Hormonal regulation of pseudocholinesterase activity in cultured rat hepatocytes.

Plasma pseudocholinesterase (PsChe) activity was examined in adult female rat hepatocytes isolated by collagenase perfusion and maintained in a chemically defined medium supplemented with dimethyl sulfoxide. Time course studies on PsChe activity in cultured hepatocytes indicate that cells maintained in a chemically defined medium lacking human GH and 17 beta-estradiol (E2) exhibit a decrease in activity after the first 3 days in culture followed by a stabilization of PsChe activity for up to 15 days. GH (0.02, 0.2, and 2 micrograms/ml) increased PsChe activity in a dose-dependent manner. Addition of E2 (10(-5)-10(-7) M) alone to hepatocyte cultures did not cause an increase in PsChe activity. The increases produced by both the 2 micrograms/ml and 0.2 micrograms/ml GH doses plus E2 (10(-7) M) were significantly greater than controls and similar to the increase produced by GH alone. The ability of the hepatocytes to express PsChe activity was not dependent upon the continuous exposure of the cells to GH, since control cultures, maintained for 12 days in medium lacking GH, were able to express a high level of PsChe activity after the addition of GH (2 micrograms/ml) on day 12. This increase was observed in hepatocytes in culture for 30 days. These results indicate that GH plays a pivotal role in the regulation of PsChe activity in vitro, and that under the conditions used in this study, E2 does not influence the ability of hepatocytes in culture to express this enzyme.

Androgen receptor dependent and independent activities of testosterone on hepatic microsomal drug metabolism.

Administration of testosterone for 6 days to intact female and castrate male BALB/cJ mice stimulated hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content by 50-75%. Testosterone also stimulated hepatic microsomal NADPH-oxidase activity, but to a lesser degree. To probe the mechanism of this effect of androgens, two antiandrogens (cyproterone acetate and flutamide) were employed. Since cyproterone acetate was a potent stimulator of hepatic microsomal ethylmorphine N-demethylase activity and cytochrome P-450 content, no antiandrogenic activity of this steroid could be detected. By contrast, flutamide alone had little effect on either ethylmorphine N-demethylase activity or cytochrome P-450 content. However, this drug effectively blocked the stimulatory effects of testosterone on ethylmorphine N-demethylase activity and cytochrome P-450 content but not on NADPH-oxidase activity. This effect was not species specific, since flutamide also prevented androgen stimulation of ethylmorphine metabolism in adult castrate and prepubertal male Fisher rats. The testosterone-induced increase of hepatic weight and microsomal protein content was not affected by the administration of flutamide. The observations are consistent with the hypothesis that androgens have two distinct effects on the liver. First, testosterone may act as a general, nonspecific stimulant of liver weight and microsomal protein content which is independent of the androgen receptor. Secondly, testosterone action in the liver may be expressed via an androgen-specific or androgen receptor-dependent mechanism which controls, in part, the cytochrome P-450-dependent demethylase system.

Epoxide hydratase and glutathione S-transferase activities in human lungs.

It has been reported human lung has cytochrome P-450 dependent monooxygenase activity which is necessary for the formation of mutagenic and carcinogenic metabolites of polycyclic aromatic hydrocarbons, such as benzo[a]pyrene. We now report epoxide hydratase and glutathione S-transferase, enzymes important in the further metabolism of certain benzo[a]pyrene metabolites formed by the monooxygenase system, have been detected in human lung tissues from patients undergoing surgery for lung cancer.

Responsiveness of an SV40-immortalized hepatocyte cell line to growth hormone.

The response of an SV40-immortalized hepatocyte cell line (CWSV-1) derived from adult male rat hepatocytes to human growth hormone (hGH) was investigated. CWSV-1 cells, which have been characterized extensively, retain certain differentiated functions of normal liver (Woodworth and Isom, Mol Cell Biol 7: 3740-3748, 1987). This cell line consists of tightly associated polygonal, mononucleated cells that grow as monolayers. These cells showed no significant morphological changes with the addition of hGH. Northern blot analysis showed that continuous treatment of the CWSV-1 cells with hGH induced the expression of insulin-like growth factor I (IGF-I) and 5 alpha-reductase RNAs. In addition, continuous exposure to hGH resulted in the induction of expression of the growth hormone receptor/growth hormone binding protein (GHR/GHBP) genes. This study indicates that the CWSV-1 cells may serve as a valuable in vitro model system for studying the signaling pathway of GH.

Mechanisms of diethyldithiocarbamate-induced loss of cytochrome P-450 from rat liver.

A single, intraperitoneal injection of diethyldithiocarbamate (DDTC) to adult, male Sprague-Dawley rats decreased hepatic cytochrome P-450 (P-450) concentrations. This effect was dose-dependent over a range of 250 to 750 mg/kg and most prominent 24-36 hr after dosing. Depletion of hepatic glutathione (GSH) by diethylmaleate (DEM) administration significantly decreased P-450 8 hr after concurrent treatment with DDTC at a dose which given alone had little effect on P-450 concentrations. When hepatic microsomes were incubated with DDTC in the presence of NADPH, P-450 was converted to cytochrome P-420 (P-420). Similar incubations employing [35S]DDTC demonstrated strict NADPH-dependent binding of labeled sulfur to microsomal membranes, suggesting that diminished P-450 concentrations are related to the metabolic activation of DDTC. Addition of reduced GSH to incubation mixtures blocked the binding of 35S to microsomal membranes, as well as conversion of P-450 to P-420. DDTC inhibited NADPH-ADP3+ mediated peroxidation of microsomal lipids in vitro, suggesting that the effect of DDTC on P-450 does not result from stimulation of lipid peroxidation, but may be influenced by the levels of hepatic GSH. DDTC treatment 1 hr after P-450 was pulse labeled by an intravenous injection of [3H]delta-aminolevulinic acid resulted in a 2-fold increase in the rate of loss of radioactivity associated with membrane-bound P-450 heme during the next 20 hr. Within this time interval, hepatic heme oxygenase (HO) activity increased and at 8 hr after dosing was 7-fold greater than control values in the livers, but was unchanged in the kidneys and spleens of DDTC-treated animals. An elevation of hepatic delta-aminolevulinic acid synthetase (delta-ALAS) activity occurred at 8 and 24 hr after DDTC treatment. Since this enzyme is rate limiting in the biosynthesis of heme, its increased activity may represent a compensatory response to offset the DDTC-mediated loss of P-450 heme.

Time-dependent induction of hepatic drug metabolism in rats by cotinine.

Cotinine, administered twice a day for 2 days in a dose of 20 or 40 mg/kg i.p., induced rat hepatic drug metabolism between 22% to 62%. Induction decreased to 26-37% after 4 days of cotinine (40 mg/kg bid). No significant induction was observed after 8 days of this dose of cotinine. Explanations are offered for decreasing induction by cotinine with time.

Effects of endogenous sex hormones on mouse liver ethylmorphine N-demethylase.

For most commonly used mouse strains there is either no sex difference in drug metabolism, or females have a higher rate of metabolism than males of the same strain. In the CRL:CD-1 strain, for example, the males have a lower Vmax and a higher Km than females for ethylmorphine N-demethylation. By contrast, kinetic analysis for this pathway of drug metabolism in the BALB/cJ mouse strain demonstrated that males have a higher Vmax and a lower Km than females. Although gonadal hormones appear to play a similar role in both the strains with respect to body weight, liver weight, microsomal protein content, and the weights of sex hormone responsive organs, a strict dependence of the sex differences in ethylmorphine (EM) metabolism on gonadal hormones could not be demonstrated. A systematic analysis of the spectral interactions of EM with cytochrome P-450 (P-450), the activities of NADPH P-450 reductase and NADPH oxidase in these mouse strains did not reveal a common regulatory site for gonadal hormones. Moreover, sex differences in EM N-demethylase activity are not a direct function of the total P-450 present in hepatic microsomes since, for both strains, males have higher P-450 content than females. We conclude, therefore, that sex differences in hepatic EM N-demethylase activity in the BALB/cJ and CRL:DC-1 mouse strains may depend on the relative quantities of the individual forms of microsomal P-450 which appear to be under genetic and/or hormonal control.