Glutathione and glutathione-related enzyme activities of male and female rat hepatocytes under various culture conditions

Abstract

The effect of culture medium on glutathione (GSH) dependent detoxification defence system of primary cultured hepatocyte from either male or female rats was studied. Intracellular reduced (GSH) and oxidized glutathione (GSSG), and six GSH-related enzyme activities, including GSH peroxidase (GSH Px), GSH reductase (GSH Rd), cytosolic GSH S-transferase (cGST), microsomal GSH S-transferase (mGST), gamma-glutamyl transpeptidase (GTP), and gamma-glutamylcysteine synthetase (GCS), were investigated during a 6-day culture. Media free of fetal bovine serum (FBS) and with 2.5 or 10% FBS were used. Whatever the medium, there was an initial decrease of intracellular GSH and GSSG, a threefold increase of GSH at day 3 and fourfold increase of GSSG at day 4, later decreasing to their original level at day 6. The activities of all six GSH-related enzymes of male and female hepatocytes remained relatively stable during the first 72h, then gradually decreased to 50-80% of initial activities. With the exception of cGST, time-course profiles of other enzyme activities were not significantly different among various media. In both sexes, higher cGST activity was maintained for cells cultured in the presence of FBS. Results of immunoblotting analysis of cytosolic GST isozymes indicate that the placental form of GST (Yp) was markedly increased after plating and the extent of increase of Yp was higher in the presence of FBS. Despite the culture medium, the level of GST isoform Ya was maintained steadily for 6 days, however, Yb was maintained during the first 3 days and then decreased. In terms of the gender difference, GSH Px and GTP activities of hepatocytes from females were significantly greater than of males over the entire culture period. Results indicate that FBS seems not to be absolutely essential in maintaining GSH level and most of the GSH-related enzyme activities in rat hepatocytes. Furthermore, GSH levels and GSH-related enzyme activities of hepatocytes from female rats were similar to those from male rats.

Sex-specific extraction of organic anions by the rat liver

“…In contrast to the lower glucuronidation rate in males just mentioned, glutathione Stransferase (GST) is variously reported to be 1.5-and 2.2-fold higher in livers of male as compared with female rats (Kanai et al, 1985;Rogiers et al, 1991), or not significantly different in rat hepatocytes (Lii et al, 1996). In our hands males exhibited a more than nine-fold higher rate of biliary BSP excretion as compared with females.…”

Suppression of Cytochrome P450 3A Protein Levels by Proteasome Inhibitors

“…The differences between these two studies may reflect differences between cultured and freshly isolated hepatocytes. However, because primary rat hepatocytes cultured for 1 to 6 days have levels of intracellular glutathione that are at least as great as those in freshly isolated cells (Lii et al, 1996), the differences between these studies are unlikely to be due to higher glutathione levels in freshly isolated cells.…”

Neonatal Selenoenzyme Expression Is Variably Susceptible to Duration of Maternal Selenium Deficiency

“…Evaluation of rodents after gonadectomy demonstrates variable results, but some prior work supports a component of estrogenic and androgenic regulation [47,69]. In vitro work in hepatocytes indicated the possible contribution of steroid-independent mechanisms [70]. Regardless, our data imply that the mechanisms responsible for the adult sex differences in GPx do not appear to be present in the neonatal period.…”