Regulation of glutathione S‐transferase gene expression by phenobarbital in cultured adult rat hepatocytes

Abstract

Previous studies, by using Northern blotting analyses, showed that phenobarbital (PB) affects the steady-state mRNA levels of glutathione S-transferase (GST) subunits l/2, 314 and 7 in both conventional cultures of adult rat hepatocytes and cotuttures, with rat liver epithelial cells [Vandenberghe ct al., 1989, FEW Lett. 2.51, 59-64;Morel et al., 1989, FEBS Lett. 258, 99-1021. To determine whether PB acts at the transcriptional level, nuclear 'run-on' experiments using cDNA probes hybridizing to GST subunits l/2, 3/4 and 7 mRNA were performed on purified nuclei isolated from control and PI3 treated hepatocytes seeded under conventional and co-culture conditions. Data from this study demonstrate that the increase in steady-state mRNA levels observed in both conventionai cuIture and co-culture after 4 days PB exposure results from an increased tmns~ptional activity of the GST genes. However, a substantial increase in steady-state mRNA levels in the absence of a commensurate increase in transcriptional activity at 12 days of co-culture, indicates that the barbiturate has also a skbilizing effect in vitro on the GST mRNAs.Glutathione S-transferase; Gene transcription; Rat hepatocyte; Primary culture; Phenobarbital 1, INTRODUCTIONPhenobarbital (PB) has previously been reported to alter the expression, regulation and activity of several liver drug metabolizing enzymes [l-3]. Most attention has been paid to its effect on cytochrome P-450 [4-61, UDP-glucuronyltransferase [7,8] and glutathione Stransferase enzymes [9-l 11, However, it is yet not clear how this compound affects the regulation of expression of these enzymes. It is not known whether PB induction of transcriptional and translational activities involves one or more receptors [12,13].It has been shown that PB does not elevate alf cytochrome P-450 enzymes to the same extent. Cytochrome P-450 IIBI and III32 are the major liver enzymes to be induced [ 14,151. The rapid increase in the rates of transcription and the appearance of elevated Ievels of nuclear pre-mRNA and cytoplasmi~ mRNAs unequivocaily demonstrated that PB elevates levels of cytochrome P-450 III& and IIBz primarily by augmenting th: rate of transcription of the respective genes Rat bcpatocytes were isolated from Sprague-Dawley rats (ISO-ZOO 11, 1348 Mont-St-Guibert, Belgium. Fax: (32) (10) 450290. g) as described by Gugucn et al. [23). I'hcy were sccdcd in convcnm result from an increased transcriptional activity as found for the intact liver. Liver glutathione ~-transferase subunits 1 and 3 especially are also induced by PB treatment Ill]. Recently, Pickett et al. [20] reported that the increase of GST mRNA in total liver after PB addition results from an elevated GST gene transcription. In cultured hepatocytes, PB treatment induces the GST enzymatic activity 121,223. It was shown in previous study [21] that, by supplementing the culture medium with 3.2 mM PB, concentrations of individual GST subunits and GST steady-state mRNA levels were changed. However, no information is available on the re...

Effects of sodium butyrate on DNA content, glutathione S-transferase activities, cell morphology and growth characteristics of rat liver nonparenchymal epithelial cells in vitro

“…The level of subunit 7, which is hardly detectable in freshly isolated liver parenchymal cells, was significantly elevated when these cells were kept in primary culture (44). In contrast to these findings, subunits of the a and ft class are down-regulated in primary cultures of liver parenchymal cells (45). On the other hand, there is immunological evidence for the presence of subunit 7 in bile duct cells (41).…”

Transcriptional‐ and post‐transcriptional‐dependent regulation of glutathione S‐transferase expression in rat hepatocytes as a function of culture conditions

“…!ndeed, they recently demonstrated that levels of liver GST 1 transcripts in adrenalectomized rats were restored by administration of glucocorticoids [37]. It was also observed by our group that the GST 1/2 transcriptional activity [38] and corresponding steady-state mRNA levels [29] were induced in vitro by treating the hepatocytes with various xenobiotics, e.g. phenobarbital.…”

Effect of cell–cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells

“…The glutathione‐S‐transferase (GST) family, a family of dimeric enzymes that catalyze the conjugation of reduced glutathiones to electrophiles, is the most commonly studied of the phase II systems. Like the cytochrome P‐450 isoenzymes, GSTs are generally more stable in coculture than pure hepatocyte culture (31, 43–46, 83, 84). Stable expression of some subunits has been noted for 12 days in coculture, with significant quantitative differences between various nonparenchymal cell sources (44, 78).…”