Role of glutathione in the accumulation of anticarcinogenic isothiocyanates and their glutathione conjugates by murine hepatoma cells

Isothiocyanates (ITCs) are abundant in the human diet, Many potently inhibit tumorigenesis induced by a wide variety of chemical carcinogens in rodents. Recently, we observed that several ITCs accumulated to very high concentrations in cultured cells and that their accumulated levels were closely related to their potencies in inducing phase II enzymes [NAD(P)H:quinone reductase and glutathione transferases] that detoxify carcinogens. To elucidate the molecular mechanism responsible for this accumulation, the intracellular chemical identities of two ITCs, sulforaphane [SF, 1-isothiocyanato-(4R,S)-(methylsulfinyl)butane] and benzyl-ITC, were investigated in murine hepatoma cells. Both ITCs accumulated very rapidly to high intracellular concentrations, but, remarkably, most of the intracellular forms of the ITCs were dithiocarbamates resulting from conjugation with reduced glutathione (GSH). For example, the intracellular concentration reached 6.4 mM when cells were exposed to 100 mu M SF for 30 min at 37 degrees C and 95% of the accumulated product was the GSH conjugate. Cellular accumulation of each ITC was accompanied by a profound reduction in cellular GSH levels. These findings, together with our previous observation that accumulation of ITCs depended on cellular GSH levels, strongly suggest that intracellular conjugation of ITCs with GSH is mainly responsible for ITC accumulation. Surprisingly, rapid accumulation to high concentrations also occurred when cells were exposed to the GSH-ITC conjugates. However, these conjugates were apparently not absorbed intact, but were hydrolyzed extracellularly to free ITCs that were taken up by the cells. This conclusion is supported by the finding that suppression of dissociation of the conjugates by excess GSH or other thiols blocks accumulation of the conjugates.