Comparison of sulfur amino acid utilization for GSH synthesis between HepG2 cells and cultured rat hepatocytes.

HepG2 cells are widely used as a model of human hepatocytes for studies of drug metabolism and toxicity. However, GSH metabolism in HepG2 cells is poorly characterized. This report describes the utilization of sulfur amino acids for GSH synthesis in HepG2 cells. In contrast to primary cultures of rat hepatocytes, which rely mostly on methionine for GSH synthesis, HepG2 cells use cystine. Their inability to utilize methionine for GSH synthesis was not due to lack of methionine uptake or low cellular ATP levels, but rather to the lack of S-adenosyl-methionine synthetase activity. When HepG2 cells were cultured overnight in medium containing cystine as the only sulfur amino acid, addition of glutamate or acivicin had minimal to no effect on cell GSH; however, addition of threonine significantly depleted cell GSH. When cystine (0.18 mM) uptake was measured, glutamate (2.5 mM), which inhibited cystine uptake in cultured rat hepatocytes, had a minimal effect in HepG2 cells. Instead, threonine (20 mM) strongly inhibited the apparent uptake of cystine by HepG2 cells. Strong inhibition by threonine of apparent cystine uptake was actually due to inhibition of cysteine uptake, which resulted from GSH-cystine mixed disulfide exchange. Radio-HPLC confirmed this. After incubating cells with [35S]cystine (0.18 mM) for 10 min, the total counts inside the cell matched the counts in the uptake medium in the form of GSH-cysteine mixed disulfide. Finally, HepG2 cells took up cysteine by both Na(+)-dependent and -independent mechanisms. The former exhibited high affinity and low capacity, whereas the latter exhibited the opposite. At a physiologic concentration of cysteine (10 microM), 68% of cysteine uptake occurred via the Na(+)-dependent system and 32% via system L1.