Glutathione (GSH) transferases (GSTs) are a superfamily of multifunctional isoenzymes that play an important role in detoxification of a wide variety of electrophilic compounds primarily by catalyzing their conjugation with GSH. Mammalian cytosolic GSTs have been grouped into several classes (e.g., alpha, mu, pi, theta etc.) based on their physicochemical properties. The Pi class human GST isoenzyme (hGSTP1-1), which is polymorphic in human populations and involves amino acid substitutions in positions 104 (isoleucine or valine) and 113 (alanine or valine), is highly effective in catalyzing the GSH conjugation of dietary isothiocyanates (ITCs). ITCs are abundant in cruciferous vegetables, and known to offer significant protection against chemically induced cancer in experimental animals. Studies have suggested that chemopreventive activity of ITCs may be influenced due to their reactivity with GSH. The present studies were designed to investigate if the amino acid substitutions in positions 104 and/or 113 of hGSTP1-1 affect its activity towards ITCs. The rationale for this investigation was based on our previous work, which indicated that hGSTP1-1 variants differ significantly in their efficacy for GSH conjugation of certain environmentally relevant carcinogens. For example, we found that the variants with valine in position 104 [hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113)] are more efficient than isoleucine 104 containing isoforms [hGSTP1-1(I104,A113) and hGSTP1-1(I104,V113)] for GSH conjugation of dihydrodiol epoxide metabolite of benzo[a]pyrene. In the present study, we used three naturally occurring and widely studied ITC compounds including allyl isothiocyanate (AITC), benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC) to determine catalytic efficiency of hGSTP1-1 for their GSH conjugation. GST-catalyzed GSH conjugation of ITCs was examined by spectrophotometry as well as reverse-phase HPLC. To our surprise, catalytic efficiencies of the hGSTP1-1 variants were comparable for each ITC compound. Consistent with these findings, intracellular accumulation of GSH conjugate of AITC, BITC or PEITC was comparable in NIH3T3 cells stably transfected with allelic variants of hGSTP1-1. For example, incubation of 1 x 106 NIH3T3-hGSTP1-1(I104,A113) and NIH3T3-hGSTP1-1(V104,V113) transfected cells with 10 μM AITC, BITC or PEITC for 15 min. at 37oC resulted in the intracellular accumulation of 6-8 nmol of GSH conjugate of respective ITC compound in both the cell lines. These results indicate that hGSTP1-1 polymorphism may not affect activity of ITC class of chemopreventive agents. Supported in part by USPHS grant ES09140, awarded by the National Institute of Environmental Health Sciences, and grant CA101753, awarded by the National Cancer Institute.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]