* Departments of Pharmacology and Toxicology and
Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205
Received September 9, 1999; accepted November 22, 1999
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ABSTRACT |
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Key Words: trichloroethylene; autoimmunity; MRL+/+; diallyl sulfide; CYP2E1; metabolism.
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INTRODUCTION |
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Trichloroethylene is extensively metabolized in both humans and laboratory animals, with the major metabolites being trichloroethanol and its glucuronide conjugate, trichloroacetic acid and trichloroacetaldyhyde (chloral). The metabolism of trichloroethylene is primarily through the cytochrome P450 monooxygenase enzymes, principally the 2E1 isozyme (CYP2E1). We have previously shown that trichloroethylene is activated to a reactive intermediate(s) that primarily binds covalently to a 50 kDa microsomal protein, probably CYP2E1 (Halmes et al., 1996, 1997a
, b
). In addition, metabolic activation of trichloroethylene is a prerequisite for hepatotoxicity associated with exposure (Buben and O'Flaherty, 1985
).
The primary concern for the health effects of trichloroethylene has been its carcinogenic potential (ATSDR, 1993). However, trichloroethylene has recently been implicated in the development of autoimmune disorders and immune system dysfunction in humans. Trichloroethylene exposure has been associated with systemic lupus erythematosis (SLE), systemic sclerosis, fasciitis, and altered T-lymphocyte ratios (Byers et al., 1988; Clark et al., 1994
; Flindt-Hansen and Isager, 1987
; Kilburn and Warshaw, 1992
; Lockey et al., 1987
; Waller et al., 1994
; Yanez Diaz et al., 1992
).
Little is known about potential mechanisms by which trichloroethylene promotes an autoimmune response. An animal model of trichloroethylene-induced acceleration of an autoimmune response has been developed in MRL+/+ mice (Khan et al., 1995). Unlike the genetically similar MRLlpr/lpr mice, which develop a spontaneous autoimmune disease early in life, mediated in part by a Th1-type immune response (Takahashi et al., 1996
), the MRL+/+ mice, although autoimmune prone, do not normally develop the clinical symptoms of autoimmunity until late in life. However, MRL+/+ mice treated with trichloroethylene have been shown to exhibit similar immune system alterations seen in the MRLlpr/lpr mice. Using the MRL+/+ mice, we reported that trichloroethylene treatment promoted expansion of the percentage of CD4+ T cells with a Th1 type cytokine profile. These mice also expressed high levels of CD44, a cell-adhesion receptor that is upregulated following T cell activation (Griffin et al., 2000
). This alteration in CD4+ T-cell activity may be involved in the accelerated autoimmune response in MRL+/+ mice exposed to trichloroethylene. Because metabolic activation of trichloroethylene is an obligatory pathway for its hepatotoxic and carcinogenic effect, the present study was undertaken to examine if metabolism by CYP2E1 is also required for the immunomodulatory effects following trichloroethylene exposure.
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MATERIALS AND METHODS |
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Animals and treatments.
Eight-week-old female MRL+/+ mice were purchased from The Jackson Laboratories (Bar Harbor, ME) and housed in polycarbonate cages, provided with hardwood-chip bedding and maintained on a 12-h light/dark cycle. The animals were provided lab chow and drinking water ad libitum and were acclimated for 1 week before treatment. The animals were given trichloroethylene in the drinking water at 0 or 2.5 mg/ml for 4 weeks, receiving approximately 0 or 300 mg/kg/day, respectively. The water was changed every 2 to 3 days to ensure maintenance of the dose. Sanders et al. determined that less than 20% of the trichloroethylene was lost when water was changed twice a week (Sanders et al., 1982). Trichloroethylene was suspended in drinking water with 1% of the emulsifier Alkamuls EL-620. Diallyl sulfide (DAS) was administered to inhibit CYP2E1 enzyme activity as previously described (Badger et al., 1995
) with the following modification: DAS, approximately 200 mg/kg/day, or saline was administered via Alzet osmotic pumps implanted subcutaneously. The mice were weighed once a week to monitor weight changes. At the end of the treatment period, the animals were anesthetized with CO2, and blood was collected from the retro-orbital plexus. The spleens were aseptically removed, and immediately processed as described in the mitogenic response section. The livers were removed immediately, weighed, and stored at 80°C until analyzed. Blood was allowed to clot at room temperature for 1 h, then centrifuged at 1000 x g for 30 min, and serum was collected and stored at 80°C until further analysis. A section of liver and kidney were fixed in neutral buffered formalin and processed for hematoxylin and eosin staining. The other section of liver was used for fraction preparation and Western-blot analysis.
Western-blotting analysis.
Liver microsomes from individual animals were prepared as previously described (Halmes et al., 1996). Microsomal protein concentration was determined by Coomassie protein assay using bovine serum albumin as the standard. Proteins from the microsomal fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then transferred to nitrocellulose as previously described (Halmes et al., 1996
). Immunoblots were stained using affinity-purified anti-dichloroacetyl antiserum as previously reported (Halmes et al., 1996
). The membranes were then stripped of antibodies according to instructions in Amersham's Enhanced Chemiluminesence (ECL) kit, and re-probed with anti-CYP2E1 antiserum (Eliasson et al., 1988
; Johansson et al., 1988
). Trichloroethylene-protein adducts and CYP2E1 were visualized using peroxidase-labeled secondary goat anti-rabbit antibodies with an ECL substrate. Immunoblots were scanned using a laser densitometer and the intensity of CYP2E1 staining for individual animals was quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Proliferative response to mitogen.
Spleens from individual animals were teased apart in RPMI 1640 and CD4+ T cells were isolated using anti-mouse CD4 magnetic beads following manufacturer's instruction (Dynal, Lake Success, NY). CD4+ T-cell purity was determined by flow cytometry to be 95%. Following purification, 2 x 105 cells per well were plated in microtiter plates in RPMI 1640 media supplemented with 2 mM L-glutamine, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 x 105 M 2-mercaptoethylene and 10% fetal calf serum. Conconavalin A was added to wells in triplicate at concentrations of 0, 0.4, 1.0, and 4.0 µg/ml. After 72 h at 37°C, the cells were pulsed with 0.5 µCi/well of 3[H]-thymidine for an additional 24 h, and the incorporated radioactivity was measured using a Matrix-96 Direct Beta counter (Packard, Meriden, CT).
Interleukin-4 secretion.
Splenic CD4+ T cells (2 x 105 per well) were incubated for 72 h at 37°C in a 96-well ELISA plate containing immobilized anti-CD3 mAb [(Hamster IgG1 clone 1452C11, Pharmingen, La Jolla, CA) 50 µl of 10 ng/ml for 24 h at 20°C] and soluble anti-CD28 mAb [(Hamster IgG1 clone 37.51, Pharmingen, La Jolla, CA) 10 µg/ml]. After 72 h, the supernatants were removed and tested using capture and detecting mAb's for IL-4 [rat IgG1 clone 11B11 and rat IgG1 clone BVD6-24G2 (Pharmingen, La Jolla, CA), respectively]. Cytokine concentrations were determined by comparison to a standard curve generated using mouse recombinant IL-4.
Clinical chemistry.
Serum levels of alanine aminotransferase were measured according to the manufacturer's instructions with a kit from Sigma Diagnostic Inc (St. Louis, MO). Renal function was determined by measuring blood urea nitrogen levels following manufacturer's instructions (Sigma Diagnostic Inc., St. Louis, MO).
Statistical analysis.
When appropriate, statistical significance was determined using one-way analysis of variance with a predetermined significance level of p 0.05. To determine statistical differences between groups, Student-Neuman-Keuls post-hoc test was used. Data is presented as mean ± standard error of the mean (SEM) for n = 5 mice per group.
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RESULTS |
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DISCUSSION |
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To confirm the effectiveness of DAS in the present study, we examined the effect of this inhibitor on the CYP2E1-mediated production of trichloroethylene-protein adducts, as a marker of metabolic activation. The trichloroethylene-protein adducts were visualized with an antibody specific for dichloroacetylated proteins. We have previously shown that several liver and lung microsomal proteins are covalently modified by a reactive metabolite of trichloroethylene (Griffin et al., 1998, 2000
; Halmes et al., 1996
, 1997a
, b
). The most predominant trichloroethylene-protein adducts are of 50 and 100 kDa. Similarly, in the present study, two major immunoreactive bands at 50 and 100 kDa were detected in a Western blot analysis of liver microsomal proteins from MRL+/+ mice treated with trichloroethylene. However, if the mice were concomitantly treated with trichloroethylene and DAS, metabolic activation of trichloroethylene to a reactive intermediate capable of covalent binding was completely blocked. Although the functional role of these protein adducts has not yet been determined, blocking the production of these adducts in the presence of DAS constituted evidence that this inhibitor was blocking metabolic activity of CYP2E1 in our system.
Inhibition of trichloroethylene-protein adducts suggests that DAS is suppressing the metabolic activity of CYP2E1. Such DAS-induced inhibition has been shown to play a role in the ability of DAS to act as a chemoprotective agent by selectively inhibiting metabolic activation of several toxic compounds (Jin and Baillie, 1997). It has been shown that inhibition of the initial metabolic event facilitated by CYP2E1 constitutes the primary protective action of DAS (Yang et al., 1994
). However, the finding that DAS inhibits protein levels of CYP2E1 suggests that DAS may also work in ours system by suppressing CYP2E1 protein levels. Along these lines, Reicks and Crankshaw (1996) reported that DAS inhibits both the activity of CYP2E1, and CYP2E1 protein expression in rats following DAS treatment. In addition to DAS, trichloroethylene also somewhat suppressed protein levels of CYP2E1. This reduction in protein expression may be related to the inhibitory effect of trichloroethylene on CYP2E1 enzyme activity as previously noted (Halmes et al., 1997b
). Taken together, this data indicates that DAS is acting to inhibit CYP2E1 mediated metabolic activation of trichloroethylene.
With evidence that DAS was effectively inhibiting CYP2E1-mediated metabolic activation of trichloroethylene, we examined the effects of CYP2E1 inhibition on trichloroethylene-induced immune system alterations. This examination centered on CD4+ T cells because we have previously shown that trichloroethylene significantly alters the activation state and cytokine profile of this lymphocyte subset (Griffin et al., 2000). The importance of CD4+ T cells in mediating trichloroethylene-induced autoimmunity is mirrored in other models of idiopathic and experimental autoimmune diseases: CD4+ T cells can often transfer autoimmune disease, while depletion of this T cell subset often suppresses disease induction and/or progression (Gilbert et al., 1999
). In addition, similar to trichloroethylene-induced autoimmunity (Griffin et al., 2000
), idiopathic systemic immune diseases such as systemic lupus erythematosus, scleroderma, rheumatoid arthritis, and Sjogren's syndrome are accompanied by an increase in the percentage of activated/memory CD4+ T cells (Gilbert et al., 1999
).
The CD4+ T cells in MRL+/+ mice, unlike the CD4+ T cells in MRL lpr/lpr mice, are normally not hyperproliferative. However, in accordance with our previous findings demonstrating increased activation of CD4+ T cells in mice treated with trichloroethylene (Griffin et al., 2000), the CD4+ T cells isolated from MRL+/+ mice treated for 4 weeks with trichloroethylene exhibited an increased mitogenic response. However, the increased responsiveness of the T cells to Con A was reversed if the mice were treated with both trichloroethylene and DAS. In addition to their increased mitogenic response, the CD4+ T cells isolated from MRL+/+ mice treated with trichloroethylene also exhibited a reduction in IL-4 production. A decrease in the percentage of CD4+ T cells that secreted IL-4, and an increase in the percentage of CD4+ T cells that secreted IFN-
were similarly observed in an earlier study, after 4 weeks of trichloroethylene treatment (Griffin et al., 2000
). This finding supports the hypothesis that, at least early in the treatment regimen, trichloroethylene promotes the expansion of Th1 cells. The fact that trichloroethylene skews the CD4+ T cell response toward a Th1-like cytokine profile may help explain the ability of the compound to promote autoimmunity since IFN-
secretion has been shown to be critical to the development of disease pathology in MRL mice (Haas et al., 1997
). It seems likely that IL-4 inhibits the Th1 cell bias needed for disease pathology, since studies on lupus-prone mice have shown that constitutive production of IL-4 through expression of an IL-4 transgene suppresses the development of lupus nephritis (Reininger et al., 1996
). In the present study, it was shown that inhibiting CYP2E1 with DAS in vivo completely reversed the decreased IL-4 production observed in CD4+T cells isolated from trichloroethylene-treated mice. The reversal of the enhanced mitogenic response, in addition to the reversal of the altered cytokine profile following inhibition of CYP2E1 with DAS in vivo suggests that metabolism of trichloroethylene is an obligatory pathway in the alterations in CD4+ T cells observed following trichloroethylene treatment.
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ACKNOWLEDGMENTS |
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NOTES |
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