Effects of glutathione depletion on cadmium-induced metallothionein synthesis, cytotoxicity, and proto-oncogene expression in cultured rat myoblasts

Cadmium (Cd) is a highly toxic metal and a known carcinogen. Although the carcinogenic mechanism of action is unknown, Cd will induce transcriptional activation of c-myc and c-jun. We have previously found that the extent of Cd-induced oncogene expression is limited by the presence of cellular metallothionein (MT) in rat L6 myoblasts. Glutathione (GSH) is thought to play an important role in protection against Cd before the onset of MT synthesis. Thus, this study examined the effects of GSH depletion on Cd-induced MT synthesis, cytotoxicity, and proto-oncogene expression in rat L6 myoblasts after pretreatment with L-buthionine sulfoximine (BSO), a potent inhibitor gamma-glutamylcysteine synthetase, which effectively depletes GSH. Exposure of L6 cells to BSO (5 or 25 mu M) resulted in a dose-dependent decrease in cellular GSH levels, GSH depletion had no effect on Cd- or zinc-induced MT synthesis. Although the depletion of GSH was not itself cytotoxic in L6 cells, BSO pretreatment, particularly at the higher dose (25 mu M), resulted in a dose-dependent increase in the sensitivity to Cd cytotoxicity, as assessed by a tetrazolium-based dye (MTT) assay. Low levels of Cd (1 mu M) slightly increased the expression of both c-myc and c-jun as assessed by increases in gene-specific mRNA levels, in accordance with previous studies. GSH depletion (5 mu M BSO) likewise caused an increase in expression of c-myc and c-jun. However, combined GSH depletion and Cd exposure decreased levels of c-myc and c-jun transcription well below control levels. These results suggest that increased cytotoxicity resulting from exposure to Cd after BSO depletion of cellular GSH abrogates the oncogene activation observed after either treatment alone. Thus proto-oncogene expression induced by Cd appears to be dependent on the absence of overt Cd-induced cytotoxicity.