3
Chem. Res. Toxicol. 1995,8, 3-21
Invited Review Metabolism and Kinetics of Trichloroethylene in Relation to Toxicity and Carcinogenicity. Relevance of the Mercapturic Acid Pathway Arnold R. Goeptar,t$SJ a n N. M. Commandeur,? Ben van Ommen,$ Peter J. van Bladeren,$ and Nico P. E. Vermeulen**t Leiden IAmsterdam Center for Drug Research, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrve Universiteit, De Boelelaan 1083, 1081 H V Amsterdam, The Netherlands, and TNO Nutrition and Food Research, P.O. Box 360, 3700 AJ Zeist, The Netherlands Received June 2, 1994
Contents I. Introduction 11. Absorption and Distribution of TRI 11.1. Routes of Absorption and Tissue Distribution 11.2. Absorption and Distribution of TRI in Rats and Mice 11.3. Absorption and Distribution of TRI in Humans 111. Excretion, Metabolism, and Toxicity of TRI 111.1. Excretion and Oxidative Metabolism of TRI 111.2. Studies in Rats and Mice 111.3. Studies in Humans 111.4. Mutagenicity of TRI and Oxidative TRIDerived Metabolites Iv. TRI Metabolism via GSH Conjugation and the Mercapturic Acid Pathway IV.l. GSH Conjugation of TRI to S-(l,2-Dichloroviny1)glutathione N.2. y-Glutamyltransferase-Catalyzed Degradation of 1,2-DCV-G to S-(l,P-Dichloroviny1)-L-cysteinylglycineS-Conjugate Iv.3. Dipeptidase-Catalyzed Degradation of the S-(1,2-Dichlorovinyl)-~-cysteinylglycineSConjugate to 1,2-DCV-Cys Iv.4. P-Lyase-Mediated Bioactivation of DCV-Cys Isomers Iv.5. N-Acetylation of DCV-Cys Isomers to DCVNac Isomers Iv.6. N-Deacetylation of DVC-Nac Isomers Iv.7. S-Oxygenating Enzymes Iv.8. Mutagenicity and Toxicity of Mercapturic Acid Pathway-Derived TRI Metabolites IV.9. The Kidney as Target Organ V. Mechanisms of TRI-Induced Toxicity and Tumor Formation in Rodents; Relevance to Humans V.l. Species Differences in TRI-Induced Lung Tumor Formation V.2. Species Differences in TRI-Induced Liver Tumor Formation V.3. Species Differences in TRI-Induced Kidney Tumor Formation
V.4. Human Toxicity and Cancer Epidemiology after Exposure to TRI VI. Conclusion VI.1. General Toxicity VI.2. Lung Tumors Induced by TRI VI.3. Liver Tumors Induced by TRI VI.4. Kidney Tumors Induced by TRI
I. Introduction Trichloroethylene (1,1,2-trichloroethene, TRIY is a colorless, highly volatile liquid (94 Torr a t 30 "C) which is used predominantly for degreasing fabricated metals with minor uses in adhesives, glass manufacture and oil extraction ( I , 2). Commercial grade TRI is generally 99.9% pure, however, occasionally up to 0.2% of stabilizers (such a s 1,2-epoxybutane) are added to commercial formulations. Because of the widespread use of TRI and because it evaporates readily, environmental exposure is mainly via the atmosphere. Nonindustrial exposure via air, food, and drinking water is negligible (3). Occupational exposure to TRI in air occurs primarily by inhalation during metal cleaning processes and is of concern because of the toxic and carcinogenic potential of the compound (1). Already more than thirty years ago, animal food based on TRI-extracted soybean was found to cause fatal aplastic anemia in cattle and to induce *To whom correspondence should be addressed, at the Leided Amsterdam Center for Drug Research, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, De Boelelaan 1083, 1081 HV Ameterdam, The Netherlands. Phone: 0204447590; Fax: 020-4447610; E-mail:
[email protected]. + LeidedAmsterdam Center for Drug Research. t: Present address: Wageningen Agricultural University, Department of Toxicology, P.O. Box 8000, 6700 EA Wageningen, The Netherlands. + TNO Nutrition and Food Research. 'Abbreviations: TRI, 1,1,2-trichloroethene; 1,2-DCV-Cys, S-(1,2dichlorovinyl)-L-cysteine;2,2-DCV-Cys, S-(2,2-dichlomvinyl)-tcysteine; GSH, glutathione; TCE, trichloroethanol; TCE-gluc, trichloroethanol glucuronide; TCA, trichloroacetic acid; OP-Nac, S-(3-oxopropyl)-Nacetvl-kcvsteine: DCA. dichloroacetic acid: HAAE.N4hvdmxvacetvl)ami6oeth"kol; AUC, area under the curve; GST, GSH S-tra&fer&e; 1,2-DCV-G, S-(1,2-dichlorovinyl)glutathione;1,2-DCV-Nac, N-acetylS-(1,2-dichlorovinyl)-~-cysteine;2,2-DCV-Nac, N-acetyl-S-(2,2-dichloGGT, 7-glutamylmvinyl)-mysteine; HCBD, hexachloro-1,3-butadiene; transferase; WL, kidneyAiver ratio; AOAA, (aminooxy)acetic acid; KMB, a-keto-y-methiolbutyric acid; BUN, blood urinary nitrogen; DCVDs-Nac, N-(trideuterioacetyl)-labeledmercapturic acids; NAG, N-acetyl,!I-D-glucosaminidase.
0893-228x/95/2708-0003$09.00/0 0 1995 American Chemical Society
Goeptar et al.
4 Chem. Res. Toxicol., Vol. 8, No. 1, 1995
severe nephrotoxicity in rodents (4, 5), most likely due to the release of S-(1,2-dichlorovinyl)-~-cysteine (1,2-DCVCys) when protein containing it was metabolized. Since then TRI and TRI-derived metabolites have been subjected to numerous toxicological studies. The cytotoxic effects of TRI can be both general and specific. Reported general effects of acute TRI inhalation are central nervous system depression in rats (6) and impaired cardiac functions in male beagles (7). Specific adverse effects of chronic TRI exposure to rodents have been described for a variety of end points. For instance, chronic exposure of mice to high doses of TRI by gavage has been shown to induce hepatocellular carcinomas (8, 9). In addition, chronic inhalation exposure of TRI showed both lung tumors and hepatomas in different strains of mice (10). In contrast to mice, chronic exposure of rats to relatively high doses of TRI by gavage caused selective nephrotoxicity and a low incidence of renal tubular adenocarcinomas (8). The identification of mercapturic acids of TRI in urine of rats (11,12) and the observation that the corresponding L-cysteine S-conjugates were potent mutagens in the Ames test (13, 14) led to the hypothesis that a glutathione (GSH) conjugation related route of metabolism (the mercapturic acid pathway) may be involved in the nephrotoxicity and/or nephrocarcinogenicity in rats. General reversible effects of TRI exposure to humans are similar to those reported in animals and include mild central nervous depression such as fatigue, lightheadedness, sleepiness, dyspnea, nausea, and headache (15). Mortality resulting from TRI exposure, due to impaired cardiac functions, has been described incidentally (7,16). Despite well-documented TRI-induced selective organ cytotoxic and carcinogenic effects in rodents, the relevance of these specific adverse effects of TRI to humans is not clear. Selden et al. (17 ) found no convincing signs of nephrotoxicity in human from TRI exposures up to 50 mg/m3. In fact, renal damage aRer acute TRI exposure has been shown to be relatively uncommon in humans (18). Likewise, acute adverse hepatic effects of TRI, when used as an anesthetic, were also rare (19). Furthermore, epidemiological studies in TRI exposed workers have not been able to identify an increased cancer risk (20-22) but are sometimes limited by confounding problems of (1) mixed exposures (23), (2) small cohorts (20), (3) inadequate latency periods (21), and (4) the healthyworker effect (22). Subsequent well-designed and wellconducted studies (24-26) provided no evidence that TRI was a human carcinogen. The molecular and cellular mechanisms by which TRI causes toxicity and/or carcinogenicity have not yet been fully elucidated. The specific adverse effects observed in experimental animals and/or in occupational settings involve processes which generally have to be distinguished in several distinct phases, i.e., (1) kinetics (absorption, distribution, elimination), (2) toxication and detoxication biotransformation, (3) reversible and irreversible interactions with cell or tissue components, (4) (bio)chemicalprotection and repair mechanisms, and (5) consequences of the cytotoxic effects for the organism (27). In this review the metabolism and kinetics of TRI in relation to its organ selective cytotoxicity and carcinogenicity, and the relevance of the mercapturic acid pathway in this process will be discussed. For this purpose, species (including human) differences in kinetics, metabolism, and mercapturic acid related nephro-
A h
i-
120
Fat
M
: O
2
Blood
100
Liver
W
-5E
60
f
40
*
Ea
80
Lungs Cerebrum Cerebellum
6
20 0
0
2
Time in h
6
Figure 1. Organ content of TRI a h r chronic daily inhalation exposure of adult rats to 200 ppm TRI for 6 Wday. Measurements were made on the 5th day. Data adapted from Savolainen et al. (33).
toxicity mechanisms of TRI will be reviewed, and the extrapolation of animal experimental data to humans will be evaluated. In addition, recent biochemical and mechanistic studies as well as mutagenicity and animal carcinogenicity data relevant to the assessment of the human carcinogenichazard of exposure to TRI will be considered. Finally, the effects of TRI in humans, as reflected by epidemiological studies, including the interpretation and their impact on health hazard assessment, will be discussed.
11. Absorption and Distribution of TRI TRI is readily absorbed through the lungs and gastrointestinal tract in all species tested (for a detailed review, see ref 1). The absorption and distribution kinetics of TRI in rat, mice, and human will be evaluated in this section. II.1. Routes of Absorption and Tissue Distribution. TRI is rapidly and extensively absorbed through the lung alveolar endothelium due to its high bloodlgas partition coefficient, which is comparable to that of anesthetic gases (28). Skin absorption of TRI vapor is negligible. Direct contact with liquid TRI may result in percutaneous absorption, however, to a minor extent (29). Because unchanged TRI is an apolar and highly lipophilic compound, it can be expected to cross the gastrointestinal mucosa easily by passive diffusion. Indeed, virtually complete absorption (93-98%) through this route has been observed in rodents dosed by gavage with [14C]TRI in corn oil (30). 11.2. Absorption and Distribution of TRI in Rats and Mice. Following pulmonary absorption, TRI is rapidly distributed throughout the body via the systemic circulation and considerable distribution into adipose tissue is found (31, 32). The storage of TRI in adipose tissue and the low amounts in other tissues reflects high solubility of TRI in lipids (Figure 1)(33). Stott et al. (34) investigated the tissue distribution of TRI by determining the 50 h post-inhalation exposure of 10 and 600 ppm [l4C1TRI in male mice and male Osborne-Mendel rats. The total body burdens were 0.24 and 9.48 mg/mouse and 1.03 and 31.09 mg/rat, respectively. Approximately 98% of the total [14C]TRI body burden of male B6C3F1 mice was excreted within 50 h. The main fraction of radioactivity in mice was found in urine and constituted 75% of the total TRI body burden (Figure 2). Low amounts of radioactivity were detected in mice skin, liver, and kidney (Figure 2). Importantly, these authors found no difference in [14C]TRI recovery between low- and highexposure levels.
Chem. Res. Toxicol., Vol. 8,No. 1,1995 5
Invited Review B6C3F1mice
Osborne-Mendel rats II ExpiredTRI
E
Carbondioxide
Q Urine
Feces
II Liver Kidney carcass
Figure 2. Recovery of radioactivity for 50 h post-inhalation exposure of male B&SFl mice and male Osborne-Mendel rats to 10 ppm [14ClTRIfor 6 h. Data adapted from Stott et al. (34).
In contrast to BGCSFl mice, the total recovery of [14C]TRI in Osborne-Mendel rats was decreased in rats exposed to 600 ppm (79% [14C]TRIbody burden) relative to those exposed to 10 ppm (98% [14C]TRIbody burden) (34).As with the mice, the main fraction of radioactivity in the rats was found in urine: 62% (Figure 2) and 55% of the [14C]TRI body burden in low- and high-dose rats, respectively. Likewise, the amounts of [14C]TRIdetected in the rat skin, liver, and kidney were relatively low (Figure 2) (34). The kinetics of distribution and elimination of TRI from the blood of Wistar rats after intravenous administration were determined using concentrations of TRI ranging from 3 to 15 mgkg (35).A disappearance half-time (tv2) of 215 min for adipose tissue was found (35).Following absorption and distribution of TRI after a single intragastric dose of 1g k g in corn oil to rats and mice, blood TRI was shown to disappear in an exponential first-order manner with a half-time (tvz)of 1.75 h for mice and 2.25 h for rats (36),indicating nearly the same elimination rate of TRI in these two species. The pulmonary uptake of TRI in rodents was, however, found not to be directly proportional to TRI concentrations in inspired air. On the contrary, the total uptake of TRI (rat versus mouse) was related to their relative body surface areas (4.5-fold) rather than to their relative body weights (9.6-fold) (1). 11.3. Absorption and Distribution of TRI in Humans. In contrast to experimental animals, little information is available about tissue distribution of TRI in humans. Estimates of the disposition of TRI in humans have been made after vapor exposure by measuring pulmonary elimination of TRI and TRI-derived metabolites excreted into urine. TRI absorbed into the human body by inhalation is directly proportional to the TRI concentration in inspired air as expressed by the equation: Y = 7.25X 5.5, where Y is the total TRI-derived metabolites in urine (mg/L) and X is TRI concentration in air (ppm) (37,38). Pulmonary uptake of TRI in humans is rapid, although at least 8 h are required for tissue equilibrium to be achieved (Figure 3) (39).Adipose tissue requires longer than 8 h for equilibrium (Figure 3). The mean body burden is approximately 75% (40). With exercise, the body burden has been shown to increase at a given inhaled air concentration (41). From a kinetic model for TRI in the human body, it has been derived that TRI distributes from the blood into three major compartments with rate constants of 17 h-l (tu2 = 2.4 min) for vessel-rich tissues (brain, heart, kidney, liver, endocrine, and digestive systems), 1.7 h-l (tu2 = 25 min) for muscle and skin, and 0.2 h-l (tu2 = 3.5-5 h) for adipose tissue (28,39,42).TRI exhibits a long residence time in adipose tissue (about 40 h), with
+
0.3
t
-
FG MG
= ---* =
ARC ALV.Air d a r t . blood
...-......-
0.2
l!
%
0.1
2
4
6
8
Exposure in h
Figure 3. TRI distribution from blood into different tissues during a n 8 h inhalatory exposure of humans to 100 ppm TRI, as derived from a kinetic model. F G fat tissue (adipose tissue); MG: muscle and skin; VRG: vessel-rich group of tissues (brain, heart, kidneys, and liver); and ALV air and arterial blood. Adapted from Fernandez et al. (39).
still detectable adipose tissue concentrations after 70 h (39).Because of the long residence time of TRI in adipose tissue, repeated daily exposure can result in accumulation of TRI until steady state is reached. It has been predicted that TRI in adipose tissue will reach equilibrium 5-7 days after daily exposure (39).
111. Excretion, Metabolism, and Toxicity of TRI Generally, elimination of TRI absorbed through the lung and gastrointestinal tract involves two major and one minor pathways. The two major pathways are (1) pulmonary excretion of unchanged TRI and (2) oxidative hepatic metabolism of TRI by cytochrome P450. The minor metabolic pathway involves conjugation of TRI with glutathione (GSH) and further metabolism by the mercapturic acid pathway. Pulmonary excretion and oxidative hepatic biotransformation of TRI will first be reviewed. Thereafter, emphasis will be specially directed to the minor metabolic pathway of TRI via GSH conjugation and mercapturic acid formation. III.1. Excretion and Oxidative Metabolism of TRI. Unmetabolized TRI is eliminated primarily by exhalation via the lungs. The principal site of TRI metabolism, however, is the liver (2,43,44).Other tissues, such as kidney, spleen, and/or small intestine, may also metabolize TRI to a certain extent, because these tissues are
6 Chem. Res. Toxicol., Vol. 8, No.1, 1995
c1-
c1
c1
CI -
Trichloethylene f TRO
/
’
(HUE)
0
0
*c-C// I HO
\
I
J J
HOCH~CH~HCOCH~OH N-(hydroxyacetyl)-aminoerhanol
Chloral
\
L :\OOH CIZCHCOCI
Goeptar et al.
Trichloroethanol
Trichloroaceric acid
CHCI~COOH~ Dichloroacetic acid (DW
OH
Oxalic acid
Figure 4. Oxidative metabolism of TRI in the liver and the formation of metabolites which are excreted in the urine. Adapted from Davidson and Belites ( I ) .
sites of cellular protein binding of TRI-derived metabolites (34-46). In the liver, TRI is primarily metabolized by cytochrome P450 [most likely cytochrome P450 2E1(47-49)1 to a proposed epoxide intermediate which spontaneously rearranges to chloral (Figure 4). Chloral is further metabolized to trichloroethanol (TCE), trichloroethanol glucuronide (TCE-gluc), and trichloroacetic acid (TCA). Minor metabolites include carbon dioxide (COZ), dichloroacetic acid (DCA), oxalic acid, and N-(hydroxyacety1)aminoethanol (HAAE)(2,43,44)(Figure 4). It has been speculated that these metabolites are formed upon hydrolysis of a TRI-epoxide intermediate. With regard to the conversion of the TRI-epoxide to chloral, this has only been seen in the presence of strong Lewis acids (50).In line with this, the bacterial enzyme methane monooxygenase forms the epoxide and its degradation products but not chloral (51).Miller and Guengerich (50)proposed an alternative pathway involving the rearrangement of the initial oxidation product within the catalytic site of cytochrome P450. IZZ.2. Studies in Rats and Mice. The excretion and metabolism of [14C]TRI have been studied in female
Wistar rats and female NMRI mice. After a single oral dose of 200 mgkg, rats exhaled 52% and mice 11%as unchanged [14C]TRI, and 2% and 6% as 14C02,respectively (Figure 5) (30). Rats excreted 41%of the recovered radioactivity in the urine, in contrast to mice where urinary excretion amounted to 76%(Figure 5). Metabolites identified in rat urine were 15%TCA, 12%free TCE, 62%TCE-gluc, 2%DCA, 1%oxalic acid, and 7%HAAE (Figure 6). In mouse urine, TCE (free and conjugated) is the main metabolite of TRI (94%),but small amounts of HAAE (4%)and oxalic acid (0.7%)are also excreted (Figure 6). Only traces of DCA (0.1%)and TCA (0.1%) were found in this species (30). In male Osborne-Mendel rats and male B&F1 mice the dose-dependent metabolism of [14C]TRI has been investigated using [14C]TRI (10-2000 mgkg) (36). As expected, the elimination of radioactivity was rapid; 8090%of the dose is excreted in the first 24 h regardless of the dose or species (Figure 7). At high doses, 500,1000, and 2000 mgkg TRI, however, in the rat there was a marked change from urinary excretion of radioactivity to exhalatory excretion of unchanged TRI in expired air (Figure 7). This dose-dependent change in the route of excretion of radioactivity was considerably less marked in the mouse (Figure 7) (36). Interestingly, the metabolism of TRI in the mouse was linear over the range of doses used (i.e., from 10 to 2000 mgkg), whereas in the rat the metabolism became saturated a t 1000 mgkg and higher (Figure 8). Blood kinetics of TRI and TRI-derived metabolites confirmed a faster rate of metabolism in the mouse than in the rat, most likely due to a higher hepatic first pass effect in the mouse than in the rat (36). Indeed, a comparison on a body weight basis indicated that the mouse metabolizes TRI 1.2 times more to TCE than the rat a t 10 ppm and 2.6 times more a t 600 ppm (34). Consequently, a t high dosage, mice are exposed to significantly higher concentrations of oxidative TRI-derived metabolites than the
NMRI mice
Human
Wistar rats
Carbondioxide Q Urine
Figure 5. Excretion of TRI in exhaled air and urine of female NMRI mice and Wistar rats after a single oral dose of 200 mgkg [l4C1TRIand of humans after inhalation exposure to 70 ppm TRI. Data adapted from Dekant et al. (30) and Monster et al. (54). NMRI mice
Wistar rats
I3 Conjugated TCE
Figure 6. Excretion of TRI-derived metabolites in urine of female NMRI mice and Wistar rats after a single dose of 200 mgkg [l4C1TRI.TRI-derived metabolites were identified by GC-MS. Data adapted from Dekant et al. (30).
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 7
Invited Review
Osborne-Mendel rats
B6C3Fl mice
calcas
$g(
Feces Urine
0 Carbonmonoxide W UnchangedTRI
'OoO
Dose
(mglkg)
2000
0
looO Dose
(mglkg) 2000
Figure 7. Disposition of a single oral dose of [14C]TRI to male BsC3F1 mice and male Osborne-Mendel rats. Data a r e expressed a s % of t h e dose and were adapted from Prout et al. (36).
0
500
lo00 1500 2000 Trichloroethylene in mg/kg
Figure 8. Dose-dependent excretion of oxidative TRI-derived metabolites i n male B&F1 and Osborne-Mendel rats after a single intragastric dose of [l4C1TRI.Taken from Prout et al. (36).
rat. As expected, peak concentrations of the principal metabolites (chloral, TCE, and TCA) were reached in the mouse within 2 h after dosing, compared to 10-12 h in the rat (Figure 9). The blood concentrations of both TCE (4-fold) and TCA (7-f01d), measured as the ratio of the areas under the curves (AUC), were significantly higher in the mouse than in the rat (Figure 9). Most remarkably, TCE and chloral were rapidly eliminated from blood (t112 = 1-2 h), while the higher concentrations of TCA were maintained for over 30 h and thereafter cleared within 48 h of dosing (36). 111.3. Studies in Humans. The kinetics and metabolism of TRI in humans have been derived from a large number of occupational and volunteer studies. Following inhalation exposure of male volunteers to 70 ppm TRI for 4 h on 5 consecutive days, TRI, TCE, and TCA were measured in exhaled air, blood, and urine (40). The uptake was approximately 7 mgkg lean body mass in 4 h. Total amount of TRI absorbed was 78%, 11% being excreted as unchanged TRI by the lung, 43% as TCE, and 24% as TCA excreted in urine (Figure 5 ) (40). Part of the unaccounted TRI dose (22%) was explained by the formation of HAAE (30),TCE-glucuronide secretion in bile (1,52),or gastrointestinal excretion (53).The concentration of TCE in human blood has been shown to increase sharply upon TRI exposure (54). The TCE concentration in blood samples reached a maximum within 20-100 min aRer exposure. In contrast, the concentration of TCA in human blood reached a maximum only on the 5th to 6th day after repeated exposure (54).
Estimates of the extent of TRI metabolism in humans, as a percentage of retained TRI, have indicated that the human capacity for oxidative metabolism of TRI is nonlimiting up to an exposure of 315 ppm (equivalent to 25 mgkg) (37,38,55). In fact, saturation of the oxidative metabolism of TRI in humans has not been observed after single nor after repeated daily exposures between 50 and 380 ppm (1). Based upon the clearance of TRI in humans [3.7 L/(h*kg)](56),it has been estimated that humans metabolize approximately 20 times less TRI on a body weight basis than rats [77 L/(h*kg)]a t similar exposure levels (57). Consequently, humans metabolize approximately 60 times less TRI on a body weight basis than mice. The kinetics of the principal TRI metabolites (i.e., chloral hydrate, TCE, and TCA) were also determined in blood and urine of volunteers (52). Immediate oxidation of choral hydrate to TCA amounting to 50% was observed. The half-time (t1I2) for TCE was 12 h, while the half-time (t112) of TCA differed greatly, ranging from 75 to 100 h (52),indicating either storage of TRI and/or TCA in adipose tissue or high plasma protein binding of TCA (58). 111.4. Mutagenicity of TRI and Oxidative TRIDerived Metabolites. Studies on the mutagenic potential of TRI and TRI-derived metabolites have been performed in bacteria, fungi, and yeast and in cultured mammalian cells. Since inhalation is the predominant route of occupational exposure of TRI, vapor data will be emphasized in this review. TRI was found not to be mutagenic in bacterial mutagenicity assays using Salmonella typhimurium TAlOO when the pure compound was tested in the vapor phase (59). No increase in his+ revertants was detected in S. typhimurium TAlOO in the presence of S9 fraction of the rat kidney with an epoxide-free TRI sample (60). Tests in the mould Aspergillus nidulans with pure TRI did show a weak mutagenic activity in the haploid strain 35, however, only when the cells were in the growing phase (61). No induction of sister chromatid exchanges was found in Chinese hamster ovary cells (62). Negative results were also reported on TRI-induced DNA repair studies in primary rat hepatocytes (63). A single hostmediated assay utilizing s. typhimurium TA98 as inoculum in mice was again negative (64). Furthermore, pure TRI was found not to be clastogenic in B&F1 mice bone marrow in vivo (65). Spermatid micronucleus analysis in mice germ cells showed no effect of TRI up to 500 ppm (66). Importantly, studies in humans did not provide evidence of chromosomal damage a t TRI levels up to 30
8 Chem. Res. Toxicol., Vol. 8, No.1, 1995
Goeptar et al.
'1
Chloral
Figure 9. Blood levels of TRI and oxidative TRI-derived metabolites after a single gavage dose of 1000 mg of TRVkg in corn oil to male Osborne-Mendel rats and male B6C3Fl mice. Taken from Prout et al. (36). Table 1. Species Differences In the Activities (in Some Cases Determined with Substrates Other Than 'MU or =-Derived Metabolites)"of the Key Enzymes Involved in Mercapturic Acid Synthesis speciesb GSH conjugation GGT dipeptidase P-lyase N-acetyltransferase acylase refs mouse + +++ + + ndc +/74,76,77,84 rat ++ +++ + +++ + + 12, 14, 74, 77, 84, 123, 128 human
+I -
+
+/-
+
nd
fl-
74-77,89
GSH conjugation to hexachIoro-l,3-butadieneand tetrachloroethylene; GGT activity assessed by liberation of p-nitroaniline from y-glutamyl-p-nitroaniline; dipeptidase activity determined by liberation of p-nitroaniline from aniline-p-nitroaniline;P-lyase activity determined with 1,2- and 2,2-DCV-Cysisomers as substrates; N-acetyltransferaseactivity determined with S-benzylcysteine and 1,2an&2,2-DCV-Cysisomers as substrates; acylase activity assessed by N-deacetylation of 1,2- and 2,2-DCV-Nac isomers. Strain and sex differences may occur (for detailed information see respective reference). nd, not determined. (I
ppm (67). Furthermore, neither TCE (68)nor TCA (68) nor DCA (68) nor chloral (69), the principal oxidative metabolites of TRI, was found to be mutagenic in 5'. typhimurium TA100, TA1535, TA1537, and TA98. Thus, there is no conclusive evidence from either animal or human studies that pure TRI is genotoxic. This conclusion is based on a n overview of a complex area that in detail is beyond the scope of this review. It should be noted that many of the mutagenicity tests reported in the literature are confounded by the use of impure TRI and/or the presence of potentially mutagenic stabilizers.
Iv. TRI Metabolism via GSH Conjugation and the Mercapturic Acid Pathway Reactive electrophilic species are known to be deactivated by conjugation to the nucleophilic sulfur atom of GSH. This conjugation reaction can occur spontaneously or may be catalyzed by GSH 5'-transferases. The GSH conjugate formed is generally further metabolized to a n N-acetyl-L-cysteine S-conjugate (or mercapturic acid), which is rapidly excreted in the urine. Although GSH conjugation is generally recognized as a detoxication pathway, evidence is now accumulating that this metabolic process is also involved in toxication reactions (70).Details of the key metabolic steps and
enzymes involved (see Table 1)in the metabolism of TRI via GSH conjugation and the mercapturic acid pathway will be reviewed in this section. N . 1 . GSH Coqjugation of TRI to S-(l,2-Dichloroviny1)glutathione. The formation of GSH conjugates is catalyzed by cytosolic and microsomal GSH S-transferases (EC 2.5.1.18; GST) (47). Substantial nonenzymatic GSH S-conjugate formation may occur with highly electrophilic substrates (71). 5'-(1,2-Dichlorovinyl)glutathione (1,2-DCV-G)is a highly toxic GSH S-conjugate of TRI (72) and is thought to be formed via an additiod elimination reaction (Figure 10). The resulting 1,2DCV-G undergoes further metabolism to yield various metabolites, the quantitatively most important of which are the mercapturic acids (Figure 11). N-Acetyl-S-(1,2-dichlorovinyl)-~-cysteine (1,2-DCVNac) has been identified as a urinary metabolite in rats given a n oral dose of 400 mgkg of TRI (11). The 1,2DCV-Nac concentration in the urine, however, accounted for less than 0.1% of the TRI dose administered (11). Only recently, TRI was shown to be metabolized in vivo to two regioisomeric mercapturic acids, namely, 1,2-DCV-Nac and 2,2-DCV-Nac (Figure 12) (11,12). Based on GC-MS fragmentation, Dekant et al. (47) claimed that the two regioisomeric mercapturic acids were the cis- (or Z-) and
Chem. Res. Toxicol., Vol. 8, No. I , 1995 9
Invited Review
H
CI
Z C i T VRI)
F2
0
HcI
0
II II HC. CHf CH2- C NH- CH- C NH-CHfO I I
H
F
COOH
H(C1).
S
Cl(H)
c1
F = c,
TRI-glutathione conjugate (I ,2-DCV-G)
Figure 10. Structure of glutathione and conjugation to TRI to a TRI-glutathione conjugate (1,2-DCV-G). fM2
P
0 0 y-'& NH CH&WH
H$?(3H2CHp2 Mi
H2Nc;H- C Mi C H m H
-f+
COOH
RS"2
Glu
(cysleinylglycinc S-conjugate)
GI,+
~ f9 l ~ c ; H ' ~ (d)H FH2
RS
(e)
(mercapturic acid)
H2Nc;H- COOH
Fl RS (cysteine-S-conjugate)
(dp-% R- SH ( I .2-DCV-)
(2.2-DCV-)
(thiol-compound)
Figure 11. Possible routes of metabolism of S-(l,2-dichloroviny1)glutathione (1,2-DCV-G). Steps a r e catalyzed by (a) y-glutamyltransferase; (b) cysteinylglycine dipeptidase; (c) Lcysteine S-conjugate P-lyase; (d) L-cysteine S-conjugate N acetyltransferase; (e) acylase.
trans- (or E-) isomers of 1,2-DCV-Nac. However, GC-MS and 13C-NMR data of synthetical DCV-Nac isomers presented in the study by Commandeur and Vermeulen (12)indicated that one of the isomers is the geminal 2,2DCV-Nac and not the vicinal 1,2-DCV-Nac. The stereochemistry of the vicinal 1,2-DCV-Nac still remains to be proven unequivocally. One may argue that the in vivo formation of the two regioisomeric mercapturic acids may have originated from two regioisomeric GSH S-conjugates of TRI, namely, the 1,2-DCV-G and 2,2-DCV-G. Unfortunately, direct experimental evidence to support this hypothesis is lacking. GSH and GST activity is present in different subcellular fractions of most tissues of mammals (73). When comparing activities of GSH conjugation to hexachloro1,3-butadiene (HCBD) in hepatic microsomes and cytosol obtained from different species (Table l), microsomal activity was comparable in livers of human, monkey, and rat (74). Much lower activities (10-fold) were observed in microsomes obtained from rabbit, hamster, mouse, and, in particular, guinea pig (74). Remarkably, studies using subcellular fractions of the human liver showed no
GSH conjugation to tetrachloroethylene (75) and perchloroethylene (76). Recently, Birner et al. (77) provided indirect evidence for GSH conjugation in humans occupationally exposed to TRI, by measuring a mixture of 1,2-DCV- and 2,2-DCV-Nac in the urine. However, details of the exposure concentration and purity of the TRI formulation were not given. It should be noted that alkaline decomposition of TRI may also produce dichloroacetylene, a product that has been shown to yield both 1,2-DCV-G and 1,2-DCV-Cys (78) identical to those presumed to be formed after conjuagtion of TRI to GSH. Taken together, these conflicting results fail to resolve the issue whether or not GSH-conjuagtion reactions are important in humans. N.2. y-Glutamyltransferase-Catalyzed Degradution of 1,2-DCV-G to S-(1,2-Dichlorovinyl)-~-cysteinylglycine S-Coqjugate. The involvement of y-glutamyltransferase (EC 2.3.22; GGT), an enzyme involved in the hydrolysis or transfer of the y-glutamyl residue of GSH S-conjugates to an appropriate acceptor (Figure 11, step a) (79, 80), in the metabolism of 1,2DCV-G to S-(1,2-dichlorovinyl)-~-cysteinylglycine S-conjugate has been demonstrated in vitro (81).AT-125 L0.25 mM, a potent inhibitor of GGT-activity in rat kidney (82, 83)]was shown to completely protect isolated proximal tubular cells against the cytotoxicity induced by 1,2DCV-G (81).In addition, when these cells were incubated in the presence of glycinylglycine (a suitable acceptor substrate for GGT-catalyzed deglutamination), the cytotoxicity of 1,2-DCV-Gwas increased, most likely due to stimulation of GGT-activity (811. Interestingly, the involvement of GGT in the bioactivation of 1,2-DCV-G was also demonstrated in vivo (72). When AT-125 was given 1h prior to the treatment of rats with 1,2-DCV-G, the nephrotoxicity of this compound was markedly reduced (72). GGT is a n ubiquitous enzyme, which is predominantly found in the kidney of mammals (84). Some enzyme is present in the cytosol, but the larger fraction is cell membrane bound (85). GGT is localized on the outer surface of both luminal and basolateral membranes of renal proximal tubules as well as on the biliary ductular epithelium (86-88). When comparing tissue distribution of GGT activities in different species (Table l ) , it appeared that the rat and mouse had very high kidneyniver (WL) ratios of GGT activity (84). Much lower WL ratios (50-fold) were observed in other species (i.e., guinea pig and macaque). Estimates of WL ratios from human liver and kidney GGT activities indicated that the relative distribution of activities resembled that of the guinea pig or macaque rather than the rat or mouse (89). The ability of GGT t o catalyze the formation of S-(1,2dichloroviny1)-L-cysteinylglycine may have important biological consequences and predispose tissues with high GGT activity to toxicity mediated through this pathway. N.3. Dipeptidase-Catalyzed Degradation of the 5(1,2-Dich2orovinyl)-Lcysteinylglycine S-Coqjugate to 2,2-DCV-Cys. The involvement of cysteinylglycine dipeptidase (EC 3.4.13.6), an enzyme known to catalyze the degration of L-cysteinylglycine S-conjugates to the corresponding L-cysteine S-conjugate (Figure 11,step b) (go), in the metabolism of S-(1,2-dichlorovinyl)-~-cysteinylglycine to 1,2-DCV-Cys has been demonstrated primarily by in vitro inhibition studies (81). Phenylalanylglycine [a competitive inhibitor of cysteinylglycine dipeptidase (91)]has been shown to protect isolated rat
10 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 A.
Goeptar et a1.
H(C1)xcl COOH
C1 (H)
'"%cl
C1 (H)
S-CH2-CH
S-CH,-CH
1.2-DCV-Cys
I
I
COOH I
I NH2
ammonia. pyluvic acid lhionoacyl chloride
COOH
B.
I
2.2-DCV-Cys
NH2
enerhiol
thioaldehyde
ammonia. pyruvic acid
Figure 12. Structures and proposed bioactivation mechanisms for the regioisomeric L-cysteine S-conjugates of TRI. (A) Vicinal (1,2-DCV-Nac) and S-(1,2-dichlorovinyl)-~-cysteine (1,2-DCV-Cys). dichlorovinyl-isomers: N-acetyl-S-(1,2-dichlorovinyl)-~-cysteine "he 1,2-DCV conjugates may have a trans- ( E - )or cis- (2-1conformation. Bioactivation of these regioisomers leads to the formation of enethiols which may tautomerize to thionoacyl chlorides or which may eliminate hydrogen chloride to yield thioketenes. (B) Geminal dichlorovinyl isomers: N-acetyl-S-(2,2-dichlorovinyl)-~-cysteine (2,2-DCV-Nac)and S-(2,2-dichlorovinyl)-L-cysteine(2,2-DCV-Cys). Bioactivation of this regioisomer leads to formation of an enethiol which may be in equilibrium with the thioaldehyde tautomer. Taken from Commandeur et al. (14). kidney cells against S-(l,2-dichlorovinyl)-~-cysteinylglycine-induced cytotoxicity (811. The highest activity of dipeptidases is found in the kidney and liver (90). In the kidney, the activity of dipeptidases is present in the renal brush border membrane (92)and in the basolateral membrane of the kidney cells (93-95). In the liver, dipeptidases are present in the canalicular membrane of hepatocytes, in the luminal membrane of the biliary epithelium (96-981, and in pancreatic secretions in the bile (87,99). When comparing tissue distribution of dipeptidases in different species (i.e,, mouse, rat, rabbit, guinea pig, pig, and macaque), the highest activity was always found in the kidney in all species (Table 1)(84). The ability of dipeptidases to catalyze the formation of 1,2-DCV-Cys may also play a role in the toxicity of TRI via the mercapturic acid pathway. N . 4 . P-Lyase-Mediated Bwactivation of DCV-Cys Isomers. The identification of N-acetyl-S-(l,2-dichloroviny1)-L-cysteine(1,2-DCV-Nac)as a metabolite in urine of rats treated with TRI (11, 12, 47, 77) led to the hypothesis that the selective nephrotoxicity and nephrocarcinogenicity of TRI in rats might be the result of bioactivation of the intermediate metabolite 1,2-DCV-Cys by L-cysteine S-conjugate ,&lyase (EC 4.4.1.13; P-lyase). P-Lyase is a pyridoxal phosphate-dependent enzyme that catalyzes the cleavage of the C-S bond of L-cysteine S-conjugates (100). Bioactivation of 1,2-DCV-Cys by
P-lyase results in the production of pyruvic acid (101, 1021,ammonia, and reactive sulfur-containing metabolites (Figure 11, step c, and Figure 12), which have been shown to bind covalently to proteins and DNA (103-105). In line with this observation, 1,2-DCV-Cysin rodents has been shown to induce severe proximal tubular necrosis (72, 106). Several in vivo and in vitro studies have suggested that P-lyase-dependent bioactivation is a key step in the nephrotoxicity of 1,2-DCV-Cys (107,108). Indeed, the observation that 1,2-DCV-Cys caused toxicity to rat kidney slices, as determined by inhibition of organic anion and cation transport, in combination with the release of equimolar amounts of pyruvic acid and ammonia, indicated the involvement of p-lyase (13). Moreover, 1,2-DCV-Cys appeared to be cytotoxic to isolated rat kidney proximal tubular cells, as assessed by lactate dehydrogenase leakage, trypan blue exclusion, and a-methylglucose uptake (81). In the presence of (aminooxylacetic acid [AOAA, a potent inhibitor of &lyase activity (7211, almost complete protection against the cytotoxicity of 1,2-DCV-Cyswas observed. The observation that the activity of peptidases was not affected by AOAA further indicated that P-lyase was involved in the toxication process (81). Similar results were also obtained with the LLC-PKI renal cell line (109). Interestingly, a significant regioselectivity was observed in the bioactivation of DCVCys isomers by @-lyase(14).For instance, the P-lyase activity toward vicinal 1,2-DCV-Cysin rat cytosol was 3
Invited Review to 4 times higher when compared to that toward geminal 2,2-DCV-Cys (14). 1,2-DCV-Cys in vivo has been shown to cause a dosedependent increase in blood urinary nitrogen (BUN) concentrations and urinary glucose excretion rates, both seen as indicators of proximal tubular damage (110).The involvement of /?-lyase in the nephrotoxicity of 1,P-DCVCys in vivo was confirmed by studies using the /?-lyase inhibitor AOAA, which at a dose of 0.5 mmovkg inhibited /?-lyase activity in rat kidney by more than 9070,already 1h &r administration (72). Additional in vivo evidence for the possible involvement of /?-lyase in the nephrotoxicity of haloalkene derived S-conjugates was obtained by dosing rats with a-methyl-L-cysteine S-conjugates (72, 111, 112). These conjugates are not metabolized by /?-lyase, because they do not possess a n a-hydrogen which can be abstracted, and consequently the a-methyl analogue of DCV-Cys was not nephrotoxic. However, next to /?-lyase activity, the nature of the electrophilic species formed upon /?-lyase-mediated bioactivation of L-cysteine S-conjugates may also play an important role in relative toxicity by determining selectivity of reactions with biological nucleophiles (14,113). Elucidation of the chemical structure of the reactive intermediates formed upon /?-elimination of DCV-Cys isomers has been hampered by their instability and high reactivity. It has been suggested that enethiol formed from vicinal 1,2-DCV-Cys is tautomerized to a highly reactive thionoacyl chloride or that it eliminates hydrogen chloride to form a thioketene intermediate (Figure 12A) (14, 114), both of which are highly reactive thioacylating compounds (115). In contrast to vicinal 1,2DCV-Cys, /?-lyase-mediated degradation of geminal 2,2DCV-Cys would lead to the formation of a n enethiol which is in equilibrium with its thioaldehyde tautomer (Figure 12B) (14). In line with this suggestion, a thioaldehyde was shown to mediate the weak mutagenicity of S42-chlorovinyl)-~-cysteine in a S. typhimurium TA2638 strain (116). Because of the lack of direct-alkylating properties of thioaldehydes, hydrolysis to chloroacetaldehyde was proposed to be a key step in the mutagenicity of S-(2-~hlorovinyl)-~-cysteine (116). Obviously, the formation of alkylating agents, such as thioketenes, with considerably higher reactivity toward cellular macromolecules (DNA and RNA) may explain the higher mutagenicity of 1,2-DCV-Cyswhen compared to that of 2,2-DCVCys (14). /?-Lyase activity has been shown to be present in several mammalian tissues (117) as well as in the intestinal microflora (118).In the rat, the highest specific activity of /?-lyase toward 1,2-DCV-Cyswas found in the kidney (117). In the rat kidney, /?-lyase is a n intracellular enzyme present mainly in cytosol (117)and to a lesser extent in the mitochondrial outer membrane (119). The cytosolic enzyme, however, has a higher activity with 1,2-DCV-Cys as substrate (14, 120, 121). By immunohistochemical techniques, rat kidney /?-lyase was shown to be located in the proximal tubular epithelium, but was absent in the glomerulus and the distal tubules (122). There are important differences in /?-lyase activity between sexes and species (Table 1). For instance, /?-lyase activity is greater in male than in female rats and in mice of both sexes (76). Cytosolic and mitochondrial j3-lyase activity has also been demonstrated in human kidney using 1,2-DCV-Cys (123,124). The highest j3-lyase activity in humans was present in the cytosolic fraction. The human p-lyase activity was similar to that
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 11 in the mouse (76). The specific activity of human kidney cytosol, however, was only 10% of that in rat kidney cytosol (123). In contrast to the rat, considerable @-lyase activity was also found in human kidney microsomes (123). Unfortunately, the nature of the microsomal /?-lyasehas not yet been investigated in detail. Moreover, the relative contribution of the cytosolic and mitochondrial fractions to 1,2-DCV-Cys-induced nephrotoxicity remains to be established. Inasmuch as mitochondria are proposed to be the initial targets of /?-lyase-mediated toxicity, the mitochondrial /?-lyase may be more important for the initial activation process (125). N.5. N-Acetylation of DCV-Cys Isomers to DCVNac Isomers. The final step in the mercapturic acid pathway is N-acetylation of L-cysteine S-conjugates to the corresponding mercapturic acids by L-cysteine S-conjugate N-acetyltransferase (EC 2.3.1.80; Figure 11, step d) (126). Previously, it has been shown that N-acetylation of 1,2-DCV-Cys to N-acetyl-S-(1,2-dichlorovinyl)-~-cysteine (1,2-DCV-Nac)was the main route of 1,2-DCV-Cys metabolism in rats (127). More recently, two regioisomeric mercapturic acids, namely, vicinal 1,2-DCV-Nac and geminal 2,2-DCV-Nac, were identified in the urine of rats treated with TRI (Figure 12) (12). The amount of 2,2-DCV-Nac in urine was found to approximate 50% of the amount of 1,2-DCV-Nac(12). It was also demonstrated using N-(trideuterioacety1)-labeled mercapturic acids (DCV-D3-Nac)that a significant amount of the corresponding L-cysteine S-conjugates formed were reacetylated; 54%(1,2-DCV-Nac)and 70% (2,2-DCV-Nac) of excreted mercapturic acids in rat urine were no longer labeled (12). Also, upon administration of bhe two L-cysteine S-conjugates to rats, a high amount of the dose was excreted as mercapturic acids, 50% in the case of 1,2-DCV-Cys and 73% in the case of 2,2-DCV-Cys (12). Taken together, these and other observations demonstrated a higher extent of N-acetylation of 2,2-DCV-Cys, probably the less nephrotoxic product of TRI metabolism via the mercapturic acid pathway. A considerable species variation has been observed in the urinary excretion of meracpturic acids (Table 1). In the rat and hamster, N-acetylation was shown to be the most important route of metabolism of S-pentyl-L-cysteine (50-80%); the guinea pig, however, was found to excrete only 2% of the dose as mercapturic acid, indicating a low activity of N-acetylation compared to the alternative routes of cysteine conjugate biotransformation (128). L-Cysteine S-conjugate N-acetyltransferase has been found a t the endoplasmic membrane in different tissues (129). N-Acetyltransferase activity has been shown in liver, kidney, and small intestine, although the specific activity in the kidney is 6- and 100-fold higher when compared to the liver and small intestine, respectively (130). In the kidney, N-acetyltransferase activity is located in cells of the proximal tubules (90). NAcetylation of nephrotoxic L-cysteine S-conjugates is believed to be a detoxification pathway. The activity of N-acetyltransferase, therefore, may be a n important determinant of the relative toxicity of L-cysteine Sconjugates. Presently, it is not known, however, to what extent these tissues contribute to the N-acetylation of DCV-Cys isomers in vivo. In studies using isolated kidney cells (131)and proximal tubules (132), N-acetylation of nephrotoxic L-cysteine S-conjugates appeared to be only minor, presumably due to the relatively high activities of acylases and P-lyase in these cells and/or due to limited
12 Chem. Res. Toxicol., Vol. 8, No.1, 1995 availability of the cofactor acetyl-coenzyme A in in vitro models (133). N.6. N-Deacetylation of DVC-Nac Isomers. Acylases [most likely acylase I (EC 3.5.1.14)l are known to catalyze N-deacetylation reactions of mercapturic acids to the respective L-cysteine S-conjugates (Figure 11, step e) (134). It has been shown that 1,2-DCV-Nacis deacetylated to 1,2-DCV-Cys in vitro (14). Also, a significant regioselectivity was observed in the deacetylation of the two regioisomeric mercapturic acids. For instance, in vitro deacetylation of geminal 2,2-DCV-Nac by a commercial acylase (acylase I from hog kidney) was 50-fold higher when compared to vicinal 1,2-DCV-Nac (12). Moreover, after intraperitoneal administration of both mercapturic acids to the rat, it was demonstrated that 1,2-DCV-Nac was excreted unchanged in urine to a significantly higher extent than 2,2-DCV-Nac, 34% uersus 17% of the dose, respectively (12). This probably reflects the lower rate of deacetylation of 1,2-DCV-Nac when compared to 2,2-DCV-Nac, which is in agreement with in vitro studies using renal fractions showing that deacetylation of 2,2-DCV-Nac by rat renal cytosol was 3 to 4 times higher than deacetylation of 1,2-DCV-Nac(14). In both rat kidney and rat liver, multiple enzymes are known to catalyze N-deacetylation reactions (Table 1). The specific activity of acylases in kidney extracts was found to be 2-fold higher than those of liver extracts (134). These enzymes antagonize the action of the N-acetyltransferases by catalyzing the deacetylation of mercapturic acids. Because the regenerated L-cysteine S-conjugates can again be bioactivated by ,&lyase, N-deacetylation may be regarded as a bioactivation step possibly contributing to toxicity. Thus the balance between N-acetylation and N-deacetylation reactions may play an important role in determining the initial toxic response of TRI via the mercapturic acid pathway (12, 14). N.7. S-Oxygenating Enzymes. Recently, it was shown that the sulfoxide of 1,2-DCV-Cysalso produced renal damage (i.e., increases in blood BUN concentrations and anuria) when administered to rats (121). 1,2-DCVCys sulfoxide was more cytotoxic in isolated rat distal tubular cells (with higher S-oxidase activity) rather than in isolated rat proximal tubular cells (with higher p-lyase activity) (121). @-Lyaseappeared not to be involved in the cytotoxicity of 1,2-DCV-Cys sulfoxide (121). These results indicate that, in addition to /3-lyase-mediated cleavage of L-cysteine S-conjugates, sulfoxidation by the cysteine conjugate S-oxidase (S-oxidase) may also play a role in 1,2-DCV-Cys-induced nephrotoxicity in rats. Sulfoxidation of L-cysteine S-conjugate has been associated with flavin-containing monooxygenases, which exhibit selectivity in the interaction with L-cysteine S-conjugates (235, 136). For the sulfoxidation of Sbenzyl-L-cysteine, it was found that microsomes of male rat kidneys were approximately 3 times more active when compared to microsomes from female rats, while overall the kidney activity was twice that of the liver activity (135). Using the cysteine conjugates of both cis- and trans-1,3-dichloropropene,it was shown that flavincontaining monooxygenases were responsible for the S-oxygenation reaction (237). These findings were confirmed using the mercapturic acid of acrolein, S-(3oxopropy1)-N-acetyl-L-cysteine(OP-Nac) (138). In this case, methimazole (an inhibitor of flavin-containing monooxygenases) was able to inhibit the cytotoxicity of OPNac (138).
Goeptar et al. The role of sulfoxidation in the disposition and toxicity of L-cysteine S-conjugates is not yet clear. Although the exact nature and extent remain t o be determined, some experimental evidence now points to a n involvement of GSH conjugation and subsequent S-oxidation of L-cysteine S-conjugates in the nephrotoxicity of a t least some xenobiotics (including TRI). As yet, no direct evidence has been presented for the in vivo sulfoxidation of 1,2DCV-Cys in the rat (127). N.8. Mutagenicity and Toxicity of Mercapturic Acid Pathway-Derived TRI Metabolites. 1,2-DCVCys is a well-known mutagenic agent in the Ames test with S. typhimurium TAlOO in the presence of rat kidney S g (115) and NADPH (13). In fact, both 1,2-DCV-Cys and 1,2-DCV-Nacwere stronger mutagens in the Ames test with S. typhimurium TA2638 when compared to 2,2DCV-Cys and 2,2-DCV-Nac (14). Similarly, both 1,2DCV-Cys and 1,2-DCV-Nacwere more cytotoxic toward isolated rat kidney proximal tubular cells than 2,2-DCVCys and 2,2-DCV-Nac. The much higher mutagenicity of 1,2-DCV-Nac when compared to 2,2-DCV-Nac could not be explained simply by a more rapid deacetylation of 1,2-DCV-Nac. In fact, the specific activity of Ndeacetylation for both 1,2-DCV-Nac and 2,2-DCV-Nac was found to be much higher in rat cytosol than the specific activity of ,&elimination of both 1,2-DCV-Cysand 2,2-DCV-Cys (14). It was concluded that the rate of deacetylation may not be the rate-limiting step for the onset of mutagenicity of either 1,2-DCV-Nacor 2,2-DCVNac. On the contrary, the rate of bioactivation of the respective L-cysteine S-conjugates by /3-lyase and the nature of the reactive intermediates formed were proposed to be more important than differences in the rate of deacetylation in determining the relative mutagenicity of the geminal (2,2-) and vicinal (1,2-) isomers of DCVNac (14). Thus 1,2-DCV-Nac,which is the most “abundant” regioisomer in urine of TRI-treated rats, and which has higher mutagenic potential than 2,2-DCV-Nac,may be more important for initiating nephrotoxicity and nephrocarcinogenicity than 2,2-DCV-Nac. The cytotoxicity of 1,2-DCV-Nactoward isolated kidney cells, however, was found to show a delayed time course when compared to 1,2-DCV-Cys(14). It has been shown previously that the uptake of 1,2-DCV-Cysin rat kidney proximal tubules was only moderately (20%) faster than the uptake of 1,2-DCV-Nac(132). Nevertheless, covalent binding was &fold higher in incubations with 1,2-DCVCys than with 1,2-DCV-Nac (132). Thus, it can be concluded that the deacetylation step rather than differences in cellular uptake may be responsible for the delayed cytotoxicity of 1,2-DCV-Nacto rat kidney proximal tubules. A delayed onset of toxicity of mercapturic acids when compared to the corresponding L-cysteine S-conjugates has also been shown previously with Sconjugates of four structurally related fluoroethylenes (131). The observation that the time course of cytotoxicity of 2,2-DCV-Nac paralleled that of 2,2-DCV-Cys, as well as the relatively high rate of N-acetylation and low rate of ,!?-lyaseactivation for this conjugate, indicates that for 2,2-DCV-Nac the /?-lyaseactivation step may be ratelimiting (14). N.9. The Kidney as Target Organ. The fact that both 1,2-DCV-G and 1,2-DCV-Cys have been shown to produce nephrotoxicity in vivo in rats (72, 110), as well as the identification of DCV-Nac isomers as urinary metabolites of TRI in rats, mice, and humans (12, 12, 47, 77) suggests that the kidney is most likely the
Invited Review primary organ for handling of the GSH conjugate of TRI. It has been suggested that glomerular filtration and further metabolic processing of 1,2-DVC-G (and/or 2,2DVC-G) by GGT and dipeptidases are followed by uptake of the 1,2-DVC-Cys (and/or 2,2-DCV-Cys) across the brush border membrane (139, 140). The DCV-Cys isomers that are transported into renal proximal tubular cells may undergo metabolism by microsomal L-cysteine S-conjugate N-acetyltransferase to form the respective mercapturic acids (1,2- and 2,2-DCV-Nac) (127), which are secreted into the lumen and excreted in the urine (11,12,30,47, 77, 78). However, the DCV-Nac isomers (notably, 1,2-DCV-Nac)may undergo enzymatic deacetylation to regenerate the respective DCV-Cys isomers (notably, 1,2-DCV-Cys) (1411, which are subsequently bioactivated by renal L-cysteine S-conjugate @lyase to ultimate nephrotoxicants (14, 1411. An alternative view that Nac-cysteine conjugates may be the species which arrive a t the kidney has also been presented (107, 142, 143). For instance, using S-carbamido[14C]methylglutathione(142), a model compound for GSH S-conjugates, the following series of events were postulated: GSH S-conjugate accumulates mainly in the kidney and is hydrolyzed into its component amino acids, presumably by GGT and some peptidase(s) on the renal brush border membranes. The cysteine S-conjugate which is formed in the tubular lumen is reabsorbed and transferred to the liver, acetylated to form N-acetylcysteine S-conjugate, and excreted in the urine. Although the study (142)was not conducted with DCV-Cys, the sequence of events imply that renal hydrolysis of GSH S-conjugate is coordinated with acetylation in the liver and with mercapturic acid biosynthesis in vivo. In fact, it was derived that exposure of the kidney to mercapturic acids might also be important for the onset of nephrotoxicity (72). In line with this, mercapturic acids have been shown to be deacetylated by renal fractions to form the corresponding cysteine conjugate, which are subsequently bioactivated by renal P-lyase (14, 141). At present, the relative contribution of cysteine versus N-acetylcysteine conjugation in exposure of the kidney to nephrotoxic S-conjugates is not clear. Dependent on the efficiency of hepatic acetylation and deacetylation, the kidney will be exposed to both cysteine S-conjugates and mercapturic acids. Both processes might be determined by the chemical nature of the sulfur substituent and might also differ strongly between different types of GSH conjugates. Moreover, whether these conjugates also reach the kidney after in vivo exposure to the parent ethylenes is a matter of speculation. An important factor might be the localization of GSH conjugation, which in turn might depend upon the route of administration of the parent ethylene. Inasmuch as formation and further processing of GSH conjugates are extremely complex, involving multiple enzymes and transport systems, several possible mechanisms have been identified which might cause nephrotoxicity or contribute to the development of nephrocarcinogenicity in animal experiments. It should be noted that these experiments were usually conducted in rats exposed t o relatively high (oral) doses of ethylenes. According to these studies, hepatic GSH conjugation and further processing of GSH conjugates are contributory factors in the onset of nephrotoxicity. However, the extrapolation of results from animal gavage studies to humans is difficult, especially when the most
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 13 appropriate route of exposure for humans is by inhalation. L-Cysteine S-conjugate @-lyase,the enzyme responsible for the ultimate bioactivation of the DCV-Cys isomers, is found in rat kidney cytosol and mitochondria and has been shown to be evenly distributed over the proximal tubular epithelium (14, 119-2221. In addition, GGT, aminopeptidase, and L-cysteine S-conjugate N-acetyltransferase activities, enzyme activities which have also been shown to play a major role in determining the nephrotoxicity of TRI, are the highest in the outer medulla (90, 144). This localization is coincident with the cytotoxicity of 1,2-DCV-Cys in isolated rat proximal tubular cells (121). In fact, the mitochondrial matrix of the rat kidney was found to be a primary target for the reactive electrophiles released upon @-eliminationof 1,2DCV-Cys by P-lyase, most likely due the lipophilic nature of 1,2-DCV-Cys (125). Using 35S-labeled substrates, Hayden et al. (145) have shown recently that mitochondrial phospholipid adducts are the major product of the p-elimination of 1,2-DCV-Cys (145). Sufficiently high covalent binding can lead to a cascade of events, including depletion of cellular nonprotein sulfhydryls, increased cellular free calcium, and lipid peroxidation, which may ultimately lead to cell damage (146). Interestingly, although all segments of the proximal tubule are affected by high doses of nephrotoxic L-cysteine S-conjugates, the S3 segment appears to be the most sensitive (122). At the same time, the significance of this effect is difficult to interpret, because the distribution of P-lyase within the proximal tubule is not consistent with the hypothesis that a higher concentration of the enzyme in the S3 segment is causually related to the greater sensitivity of the S3 segment to nephrotoxic L-cysteine S-conjugates when compared to the SI and SZsegments (122). Therefore, factors other than the amount ofp-lyase in the site specific nephrotoxicity of L-cysteine S-conjugates cannot be ruled out.
V. Mechanisms of TRI-Induced Toxicity and Tumor Formation in Rodents; Relevance to Humans TRI has been shown to increase the incidence of tumor formation both in the mouse and in the rat. The principle tumor sites in the mouse are the lung and the liver, whereas in the rat the kidney is the primary target organ (Table 2). In humans, these selective organ toxicities of TRI are rare (Table 2). The incidence and the mechanism of tumor formation in rats and mice and its relevance for humans are the subject of the following discussion. V.1. Species Differences in TRI-Induced Lung Tumor Formution. Increased incidence of lung tumors (lung adenocarcinomas and adenomas) induced by TRI have been observed in female ICR mouse (147) and in female B&F1 and male Swiss mouse (10). The ICR mice (49/group)were exposed, by inhalation for 6 Wday, 5 days/ week for 104 weeks, to 0,50,150, or 450 ppm TRI (147). In female ICR mice, the incidence of lung adenocarcinomas was observed only a t the 150 ppm (16%) and 450 ppm (15%) exposures (147). Furthermore, in a series of studies, groups of 90 male and female BBCBFIand Swiss mice were exposed, by inhalation 7 hfday, 5 dayslweek for 8 and 78 weeks, to epoxide-free TRI a t 0, 100, 300, and 600 ppm and observed for lifetime (10). A small incidence of lung tumor (essentially adenomas) was observed in female BeCSF1 (17%) and male Swiss (30%)
14 Chem. Res. Toxicol., Vol. 8, No. 1, 1995
Goeptar et al.
Table 2. Effects Related to TRI andor TRI-Derived Metabolites compoundu
effect
TRI TRLkhloral
lung adenocarcinomas and adenomas vacuolation of Clara cells
TRI TCAiDCA TRU"CA/DCA TRI TCA TRVTCA
hepatocellular carcinomas and adenomas hepatocellular carcinomas and adenomas peroxisome proliferation DNA damage lipid peroxidation inhibition of intercellular communication
TRI TRI TRI 1,2-DCV-Cys 1,2-DCV-Cys 1,2-DCV-Cys 1,2-DCV-Nac 2,2-DCV-Nac
nephrotoxicity protein droplet nephropathy nephrotoxicity and nephrocarcinogenicity DNA strand breaks nephrotoxicity nephrocarcinogenicity excreted in urine excreted in urine
mouseb
+ + + + + + + + -
nd
+ +
+/-
ratb
human
-
-
+
ndc -
nd -
nd nd
-
+ + + + + +I+ +
-
nd nd nd nd
+
+/-
Dose and exposure schedules are indicated in the respective references. (-) negative; (+) positive. occur (for detailed information see respective reference). nd, not determined. a
mice only a t the highest concentration (600 ppm) (10). No increase in lung tumors was observed in male B&$1 or in female Swiss. The possible significance of these findings for tumor development in mice has been further examined in a series of in vivo and in vitro studies (148). A single 6 h exposure of CD-1 mice to TRI a t concentrations ranging from 20 to 2000 ppm caused a dose-related cytotoxicity, characterized by vacuolation of Clara cells (a specific cell type in the lung). Moreover, mice exposed to 100 ppm TRI and higher showed a marked reduction of cytochrome P450 activity in Clara cells, while GST activities were generally unaffected (148). Metabolism studies using isolated mouse Clara cells showed a n oxidative metabolic pathway for TRI similar to that known in the liver, i.e., via TRI to chloral (Figure 4). In fact, mouse lung Clara cells contain high levels of cytochrome P450 (148). However, mouse Clara cells were found deficient in metabolizing chloral to TCE (Figure 4) and further conjugation of TCE with glucuronic acid. Thus, the accumulation of chloral in mouse lung Clara cells most likely explains lung tumor formation in the mouse. The rat lung, however, contains fewer Clara cells and lower levels of cytochrome P450 than the mouse lung, which may explain the lack of a lung cell proliferation response of TRI in rats. Clara cells found in human lung tissue show no smooth endoplasmic reticulum (149)and are therefore presumed not to possess significant cytochrome P450 activities. Thus, the chloral-dependent lesions observed in the mouse lung Clara cells would not be expected to occur in humans a t foreseeable low level of occupational exposure to TRI. V.2. Species Differences in TRI-Induced Liver T u m o r Formation. The occurrence of increased incidences of mouse liver tumors (hepatocellular carcinomas and adenomas) is a significant observation in lifetime cancer bioassays of TRI and is frequently reported. For instance, in a study conducted by the National Toxicology Program (NTP), in both male and female B&F1 mice, exposed by gavage to 1000 mgkg epoxide-free TRI (in corn oil), 5 dayslweek for 2 years, increased incidences of hepatocellular adenomas and carcinomas were observed in both sexes (8). Increased incidence of hepatocellular adenomas has also been observed in male and female Swiss mice following inhalation exposure to 300600 ppm amine-stabilized, epoxide-free TRI (101,but not in NMRI mice (150),nor in male and female Ha:ICR mice
refs 10,24-26,147 148,149 8,10,24-26,169,170 156,160,161 152,154,157 34,63,67, 154, 155, 157 156,157 158 8,17,18,24-26,162 163 8,10,24-26,162 166 72,106,167 11,107,108 12,43,47, 77, 163 12,77 Strain and sex differences may
(1511, nor in other species, such as rats (8,10) and hamsters (150)exposed to TRI. There is strong evidence that liver tumors observed in mice exposed to TRI are a result of the oxidative metabolism of TRI by cytochrome P450 to TCA (Figure 4). In line with this suggestion, TCA has been shown to cause peroxisome proliferation in the mouse liver (1521, a biochemical response associated with cancer induction in rodents (153). TRI-induced peroxisome proliferation could not be demonstrated in rats (1521,most likely due to saturation of the oxidative metabolism of TRI in this species (Figure 81, thereby limiting the maximal (threshold) levels of TCA (Figure 9) to below those required to induce this effect. An in vitro comparison of the oxidative metabolism of TRI to TCA in rat, mouse, and human hepatocytes further confirmed the species differences observed in vivo between rats and mice and showed low rates of TCA formation in human hepatocytes (152). Importantly, TCA-induced peroxisome proliferation, as observed in rat and mouse hepatocytes, could not be induced in human hepatocytes (152). It is, therefore, concluded that liver tumors seen in mice are unlikely to occur in either rats or humans. Increases in hepatic DNA synthesis and mitosis, but not unscheduled DNA synthesis, have also been reported in mice dosed with TRI either by gavage or by inhalation (34, 154, 155). Moreover, TCA has been shown to increase lipid peroxidation in the mouse liver, suggesting that the production of free radicals may be responsible for DNA damage, and consequently for mouse liver tumor formation (156,157). Indeed, the carcinogenic effects of TRI appeared to be closely related to L3H1thymidine incorporation in DNA, indicating increased DNA synthesis and/or repair (157). Importantly, the negative results on TRI-induced chromosomal damage in rats (63) and in humans (67) make the relevance of the mouse findings to both rat and human questionable. The effects of TRI and TRI-derived metabolites (notably, TCA, TCE, and chloral) on gap junction-mediated intercellular communication in cultured B6C3F1 mouse and Fischer 344 rat hepatocytes were also studied (158). This phenomenon is exhibited by a number of nongenotoxic carcinogens and tumor promoters. TRI and TCA were found to inhibit intercellular communication in mouse hepatocytes but not in rat hepatocytes (158). Whereas TRI inhibited intercellular communication in a cytochrome P450-dependent manner in mouse hepa-
Invited Review tocytes, TCA did not. One may argue that the speciesdependent effect of TRI on intercellular communication may be related to the different rates and the extents of metabolism of TRI by rat and mouse hepatocytes. However, the inhibitory effect of TCA on primarily mouse hepatocytes strongly suggests that other, as yet unknown factors in the mouse may also play a role in making mice more susceptible to these effects of TRI and TCA. These findings may account, in part, for the observed difference in susceptibility to TRI-induced liver carcinogenesis between rats and mice. Unfortunately, the effects of TRI and TRI-derived metabolites on gap junction-mediated intercellular communication in cultured human hepatocytes have not been investigated, thereby hampering definitive conclusions. The relative roles of TCA and DCA in the induction of mouse liver tumors have been compared (9,159). Both TCA and DCA have been shown to cause liver tumors (hepatocellular adenomas and carcinomas) when administered to male BGC3Fl mice in drinking water (concentration ranging from 1 to 5 g/L for 52-61 weeks), but not in male Fisher 344 rats at equivalent dose (0.05-5 g/L for 100-104 weeks) levels (156,160,161). It has been suggested that both peroxisome proliferation and DNA synthesis are required to induce tumors in mice upon TCA exposure (152,157). The apparent strain differences between mice in the hepatocarcinogenicity of TRI may be explained by strain differences in the oxidative metabolism to TCA. In Swiss and B&Fl mice where liver tumors were observed (8, IO),TCA accounted for 7-12% of the dose (441,whereas in NMRI mice where no liver tumors were found (1501, this metabolite was only 0.1% of the dose of TRI (30). Similarly, the levels of DCA differed, being 2% of the dose in Swiss and B ~ C ~ mice F I and 0.1% in NMRI mice. V.3. Species Differences in TRI-Induced Kidney Tumor Formution. TRI has been reported to cause a low incidence (3%) of kidney adenocarcinoma in male Sprague-Dawley rats upon inhalation exposure to 600 ppm amine-stabilized, epoxide-free TRI for 7 Wday, 5 dayslweek, during 104 weeks (10). Chronic exposure of male Fischer 344 and Osborne-Mendel rats to 1000 mgl kg epichlorohydrin-free TRI by gavage for 2 years caused severe nephrotoxicity (70-90%) and a low incidence (26%) of adenocarcinomas (8,162).The nephrocarcinogenic effects of TRI were reported neither for the mouse nor for female rats, either upon inhalation exposure or upon oral dosing (8,lO). A number of potential mechanisms, notably, chronic nephrotoxicity, protein droplet nephropathy, and mercapturic acid pathway metabolism of TRI, which are all known to be involved in the development of rat specific renal tumors, have been investigated for TRI (163). Studies performed in vivo using [35Sl-S-(1,2-dichloroviny1)-L-cysteine support a mechanism for damage and nephrogenic repair composed of (1) interaction of the toxin with the target cells, (2) necrosis and exfoliation, (3)loss of differentiation and cell growth, (4) recovery of the damaged area and cessation of cell growth, and (5) differentiation of the quiescent cells (164). In fact, nephrogenic repair may have similarities with the differentiation of the tubular epithelium during development (164). In addition, the possibility that hyaline droplet nephropathy (upon a-2p-globulin accumulation in renal proximal tubular cells), which is frequently observed in ethylene-induced animal cancer studies, is causally related to the development of renal tumors in
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 15 male rats cannot be ignored (163). In fact, there is a strong correlation between this epigenic effect and the development of male rat-specific kidney tumor ( I 65). Tumors have been observed in rat kidneys aRer chronic exposure to high doses of TRI. Also, in 1,2-DCV-Cys incubated renal tubules, DNA strand breaks were observed both in vivo and in vitro (166). At the concentrations necessary for tumor induction, however, TRI also produced severe nephrotoxicity. These tumors are probably caused by nonspecific factors associated with the onset of nephrotoxicity. In this case, kidney damage upon TRI exposure and the resulting cell division may facilitate the expression of the genotoxic potential of 1,2DCV-Cys, or alternatively, kidney damage may alone lead to tumors by a nongenotoxic mechanism. A key aspect of this process is that nephrocarcinogenicity has never been observed in the absence of severe and chronic kidney damage. Analysis of urine from rats and mice dosed by gavage, and from humans occupationally exposed to TRI, identified trace amounts of vicinal 1,2-DCV-Nac (12,43,47, 77, 1631, confirming the formation of this potentially nephrotoxic and potentially mutagenic metabolite by the mercapturic acid pathway. The less mutagenic geminal 2,2-DCV-Nac was also detected in rat urine (12).The urinary levels of these regioisomeric mercapturic acids in rat and mouse urine were extremely small, i.e., lower than 0.1% of the dose, even at the extremely high TRI dose levels used in these studies (77,163).Remarkably, in a study by Birner et al. (77)the levels of mercapturic acids (i.e., 1,2- and 2,2-DCV-Nac) in mouse urine were reported to be higher than those in rat urine (14.5 pM versus 10.1 pM, respectively), which would suggest (1) that formation of the DCV-Nac isomers are not causally related to the occurrence of rat-specific renal tumors and (2) that there exists a higher susceptibility of mice to develop renal tumors. However, when male Swiss Webster mice were dosed with 0.01-0.1 mg of 1,2-DCV-Cys/ mL in drinking water for up to 37 weeks, only nephrotoxicity was observed but no nephrocarcinogenicity (167). A role for the mercapturic acids of TRI in the nephrotoxicity and/or nephrocarcinogenicity by a genotoxic mechanism has also been suggested by Birner et al. (77). In line with this suggestion, traces of 1,2- and 2,2-DCVNac isomers were identified in urine of four metal cleaning workers exposed to TRI of undefined purity during a n 8 h work shift (77).However, details of the exposure were not given. Moreover, the principal compound on which the argument was based, namely 1,2DCV-Nac, was not quantified. It should be noted that 1,2-DCV-Cys, presumably formed after deacetylation of 1,2-DCV-Nac,is a better substrate for renal p-lyase than the corresponding 2,2-DCV-Cysisomer (14).In addition, bioactivation of the 1,2-DCV-Cysisomer by renal p-lyase may ultimately lead to the formation of chlorothioketene and the 2,2-DCV-Cys isomer to the less cytotoxic thioaldehyde (Figure 12) (14).Comparison of the relative levels of the DCV-Nac isomers with those of TCA in urine led Birner et al. (77)to conclude that the mercapturic acid pathway in the metabolism of TRI is more important in humans than in rodents. It should be noted, however, that urine was only collected for 16 h post-exposure despite the extremely long half-time of TCA in humans (t112 = 75-100 h) (52)and despite a s yet unknown halftimes of the 1,2- and 2,2-DCV-Nac isomers, thereby precluding a proper comparison of the relative extents
Goeptar et al.
16 Chem. Res. Toxicol., Vol. 8,No. 1, 1995 of the oxidative and glutathione conjugation pathway of TRI. 1,2-DCV-Cys and to a lesser extent 2,2-DCV-Cys are the likely precursor metabolites presumably involved in nephrotoxicity and nephrocarcinogenicity in rats. All evidence presented as yet suggests, however, that kidney tumors in the rat will not occur in the absence of severe and chronic kidney damage. The onset of kidney damage may only occur when the cytochrome P450 pathway in TRI metabolism becomes saturated. In this context, GSH conjugation of TRI in the rat has been shown to be a significant pathway only at high dose levels causing saturation of the cytochrome P450 pathway (Figure 8) (163).Lack of saturation of the cytochrome P450 pathway could explain why the 1,2-DCV-Cysisomer does not play a role in the TRI-induced nephrotoxicity in the mouse (Figure 8). In a similar vein, it would appear unlikely that humans are exposed to TRI doses approaching saturation of the cytochrome P450 metabolism. Furthermore, hyaline droplet nephropathy is only seen in male rats but not in female rats nor in mice of either sex and is generally considered not to be relevant to humans (165). V.4. H u m a n Toxicity and Cancer Epidemiology after Exposure to TRI. N-Acetyl-p-D-glucosaminidase (NAG, P-hexosaminidase), a lysosomal enzyme located mainly in the proximal tubules, is believed to be a sensitive indicator of renal injury (97,168).In a recent cross-sectional study of metal degreasers in central Sweden (17),86% of 8 h TRI air concentrations were well below 50 mg/m3. Normal levels of urinary NAG (0.19 unit"mo1 of creatinine) were found in urine samples from 29 workers when compared to a reference group. The authors, therefore, concluded that TRI was not nephrotoxic a t chronic low exposure levels (17).Thus metabolism of TRI to the nephrotoxic metabolites (including DCV-Cys isomers) in human occurs a t best at levels which are not significant from a toxicological point of view. A number of epidemiological studies in humans concerning the possible carcinogenicity of TRI have been performed. The first epidemiological study was conducted by Axelson et al. (20),and subsequently two cohort studies (21,221, two liver cancer studies (169,1701, one small cohort study (171),and one colon cancer study (172) on TRI were reported. Two of these studies (21,171) included highly exposed populations for which repeated instances of acute nongenotoxic effects were reported. In two Scandinavian cohort studies (20,21)average inhalatory air exposures were reported to be below 50 ppm of TRI. None of the studies described above indicated any link between exposure to TRI and cancer mortality or the incidence of liver cancer or colon cancer. However, all these studies suffered from shortcomings such as a poor study design or small cohorts, thereby hampering definitive conclusions. Moreover, past exposure levels could never be exactly quantified, coexposure to other chemicals ke., impurities) was usually not accounted for, and other personal factors (Le., tobacco consumption, diet, social-economic factors) also could not be evaluated. Finally, interpretation was often hampered by the socalled healthy-worker effect. Recently, several well-designed and well-conducted studies became available. Spirtas et al. (24,25)studied mortality in a cohort of 6929 workers with a followup of up to 30 years. The study included a n extensive exposure assessment which indicated very high TRI exposures (as
high as 600 ppm), particularly in the first part of the study period. Axelson et al. (26)reported mortality as well as morbidity in a n extension to 1670 of the initial cohort, with a n increased followup period of 37 years. Again, none of these studies showed any association between exposure to TRI and cancer in general or any specific type of cancer.
VI. Conclusion VI.1. General Toxicity. The solvent properties of TRI have resulted in its widespread use in metal degreasing and a wide variety of other industrial applications. TRI has now been in common use for more than 50 years. During this period of time, workers have been exposed to a wide range of concentrations, in some cases for periods of 25 years or longer. This has allowed the compilation of a great data base about the effects of TRI on human health. Moreover, information has been supplemented by numerous studies in experimental animals. Epidemiological studies on more than 15 000 individuals with a followup of more than 25 years have shown no evidence of a n association between human exposure to TRI and increased incidence of cancer or cancer mortality. However, several of these studies had more or less serious shortcomings. VI.2. Lung TumorsInduced by TRI. An increased incidence of lung tumors has been reported in female B6C3F1 and male Swiss mice exposed to TRI by inhalation. The effect was not observed in male BBCSF~ nor in female Swiss mice nor in rats. This apparent strain-, sex-, and lung-specific response fails to resolve the issue of whether or not TRI is a carcinogenic hazard to man. Mechanistic studies on mouse lung tumor formation have explained the sex and species differences. In this context, chloral formation (Figure 4) in Clara cells, containing relatively high cytochrome P450 concentrations, has been identified to be responsible for the development of mouse lung tumors. Importantly, lung tumors have not been found in humans after long-term occupational exposure to TRI (Table 2). Moreover, the finding that the micespecific lesions in the lung have not been observed in humans (Table 2) rules out this adverse effect of TRI on the human respiratory tract a t forseeable low levels of exposure. Therefore, the relevance of the observed TRIinduced mouse-specific lung tumor formation is questionable for human health hazards. VI.3. Liver Tumors Induced by TRI. TRI causes a n increase in the incidence of liver cancer in both sexes of B6C3F1and Swiss mice following either gavage or inhalatory exposure, but not in NMRI and Ha:ICR mice nor in rats. A rodent-specific link between peroxisome proliferation, DNA synthesis, inhibition of intercellular communication, and cancer (Table 2) suggests that these responses are the basis of the hepatocarcinogenicity induced by TRI. The identification of TCA in cancer bioassays as the responsible metabolite for these effects confirmed this hypothesis. However, when TCA was administered to both rats and mice, liver cancer was only observed in mice and not in rats. The reason for this species selectivity in liver effects is explained by the kinetic behavior of TRI and TCA in rodents. Both rats and mice have a considerable capacity to metabolize TRI to TCA and TCE, the maximal capacities being closely related to the relative surface areas rather than to their body weights. Oxidative metabolism of TRI in rats is
Invited Review
linearly related to dose at lower dose levels, but it becomes saturated at higher dose levels (Figure 8). Thus, an important difference between rats and mice is the lower saturation concentration in the former species (Figure 8). Therefore, it is tempting to suggest that TRIinduced liver tumors in mice is a threshold phenomenon. Inasmuch as oxidative TRI metabolism in rat becomes saturated at relatively low dose levels (Figure 81, the threshold concentration of TCA to induce liver tumors is not easily exceeded in this species. This aspect of the species difference is clearly seen a t the blood levels of TCA, being 7 times higher in the male mouse than in the male rat when exposed by gavage to 1000 mgkg TRI (Figure 9). The threshold response of TCA for the induction of liver cancer in mice is also consistent with a nongenotoxic mechanism of TRI-induced hepatocarcinogenicity. Carcinogenicity studies also revealed that NMRI and Ha: ICR mice are not as susceptible to TRI-induced liver cancer in contrast to B6C3F1 and Swiss mice. The apparent strain difference is explained by the kinetic behavior of TCA. TCA levels in NMRI mice are 100-fold lower than in B&F1 mice, which corresponds to the higher susceptibility of B&F1 mice to develop liver tumors. The relevance of the mechanisms of liver tumor formation in B6C3F1 and Swiss mice for humans exposed to TRI has been assessed in studies comparing metabolic rates in mice, rats, and human. In contrast to the rat, the oxidative metabolism of TRI to TCA in humans is not limited by saturation. In this respect, humans resemble the mouse and might be abIe to produce sufficient TCA to induce peroxisome proliferation and consequently liver cancer. However, there are significant differences between mice and humans. First, humans metabolize approximately 60 times less TRI on a body weight basis than mice at similar exposure levels. Second, TCA has been shown to induce peroxisome proliferation in mouse hepatocytes but not in human hepatocytes (Table 2). Consequently, the combination of extensive oxidative metabolism of TRI to TCA and the ability of TCA to induce peroxisome proliferation appear to be unique to B6C3FI and Swiss mice. v1.4. Kidney Tumors Induced by TRI. TRI-induced renal toxicity and tumors were found in Sprague-Dawley, Fischer 344, and Osborne-Mendel rats. These nephrocarcinogenic effects of TRI were specific to male rats and were not seen in female rats nor in mice of either sex. 1,2-DCV-Cys,formed from TRI via the mercapturic acid pathway (Figure ll),has been identified as a likely metabolite involved in the observed renal toxicity and probably also in the renal carcinogenicity in rats. TRI is metabolized by a minor pathway involving initial hepatic GSH conjugation of TRI (Figure 10). The resulting DCV-G is further metabolized (Figure 11) and excreted in urine as two regioisomeric mercapturic acids, namely, vicinal 1,2-DCV-Nacand geminal 2,2-DCV-Nac (Figure 12). 1,2-DCV-Cys (the precursors of 1,P-DCVNac) is a substrate for the renal L-cysteine S-conjugate ,!?-lyase,and it is more mutagenic and cytotoxic than 2,2DCV-Cys (the precursors of 2,2-DCV-Nac). Metabolism of TRI via the mercapturic acid pathway is consistent with the male rat as susceptible species, since GSH conjugation, GGT-activity, and ,!?-lyaseactivity (Table 1) are considerably higher in male rats than in female rats or in mice of either sexes. However, even in the male rat, the metabolism of TRI via the mercapturic acid pathway is extremely small and is only significant when
Chem. Res. Toxicol., Vol. 8, No.1, 1995 17 the oxidative metabolism of TRI is saturated (Figure 8). The bioactivation of 1,2-DCV-Cys(Figure 12) may be considered as the initiating step in the onset of nephrotoxicity in the rat, although the precise biological mechanisms by which these metabolites exert their nephrocarcinogenic effects are not yet fully understood. A key aspect in the onset of nephrocarcinogenicity in rats, however, is that it will not occur in the absence of severe and chronic nephrotoxicity. This suggests that the alkylating effects of the reactive metabolites (most likely thioketenes) derived from bioactivation of 1,2-DCV-Cys by j3-lyase (Figure 12) may not be sufficient to cause kidney tumors. In line w i t h this, male rat-specific protein droplet nephropathy may also be considered as a contributory factor in kidney tumor development (Table 2). An attractive hypothesis is that kidney damage and resulting increased cell division will facilitate the expression of the genotoxic potential of the DCV-Cys-derived metabolites. Of course, the exact role of kidney damage in the possible carcinogenesis of 1,2-DCV-Cys remains to be established. The specific activity of j3-lyase, the enzyme most likely involved in the bioactivation of DCV-Cys isomers, is similar in humans to that in the mouse and only 10% of that in the rat (Table 1). Moreover, human TRI metabolism via the mercapturic acid pathway resembles that of the mouse. It is, therefore, questionable whether humans are able to produce sufficient DCV-Cys isomers from TRI to cause first nephrotoxicity and subsequent nephrocarcinogenicity. The link between a high &lyase activity (Table 1)and the occurrence of kidney damage in the male rat (Table 2) a t relatively high TRI dose levels is supported by the facts that TRI is neither nephrotoxic nor nephrocarcinogenic in either humans or in mice. However, each other step in the metabolism of TRI via the mercapturic acid pathway (notably GSH conjugation, GGT- and dipeptidase-catalyzed degradation of 1,2-DCVG, and the balance between N-acetylation and N-deacetylation) may also be essential for the onset of nephrotoxicity and nephrocarcinogenicity in the rat (Figure 11). Moreover, chronic and severe nephrotoxicity and protein droplet accumulation may be additional factors in kidney tumor development (Table 2). It is, therefore, tempting to suggest that the nephrocarcinogenic effects of TRI arise from a combination of events which seem to be unique to the male rat. An important finding is also that the occurrence of nephrotoxicity and nephrocarcinogenicity in the male rat is dose-dependent. More specifically, cytotoxic kidney damage is a feature of high and continuous exposure to TRI over prolonged periods of time. This is unlikely to occur in humans during occupational exposure. In fact, TRI has been found not to be nephrotoxic in humans chronically exposed to low levels of TRI (50 mg/m3). Moreover, protein droplet nephropathy has been shown to be a threshold phenomena and as such not to be relevant to either mice or humans. Consequently, it is unlikely that the renal tumors which are seen in rats a t nephrotoxic dose levels are relevant to human health hazards a t reasonably foreseeable low levels of exposure.
References (1) Davidson, I. W. F., and Belites, R. P. (1991) Consideration ofthe
target organ toxicity of trichloroethylene in terms of metabolite toxicity and pharmacokinetics. Drug Metab. Rev. 23, 493-599. ( 2 ) Bruckner, J. V., Davis, B. D., and Blancato, J. N. (1989) Metabolism, toxicity, and carcinogenicity of trichloroethylene. CRC Crit. Rev. Toxicol. 20, 31-50.
18 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 (3) Brown, L. P., Farrar, D. G., and De Rood, C. G. (1990) Health risk assessment of environmental exposure to trichloroethylene. Regul. Toxicol. Pharmacol. 11, 24-41. (4) McKinney, L. L., Picken, J. C., Jr., Weakly, F. B., Eldridge, A. C., Campbell, R. E., Cowan, J. C., and Biester, H.E. (1959) Possible toxic factor of trichloroethylene-exctraded soybean oil meal. J . Am. Chem. Soc. 81,909-915. ( 5 ) Schulze, M. O., Derr, R. F., Mizuno, N. S.,Joel, D. D., and Sautter, I. (1962) Effect of phenylalanine on toxicity of S4dichlorovinyl)L-cysteine in the rat and calf. Proc. SOC. Exp. Biol. Med. 111,499506.
(6) Schumacher, H., and Grandjean, E. (1960) Comparative studies on the narcotic activity and acute toxicity of nine solvents. Arch. Gewerbepathol. Gewerbehyg. 18, 109-119. (7) Reinhardt, C. F., Mullin, L. S., and Maxfield, M. E. (1973) Epineprine-inducedcardiac arrhythmia potential of some common industrial solvent. J. Occup. Med. 16, 953-955. (8)National Toxicology Program (1986) Carcinogenesis Studies of Trichloroethylenes(without Epichlorohydrin) (CAS No. 79-01-6) in F344 Rats and B&$I Mzce (Ciauage Studies), NIH Publication NO. 83-1799, NTP TR-243. (9) Larson, J. L., and Bull, R. J. (1992) Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate. Toxicol. Appl. Pharmacol. 116,278-285. (10) Maltoni, C., Lefemine, G., and Lotti, G. (1986) Experimental Research on Trichloroethylene Carcinogenesis. In Archives of Research of Industrial Carcinogenesis (Maltoni,C., and Mehlman, M. A., Eds.) pp 1-393, Princeton Scientific Publishing Co., Princeton. (11) Dekant, W., Metzler, M., and Henschler, D. (1986) Identification of S-(1,Z-dichloroviny1)-N-acetyl-cysteine as a urinary metabolite of trichloroethylene: A possible explanation for ita nephrocarcinogenicity in male rats. Biochem. Pharmacol. 36, 2455-2458. (12) Commandeur, J. N. M., and Vermeulen, N. P. E. (1990) Identification of N-acetyl-(2,2-dichlorovinyl)-and N-acetyl-(1,2-dichloroviny1)-L-cysteine as two regioisomeric mercapturic acids of trichloroethylene in the rat. Chem. Res. Toxicol. 3, 212-218. (13) Green, T., and Odum, J. (1985) Structure/activity studies of the nephrotoxic and mutagenic action of cysteine conjugates of chloro and fluoroalkenes. Chem.-Biol. Interact. 64, 15-31. (14) Commandeur, J. N. M., Boogaard, P. J., Mulder, G. J., and Vermeulen, N. P. E. (1991) Mutagenicity and cytotoxicity of two regioisomeric mercapturic acids and cysteine-S-conjugates of trichloroethylene. Arch. Toxicol. 66,373-380. (15) Phoon, W. H., Chan, M. 0. Y., Rajan, V. S.,Tan, J. K., Thirumorthy, T., and Goh, C. L. (1984) StevensJohnson syndrome associated with occupational exposure to trichloroethylene. Contact Dermatitis 10, 270-276. (16) Dhuner, K G., Nordquist, P., and Renstrom, R. (1957) Cardiac irregularities due to trichlorethylene poisoning. Acta Anaesth. Scand. 1, 121-135. (17) Selden, A., Hultberg, B., Ulander, A., and Ahlborg, G., Jr. (1993) Trichloroethylene exposure in vapour degreasing and the urinary excretion of N-acetyl-B-D-glucosaminidase. Arch. Toxicol. 67,224226. (18) Theile, D. L., Eigenbrodt, E. H., and Ware, A. J. (1982) Cirrhosis after repeated trichloroethylene and l,l,l-trichloroethane exposure. Gastroenterology83, 926-929. (19) Ayre, P. (1945)Accute yellow atrophy after trilene anaesthia. Br. Med. J. 2, 784. (20) Axelson, O., Anderson, K., Hogstedt, C., Holmbert, B., Molina, G., and de Verdier, A. (1978)A cohort study on trichloroethylene exposure and cancer mortality. J. Occup. Med. 20, 194-196. (21) Tola, S., Vilhunen, R., Jarvinen, E., and Korkala, M. L. (1980) A cohort study on workers exposed to trichloroethylene. J . Occup. Med. 22,737-740. (22) Shindel, S., and Ulrich, S. (1985) A cohort study of employees of a manufacturing plant using trichloroethylene. J. Occup. Med. 27,577-579. (23) Blair, A., Decode, P., and Grauman, D. (1979) Causes of death among laundry and dry cleaning workers. Am. J. Public Health 69,509-511. (24) Spirtas, R., Stewart, P., and Lee, S. (1991) Retrospective cohort mortality study of workers a t a n aircraft maintenance facility. I. Epidemiological results. Br. J. Ind. Med. 48, 515-530. (25) Spirtas, R., Lee, S., and Marano, D. (1991) Retrospective cohort mortality study of workers at an aircraft maintenance facility. 11. Exposures and their assessment. Br. J . Ind. Med. 48, 531537. (26) Axelson, O., Selden, A,, Andersson, K., and Hogstedt, C. (1994) Updated and expanded Swedish cohort study on trichloroethylene and cancer risk. J. Occup. Med. (in press). (27) Vermeulen, N. P. E., Donn6-Op den Kelder, G., and Commandeur, J. N. M. (1993)Formation of and protection against toxic reactive
Goeptar et al. intermediates. In Perpsectives in Medicinal Chemistry (Testa, B., Kyburz, E., Fuhrer, W., and Giger, R., Eds.) pp 573-593, Verlag Helvetica Chimica Acta, Basel. (28) Sato, A., Nakajima, T., Fujiwara, Y., and Murayama, N. (1977) A pharmacokinetic model to study the excretion of trichloroethylene and ita metabolites after inhalation exposure. Br. J. Ind. Med. 34,55-63. (29) Stewart, R. D., and Dodd, H. C. (1964) Absorption of carbon tetrachloride, trichloroethylene, tetrachloroethylene, methyl chloride and l,l,l-trichloroethane through the human skin. Am. Ind. Hyg. ASSOC.J. 26, 439-446. (30) Dekant, W., Metzler, M., and Henschler, D. (1984) Novel metabolites of trichloroethylene through dechlorination reactions in rats, mice and humans. Biochem. Pharmacol. 33, 2021-2027. (31) Eger, I., and Larson, C. P. (1964) Anaesthetic solubility in blood and tissues: Values and significance. Br. J. Anaesth. 36, 140149. (32) Powel, J. F. (1947) Solubility and distribution coefficient of trichloroethylene in water, whole blood and plasma. Br. J. Ind. Med. 4,233-236. (33) Savolainen, H.,P f a i , P., Tengen, M., and Vaino, H. (1977) Trichloroethyleneand l,l,l-trichlomethane: Effects on brain and liver aRer five days intermittent inhalation. Arch. Toxicol. 38, 229-237. (34) Stott, W. T., Quast, J. F., and Watanabe, P. G. (1982) The pharmacokinetics and macromolecular interactions of trichloroethylene in mice and rats. Toxicol. Appl. Pharmacol. 62, 137151. (35) Withey, J. R., and Collins, B. T. (1980) Chlorinated aliphatic hydrocarbons used in the food industry: The comparative pharmacokinetics of methylene chloride, 1,2-dichloroethane, chloroform, and trichloroethylene after i.v. administration in the rat. J. Enuiron. Pathol. Toxicol. 3, 313-332. (36) Prout, M. S., Provan, W. M., and Green, T. (1985) Species differences in response to trichloroethylene. I. Pharmacokinetics in rata and mice. Toxicol. Appl. Pharmacol. 79, 389-400. (37) Ikeda, M., Ohtsuji, H., Imara, T., and Komoike, Y. (1972) Urinary excretion of total trichloro-compounds, trichloroethanol, and trichloroacetic acid as a measure of exposure to trichloroethylene and tetrachloroethylene. Br. J. Ind. Med. 29, 328-333. (38) Ikeda, M. (1977) Metabolism of trichloroethylene and tetrachloroethylene in human subjects. Enuiron. Health Perspect. 21,239245. (39) Fernandez, J. G., Droz, P. O., Humbert, B. E., and Caperos, J. R. (1977) Trichloroethylene exposure: Simulation of uptake, excretion, and metabolism using a mathematical model. Br. J. Ind. Med. 34,43-55. (40) Monster, A. C. (1979) Difference in uptake, elimination, and metabolism in exposure to trichloroethylene, l,l,l-trichloroethane, and tetrachloroethylene. Int. Arch. Occup. Enuiron. Health 42,311-317. (41) Astrand, I., and h u m , P. (1976) Exposure to trichloroethylene. I. Uptake and distribution in man. Scand. J. Work, Enuiron. Health 2, 199-211. (42) Sato, A., Nakajima, T., and Koyama, Y. (1981) Dose related effecta of a single dose of ethanol on the metabolism in rat liver of some aromatic and chlorinated hydrocarbons.Toxicol. Appl. Pharmacol. 60,8-15. (43) Dekant, W., Schultz, A., Metzler, M., and Henschler, D. (1986) Absorption, elimination and metabolism of trichloroethylene: A quantitative comparison between rats and mice. Xenobiotica 16, 143-152. (44) Green, T., and Prout, M. S. (1985) Species differences in response to trichloroethylene 11. Biotransformation in rats and mice. Toxicol. Appl. Pharmacol. 79, 401-411. (45) Bolt, H. M., and Filser, J. G. (1977) Irreversible binding of chlorinated ethylenes to macromolecules. Enuiron. Health Perspect. 21, 107-112. (46) Banerjee, S.,and van Duuren, B. L. (1978) Covalent binding of the carcinogenic trichloroethylene to hepatic microsomal proteins and to exogenous DNA in vitro. Cancer. Res. 38, 776-780. (47) Dekant, W., Koob, M., and Henschler, D. (1990) Metabolism of trichloroethylene. In vivo and in vitro; Evidence for activation by glutathione conjugation. Chem.-Biol.Interact. 73, 89-101. (48) Rauncy, J. L., Kramer, J. C., and Lasker, J. M. (1993) Bioactivation of halogenated hydrocarbons. CRC Crit. Rev. Toxicol. 23, 1-20. (49) Guengerich, F. P., Kim, D. H., and Iwasaki, H.(1991) Role of human cytochrome P-450IIE1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168179. (50) Miller, R. E., and Guengerich, F. P. (1982) Oxidation of trichloroethylene by liver microsomal cytochrome P450: Evidence for chlorine migration in a transition state not involving trichloroethyleneoxide. Biochemistry 21, 1090-1097.
Invited Review (51) Fox, B. G., Borneman, J. G., Wackett, L. P., and Lipscomb, J. D. (1990) Haloalkene oxidation by the soluble methane monooxygenase from Methylosinus trichosporium OB3b: Mechanistic and environmental implications. Biochemisty 29, 6419-6427. (52) Muller, G., Spassovski, M., and Henschler, D. (1974)Metabolism of trichloroethylene in man. 11. Pharmacokinetics of metabolites. Arch. T~ricol.32, 283-295. (53) Bartonicek, V. (1962) Metabolism and excretion of trichloroethylene after inhalation by human subjects. Br. J . Ind. Med. 19, 134-141. (54) Monster, A. C., Boersma, G., and Duba, W. C. (1976) Kinetics of trichloroethylene in repeated exposure of volunteers. Int. Arch. Occup. Environ. Health 42, 283-292. ( 5 5 ) Nomiyama, K., and Nomiyama, H. (1977) Dose-response relationship for trichloroethylene in man. Znt. Occup. Environ. Health 39,237-248. (56) Monster, A. C., Boersma, G., and Duba, W. C. (1976) Pharmakokinetics of trichloroethylene in volunteers: Influences of workload and exposure concentration. Int. Arch. Occup. Environ. Health 38, 87-102. (57) Filser, J. G., and Bolt, H. M. (1979) Pharmacokinetics of halogenated ethylenes in rats. Arch. Toxicol. 42, 123-136. (58) Muller, G., Spassovski, M., and Henschler, D. (1972) Trichloroethylene exposure and trichloroethylene metabolites in urine and blood. Arch. To~icol.29, 335-340. (59) Crebelli, R., Bignami, M., Conti, L., and Carere, A. (1982) Mutagenicity of trichloroethylene in Salmonella typhimurium TA100. Ann. Ist. Super. Sanitci 18, 117-122. (60) Simmon,V. F., Kauhanen, K., and Tardiff, R. G. (1977)Mutagenic activity of chemicals identified in drinking water. Prog. Genet. Toxicol. (Proc. Int. Conf.) 2, 249-258. (61) Crebelli, R., Conti, G., Conti, L., and Carere, A. (1985) Mutagenicity of trichloroethylene, trichloroethanol and chloral hydrate in Aspergillus nidulans. Mutat. Res. 155, 105-111. (62) White, A. E., Eger, E. I., Wolff, S., and Stevens, W. C. (1979) Sister chromatic exchange induced by inhalated anesthetics. Anaestheswlogy 50, 426-430. (63) Shimada, T., Swanson,A. F., Leber, P., and Williams, G. M. (1985) Activities of chlorinated ethane and ethylene compounds in the Salmonellahat microsome mutagenesis and rat hepatocytelDNA repair assays under vapour phase exposure conditions. Cell. Biol. Toxicol. 1, 159-179. (64) Belites, R. P., Brusick, D. J., and Mecler, F. J. (1980)Teratogenicmutagenic risk of workplace contaminants: Trichloroethylene, perchloroethylene, and carbon disulfide. NIOSH Contract Report No. 210-77-0047from Litton Benetics, NTIS Publication No. PB82-185-075,National Technical Information Service, Springfield, VA. (65) Sbrana, I., Lascialfari, D., and Loprieno, N. (1985) TCE induces micronuclei but not chromosomal abberations in mouse bone marrow cells. N ICEM; Stockholm, Abstract, p 163. (66) Allen, J. W., Collins, B. W., and Evansky, P. A. (1994) Spermatid micronucleus analyses of trichloroethylene and chloral hydrate in mice. Mutat. Res. 323, 81-88. (67) Nagaya, T., Ishikawa, N., and Hata, H. (1989) Sister chromatid exchanges in lymphocytes of workers exposed to trichloroethylene. Mutat. Res. 222, 279-282. (68) Waskell, L. (1978) A study of the mutagenicity of anaesthetics and their metabolites. Mutat. Res. 57, 141-153. (69) Leuschner, J., and Leuschner, F. (1991) Evaluation of the mutagenicity of chloral hydrate in vitro and in vivo. Arzneim. Forsch.lDrug Res. 41, 1101-1103. (70) van Bladeren, P. J. (1988) Formation of toxic metabolites from drugs and other xenobiotics by glutathione conjugation. Trends Pharmacol. Sci. 9, 295-298. (71) Elfarra, A. A., and Dohn, D. R. (1988) Biosynthesis and biotransformation of glutathione S-conjugates to toxic metabolites. CRC Crit. Rev. Toxicol. 18, 311-341. (72) Elfarra, A. A., Jakobson, I., and Anders, M. W. (1986) Identification of S-(1,2-dichlorovinyl)glutathione-inducednephrotoxicity. Biochem. Pharmacol. 35,283-288. (73) Cohen, G. M., and Moss, E. J. (1987) Tissue distribution of drug metabolizing enzymes in relation to toxicity. In Drug Metabolism-From Molecules to man (Benford, D.J., Bridges, J. W., and Gibson, G. G., Eds.) pp 690-707, Taylor & Francis, London. (74) Oesch, F., and Wolf, C. R. (1989) Properties of the microsomal and cytosolic glutathione transferase involved in hexachloro-1: 3-butadiene conjugation. Biochem. Pharmacol. 38, 353-359. (75) Green, T. (1990) Chloroethylenes: A mechanistic approach t o human risk evaluation. Annu. Rev. Pharmacol. Toxicol. 30, 7389. (76) Green, T., Odum, J., Nash, J. A., and Foster, J. R. (1990) Perchloroethylene induced rat kidney tumour. An investigation
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 19 of the mechanisms involved and their relevance to man. Toxicol. Appl. Pharmacol. 103, 77-89. (77) Birner, G., Vamvakas, S., Dekant, W., and Henschler, D. (1993) Nephrotoxic and genotoxic N-acetyl-S-dichlorovinyl-L-cysteine is a urinary metabolite after occupational 1,1,2-trichloroethene exposure in humans: Implications for the risk of trichloroethene exposure. Environ. Health Perspect. 99, 281-284. (78) Kanhai, W., Dekant, W., and Henschler, D. (1989) Metabolism of the nephrotoxin dichloroacetyleneby glutathione conjugation. Chem. Res. Toxicol. 2, 51-56. (79) Meister, A., and Tate, S. S. (1976) Glutathione and related y-glutamyl compounds: Biosynthesis and utilization. Annu. Rev. Biochem. 45, 559-604. (80) Cyrthoys, N. P., and Hughey, R. P. (1979) Characterization and physiological function of rat renal y-glutamyltranspeptidase. Enzyme 24,383-403. (81) Lash, L. H., and Anders, M. W. (1986) Cytotoxicity of S-(1,2dichloroviny1)glutathioneand S-(l,2-dichlorovinyl)-L-cysteinein isolated rat kidney cells. J . Biol. Chem. 261, 13076-13081. (82) Monks, T. S., Lau, S. S., Highet, R. J., and Gillette, J. R. (1985) Glutathione conjugates of 2-bromohydroquinoneare nephrotoxic. Drug Metab. Dispos. 13, 553-559. (83) Kramer, R. A., Foureman, G., Green, K. E., and Reed, D. J. (1987) Nephrotoxicity of S-(2-chloroethyl)glutathione in the Fischer r a t Evidence for y-glutamyltranspeptidase-independentuptake by the kidney. J. Pharmacol. Exp. Ther. 242, 741-748. (84) Hinchman, C. A., and Ballatori, N. (1990)Glutathione-degrading capacities of liver and kidney in different species. Biochem. Pharmacol. 40, 1131-1135. (85) Horiuchi, S., Inoue, M., and Morino, Y. (1978) y-Glutamyl transpeptidase. Sideness of its active site and renal brush border membranes. Eur. J . Bwchem. 87, 429-437, (86) Abott, W. A., and Meister, A. (1986) Intrahepatic transport and utilization of biliary glutathione and its metabolites. Proc. Natl. Acad. Sci. U S A . 83, 1246-1250. (87) Abott, W. A., Bridges, R. J., and Meister, A. (1984) Extracellular metabolism of glutathione accounts for its disappearance from the basolateral circulation of the kidney. J. Bwl. Chem. 259, 15393-15400. (88) Anderson, M. E., and Meister, A. (1986) Inhibition of y-glutamyl transpeptidase and induction of glutathione by y-glutamyl amino acids. Proc. Natl. Acad. Sci. U S A . 83, 5029-5032. (89) Shaw, L. M., London, J. W., and Petersen, L. E. (1978) Isolation of y-glutamyltransferase from human liver and comparison with the enzyme from human kidney. Clin. Chem. 24, 905-914. (90) Hughey, R., Rankin, B., Elce, J., and Curthoys, N. P. (1978) Specificity of a particulate rat renal peptidase and its localization along with other enzymes of mercapturic acid synthesis. Arch. Biochem. Biophys. 186, 211-217. (91) Jones, D. P., MoldBus, P., Stead, A. H., Ormstad, K., Jornvall, H., and Orrenius, S. (1979) Metabolism of glutathione and glutathione conjugate by isolated kidney cells. J . Bwl. Chem. 254, 2787-2792. (92) Curthoys, N. P., and Shapiro, R. (1975)y-Glutamyltranspeptidase in intestinal brush border membranes. FEBS Lett. 58,230-233. (93) Dass, P. D., Misra, R. P., and Welbourne, T. C. (1981) Presence of y-glutamyltransferase in the renal microvascular compartment. Can. J. Biochem. 59,383-386. (94) Sochor, M., El Sheik, 0. K., and Mclean, P. (1980) y-Glutamyltransferase in glomeruli and tubulus of rat kidney cortex: Effect of experimental diabetes. Enzyme 25, 205-208. (95) Spater, H. W., Poruchynsky, M. S., Quintana, N., Inoue, M., and Novikoff, A. B. (1982) Immunocytochemical localization of y-glutamyltransferase in rat kidney with protein A-horseradish peroxidase. Proc. Natl. Acad. Sci. U.SA. 79, 3547-3550. (96) Inoue, M., Kinne, R., Tran, T., Biempica, L.. and Arias. I. M. (1983) Rat liver candicular membrane vesicles. J . Biol. Chem. 258, 5183-5188. (97) Meier, P. J., Sztul, E. S., Reuben, A., and Boyer, J. L. (1984) Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver. J . Cell Biol. 98, 991-1000. (98) Ballatori, N., Jacob, R., and Boyer, J. L. (1986) Intrabiliary glutathione hydrolysis. A source of glutamate in bile. J. Biol. Chem. 261, 7860-7865. (99) Hirata, E., and Takahashi, H. (1981) Degradation of methyl mercury glutathione by the pancreatic enzymes in bile. Toxicol. Appl. Pharmacol. 58, 483-491. (100)Tateishi, M. (1983) Methylthiolated metabolites. Drug Metab. Rev. 14,1207-1234. (101) Stijntjes, G. J., te Koppele, J. M., and Vermeulen, N. P. E. (1992) HPLC-fluorescence assay of the pyruvic acid to determine cysteine conjugate 8-lyase activity. Anal. Biochem. 206, 334343.
20 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 Yamauchi, A., Stijntjes, G. J., Commandeur, J. N. M., and Vermeulen, N. P. E. (1993) Purification of glutamine transaminase Wcysteine conjugate 8-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC. Protein Expression Purif, 4, 552-562. Anderson, P. M., and Schultze, M. 0. (1965) Cleavage of S-(1,2dichloroviny1)-L-cysteineby an enzyme of bovine origin. Arch. Biochem. Biophys. 111,593-602. Bhattacharya, R. IC,and Schultze, M. 0. (1967) Enzymes from bovine and turkey kidneys which cleave S-(1,2-dichlorovinyl)L-cysteine. Comp. Biochem. Physiol. 22, 723-735. Bhattacharya, R. K., and Schultze, M. 0. (1972) Properties of DNA treated with S-(1,2-dichlorovinyl)-L-cysteineand a lyase. Arch. Bwchem. Biophys. 153, 105-115. Terracini, B., and Parker, V. H. (1965) A pathological study on the toxicity of S-dichlorovinyl-L-cysteine. Food Cosmet. Toxicol. 3, 67-74. Lock, E. A. (1988) Studies on the mechanism of nephrotoxicity and nephrocarcinogenicity of halogenated alkenes. CRC Crit. Rev. Toxicol. 19, 23-42. Dekant, W., Vamvakas, S., and Anders, M. W. (1989) Bioactivation of nephrotoxic alkenes by glutathione conjugation: Formation of toxic and mutagenic intermediates by cysteine conjugate @-lyase.Drug Metab. Rev. 20, 43-83. Stevens, J., Hayden, P., and Taylor, G. (1986) The role of glutathione conjugate metabolism and cysteine conjugate ,%lyase in the mechanism of S-cysteine conjugate toxicity in LLC-PKI cells. J. Biol. Chem. 261, 3325-3332. Monks, T. J., Anders, M. W., Dekant, W., Stevens, J. L., Lau, S. S., and van Bladeren, P. J. (1990) Contemporary issues in toxicolom. Glutathione coniueate mediated toxicitv. Toxicol. Appl. Prarmacol. 106, 1-19'. Finkelstein. M. B., Baggs. R. B.. and Anders. M. W. (1992) Nephrotoxicity of the &tathione and cysteine coqjugates of 2-bromo-2-chloro-l,l-difluoroethene. J. Pharmacol. Exp. Ther. 261, 1248-1252. Dohn, D. R., Leininger, J. R., Lash, L. H., Quebbemann, A. J., and Anders, M. W. (1985) Nephrotoxicity of S-(2-chloro-l,1,2trifluoroethy1)-L-glutathioneand S-(2-chloro-l,l,2-trifluoroethy1)-L-cysteine, the glutathione and cysteine conjugates of chlorotrifluoroethylene.J. Phrmucol. Exp. Ther. 235,851-857. Coles, B. (1985) Effects of modifying structure on electrophilic reactions with biological nucleophiles. Drug Metab. Rev. 15, 1307-1334. Dekant, W., Urban, G., Gdrsmann, C., and Anders, M. W. (1991) Thioketene formation from a-haloalkenyl 2-nitrophenyl disulfides: Models for biological reactive intermediates of cytotoxic S-conjugates. J . Am. Chem. SOC.113, 5120-5122. Dekant, W., Vamvakas, S., Berthold, K., Smidt, S., Wild, D., and Henschler, D. (1986) Bacterial @-lyasemediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene. Chem.-Biol. Interact. 60, 31-45. Vamvakas, S., Berthold, K., Dekant, W., and Henschler, D. (1988) Bacterial cysteine conjugate ,%lyase and metabolism of cysteine conjugates: Structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates. Chem.-Biol.Interact. 65, 59-71. Stevens, J . L., Robbins, J . D., and Byrd, R. A. (1986) A purified cysteine conjugate 8-lyase from rat kidney cytosol Requirement for an a-keto acid or an amino acid oxidase for activity, and identity with soluble glutamine transaminase K. J . Biol. Chem. 261, 15529-15537. Suzuki, S., Tomisawa, H., Ichihara, H., Fakazawa, H., and Tateishi, M. (1981) Purification and characterization of a rat liver enzyme catalyzing N-deacetylation of mercapturic acid conjugates. Drug Metab. Dispos. 9, 573-577. Lash, L. H., Elfarra, A. A., and Anders, M. W. (1986) Renal cysteine conjugate ,!?-lyase.Bioactivation of nephrotoxic cysteine S-conjugates in mitochondrial outer membrane. J.Biol. Chem. 261, 5930-5935. Stevens, J. L. (1988) Cysteine conjugate @-lyaseactivities in rat kidney cortex: Subcellular localization and relationship to the hepatic enzyme. Biochem. Biophys. Res. Commun. 129, 499504. Lash, L. H., Sausen, P. J., Duescher, R. J., Cooley, A. J., and Elfarra, A. A. (1994) Roles of cysteine conjugate @-lyase and S-oxidase in nephrotoxicity studies with S-(1,2-dichlorovinyl)sulfoxide. J. PharLcysteine and S-(l,Z-dichlorovinyl)-Lcyste~e m o l . Exp. Ther. 269, 374-383. Jones, T. W., gin, C., Schaeffer, V. H., and Stevens, J. L. (1988) Immunohistochemical localization of glutamine transaminase K, a rat kidney cysteine conjugate 8-lyase, and the relationship to the segment specificity of cysteine conjugate nephrotoxicity. Mol. Pharmacol. 34, 621-627.
-
Goeptar et al. Lash, L. H., Nelson, R. M., van Dijke, R. A., and Anders, M. W. (1990) Purification and characterization of human kidney cytosolic cysteine conjugate @-lyaseactivity. Drug Metab. Dispos. 18, 50-54. Buckbemy, L. D., Blagbrough, I. S., Bycroft, B. W., and Shaw, P. N. (1992) Kynurenine aminotransferase activity in human liver: Identity with human C-S lyase activity and a physiological role for this enzyme. Toxicol. Lett. Bo, 241-246. Hayden, P. J., and Stevens, J. L. (1990) Cysteine conjugate toxicity, metabolism, and binding to macromolecules in isolated rat kidney mitochondria. Mol. Pharmacol. 37, 468-476. Green, R. M., and Elce, J. S. (1975) Acetylation of S-substituted cysteines by a rat liver and kidney microsomal N-acetyltransferase. Bwchem. J. 147, 283-289. Derr, R. F., and Schultze, M. 0. (1963) The metabolism of 3%(1,2-dichlorovinyl)-L-cysteine in the rat. Biochem. Pharmacol. 12,465-474. James, S. P., and Needham, D. (1973) Some metabolites of S-pentyl-Lcysteine in the rabbit and other species. Xenobiotica 4, 207-218. Okajima, K., Inoue, M., Morino, Y., and Itoh, K. (1984) Topological aspects of microsomal N-acetyltransferase, an enzyme responsible for the acetylation of cysteine-S-conjugates of xenobiotics. Eur. J . Bwchem. 142, 281-286. Inoue, M., Okajima, K., Nagase, S., and Morino, Y. (1987) Interorgan metabolism and transport of a cysteine-S-conjugate of xenobiotics in normal and mutant analbuminemic rats. Biochem. Pharmacol. 36, 2145-2150. Boogaard, P. J., Commandeur, J. N. M., Mulder, G. J., Vermeulen, N. P. E., and Nagelkerke, J. F. (1989) Toxicity of the cysteine-S-conjugates and mercapturic acids of four structurally related difluoroethylenes in isolated proximal tubular cells from rat kidney. Uptake of the conjugates and activation to toxic metabolites. Biochem. Pharmacol. 38, 3731-3741. Zhang, G., and Stevens, J. L. (1989) Transport and activation of S-(1,2-dichlorovinyl)-L-cysteine and N-acetyl-S-(1,2-dichloroviny1)-L-cysteinein rat kidney proximal tubules. Toxicol. Appl. Pharmacol. 100, 51-61. Commandeur, J. N. M., De Kanter, F. J. J., and Vermeulen, N. P. E. (1989) Bioactivation of the cysteine-S-conjugate and mercapturic acid of tetrafluroethylene to acetylating reactive intermediates in the r a t Dependence of activation and deactivation on acetyl coenzymeA available. Mol. Pharmacol. 36,654663. Commandeur, J. N. M., Stijntjes, G. J., Wijngaard, J., and Vermeulen, N. P. E. (1991) Metabolism of L-cysteine S-conjugates and N-(trideuteroacety1)-L-cysteineS-conjugates of four fluoroethylenes in rat. Role of balance of deacetylation and acetylation in relation to nephrotoxicity of mercapturic acids. Biochem. Pharmacol. 42, 31-38. Sausen, P. J., Duescher, R. J., and Elfarra, A. A. (1993) Further characterization and purification of the flavin-dependent Sbenzyl-L-cysteine S-oxidase activities of rat liver and kidney microsomes. Mol. Pharmacol. 43, 388-396. Sausen, P. J., and Elfarra, A. A. (1990) Cysteine conjugate S-oxidase. Characterization of a novel enzymatic activity in rat hepatic and renal microsomes. J. Biol. Chem. 265, 6139-6145. Park, S. B., Osterloh, J. D., Vamvakas, S., Hasmi, M., Anders, M. W., and Cashman, J. R. (1992) Flavin-containing monooxygenase-dependent stereoselective S-oxygenation and cytotoxicity of cysteine S-conjugates and mercapturates. Chem. Res. Toricol. 5, 193-210. Hasmi, M., Vamvakas, S., and Anders, M. W. (1992) Bioactivation mechanism of S-(3-oxopropyl)-N-acetyl-~-cysteine, the mercapturic acid of acrolein. Chem. Res. Toxicol. 5 , 360-365. Lash, L. J., and Jones, D. P. (1984) Renal glutathione transport. Characterization of the sodium-dependent system in the basallateral membrane. J. Biol. Chem. 259, 14508-14514. Ullrich, K J., Rumrich, G., Wieland, T., and Dekant, W. (1989) Contraluminal para-biaminohippurate (PAH) transport in the proximal tubule of the rat kidney. Pfluegers Arch. 415, 342350. Vamvakas, S., Dekant, W., Berthold, K., Schmidt, S., Wild, D., and Henschler, D. (1987) Enzymic transformation of mercapturic acids derived from halogenated alkenes to reactive and mutagenic intermediates. Biochem. Pharmacol. 36, 2741-2748. Inoue, M., Okajima, K., and Morino, Y. (1982) Metabolic coordination of liver and kidney in mercapturic acid biosynthesis in vivo. Hepatology 2, 311-316. Lock, E. A., and Ishmael, J. (1985) Effect of the organic acid transport inhibitor probenecid on renal cortical uptake and proximal tubular toxicity of hexachloro-l,3-butadieneand its conjugates. Toxicol. Appl. Pharmacol. 81, 32-42. Mohandas, J., Marshall, J. J., Duggin, G. G., Horvath, J . S., and Tiller, D. J . (1984) Differential distribution of glutathione and
Invited Review glutathione-related enzymes in rabbit kidney. Possible implications in analgesic nephropathy. Biochem. Pharmacol. 33,18011807. (145)Hayden, P. J., Welsh, C. J.,Yang, Y., Schaeffer, W. H., Ward, A. J. I., and Stevens, J. L. (1992)Formation of mitochondrial phospholipid adducts by nephrotoxic cysteine conjugate metabolites. Chem. Res. Toxicol. 5, 231-237. (146)Chen, Q,? Yu,K., and Stevens, J. L. (1992)Regulation of the cellular response by reactive electrophiles. The role of covalent binding and cellular thiols in transcriptional activation of the 70-kilodalton heat shock protein gene by nephrotoxic cysteine. J. Bwl. Chem. 267,24322-24327. (147)Fukuda, K., Takemoto, K., and Tsuruta, H. (1983)Inhalation carcinoeenicitv of trichloroethvlene in mice and rats. Ind. Health 21,24c-254.(148) Odum. J.. Foster. J. R.. and Green, T. (1992)A mechanism for the development’of Clara cell lesions in the mouse lung after exposure to trichloroethylene. Chem.-Biol.Interact. 83,135-153. (149)Smith, M.N., Greenberg, S. D., and Spjut, H. J. (1979)The Clara cell: A comparative ultrastructural study in mammals. Am. J. Anat. 166,15-30. (150)Henschler, D., Romen, W., Elsasser, H. M., Reichert, D., Eder, E., and Radwan, Z. (1980)Carcinogenicity study of trichloroethylene by longterm inhalation in three animal species. Arch. To~icol.43,237-248. (151)Henschler, D., Elsasser, H. M., Romen, W., and Eder, E. (1984) Carcinogenicity study of trichloroethylene with and without epoxide stabilisers in mice. J.Cancer Res. Clin. Oncol. 104,149156. (152) Elcombe, C. R. (1985)Species differences in carcinogenicity and peroxisome proliferation due to trichloroethylene. A biochemical human hazard assessment. Arch. Toxicol., Suppl. 8,6-17. (153)Reddy, J. K.,Azarnoff, D. L., and Hignite, C. E. (1980)Hypolipidemic hepatic peroxisome proliferators form a novel class of chemical carcinogens. Nature 283,397-398. 154) Mirsalis, J. C., Tyson, C. IC,h h , E. N., Steinmetz, K. L., Bakke, J. P., Hamilton, C. M., Spak, D. K., and Spalding, J. W. (1985) Induction of hepatic cell proliferation and unscheduled DNA synthesis in mouse hepatocytes following in vivo treatment. Carcinogenesis 6 , 1521-1524. 155) Dees, C.,and Travis, C. (1993)The mitogenic potential of trichloroethylene in B&Fl mice. Toxicol. Lett. 69,129-137. (156)Bull, R. J., Sanchez, I. M., Nelson, M. A., Larson, J. L., and Lansing, A.J. (1990)Liver tumor induction in B&F1 mice by dichloroacetate and trichloroacetate. Toxicology 63, 341-359. (157)Sanchez, I. M.,and Bull, R. J. (1990) Early induction of reparative hyperplasia in the liver of BsCsFl mice treated with dichloroacetate and trichloroacetate. Toxicology 64, 33-46. (158)Klaunig, J. E., Ruch, R. J., and Lin, E. L. (1989)Effects of trichloroethylene and its metabolites on rodent hepatocyte intercellular communication. Toxicol.Appl. Pharmacol. 99,454465. (159)Bull, R. J., Templin, M., Larson, J. L., and Stevens, D. K. (1993) The role of dichloroacetate in the hepatocarcinogenicity of trichloroethylene. Toxicol. Lett. 68, 203-21 1.
Chem. Res. Toxicol., Vol. S, No. 1, 1995 21 (160)Herren-Freund, S.L., Periera, M. A., and Olson, G. (1987)The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver. Toxicol. Appl. Pharmacol. 90,183-189. (161)DeAngelo, A. B., Daniel, F. B., Stober, J. A,, and Olson, G. R. (1991)The carcinogenicity of dichloroacetic acid in the male B&Fl mouse. Fundam. Appl. Toxicol. 16,337-347. (162)NTP (National Toxicology Program) (1987)NTP Technical Report on the Toxicology and carcinogenesis studies of Trichloroethylene (CAS No. 79-01-6) in Four strains of Rats (ACI, August, Marshall, Osborne-Mendel) (Gauage Studies); National Toxicology Program. (163)Green, T., Odum, J., Nash, J. A., Foster, J. R., and Gore, W. C. (1990)Trichloroethylene induced rat kidney tumours: The mechansims involved and their relevance to humans. CTL Report No. CTLIRl1037. (164)Wallin, A., Zhang, G., Jones, T. W., Jaken, S., and Stevens, J. L. (1992)Mechanism of the nephrogenic repair response. Studies on proliferation and vimetin expression after s-35-1,2-dichlorovinyl-Gcytseine nephrotoxicity in vivo and in cultered proximal tubule epithelial cells. Lab. Znuest. 66, 474-784. (165)Goldworthy, T. L., Lyght, O., Burnett, V. L., and Popp, J. A. (1988)Potential role of a-’&-globulin, protein droplet accumulation and cell replication in the renal carcinogenicity of rats exposed to trichloroethylene, perchloroethylene and pentachloroethane. Toxicol. Appl. Pharmacol. 96,367-379. (166)J d e , D. R., Hassal, C. D., Gandolfi, A. J., and Brendel, K. (1985) Production of DNA single strand breaks in rabbit renal tissue after exposure to 1,2-dichlorovinylcysteine.Toxicology 35,2533. (167)Jaffe, D. R., Gandolfi, A. J., and Nagle, R. B. (1984)Chronic toxicity of S-(trans-l,2-dichlorovinyl)-L-cysteine in mice. J.Appl. Toxicol. 4,315-319. (168)Price, R. G. (1982)Urinary enzymes, nephrotoxicity and renal disease. Toxicology 23,99-134. (169)Novotna, E., David, A., and Malek, B. (1979)An epidemiological study of the hepatic tumor incidence in persons working with trichloroethylene. I. The negative result of retrospective investigations in persons with primary liver carcinoma. Prac. Lek. 31,121-123. (170)Paddle, G. M. (1983)Incidence of liver cancer and trichloroethylene manufacture: Joint study by industry and a cancer registry. Br. Med. J. 286,846. (171)Malek, B., Kremarova, B., and Rodova, 0. (1979)An epidemiological study of hepatic tumor incidence in subjects working with trichloroethylene. 11. Negative result of retrospective investigations of dry cleaners. Prac. Lek. 31,124-126. (172)Frederiksson, M., Bengtsson, N. O., Hardell, L., and Axelson, 0. (1989)Colon cancer, physical activity, and occupational exposure. Cancer 63,1838-1842.
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