Rapid decrease of cytochrome P-450IIE1 in primary hepatocyte culture and its maintenance by added 4-methylpyrazole.
Studies were conducted to evaluate the possible induction or the maintenance of cytochrome P-450IIE1 in primary hepatocyte cultures by the inducing agent 4-methylpyrazole. Hepatocytes were isolated from control (noninduced) rats and from rats treated in vivo with either pyrazole or 4-methylpyrazole to induce P-450IIE1. The content of P-450IIE1 was determined by Western blots with antipyrazole P-450 IgG, and catalytic activity was assessed by assays of dimethylnitrosamine demethylase activity. The treatment with 4-methylpyrazole in vivo increased the content of P-450IIE1 and dimethylnitrosamine demethylase activity sevenfold and fourfold, respectively. In cultures prepared from noninduced hepatocytes, P-450IIE1 levels fell to values of 76%, 65%, 31% and 1% of freshly isolated hepatocytes after 1, 3, 6 and 9 days in culture. A similar decrease in dimethylnitrosamine demethylase was observed during this time. In cultures prepared from induced hepatocytes, the decline in P-450IIE1 was more rapid as levels fell to 77%, 31%, 3% and 3% of initial values after 1, 3, 6 and 9 days in culture. Again, the fall in dimethylnitrosamine demethylase activity paralleled the decline in content of P-450IIE1 and was more rapid with the induced hepatocytes. With cultures prepared from noninduced or induced hepatocytes, the addition of 4-methylpyrazole in vitro did not increase the content of P-450IIE1 or the activity of dimethylnitrosamine demethylase over the initial values. However, 4-methylpyrazole appeared to stabilize the P-450IIE1 and to decrease its rate of decline in culture. In noninduced cultures, the percent remaining content of P-450IIE1 after 6 days was 31% in the absence of and 52% in the presence of 5 mol/L 4-methylpyrazole. In cultures from 4-methylpyrazole-induced hepatocytes, the percent remaining P-450IIE1 after 3 days was 31% in the absence of inducer and 59% with 4-methylpyrazole added in vitro. Similarly 4-methylpyrazole helped to prevent the rapid decline of dimethylnitrosamine demethylase activity in induced and noninduced cultures. Viability of the induced and noninduced cultures in the absence or presence of added 4-methylpyrazole was similar. Levels of mRNA for P-450IIE1 were similar for livers from control rats and from rats treated in vivo with 4-methylpyrazole. The mRNA levels rapidly declined in induced and noninduced cultures, and this decline, unlike the fall in P-450IIE1 or dimethylnitrosamine demethylase activity, could not be prevented by the addition of 4-methylpyrazole in vitro to the cultures.(ABSTRACT TRUNCATED AT 400 WORDS)
Wu
D F
DF
Department of Biochemistry, Mount Sinai School of Medicine, New York, New York 10029.
Clejan
L
L
Potter
B
B
Cederbaum
A I
AI
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D019392:Cytochrome P-450 CYP2E1; D004790:Enzyme Induction; D000077604:Fomepizole; D008099:Liver / Q000201:enzymology*; D008297:Male; D008858:Microscopy, Phase-Contrast; D008862:Microsomes, Liver / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains
D011720:Pyrazoles; D012333:RNA, Messenger; C031280:pyrazole; D000077604:Fomepizole; D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating