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Covalent binding of trichloroethylene to proteins in human and rat hepatocytes
Toxicology Letters 95 (1998) 173 – 181
Covalent binding of trichloroethylene to proteins in human and rat hepatocytes Joseph M. Griffin a, John C. Lipscomb b, Neil R. Pumford a,* a
Uni6ersity of Arkansas for Medical Science, Department of Pharmacology and Toxicology, Mail Slot c 638, 4301 W. Markham Street, Little Rock, AR, USA b Armstrong Laboratory OL-AL HSC/OETA, Occupational and En6ironmental Health Directorate, Toxicology Di6ision, 2856 G Street, Bldg. 79, Wright-Patterson AFB, Cincinnati, OH, USA Received 16 February 1998; received in revised form 14 April 1998; accepted 15 April 1998
Abstract The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte cultures were exposed to vaporized trichloroethylene at 0 – 10000 parts per million for up to 2 h. Immunochemical detection of three major dose- and time-dependent trichloroethylene protein adducts at 50, 52 and 100 kDa was demonstrated in the rat hepatocytes, while a single, distinctively different 47 kDa adduct was detected in human hepatocytes. The 50 kDa adduct in rat hepatocytes was found to comigrate on SDS-PAGE with cytochrome P450 2E1 (CYP2E1), while the adduct found in humans did not comigrate with CYP2E1. These data show that reactive metabolites of trichloroethylene can be formed in human and rat hepatocytes and bind covalently to discrete hepatic proteins, and suggests that in rats, but not humans, that one of the targets is CYP2E1. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trichloroethylene; Covalent binding; Reactive intermediates; Cytochrome P450 2E1
1. Introduction 1,1,2-Trichloroethylene is a volatile organic compound that has been extensively used in industry as a metal degreaser, a fumigant, an ex* Corresponding author. Tel.: + 1 501 6865467; fax: +1 501 6868970; e-mail: [email protected]
tractant in food processing and a dry cleaning agent. It is estimated that 3.5 million people in the United States are annually exposed to trichloroethylene. At least 100000 workers are exposed full-time with 67% of these are working under conditions of inadequate control measures (National Institute for Occupational Safety and Health, 1978). Because of its widespread commer-
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cial use, trichloroethylene has become one of the most abundant organic contaminants at many of the superfund sites (Westrick et al., 1984). Trichloroethylene has been found in surface water, ground water, ambient air and soil (Bruckner et al., 1989). The primary concern of human exposure to trichloroethylene is the carcinogenic potential of the chemical, as well as an increasing interest in the association of trichloroethylene with several autoimmune related diseases. Chronic exposure to trichloroethylene has been shown to cause cancer in mice and rats (Green, 1990; Davidson and Beliles, 1991; Goeptar et al., 1995). In addition, there is an association between trichloroethylene and liver cancer in humans such that trichloroethylene is classified as a suspect human carcinogen (IARC, 1995; Weiss, 1996). Trichloroethylene is extensively metabolized in humans and rodents, with the major metabolites being trichloroethanol and its glucuronide conjugate, trichloroacetic acid and chloral (trichloroacetaldehyde). The metabolic conversion of trichloroethylene to a reactive intermediate is primarily accomplished by cytochromes P450, specifically the 2E1, 2C11/6, 2B1/2 and 1A1/2 isotypes (Nakajima et al., 1992; Lipscomb et al., 1997) and is an essential pathway for the carcinogenic and cytotoxic effects (Buben and O’Flaherty, 1985). Carcinogenesis may involve either peroxisomal proliferation caused by the trichloroacetic acid metabolite or by metabolism through the mercapturic acid pathway (Nelson, 1980; DeAngelo et al., 1989; Goeptar et al., 1995). An association has been established between exposure to trichloroethylene and autoimmune diseases like systemic lupus erythematosus, fasciitis and systemic sclerosis (Lockey et al., 1987, Kilburn and Warshaw, 1992; Yanez Diaz et al., 1992; Waller et al., 1994). Covalent binding of reactive trichloroethylene intermediates to protein may be responsible for the initiation of an immune response that is directed against trichloroethylene as a hapten, similar to that shown by halothane (Pohl, 1993). In addition, chemicals can cause a response directed against native proteins including the cytochromes P450 (Pumford and Halmes, 1997). We have recently developed immunochemical techniques to detect
covalently modified proteins in mice treated with trichloroethylene (Halmes et al., 1996) and have suggested that the major protein adduct is CYP2E1 (Halmes et al., 1996, 1997a,b). The present studies were undertaken to determine if human hepatocytes could produce reactive trichloroethylene intermediates that covalently bind to cellular proteins. The exposure of trichloroethylene to rat hepatocytes was performed for a comparison to human hepatocytes. The demonstration of adducted proteins in human hepatocytes exposed to trichloroethylene is novel and may be important in understanding the mechanisms of toxicities associated with acute and chronic exposure to this abundant environmental contaminant. The demonstration of similar biochemical mechanisms in rodents and humans will aid in the extrapolation of effects from rodents and help improve the human health risk assessment for trichloroethylene.
2. Methods
2.1. Animals To maintain consistency with several other studies on the metabolism and toxicity of trichloroethylene and its metabolites (Larson and Bull, 1992) male inbred rats (CDFR(F-344)/ CrlBR) weighing 190–230 g were procured from the Raleigh, NC production facility of Charles River Laboratories (Wilmington, MA). They were housed in polycarbonate cages, provided with hardwood chip bedding and maintained on a 12-h light:dark cycle at a constant temperature of 209 2°C and 459 5% relative humidity. The rats were provided with fresh conditioned (reverse osmosis) water and rodent chow (c 5008, Purina Mills, St. Louis, MO) ad libitum. The animals were not fasted prior to euthanasia. All studies involving live animals were conducted under a program of animal care accredited by the American Association for the Accreditation of Laboratory Animal Care and in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals’’, NIH Publication No. 86–23 (1985).
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2.2. Rat hepatocyte preparations Hepatocytes were isolated under modifications of Berry and Friend (1969) as detailed by DelRaso (1992). Briefly, rats were anesthetized by an intraperitoneal injection (1 ml/kg) containing a mixture of ketamine (70 mg/kg) and xylazine (6 mg/kg). Their livers were perfused with perfusion media c 1: pH 7.4 (10× Hank’s Balanced Salt Solution (HBSS), distilled water, fraction V bovine serum albumin, sodium bicarbonate, HEPES and heparin) and then perfusion media c 2: digestion media containing same except heparin, and supplemented with phenol red and collagenase Type IV at 4°C. The livers were removed and the cells were released from the tissue through incisions and combing (2×) of the lobes. The cells were washed twice by centrifugation for 3 min at 50 × g in HBSS-albumin containing 4.29 mg/ml HEPES, 0.5 mg/ml BSA and 1.5 mg/ml glucose.
2.3. Human hepatocyte studies Human hepatocytes were obtained from the Human Cell Culture Center, Columbia, MD. The cells usually arrived 24 – 36 h after the donor’s expiration and 6–12 h postisolation. Immediately upon arrival, A trypan blue viability test was performed on the hepatocytes. After viability was determined (70% positive by trypan blue exclusion), the cells were resuspended in Chee’s Buffer (modified MEM, Gibco BRL, Gaithersburg MD) at a cell concentration of 2×106 cells/ml.
2.4. Exposure methodology Rat and human hepatocytes were placed in individual 40 ml Erlenmeyer flasks whose exact volume was gravimetrically determined. Each flask was sealed with a screw cap and Teflon lined rubber septum. Trichloroethylene (Aldrich, Milwaukee, WI) was vaporized in a Tedlar bag to a final concentration in air of 30000 ppm. Samples of flask headspace (n =2/flask) were injected on a Hewlett–Packard (Avondale, PA) Model 5890 Series II gas chromatograph equipped with a Supelco 2-5320 Vocol™ capillary column (0.53
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mm× 30 m) and interfaced with a flame ionization detector. The concentration of trichloroethylene in flasks was verified by comparing area counts with those from an external standard curve of authentic trichloroethylene volatilized directly in crimp-sealed serum vials and analyzed simultaneously by the same system. Dilutions from trichloroethylene bags were calculated for individual flasks and the appropriate volume of air was first removed from each flask and replaced immediately with the same volume of trichloroethylene in air using a glass and Teflon, gas-tight syringe. Flasks were immediately placed in a Fisher Scientific Model 224 Versa Bath oscillating at 50 rpm at 37°C and allowed to incubate for 30, 60, 90 or 120 min.
2.5. Immunochemical detection of trichloroethylene adducts Samples from each experimental group were diluted to a final concentration of 20 mg/ml and 10 ml of total cellular protein was separated by SDS-PAGE under reducing conditions as previously described (Halmes et al., 1996). Following transfer to nitrocellulose, trichloroethylene adducts were detected with affinity purified antidichloroacetyl (DCA) serum antibodies, which recognize trichloroethylene-protein adducts, using a peroxidase detection system and visualized by enhanced chemiluminescence (ECL) from Pierce (Rockford, IL), as described (Halmes et al., 1996). Briefly, the immunogen was KLH treated with dichloroacetic anhydride and the antibodies produced were shown to react with the solid-phase antigen dichloroacetyled rabbit serum albumin (DCA-RSA) in an ELISA. The specificity was determined in a competitive ELISA, which found that dichloroacetyl lysine was the most potent inhibitor followed by trichloroacetyl and monochloroacetyl lysine. For human samples, the anti-DCA antibodies were pre-absorbed with 1% rat homogenate from control rat to reduce nonspecific binding (Kenna et al., 1984). To determine if the major adduct could be CYP2E1, nitrocellulose blots were stripped according to protocol in ECL kit from Amersham (Buckinghamshire, UK) and reprobed with anti-CYP2E1
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Fig. 1. Western blot and densitometric analysis of dose-responsive formation of trichloroethylene-protein adducts in rat hepatocytes. Left panel: Primary cultures of rat hepatocytes exposed to vaporized trichloroethylene from 0 to 5000 ppm. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Arrows indicate positions of molecular weight standards. Right Panel: Densitometric analysis of the 50 kDa protein adduct.
antibody and detected as described above. Densitometry analysis was performed on a BIO-RAD imaging densitometer model GS-670, the software package was BIO-RAD Molecular Analyst. Western blot analysis was preformed a minimum of three times for each test group and the blot presented is representative.
2.6. Clinical chemistry Viability controls were included in each experiment to assess the effect of incubation and the cytotoxicity of trichloroethylene. Measurements included intracellular potassium content and leakage of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from cells into medium. Potassium content of sonicated samples was determined using an AVL 982-S Electrolyte Analyzer (Roswell, GA). The acceptable level of intracellular potassium content was set at a greater than 35 mmoles K + /g wet weight sample. ALT and AST levels were determined using a Kodak Ektachem Analyzer (model 700XR) and LDH levels determined using a DuPont acaV. The acceptable control enzyme leakage level for metabolic studies was
less than 25% of total or a level not in excess of the control (no trichloroethylene) incubation.
3. Results In primary cultures of rat hepatocytes exposed to trichloroethylene from 0 to 5000 ppm for 2 h and probed in a Western blot with anti-DCA antibodies, we detected three major adducts of rat hepatic protein (50, 52 and 100 kDa; Fig. 1, left panel). The intensity of all the bands increased in a dose-dependent manner. Densitometry analysis in the right panel of Fig. 1 shows the dose-responsive increase in relative intensity of the 50 kDa adduct. When rat hepatocytes were treated with 5000 ppm for 0, 30 or 120 min (Fig. 2, left panel), three major adducts of 50, 52 and 100 kDa were seen, similar to the three adducts shown in the dose-response experiment. There were three minor bands at 60, 70 and 75 kDa. The right panel of Fig. 2 shows the time dependent increase in intensity of the 50 kDa adduct by densitometric analysis. Similar experiments were performed to determine if trichloroethylene adducts could be detected in human hepatocytes in primary culture.
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Fig. 2. Western blot and densitometric analysis of time-responsive formation of trichloroethylene-protein adducts in rat hepatocytes. Left panel: Primary cultures of rat hepatocytes exposed to 5000 ppm vaporized trichloroethylene for 0, 30 and 120 min. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Position of molecular weight Standards are indicated by arrows. Right Panel: Densitometric analysis of the 50 kDa protein adduct.
Human hepatocytes were exposed to vaporized trichloroethylene with exposure concentrations ranging from 0 to 10000 ppm for 2 h. Western blot analysis of total cellular proteins (Fig. 3, left panel) revealed only one trichloroethylene-protein adduct at 47 kDa. The densitometry analysis showed an increase in intensity up to 3000 ppm with higher doses showing no further increases (Fig. 3, right panel). Time course analysis of human hepatocytes also showed a single band at 47 kDa. The intensity of staining increase from 30 to 90 min with no further increases seen (Fig. 4). To test the possibility that one of the three major adducts detected in the rat hepatocytes was CYP2E1 the nitrocellulose blot used to produce the time course response (Fig. 2, 120 min exposure) was stripped of antibodies. Removal of antibodies was confirmed by reprobing with peroxidase-labeled rabbit anti-IgG antibodies and detecting as described in Section 2. The nitrocellulose was then reprobed with anti-CYP2E1 antibody (Fig. 5, left panel). The CYP2E1 comigrated with the 50 kDa trichloroethylene-protein adduct in the rat. Evidence that the 50 kDa protein is CYP2E1 is strengthened by the fact that the bands detected with both anti-DCA and anti-
CYP2E1 were identical when the two films were overlaid. Similar experiments were performed on the human hepatocytes. The nitrocellulose blot used to produce the dose response (Fig. 3, 3000 ppm exposure) was stripped and re-probed with anti-CYP2E1 antibody. As shown in the right panel of figure, the major trichloroethylenemodified protein did not comigrate with the CYP2E1 protein. At all treatment concentrations and times, measurements of intracellular potassium content and leakage of enzymes including LDH, ALT and AST from the cells into the media showed no significant change from that measured in the controls (no trichloroethylene; data not shown).
4. Discussion We have previously shown in rats (Halmes et al., 1997a) and mice (Halmes et al., 1996) that trichloroethylene is metabolized in vivo to a reactive intermediate that will bind to specific hepatic proteins and have suggested that one of the primary targeted proteins is CYP2E1. The present studies were undertaken to determine if human
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Fig. 3. Western blot and densitometric analysis of dose-responsive formation of trichloroethylene-protein adducts in human hepatocytes. Left panel: Primary cultures of human hepatocytes exposed to vaporized trichloroethylene from 0 to 10000 ppm. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Position of molecular weight standards are indicated by arrows. Right Panel: Densitometric analysis of the 47 kDa protein adduct.
hepatocytes metabolize trichloroethylene to a reactive intermediate capable of binding to proteins similar to those found in rodents. Halmes et al. (1996) detected two major protein adducts at 50 and 100 kDa in vivo in mice and only a 50-kDa adduct in vivo in the rat. These adducts were shown to be both dose and time dependent. The data presented here are similar in that rat hepatocytes show a dose and time dependent increase in trichloroethylene-protein adducts at 50, 52 and 100 kDa (Figs. 1 and 2). The appearance of a minor adduct at 52 kDa is novel and its importance is not yet known. Nakajima et al. (1992) showed that multiple isozymes of cytochrome P450 (i.e. 2E1, 2C11/6, 2B1/2 or 1A1/2) in rats are responsible for the metabolism of trichloroethylene. The appearance of the 52 kDa trichloroethylene-adduct may be an isozyme other than CYP2E1. The significance of the 100 kDa trichloroethylene-adduct seen in the rat hepatocytes is unknown at this time, the possibility that this is a dimer two of 50 kDa adducts is unlikely since it was neither recognized by the antiCYP2E1 antibody nor observed in vivo. Interestingly, the human hepatocytes treated with trichloroethylene formed only a single detectable adduct at 47 kDa (Figs. 3 and 4). The
overall staining was considerably less intense when compared rat hepatocytes suggesting much lower levels of proteins modified by trichloroethylene. This is consistent with the quantitatively lower cytochrome P450-dependent trichloroethylene metabolism in humans (Garrett et al., 1996). In the human hepatocyte dose-response experiment (Fig. 3), the intensity of trichloroethyleneadducts appears to plateau between 1000 and 3000 ppm. One explanation of the initial increase in intensity followed by an apparent saturation at higher doses is that the binding may be related to the ability of trichloroethylene to inactivate cytochrome P450 by a mechanism-based process (Halmes et al., 1997b). That is, a trichloroethylene-cytochrome P450 intermediate complex, which is formed during metabolism, can rearrange to a reactive intermediate that can covalently bind to the heme prosthetic group and/or the apoprotein. Taken collectively, the increase in intensity followed by a plateau and the overall lower level of intensity seen in the human hepatocytes may be explained by the lower capacity of humans to metabolize trichloroethylene and the data showing a mechanism-based inhibition of cytochrome P450 by trichloroethylene. The sug-
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Fig. 4. Western blot and densitometric analysis of time-responsive formation of trichloroethylene-protein adducts in human hepatocytes. Left panel: Primary cultures of human hepatocytes exposed to 5000 ppm vaporized trichloroethylene for 0, 30, 60, 90 or 120 min. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Position of molecular weight standards are indicated by arrows. Right Panel: Densitometric analysis of the 47 kDa protein adduct.
gestion that the trichloroethylene is a suicide inhibitor of CYP2E1 in rats is also supported by the data presented in Fig. 5 left panel, which shows that the 50 kDa trichloroethylene-protein adduct in rat hepatocytes comigrates with a protein recognized by an antibody raised against CYP2E1. In the human hepatocytes this is not the case, since the major trichloroethylene adduct migrated to an appreciably different position than CYP2E1 (Fig. 5, right panel). These results taken collectively suggest that trichloroethylene is metabolized to a reactive intermediate which can bind covalently to cellular proteins and that one of the major targets of this metabolite in rats is likely CYP2E1. Binding to CYP2E1 is consistent with existing data showing a mechanism-based inhibition of this enzyme by trichloroethylene. However, the human hepatocytes exposed to trichloroethylene did not show the same pattern of adduct formation. It has been shown that there can be considerable variation in the levels of CYP2E1 among humans (Guengerich et al., 1991; Lipscomb et al., 1997), and the donor of these hepatocytes may have had very low levels of expressed CYP2E1. Because it is accepted that the range of molecular weights of the cytochromes
P450 is between 47 and 55 kDa, the adduct found in these particular human hepatocytes may be a different isozyme of the cytochromes P450. The pattern of the intensity shown in the human hepatocytes can be explained by the suggested mechanism-based inhibition of cytochromes P450 by trichloroethylene. Additional work needs to be done to identify the proteins covalently modified by a metabolite of trichloroethylene in humans. This can be done through immunoaffinity or other forms of chromatography (Pumford and Halmes, 1997). The ability to detect and identify the proteins modified by trichloroethylene can be a useful tool to enable further understanding of the underlying mechanisms of acute and chronic trichloroethylene toxicity as well as the carcinogenicity that has been associated with trichloroethylene exposure. The covalent binding of trichloroethylene to proteins may cause damage to proteins such as tubulin, histone proteins, topoisomerase II, or other DNA associated proteins which may potentially lead to DNA damage and this may be important in trichloroethylene carcinogenesis. Since we do not know the mechanism of carcinogenesis this and other possibilities cannot be ruled out. In addi-
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tion, regeneration of tissues following cell death could also be considered an epigenetic mechanism of carcinogenesis. The covalent binding of trichloroethylene could cause acute toxicity and potentially lead to tissue regeneration and incorporation of any mutations that may have occurred. At this time we do not know what if any role covalent binding to proteins has in the carcinogenesis of trichloroethylene. This is the first report of the production of a reactive metabolite capable of covalently modifying cellular protein in human cells exposed to trichloroethylene. The demonstration of covalent binding of a reactive intermediate of trichloroethylene in human hepatocytes along with the previously reported data in rodents suggests that differences exist in metabolism between humans and rodents which may be important in extrapolation and risk assessment.
Fig. 5. Comigration study of trichloroethylene-protein adduct with CYP2E1 in human and rat hepatocytes. Left Panel: Left lane: 2 h exposure of human hepatocytes to 5000 ppm vaporized trichloroethylene probed with anti-trichloroethylene antibody. Right lane: Same lane that has been stripped and reprobed with anti-CYP2E1 antibody. Right Panel: Left lane: 2 h exposure of rat hepatocytes to 5000 ppm vaporized trichloroethylene probed with anti-trichloroethylene antibody. Right lane: Same lane that has been stripped and reprobed with anti-CYP2E1 antibody.
Acknowledgements This work was supported in part by the Department of Energy (DE-FG01-92EW50625), the Strategic Environmental Research and Development Program (SERDP), the UAMS Graduate Student Research Fund, and the U.S. Environmental Protection Agency. We gratefully acknowledge the technical assistance of Patricia Confer.
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J.M. Griffin et al. / Toxicology Letters 95 (1998) 173–181 in mice treated with trichloroethylene. Chem. Res. Toxicol. 9, 451 – 456. Halmes, N.C., Perkins, E.J., McMillan, D.C., Pumford, N.R., 1997a. Detection of trichloroethylene-protein adducts in rat liver and plasma. Toxicol. Lett. 92, 187–194. Halmes, N.C., Samokyszyn, V.M., Pumford, N.R., 1997b. Covalent binding and inhibition of cytochrome P4502E1 by trichloroethylene. Xenobiotica 27, 101–110. IARC, 1995. World Health Organization, International Agency for Research on Cancer, Lyon, France, vol. 63, pp. 75 – 158 . Kenna, J.G., Neuberger, J., Williams, R., 1984. An enzymelinked immunosorbent assay for detection of antibodies against halothane-altered hepatocyte antigens. J. Immunol. Methods 75, 3–14. Kilburn, K.H., Warshaw, R.H., 1992. Prevalence of symptoms of systemic lupus erythematosus (SLE) and of fluorescent antinuclear antibodies associated with chronic exposure to trichloroethylene and other chemicals in well water. Environ. Res. 57, 1–9. Larson, J.L., Bull, R.J., 1992. Species differences in the metabolism of trichloroethylene to the carcinogenic metabolites trichloroacetate and dichloroacetate. Toxicol. Appl. Pharmacol. 115, 278–285. Lipscomb, J.C., Garret, C.M., Snawder, J.E., 1997. Cytochrome P450-dependent metabolism of trichloroethylene— interindividual differences in humans. Toxicol. App. Pharmacol. 142, 311–318. Lockey, J.E., Kelly, C.R., Cannon, G.W., Colby, T.V., Aldrich, V., Livingston, G.K., 1987. Progressive systemic sclerosis associated with exposure to trichloroethylene. J. Occup. Med. 29, 493–496.
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Nakajima, T., Wang, R.S., Elovaara, E., Park, S.S., Gelboin, H.V., Vainio, H., 1992. A comparative study on the contribution of cytochrome P450 isozymes to metabolism of benzene, toluene and trichloroethylene in rat liver. Biochem. Pharmacol. 43, 251 – 257. National Institute for Occupational Safety and Health, 1978. Special Occupational Hazard Review of Trichloroethylene. DHEW Publ. No. 78 – 130, Washington, DC. Nelson, E.B., 1980. The pharmacology and toxicology of meta-substituted acetanilide: I. Acute toxicity of 3HAA in mice. Res. Commun. Chem. Pathol. Pharmacol. 28, 447 – 456. Pohl, L.R., 1993. An immunochemical approach of identifying and characterizing protein targets of toxic reactive metabolites. Chem. Res. Toxicol. 6, 786 – 793. Pumford, N.R., Halmes, N.C., 1997. Protein targets of reactive intermediates of xenobiotics. Annu. Rev. Pharmacol. Toxicol. 37, 91 – 117. Waller, P.A., Clauw, D., Cupps, T., Metcalf, J.S., Silver, R.M., LeRoy, E.C., 1994. Fasciitis (not scleroderma) following prolonged exposure to an organic solvent (trichloroethylene). J. Rheumatol. 21, 1567 – 1570. Weiss, N.S., 1996. Cancer in relation to occupational exposure to trichloroethylene. Occup. Env. Med. 53, 1 – 5. Westrick, J.J., Mello, J.W., Thomas, R.F., 1984. The ground water supply survey. J. Am. Water Works Assoc. 76, 52 – 59. Yanez Diaz, S., Moran, M., Unamuno, P., Armijo, M., 1992. Silica and trichloroethylene-induced progressive systemic sclerosis. Dermatology 184, 98 – 102.
Covalent binding of trichloroethylene to proteins in human and rat hepatocytes Joseph M. Griffin a, John C. Lipscomb b, Neil R. Pumford a,* a
Uni6ersity of Arkansas for Medical Science, Department of Pharmacology and Toxicology, Mail Slot c 638, 4301 W. Markham Street, Little Rock, AR, USA b Armstrong Laboratory OL-AL HSC/OETA, Occupational and En6ironmental Health Directorate, Toxicology Di6ision, 2856 G Street, Bldg. 79, Wright-Patterson AFB, Cincinnati, OH, USA Received 16 February 1998; received in revised form 14 April 1998; accepted 15 April 1998
Abstract The environmental contaminant and occupational solvent trichloroethylene is metabolized to a reactive intermediate that covalently binds to specific hepatic proteins in exposed mice and rats. In order to compare covalent binding between humans and rodents, primary hepatocyte cultures were exposed to vaporized trichloroethylene at 0 – 10000 parts per million for up to 2 h. Immunochemical detection of three major dose- and time-dependent trichloroethylene protein adducts at 50, 52 and 100 kDa was demonstrated in the rat hepatocytes, while a single, distinctively different 47 kDa adduct was detected in human hepatocytes. The 50 kDa adduct in rat hepatocytes was found to comigrate on SDS-PAGE with cytochrome P450 2E1 (CYP2E1), while the adduct found in humans did not comigrate with CYP2E1. These data show that reactive metabolites of trichloroethylene can be formed in human and rat hepatocytes and bind covalently to discrete hepatic proteins, and suggests that in rats, but not humans, that one of the targets is CYP2E1. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Trichloroethylene; Covalent binding; Reactive intermediates; Cytochrome P450 2E1
1. Introduction 1,1,2-Trichloroethylene is a volatile organic compound that has been extensively used in industry as a metal degreaser, a fumigant, an ex* Corresponding author. Tel.: + 1 501 6865467; fax: +1 501 6868970; e-mail: [email protected]
tractant in food processing and a dry cleaning agent. It is estimated that 3.5 million people in the United States are annually exposed to trichloroethylene. At least 100000 workers are exposed full-time with 67% of these are working under conditions of inadequate control measures (National Institute for Occupational Safety and Health, 1978). Because of its widespread commer-
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cial use, trichloroethylene has become one of the most abundant organic contaminants at many of the superfund sites (Westrick et al., 1984). Trichloroethylene has been found in surface water, ground water, ambient air and soil (Bruckner et al., 1989). The primary concern of human exposure to trichloroethylene is the carcinogenic potential of the chemical, as well as an increasing interest in the association of trichloroethylene with several autoimmune related diseases. Chronic exposure to trichloroethylene has been shown to cause cancer in mice and rats (Green, 1990; Davidson and Beliles, 1991; Goeptar et al., 1995). In addition, there is an association between trichloroethylene and liver cancer in humans such that trichloroethylene is classified as a suspect human carcinogen (IARC, 1995; Weiss, 1996). Trichloroethylene is extensively metabolized in humans and rodents, with the major metabolites being trichloroethanol and its glucuronide conjugate, trichloroacetic acid and chloral (trichloroacetaldehyde). The metabolic conversion of trichloroethylene to a reactive intermediate is primarily accomplished by cytochromes P450, specifically the 2E1, 2C11/6, 2B1/2 and 1A1/2 isotypes (Nakajima et al., 1992; Lipscomb et al., 1997) and is an essential pathway for the carcinogenic and cytotoxic effects (Buben and O’Flaherty, 1985). Carcinogenesis may involve either peroxisomal proliferation caused by the trichloroacetic acid metabolite or by metabolism through the mercapturic acid pathway (Nelson, 1980; DeAngelo et al., 1989; Goeptar et al., 1995). An association has been established between exposure to trichloroethylene and autoimmune diseases like systemic lupus erythematosus, fasciitis and systemic sclerosis (Lockey et al., 1987, Kilburn and Warshaw, 1992; Yanez Diaz et al., 1992; Waller et al., 1994). Covalent binding of reactive trichloroethylene intermediates to protein may be responsible for the initiation of an immune response that is directed against trichloroethylene as a hapten, similar to that shown by halothane (Pohl, 1993). In addition, chemicals can cause a response directed against native proteins including the cytochromes P450 (Pumford and Halmes, 1997). We have recently developed immunochemical techniques to detect
covalently modified proteins in mice treated with trichloroethylene (Halmes et al., 1996) and have suggested that the major protein adduct is CYP2E1 (Halmes et al., 1996, 1997a,b). The present studies were undertaken to determine if human hepatocytes could produce reactive trichloroethylene intermediates that covalently bind to cellular proteins. The exposure of trichloroethylene to rat hepatocytes was performed for a comparison to human hepatocytes. The demonstration of adducted proteins in human hepatocytes exposed to trichloroethylene is novel and may be important in understanding the mechanisms of toxicities associated with acute and chronic exposure to this abundant environmental contaminant. The demonstration of similar biochemical mechanisms in rodents and humans will aid in the extrapolation of effects from rodents and help improve the human health risk assessment for trichloroethylene.
2. Methods
2.1. Animals To maintain consistency with several other studies on the metabolism and toxicity of trichloroethylene and its metabolites (Larson and Bull, 1992) male inbred rats (CDFR(F-344)/ CrlBR) weighing 190–230 g were procured from the Raleigh, NC production facility of Charles River Laboratories (Wilmington, MA). They were housed in polycarbonate cages, provided with hardwood chip bedding and maintained on a 12-h light:dark cycle at a constant temperature of 209 2°C and 459 5% relative humidity. The rats were provided with fresh conditioned (reverse osmosis) water and rodent chow (c 5008, Purina Mills, St. Louis, MO) ad libitum. The animals were not fasted prior to euthanasia. All studies involving live animals were conducted under a program of animal care accredited by the American Association for the Accreditation of Laboratory Animal Care and in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals’’, NIH Publication No. 86–23 (1985).
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2.2. Rat hepatocyte preparations Hepatocytes were isolated under modifications of Berry and Friend (1969) as detailed by DelRaso (1992). Briefly, rats were anesthetized by an intraperitoneal injection (1 ml/kg) containing a mixture of ketamine (70 mg/kg) and xylazine (6 mg/kg). Their livers were perfused with perfusion media c 1: pH 7.4 (10× Hank’s Balanced Salt Solution (HBSS), distilled water, fraction V bovine serum albumin, sodium bicarbonate, HEPES and heparin) and then perfusion media c 2: digestion media containing same except heparin, and supplemented with phenol red and collagenase Type IV at 4°C. The livers were removed and the cells were released from the tissue through incisions and combing (2×) of the lobes. The cells were washed twice by centrifugation for 3 min at 50 × g in HBSS-albumin containing 4.29 mg/ml HEPES, 0.5 mg/ml BSA and 1.5 mg/ml glucose.
2.3. Human hepatocyte studies Human hepatocytes were obtained from the Human Cell Culture Center, Columbia, MD. The cells usually arrived 24 – 36 h after the donor’s expiration and 6–12 h postisolation. Immediately upon arrival, A trypan blue viability test was performed on the hepatocytes. After viability was determined (70% positive by trypan blue exclusion), the cells were resuspended in Chee’s Buffer (modified MEM, Gibco BRL, Gaithersburg MD) at a cell concentration of 2×106 cells/ml.
2.4. Exposure methodology Rat and human hepatocytes were placed in individual 40 ml Erlenmeyer flasks whose exact volume was gravimetrically determined. Each flask was sealed with a screw cap and Teflon lined rubber septum. Trichloroethylene (Aldrich, Milwaukee, WI) was vaporized in a Tedlar bag to a final concentration in air of 30000 ppm. Samples of flask headspace (n =2/flask) were injected on a Hewlett–Packard (Avondale, PA) Model 5890 Series II gas chromatograph equipped with a Supelco 2-5320 Vocol™ capillary column (0.53
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mm× 30 m) and interfaced with a flame ionization detector. The concentration of trichloroethylene in flasks was verified by comparing area counts with those from an external standard curve of authentic trichloroethylene volatilized directly in crimp-sealed serum vials and analyzed simultaneously by the same system. Dilutions from trichloroethylene bags were calculated for individual flasks and the appropriate volume of air was first removed from each flask and replaced immediately with the same volume of trichloroethylene in air using a glass and Teflon, gas-tight syringe. Flasks were immediately placed in a Fisher Scientific Model 224 Versa Bath oscillating at 50 rpm at 37°C and allowed to incubate for 30, 60, 90 or 120 min.
2.5. Immunochemical detection of trichloroethylene adducts Samples from each experimental group were diluted to a final concentration of 20 mg/ml and 10 ml of total cellular protein was separated by SDS-PAGE under reducing conditions as previously described (Halmes et al., 1996). Following transfer to nitrocellulose, trichloroethylene adducts were detected with affinity purified antidichloroacetyl (DCA) serum antibodies, which recognize trichloroethylene-protein adducts, using a peroxidase detection system and visualized by enhanced chemiluminescence (ECL) from Pierce (Rockford, IL), as described (Halmes et al., 1996). Briefly, the immunogen was KLH treated with dichloroacetic anhydride and the antibodies produced were shown to react with the solid-phase antigen dichloroacetyled rabbit serum albumin (DCA-RSA) in an ELISA. The specificity was determined in a competitive ELISA, which found that dichloroacetyl lysine was the most potent inhibitor followed by trichloroacetyl and monochloroacetyl lysine. For human samples, the anti-DCA antibodies were pre-absorbed with 1% rat homogenate from control rat to reduce nonspecific binding (Kenna et al., 1984). To determine if the major adduct could be CYP2E1, nitrocellulose blots were stripped according to protocol in ECL kit from Amersham (Buckinghamshire, UK) and reprobed with anti-CYP2E1
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Fig. 1. Western blot and densitometric analysis of dose-responsive formation of trichloroethylene-protein adducts in rat hepatocytes. Left panel: Primary cultures of rat hepatocytes exposed to vaporized trichloroethylene from 0 to 5000 ppm. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Arrows indicate positions of molecular weight standards. Right Panel: Densitometric analysis of the 50 kDa protein adduct.
antibody and detected as described above. Densitometry analysis was performed on a BIO-RAD imaging densitometer model GS-670, the software package was BIO-RAD Molecular Analyst. Western blot analysis was preformed a minimum of three times for each test group and the blot presented is representative.
2.6. Clinical chemistry Viability controls were included in each experiment to assess the effect of incubation and the cytotoxicity of trichloroethylene. Measurements included intracellular potassium content and leakage of lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) from cells into medium. Potassium content of sonicated samples was determined using an AVL 982-S Electrolyte Analyzer (Roswell, GA). The acceptable level of intracellular potassium content was set at a greater than 35 mmoles K + /g wet weight sample. ALT and AST levels were determined using a Kodak Ektachem Analyzer (model 700XR) and LDH levels determined using a DuPont acaV. The acceptable control enzyme leakage level for metabolic studies was
less than 25% of total or a level not in excess of the control (no trichloroethylene) incubation.
3. Results In primary cultures of rat hepatocytes exposed to trichloroethylene from 0 to 5000 ppm for 2 h and probed in a Western blot with anti-DCA antibodies, we detected three major adducts of rat hepatic protein (50, 52 and 100 kDa; Fig. 1, left panel). The intensity of all the bands increased in a dose-dependent manner. Densitometry analysis in the right panel of Fig. 1 shows the dose-responsive increase in relative intensity of the 50 kDa adduct. When rat hepatocytes were treated with 5000 ppm for 0, 30 or 120 min (Fig. 2, left panel), three major adducts of 50, 52 and 100 kDa were seen, similar to the three adducts shown in the dose-response experiment. There were three minor bands at 60, 70 and 75 kDa. The right panel of Fig. 2 shows the time dependent increase in intensity of the 50 kDa adduct by densitometric analysis. Similar experiments were performed to determine if trichloroethylene adducts could be detected in human hepatocytes in primary culture.
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Fig. 2. Western blot and densitometric analysis of time-responsive formation of trichloroethylene-protein adducts in rat hepatocytes. Left panel: Primary cultures of rat hepatocytes exposed to 5000 ppm vaporized trichloroethylene for 0, 30 and 120 min. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Position of molecular weight Standards are indicated by arrows. Right Panel: Densitometric analysis of the 50 kDa protein adduct.
Human hepatocytes were exposed to vaporized trichloroethylene with exposure concentrations ranging from 0 to 10000 ppm for 2 h. Western blot analysis of total cellular proteins (Fig. 3, left panel) revealed only one trichloroethylene-protein adduct at 47 kDa. The densitometry analysis showed an increase in intensity up to 3000 ppm with higher doses showing no further increases (Fig. 3, right panel). Time course analysis of human hepatocytes also showed a single band at 47 kDa. The intensity of staining increase from 30 to 90 min with no further increases seen (Fig. 4). To test the possibility that one of the three major adducts detected in the rat hepatocytes was CYP2E1 the nitrocellulose blot used to produce the time course response (Fig. 2, 120 min exposure) was stripped of antibodies. Removal of antibodies was confirmed by reprobing with peroxidase-labeled rabbit anti-IgG antibodies and detecting as described in Section 2. The nitrocellulose was then reprobed with anti-CYP2E1 antibody (Fig. 5, left panel). The CYP2E1 comigrated with the 50 kDa trichloroethylene-protein adduct in the rat. Evidence that the 50 kDa protein is CYP2E1 is strengthened by the fact that the bands detected with both anti-DCA and anti-
CYP2E1 were identical when the two films were overlaid. Similar experiments were performed on the human hepatocytes. The nitrocellulose blot used to produce the dose response (Fig. 3, 3000 ppm exposure) was stripped and re-probed with anti-CYP2E1 antibody. As shown in the right panel of figure, the major trichloroethylenemodified protein did not comigrate with the CYP2E1 protein. At all treatment concentrations and times, measurements of intracellular potassium content and leakage of enzymes including LDH, ALT and AST from the cells into the media showed no significant change from that measured in the controls (no trichloroethylene; data not shown).
4. Discussion We have previously shown in rats (Halmes et al., 1997a) and mice (Halmes et al., 1996) that trichloroethylene is metabolized in vivo to a reactive intermediate that will bind to specific hepatic proteins and have suggested that one of the primary targeted proteins is CYP2E1. The present studies were undertaken to determine if human
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Fig. 3. Western blot and densitometric analysis of dose-responsive formation of trichloroethylene-protein adducts in human hepatocytes. Left panel: Primary cultures of human hepatocytes exposed to vaporized trichloroethylene from 0 to 10000 ppm. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Position of molecular weight standards are indicated by arrows. Right Panel: Densitometric analysis of the 47 kDa protein adduct.
hepatocytes metabolize trichloroethylene to a reactive intermediate capable of binding to proteins similar to those found in rodents. Halmes et al. (1996) detected two major protein adducts at 50 and 100 kDa in vivo in mice and only a 50-kDa adduct in vivo in the rat. These adducts were shown to be both dose and time dependent. The data presented here are similar in that rat hepatocytes show a dose and time dependent increase in trichloroethylene-protein adducts at 50, 52 and 100 kDa (Figs. 1 and 2). The appearance of a minor adduct at 52 kDa is novel and its importance is not yet known. Nakajima et al. (1992) showed that multiple isozymes of cytochrome P450 (i.e. 2E1, 2C11/6, 2B1/2 or 1A1/2) in rats are responsible for the metabolism of trichloroethylene. The appearance of the 52 kDa trichloroethylene-adduct may be an isozyme other than CYP2E1. The significance of the 100 kDa trichloroethylene-adduct seen in the rat hepatocytes is unknown at this time, the possibility that this is a dimer two of 50 kDa adducts is unlikely since it was neither recognized by the antiCYP2E1 antibody nor observed in vivo. Interestingly, the human hepatocytes treated with trichloroethylene formed only a single detectable adduct at 47 kDa (Figs. 3 and 4). The
overall staining was considerably less intense when compared rat hepatocytes suggesting much lower levels of proteins modified by trichloroethylene. This is consistent with the quantitatively lower cytochrome P450-dependent trichloroethylene metabolism in humans (Garrett et al., 1996). In the human hepatocyte dose-response experiment (Fig. 3), the intensity of trichloroethyleneadducts appears to plateau between 1000 and 3000 ppm. One explanation of the initial increase in intensity followed by an apparent saturation at higher doses is that the binding may be related to the ability of trichloroethylene to inactivate cytochrome P450 by a mechanism-based process (Halmes et al., 1997b). That is, a trichloroethylene-cytochrome P450 intermediate complex, which is formed during metabolism, can rearrange to a reactive intermediate that can covalently bind to the heme prosthetic group and/or the apoprotein. Taken collectively, the increase in intensity followed by a plateau and the overall lower level of intensity seen in the human hepatocytes may be explained by the lower capacity of humans to metabolize trichloroethylene and the data showing a mechanism-based inhibition of cytochrome P450 by trichloroethylene. The sug-
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Fig. 4. Western blot and densitometric analysis of time-responsive formation of trichloroethylene-protein adducts in human hepatocytes. Left panel: Primary cultures of human hepatocytes exposed to 5000 ppm vaporized trichloroethylene for 0, 30, 60, 90 or 120 min. Lane 1: Total protein stained with Coomassie Brilliant Blue (CBB). Position of molecular weight standards are indicated by arrows. Right Panel: Densitometric analysis of the 47 kDa protein adduct.
gestion that the trichloroethylene is a suicide inhibitor of CYP2E1 in rats is also supported by the data presented in Fig. 5 left panel, which shows that the 50 kDa trichloroethylene-protein adduct in rat hepatocytes comigrates with a protein recognized by an antibody raised against CYP2E1. In the human hepatocytes this is not the case, since the major trichloroethylene adduct migrated to an appreciably different position than CYP2E1 (Fig. 5, right panel). These results taken collectively suggest that trichloroethylene is metabolized to a reactive intermediate which can bind covalently to cellular proteins and that one of the major targets of this metabolite in rats is likely CYP2E1. Binding to CYP2E1 is consistent with existing data showing a mechanism-based inhibition of this enzyme by trichloroethylene. However, the human hepatocytes exposed to trichloroethylene did not show the same pattern of adduct formation. It has been shown that there can be considerable variation in the levels of CYP2E1 among humans (Guengerich et al., 1991; Lipscomb et al., 1997), and the donor of these hepatocytes may have had very low levels of expressed CYP2E1. Because it is accepted that the range of molecular weights of the cytochromes
P450 is between 47 and 55 kDa, the adduct found in these particular human hepatocytes may be a different isozyme of the cytochromes P450. The pattern of the intensity shown in the human hepatocytes can be explained by the suggested mechanism-based inhibition of cytochromes P450 by trichloroethylene. Additional work needs to be done to identify the proteins covalently modified by a metabolite of trichloroethylene in humans. This can be done through immunoaffinity or other forms of chromatography (Pumford and Halmes, 1997). The ability to detect and identify the proteins modified by trichloroethylene can be a useful tool to enable further understanding of the underlying mechanisms of acute and chronic trichloroethylene toxicity as well as the carcinogenicity that has been associated with trichloroethylene exposure. The covalent binding of trichloroethylene to proteins may cause damage to proteins such as tubulin, histone proteins, topoisomerase II, or other DNA associated proteins which may potentially lead to DNA damage and this may be important in trichloroethylene carcinogenesis. Since we do not know the mechanism of carcinogenesis this and other possibilities cannot be ruled out. In addi-
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tion, regeneration of tissues following cell death could also be considered an epigenetic mechanism of carcinogenesis. The covalent binding of trichloroethylene could cause acute toxicity and potentially lead to tissue regeneration and incorporation of any mutations that may have occurred. At this time we do not know what if any role covalent binding to proteins has in the carcinogenesis of trichloroethylene. This is the first report of the production of a reactive metabolite capable of covalently modifying cellular protein in human cells exposed to trichloroethylene. The demonstration of covalent binding of a reactive intermediate of trichloroethylene in human hepatocytes along with the previously reported data in rodents suggests that differences exist in metabolism between humans and rodents which may be important in extrapolation and risk assessment.
Fig. 5. Comigration study of trichloroethylene-protein adduct with CYP2E1 in human and rat hepatocytes. Left Panel: Left lane: 2 h exposure of human hepatocytes to 5000 ppm vaporized trichloroethylene probed with anti-trichloroethylene antibody. Right lane: Same lane that has been stripped and reprobed with anti-CYP2E1 antibody. Right Panel: Left lane: 2 h exposure of rat hepatocytes to 5000 ppm vaporized trichloroethylene probed with anti-trichloroethylene antibody. Right lane: Same lane that has been stripped and reprobed with anti-CYP2E1 antibody.
Acknowledgements This work was supported in part by the Department of Energy (DE-FG01-92EW50625), the Strategic Environmental Research and Development Program (SERDP), the UAMS Graduate Student Research Fund, and the U.S. Environmental Protection Agency. We gratefully acknowledge the technical assistance of Patricia Confer.
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