Xenobiotic-modulated expression of hepatic glutathione S-transferase genes in primary rat hepatocyte culture

Abstract

CYP 2B1/B2 and 1A1 expression in primary rat hepatocytes plated on a substratum of Vitrogen using Chee's Essential Medium has been reported to be responsive to xenobiotic treatment (Jauregui, H.O., Ng, S.F., Gann, K.L. and Waxman, D.J. (1991) Xenobiotica 21, 1091–1106). Class α, μ and π glutathione S-transferase (GST) gene expression in response to xenobiotic treatment using this primary hepatocyte culture system was examined and the results compared with those obtained for P4502B1/B2 and 1A1 expression. Cytosolic GST activity decreased approx. 75% during the first 48 h of culture relative to freshly isolated hepatocytes and subsequently, increased, attaining a level at 96 h that was 134% of the activity at 48 h post-plating. Treatment of the hepatocyte cultures with phenobarbital (2 mM) or 3-methylcholanthene (5 μM) for 24, 48, or 72 h, beginning 24 h after plating, resulted in significant increases in glutathione S-transferase activity relative to control, with maximal increases of 158 and 164% measured at 72 h following phenobarbital or 3-methylcholanthrene treatment, respectively. SDS-PAGE analysis of cytosolic proteins showed a substantial increase in the intensities of protein bands migrating in the region of the GSTs following phenobarbital, β-naphthoflavone or 3-methylcholanthrene treatment. Immunoblot analysis of cytosolic fractions using affinity-purified class-specific GST IgGs confirmed that α, μ and π-class GST isozymes were elevated approx. 1.5- to 2-fold following phenobarbital, or β-naphthoflavone treatment; 3-methylcholanthrene was less effective in enhancing GST expression in cultured hepatocytes as compared to phenobarbital or β-naphthoflavone. Although GST π was below the limit of detection in freshly-isolated hepatocytes, enhanced expression of this form was observed in untreated hepatocytes cultured for longer than 72 h. Immunoblot analysis of microsomal fractions revealed that cytochrome P-4502 B1 2 B2 and 1A1 levels were increased significantly in hepatocyte cultures treated with phenobarbital or 3-methylcholanthrene, respectively, relative to the undetectable levels found in untreated controls. Northern blot analysis of poly(A) + mRNA isolated from cultures that had been treated with phenobarbital or 3-methylcholanthrene showed an approx. 2- and 4-fold increase in the expression of α and π class glutathione S-transferase mRNAs, respectively, as compared to untreated cells. The level of P-4501A1 or 2B1 mRNA was also markedly elevated following 3-methylcholanthrene or phenobarbital treatment, respectively. The results of this study demonstrate, for the first time, that expression of α, μ and π-class glutathione S-transferase genes is effectively modulated in primary hepatocyte culture system by different classes of xenobiotics.