Inducer-dependent transcriptional activation of the P4501A2 gene in vivo and in isolated hepatocytes.

In vitro nuclear run-on transcription analysis using probes directed against different regions of CYP1A2 revealed that the 70-100-fold induction of CYP1A2 mRNA by polycyclic aromatic compounds is associated with a corresponding increase in the transcriptional activation of this gene in rat liver. Probes from regions of the 1st, 2nd, and 4th introns detected approximately 50-100-fold higher CYP1A2 run-on transcription in liver nuclei from inducer-treated animals than in nuclei from untreated animals. The run-on signals from untreated rats were 3-5-fold above background signals. Additional experiments using single-stranded DNA probes and a probe from a region 5' to the CYP1A2 transcription start site revealed that the inducer-dependent transcripts were colinear with the CYP1A2 mRNA and that they did not result from read through of an initiation event 5' to CYP1A2. Run-on transcription analyses were also carried out with nuclei from isolated hepatocytes using the same series of probes spanning CYP1A2. These analyses indicated that the inducer-dependent accumulation of CYP1A2 mRNA in hepatocytes is associated with at least a 20-fold increase in CYP1A2 transcription. In contrast to liver and hepatocytes, these probes failed to detect run-on transcripts from kidney nuclei, indicating that the lack of CYP1A2 mRNA in this tissue is due to the lack of transcriptional activation of this gene by polycyclic aromatic compounds.

Transcriptional and post-transcriptional regulation of the genes encoding cytochromes P-450c and P-450d in vivo and in primary hepatocyte cultures.

In both primary cell cultures of rat hepatocytes and in liver, polycyclic aromatic hydrocarbons (PAHs) were found to influence the accumulation of the cytochrome P-450c and P-450d mRNAs by both transcriptional and post-transcriptional mechanisms. Following treatment with PAHs, cytochrome P-450c mRNA levels increased approximately 100-fold in both hepatocyte cultures and in liver, while transcription rates, measured by run-on transcription of isolated nuclei, increased 3-fold in hepatocyte cultures and 10-fold in liver. The difference in the -fold increases of mRNA level and transcription rate suggests that post-transcriptional, as well as transcriptional, mechanisms contributed to the regulation of cytochrome P-450c mRNA levels. Following treatment with PAHs, cytochrome P-450d mRNA levels increased 200-fold in hepatocyte cultures and 70-fold in liver, while transcription rates remained unchanged in hepatocyte cultures and increased only 1.7-fold in liver. This suggests that post-transcriptional mechanisms were of primary importance in regulating cytochrome P-450d mRNA levels. The newly developed hepatocyte primary cell culture system used in these studies differs from previously reported systems in that the cytochrome P-450d gene, as well as the cytochrome P-450c gene, were expressed in response to PAHs. In this cell culture system the regulation of these two genes was quite similar, although not identical, to that found in liver. The mechanisms controlling the tissue-specific expression of the genes encoding cytochromes P-450c and P-450d were also examined. The cytochrome P-450c mRNA was found in kidney, heart, and lung, as well as in liver, of PAH-treated rats, while the mature cytochrome P-450d mRNA was detected only in liver. The substantial increase in cytochrome P-450c mRNA in kidney in response to beta-napthoflavone was not associated with a detectable change in the transcription rate of cytochrome P-450c gene, indicating that cytochrome P-450c mRNA levels must be regulated primarily post-transcriptionally in kidney. Even though mature cytochrome P-450d mRNA could not be detected in kidney, the cytochrome P-450d gene was transcribed at a substantial rate in this tissue; therefore, the lack of accumulation of mature cytochrome P-450d mRNA in kidney must have been due to post-transcriptional control.