Effect of pyrazole, cobalt and phenobarbital on mouse liver cytochrome P-450 2a-4/5 (Cyp2a-4/5) expression.
Pyrazole, cobalt and phenobarbital increase the activity of coumarin 7-hydroxylase (COH) in mouse liver. To study the mechanism of this increase, we measured the expression of the cytochrome P-450 2a-4/5 (Cyp2a-4/5) complex, which mediates testosterone 15 alpha-hydroxylase and COH activities, as a function of dose and time after the treatment of C57BL/6 (B6) and DBA/2 (D2) male mice with the inducers. COH activity and Cyp2a-4/5 steady-state mRNA levels were increased in both strains in response to the inducers. No marked effect occurred with testosterone 15 alpha-hydroxylase or activities associated with Cyp1a-1 or Cyp2e-1. A 2-7-fold increase in response to the inducers was seen in the amount of P-450Coh (cytochrome P-450 isoenzyme catalysing coumarin 7-hydroxylation) protein in Western immunoblots. PCR amplification of a 1 kb region in Cyp2a-4/5-mRNA-derived cDNA, followed by cutting at the diagnostic PstI site, showed that most of the steady-state mRNA consisted of Cyp2a-5, which is also the form most affected by pyrazole. Nuclear run-off analysis revealed no increase in the transcription rate of Cyp2a-4/5 after pyrazole or cobalt treatment, whereas a 2-3-fold increase occurred after phenobarbital pretreatment in B6 mice. Together with previous reports [Aida & Negishi (1991) Biochemistry 30, 8041-8045], the current data suggest that both pyrazole and cobalt increase COH catalytic activity by affecting Cyp2a-5 by post-transcriptional mechanisms in mice.
Hahnemann
B
B
Department of Pharmacology and Toxicology, University of Oulu, Finland.
Salonpää
P
P
Pasanen
M
M
Mäenpää
J
J
Honkakoski
P
P
Juvonen
R
R
Lang
M A
MA
Pelkonen
O
O
Raunio
H
H
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D003035:Cobalt / Q000494:pharmacology*; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D007527:Isoenzymes / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010634:Phenobarbital / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000096:biosynthesis* / Q000235:genetics
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D011720:Pyrazoles; D012333:RNA, Messenger; D003035:Cobalt; C031280:pyrazole; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; C050356:steroid 15-alpha-hydroxylase; C018021:cobaltous chloride; D010634:Phenobarbital