase activity appears not to reflect the in vivo nephrotoxicity of DCVC. p-lyase activity in the rat kidney was ten-fold greater than that in the mouse yet the

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mouse was ten-fold more sensitive to DCVC in vivo. This difference between in vitro enzyme activity and in vivo toxicity may be due to delivered dose or differences in renal processing between rats and mice. It appears not to be related to N-acetyl transferase activity which was similar in both species. Trichloroethylene is a weak acute nephrotoxin in the rat. Very high dose levels of 2000 mg/kg administered daily for 42 days failed to cause any morphological damage in the kidney and gave relatively minor changes in biochemical markers of kidney damage. There was no evidence of protein droplet nephropathy, a well characterised response associated with a number of male rat kidney carcinogens [34], including tetrachloroethylene [28,35]. Thus, even in the rat, kidney damage is a function of chronic exposure and is not observed after acute exposure, even at very high dose levels. Others have similarly failed to find evidence of kidney damage or increased cell replication in rats dosed with 1000 mg/kg trichloroethylene for up to three weeks [35,36]. Consistent with the findings of Eyre et al. [21], DCVC was significantly more potent as a nephrotoxin in the mouse than the rat based on both morphological or biochemical endpoints. In fact, based on alkaline phosphatase and transaminase measurements, DCVC was more potent as an hepatotoxin than a nephrotoxin in the rat. In the present study the no-effect level for DCVC in all target organs in the rat was 5-10 mg/kg, which is three orders of magnitude higher than the levels of N-acetyl DCVC, lo-20 pg/kg, found in any of the species of interest after exposure to trichloroethylene. The latter dose assumes that the amount of conjugate excreted in urine is approximately half of the total produced in vivo [13]. Based on the relative activities of D-lyase and N-acetyl transferase in rodents, the amount metabolised through the /3-lyase pathway would be significantly lower than this, in the order of 0.1-0.2 pg/kg. The ability of such low doses of DCVC to cause either kidney damage or tumours must be questioned, particularly in view of the Terracini and Parker [23] study, in which, DCVC was administered at 10 mg/kg per day for 46 weeks and failed to cause tumours by 87 weeks. It is also interesting to note that Eyre et al. [21] also found that trichloroethylene induced an increase in cell proliferation in the mouse kidney but not the rat. Thus, the mouse kidney is more sensitive to the acute effects of both trichloroethylene and DCVC than the rat kidney. In conclusion, the very low rates of conjugation of trichloroethylene with glutathione in vitro reflect the very minor nature of this pathway and thus confirm the findings in vivo. The carefully controlled experimentation required to measure these rates exemplifies the difficulties in measuring metabolic pathways which account for less than 0.01% of the administered dose. The potential for measuring artefactually high rates is substantial, particularly when none of the test materials had a purity of greater than 99%. Comparable experiments measuring the metabolism of trichloroethylene by the cytochrome P-450 pathway in vitro give metabolic rates which are typically at three orders of magnitude greater than those seen here for the glutathione pathway [37]. The current data question the role of the glutathione//3_lyase pathway in the development of nephrotoxicity and, subsequently, kidney tumours in both rats and

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humans. Based on either the urinary excretion of N-acetyl DCVC [18] or the in vitro conjugation of trichloroethylene in the liver, the pathway is less important in humans than in rats. It is surprising therefore that in the exposed human population studied by Henschler et al. [ll], the tumour incidence was higher than that in rats exposed to a maximum tolerated dose for a lifetime, a most unusual finding in toxicology. Perhaps more importantly, based on the data available, the pathway does not correlate with the species differences in renal carcinogenicity observed in rats and mice, the pathway occurring to a greater extent in mice, and this species being more susceptible in vivo to both DCVC, and in the studies by Eyre et al. [20,21], to trichloroethylene. Additionally there must also be a question about the ability of the very low levels of DCVC produced by this pathway to cause tumours in either rats or humans. Although the comparisons made in this paper and those of others have failed to find a clear explanation for the rat specific tumours, it is entirely possible that other factors make the rat kidney more susceptible to the development of tumours than the kidneys of mice. The /?-lyase pathway is extremely complex [14] and critical factors may not yet have been taken into consideration. Equally the answer may lie in species differences in DNA repair or some other aspect of the tumour development mechanism. Finally, there is the possibility that an entirely different, as yet unknown, mechanism is responsible for these tumours. Overall, there are sufficient uncertainties to suggest that further studies are required to fully evaluate whether this minor pathway is indeed responsible for the observed toxic response in the rat kidney.

Acknowledgements These studies were sponsored Chlorinated Solvents Association.

by the member

companies

of the European

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