Phenobarbital induction of CYP1A1 gene expression in a primary culture of rainbow trout hepatocytes.
In mammals, phenobarbital (PB) is an in vivo inducer of the cytochrome P4502B (CYP2B) family, whereas in teleosts PB induction of cytochrome P450 is unclear. We show that teleost cytochrome P4502K1 (CYP2K1) protein levels and 7-pentoxyresorufin-O-deethylase activity were not induced by exposure of primary cultures of rainbow trout hepatocytes to PB. Instead, cytochrome P4501A1 (CYP1A1) gene expression was strongly induced by PB, based upon observations of marked increases in CYP1A1 mRNA, CYP1A1 protein, and 7-ethoxyresorufin-O-deethylase activity. In accordance with these data we provide a temporal study employing antibodies for the aromatic hydrocarbon (Ah) receptor that showed an increase in Ah receptor in nuclear extracts prepared from cells exposed to PB. Employment of the electrophoretic mobility shift assay (EMSA) showed PB to cause activation or "transformation" of the Ah receptor in nuclear extracts. Studies employing actinomycin D and cycloheximide indicated that PB induction of CYP1A1 was regulated at both the transcriptional and post-transcriptional levels. Nuclear run-off experiments confirm that PB causes an increase in CYP1A1 transcription. Inhibition of protein synthesis led to the superinduction of CYP1A1 mRNA, suggesting the regulation of teleost CYP1A1 may involve a labile repressor protein. These findings suggest that PB induction of the CYP1A1 gene involves the Ah receptor and is via transcriptional activation.
Sadar
M D
MD
Department of Zoophysiology, University of Göteborg, S 40031 Göteborg, Sweden.
Ash
R
R
Sundqvist
J
J
Olsson
P E
PE
Andersson
T B
TB
D015660:7-Alkoxycoumarin O-Dealkylase / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D019362:Cytochrome P-450 CYP2B1; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D003600:Cytosol / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D029941:Fish Proteins; D007527:Isoenzymes / Q000235:genetics*; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D017686:Oncorhynchus mykiss / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics; D010634:Phenobarbital / Q000494:pharmacology*; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000235:genetics; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D029941:Fish Proteins; D007527:Isoenzymes; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D013250:Steroid Hydroxylases; D015660:7-Alkoxycoumarin O-Dealkylase; D001189:Aryl Hydrocarbon Hydroxylases; C088323:CYP2K1 protein, Oncorhynchus mykiss; D019362:Cytochrome P-450 CYP2B1; D000072467:Cytochrome P450 Family 2; D010634:Phenobarbital