In Vivo and in Vitro Hepatotoxicity and Glutathione Interactions of N-Methyldithiocarbamate and N,N-Dimethyldithiocarbamate in the Rat

Rodney W. Thompson 0 Holly L. Valentine 0 William M. Valentine 0 0 Department of Pathology and Center in Molecular Toxicology, Vanderbilt University Medical Center, MCN U4213 , 1161 21st Ave. South, Nashville, Tennessee 37232 The ability of N-methyldithiocarbamate (NMDC) to generate methylisothiocyanate and HS- together with its greater acid stability suggest that NMDC may exert greater acute toxicity following oral exposure than its dialkyl analog, N,N-dimethyldithiocarbamate (DMDC). To assess this possibility, cell culture, perfused liver, and in vivo studies were performed to delineate differences in the hepatotoxicity and thiol interactions of NMDC and DMDC in the rat. The role of methylisothiocyanate and HS- in NMDCinduced hepatotoxicity was evaluated and glutathione interactions characterized through analysis of reduced glutathione (GSH), glutathione disulfide (GSSG), and S-methylthiocarbamoylglutathione (GSMITC) using HPLC and liquid chromatography tandem mass spectrometry (LC/MS/MS). Following oral administration, centrilobular hepatocyte necrosis and enzyme leakage was observed for NMDC but not for DMDC. Dose dependent decreases of intracellular GSH were produced by both dithiocarbamates in primary hepatocytes but DMDC appeared to deplete GSH through the generation of GSSG whereas NMDC produced GSMITC consistent with the generation of a methylisothiocyanate intermediate. In primary hepatocytes, both NMDC and DMDC cytotoxicity was increased by prior depletion of intracellular GSH and diminished by prior supplementation of GSH. The results obtained using perfused livers were similar for NMDC in that elevated levels of GSMITC were detected in the bile; however, DMDC produced only a modest increase of GSSG over controls that was not significantly different to that produced by NMDC. Results obtained from isolated liver mitochondria and primary hepatocytes were not consistent with NMDC producing HS--mediated inhibition of mitochondrial respiration. These data support a greater potential for hepatotoxicity to result following oral exposure to NMDC relative to DMDC and that glutathione may play a role in cytoprotection for NMDC, presumably through detoxification of a methylisothiocyanate metabolite. - Human exposure to dithiocarbamates and their decomposition products occurs due to their many uses (WHO, 1998). 1 To whom correspondence should be addressed. Fax: (615) 343-9825. E-mail: . Populations that are most susceptible to high level exposures include those utilizing dithiocarbamates in the occupational setting, e.g., orchard workers, and individuals administered dithiocarbamates therapeutically (Brewer, 1993; Shinobu et al., 1983). In addition to human exposure, plant and animal wildlife are also exposed to dithiocarbamates through their use as pesticides and release into the environment as occurred with the 1991 spill of metam-sodium into the Sacramento River in California (CEPA, 1992). Two classes of commonly used dithiocarbamates include the monoalkyl and dialkyl dithiocarbamates that are distinguished by the number of alkyl substituents on the nitrogen. The sodium salt of N-methyldithiocarbamate (NMDC), metam-sodium, is a prevalent monoalkyl dithiocarbamate used principally as a soil fumigant (Keil et al., 1996). N,N-diethyldithiocarbamate (DEDC) and N,N-dimethyldithiocarbamate (DMDC), prepared as salts of various metals or as bis disulfides, are common dialkyl dithiocarbamates used in a variety of agricultural, medical, and industrial applications. Nitrogen substitution influences the rates of decomposition, the decomposition products generated, and the metabolic pathways observed for dithiocarbamates. Both monoalkyl and dialkyl dithiocarbamates share the ability to undergo acid-promoted decomposition to parent amine and CS2, as well as biotransformation through methylation and subsequent oxidation to monothiocarbamate sulfone and sulfoxide metabolites (Joris et al., 1970; Staub et al., 1995). An additional pathway unique to monoalkyl dithiocarbamates is generation of an alkyl isothiocyanate under physiological conditions, presumably through release of sulfhydryl ion (HS) (Drobnica et al., 1977; Joris et al., 1970). Both the isothiocyanate and the HS generated are biologically active species with the potential to acylate nucleophiles and inhibit mitochondrial respiration, respectively (Lam et al., 1993). Generation of the potent broadspectrum cytotoxicant, methyl isothiocyanate, is generally given credit for the effectiveness of NMDC as a soil sterilant (Pruett et al., 2001). Thus the unique ability of monoalkyl dithiocarbamates to generate the HS and isothiocyanate suggests that monoalkyl dithiocarbamates may present hazards not associated with the dialkyl dithiocarbamates. Dialkyl dithiocarbamates have been demonstrated in vitro to modulate apoptosis, enzyme function, transcription, and oxidation status depending upon the experimental conditions and cells used (Chinery et al., 1997; Hosni et al., 1992; Nobel et al., 1995, 1997). Although the biological consequences and target organs of dithiocarbamates are still being delineated in vivo, neurotoxicity has been associated with subchronic administration of DEDC in rabbits, rats, sheep, and hens while the use of disulfiram, the disulfide of DEDC, in alcohol aversion therapy in humans has produced two well recognized sequelae, neurotoxicity and hepatotoxicity (Edington and Howell, 1969; Forns et al., 1994; Johnson et al., 1998). Whereas hepatotoxicity appears to be an idiosyncratic reaction possibly mediated through an auto-immune mechanism, neurotoxicity appears to be dose dependent. Thus the potential for acute toxicity following common dialkyldithiocarbamate exposures appears to be low with adverse effects requiring repeated exposures. In contrast, there is considerably less toxicological data available for NMDC in mammals, with the data available consisting primarily of reports on workers and wildlife following the metam-sodium spill in California and immune system effects produced in rodents (CEPA, 1992; Keil et al., 1996). The chemical properties of its unique decomposition products combined with its greater acid stability suggest that NMDC may exert greater acute toxicity following oral exposure relative to its dialkyl analog, DMDC. In the study presented here the potential for NMDC to produce hepatotoxicity in the rat following acute oral exposure was examined. The role of methylisothiocyanate and HS in NMDC induced hepatotoxicity was evaluated and the major interactions of NMDC with intracellular thiols were characterized through the analysis of reduced glutathione (GSH), glutathione disulfide (GSSG), and glutathione conjugates in primary rat hepatocytes and intact rat liver perfusion systems. Reactions and biological effects unique to the mono substituted NMDC were identified through comparison to results obtained for its dialkyl analog, DMDC. MATERIALS AND METHODS Caution: Carbon disulfid (...truncated)