Modulation of gamma-glutamylcysteine synthetase large subunit mRNA expression by butylated hydroxyanisole

Dietary 2(3)-tert-butyl-4-hydroxyanisole (BHA) treatment has been shown to increase hepatic glutathione (GSH) content in rats and mice. Subsequent studies in our laboratory have demonstrated that hepatic gamma-glutamylcysteine synthetase (GCS) activity is increased in mice treated with dietary BHA. To test whether this increase in GCS activity follows an increase in hepatic messenger RNA for the large subunit of GCS (GCS-LS mRNA), a 390-base pair fragment corresponding to a region near the 5' end of the rat GCS-LS cDNA sequence was amplified using the PCR reaction and used to detect GCS-LS mRNA on Northern blots. Hepatic GSH, GCS activity, and GCS-LS mRNA levels were determined either in mice treated with BHA in the diet for 12 days or mice injected with diethyl maleate (DEM), phorone, and/or DL-buthionine-[S,R]-sulfoximine (BSO) over a 24 hr period. BHA caused a 1.5-fold increase in GSH levels, a 1.7-fold increase in hepatic GCS activity by Day 12, and a rapid 5-fold increase in hepatic GCS mRNA levels reaching maximal levels after 2-3 days. Partial depletion of GSH with either phorone (70%) or DEM (50%) resulted in a 4- to 5-fold increase in hepatic GCS-LS mRNA levels by 9 hr and a 1.5- to 2-fold increase in hepatic GSH and GCS activity by 24 hr. Depletion of GSH with the GCS enzyme inhibitor BSO had no effect on GCS mRNA expression, even though GSH was depleted to 30%. When BSO was combined with the phorone treatment GSH levels were depleted to < 10%, but the large increase in GCS-LS mRNA seen with phorone alone was greatly attenuated. These data suggest that depletion of GSH per se, is not sufficient to induce elevation of GCS-LS mRNA levels, but that the formation of GSH conjugates may be required to trigger GCS-LS mRNA induction. The increase in GCS-LS mRNA levels may account for the increase in GCS activity and elevation of GSH observed following BHA treatment, as well as the "rebound" of GSH above control levels observed 18-24 hr following depletion of GSH by other chemicals. These results are consistent with the Michael acceptor, hypothesis by Talalay.