Utilization of methionine as a sulfhydryl source for metallothionein synthesis in rat primary hepatocyte cultures

Metallothioneins (MT) contain a high concentration of cysteine which bind heavy metals. Exposure of liver cells to metals induces the synthesis of MT and thus causes the cells to draw upon their sulfhydryl (SH) pools. The utilization of methionine as compared with that of cysteine as a source of SH for the synthesis of MT has not been shown. Therefore, studies were designed to determine whether methionine, in addition to cysteine, serves as an SH donor for Zn-induced synthesis of MT in rat hepatocyte cultures. Hepatocytes were able to synthesize only low levels of MT when the concentration of both amino acids was extremely low; however, when either of the amino acids was present at a high concentration, production of MT was independent of the other amino acid concentration. Subsequently, induction of MT was compared in four media: complete (0.5 mM methionine, 0.5 mM cysteine), Met (0.5 mM methionine), Cys (0.5 mM cysteine), and SH free (-SH). Somewhat higher concentrations of MT were produced by the hepatocytes in the Met than in the Cys media and no differences were observed between the Met and the complete media. By contrast, GSH synthesis was much more dependent on methionine than on cysteine for its synthesis. Incorporation studies with 35S-labeled cysteine and methionine indicated that lower concentrations of MT found in hepatocytes in the Cys media may be due to less accumulation of cysteine by the hepatocytes. Cellular accumulation of cysteine was initially rapid and then reached a plateau, whereas the rate for methionine accumulation was more constant and eventually obtained higher cellular levels. To provide additional evidence for the role of methionine in MT production, a known inhibitor of the cystathionine pathway, DL-propargylglycine (PPG), was added to each of the four media. Reductions in MT levels were not observed in the cells cultured in the complete and Cys media; however, a 95% reduction was observed in the cells cultured in the Met media. In summary, the present results suggest that both cysteine and methionine can serve as a SH source for MT synthesis, and that the availability of SH in most culture mediums would not limit the synthesis of MT. Whereas methionine is a much better SH source than cysteine for GSH synthesis in hepatocyte cultures, it is only slightly better for MT synthesis.