The formation of both a mono- and a bis-substituted glutathione conjugate of hexachlorobutadiene by isolated hepatocytes and following in vivo administration to the rat.

The nephrotoxicity of hexachloro-1,3-butadiene (HCBD) appears to depend on the initial formation of a glutathione (GSH) conjugate in the liver. In the present study we have examined the hepatic metabolism of HCBD using isolated hepatocytes and following in vivo administration. Exposure of isolated hepatocytes to HCBD resulted in a dose-dependent depletion of GSH. HPLC analysis of the incubation medium demonstrated the formation of two products. When isolated hepatocytes containing [3H]GSH were exposed to [14C]HCBD, coincident elution of 3H and 14C corresponding to the previously recognized HPLC peaks was observed. Both products were sensitive to treatment with gamma-glutamyl transpeptidase (gamma-GT), providing additional support for their identification as GSH conjugates. The ratio of 3H to 14C in the two peaks indicated the formation of both a mono- and a bis-substituted GSH conjugate of HCBD. The identification of the mono- and bis-GSH conjugates was further confirmed by the preparation of synthetic standards which displayed retention times by HPLC identical to the biological products. The production of the total and individual GSH conjugates displayed both dose and time dependence. The production of the total as well as the ratio of mono- to bis-conjugate was found to depend on the availability of GSH. At low HCBD exposure levels the bis-substituted conjugate accounted for more than 20% of the total conjugate produced by isolated hepatocytes. This value decreased at higher HCBD concentrations. Analysis of bile collected from rats following intraportal administration of [14C]HCBD revealed the presence of both the mono- and bis-substituted GSH conjugates of HCBD as well as additional 14C-containing metabolites. The results of the present study clearly demonstrate the production of both a mono- and a bis-substituted GSH conjugate of HCBD. The potential importance of this finding in terms of the nephrotoxicity of HCBD is discussed.