PubMed1993
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 alpha, mu and pi 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 microM) 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, beta-naphthoflavone or 3-methylcholanthrene treatment. Immunoblot analysis of cytosolic fractions using affinity-purified class-specific GST IgGs confirmed that alpha, mu and pi-class GST isozymes were elevated approx. 1.5- to 2-fold following phenobarbital, or beta-naphthoflavone treatment; 3-methylcholanthrene was less effective in enhancing GST expression in cultured hepatocytes as compared to phenobarbital or beta-naphthoflavone. Although GST pi 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-4502B1/2B2 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 alpha and pi 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 demonor the first time, that expression of alpha, mu and pi-class glutathione S-transferase genes is effectively modulated in primary yet culture system by different classes of xenobiotics.