Induction, regulation and messenger half‐life of cytochromes P450 IA1, IA2 and IIIA6 in primary cultures of rabbit hepatocytes

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Abstract
A study on the regulation and induction of expression of cytochromes P450-IA1, IA2 and IIIA6 genes has been undertaken using primary cultures of adult rabbit hepatocytes grown in a serum-free chemically and hormonally defined medium. In 72-h-old cultures, 50 μM β-naphthoflavone induced both IA1 and IA2 mRNA, the maximum level being reached after 4 h and 12 h, respectively. This was shown to result from an increase in the rate of transcription of gene IA1. In contrast, gene IA2 was constitutively transcribed in untreated cells, but mRNA only accumulated in the presence of β-naphthoflavone which, however, did not affect the rate of transcription. Actinomycin D fully blocked induction of both IA1 and IA2 mRNA in response to their inducer. In untreated cells the presence of cycloheximide allowed a ‘constitutive’ expression of gene IA1, while in β-naphthoflavone-treated cells, it produced a super-induction of IA1 but no modification of IA2 gene expression. Rifampicin (50 μM) strongly increased the IA1 mRNA level and rate of transcription only in cycloheximide-treated cells. Rifampicin and dexamethasone, two prototypical inducers of P450-IIIAs, induced both large and small IIIA6 mRNAs in a time-dependent fashion, the maximum level being reached after 24 h. This was related to a large increase in the rate of transcription of the gene. Cycloheximide significantly decreased the accumulation of both IIIA6 mRNAs in response to rifampicin, while actinomycin D fully blocked induction. The half-lives of IA1, IA2 and IIIA6 mRNAs were determined by two different methods, namely actinomycin D and [3H]uridine-chase experiments. In untreated cells, the half-lives for IA1, IA2 and IIIA6 mRNAs were 14 h, 16 h and 19 h, respectively when determined by the uridine chase and 18 h, 25 h and 22 h when determined by the actinomycin-D chase. These values were not modified significantly in cells treated with β-naphthoflavone or rifampicin, indicating that neither of these inducers affected the stability of IA1 and IA2 or IIIA6 messages, respectively. We conclude that: (a) gene IA1 is primarily regulated at the transcriptional level in response to β-naphthoflavone, derepression of this gene being the most critical step controlling its expression; (b) gene IA2 expression does not require protein synthesis and appears to be placed under the control of both transcriptional and post-transcriptional effects (stabilization of cytoplasmic IA2 mRNA by the inducer, suspected by several authors to account for message accumulation, was not detected in this work); (c) gene IIIA6 expression is primarily regulated at the transcriptional level in response to rifampicin and dexamethasone and requires protein synthesis. Finally, CYP 1A1 and 1A2 were located on rabbit chromosome 17 p1.2 and CYP 3A6 on 6 q-ter.