Selective potent restriction of P450b- but not P450e-dependent 7,12-dimethylbenz[a]anthracene metabolism by the microsomal environment.
The prototypic members of the rat liver cytochrome P450IIB subfamily, P450b and P450e, differ by only 13 amino acids and yet purified P450b is considerably more active than P450e for all known substrates. A unique regioselectivity difference between cytochromes P450b and P450e for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and a genetic deficiency in P450e expression in the Marshall (M520/N) rat strain have been exploited to determine the microsomal contributions of the respective forms toward the metabolism of DMBA. The total contribution to metabolism by each isozyme has been assessed based on the sensitivity to rabbit anti-P450b/e IgG and comparison with microsomal P450b and P450e content as measured by Western blots. Liver microsomes from untreated M520/N rats do not express detectable levels of P450e but express P450b at a level that is 2-fold higher than that of P450e in liver microsomes from untreated F344 rats (50 pmol/mg). However, only 4% of the constitutive DMBA metabolizing activity of liver microsomes from the M520/N rat strain could be inhibited by anti-P450b/e IgG. A 30-fold induction of hepatic P450b by phenobarbital (PB) was also completely ineffective in increasing P450b-dependent DMBA metabolism. PB treatment had no appreciable effect on either the levels of expression of P450b protein or P450b-dependent DMBA metabolism, in M520/N lung and adrenal microsomes. In contrast, PB treatment of F344 rats considerably increased P450b/e-dependent metabolism by liver, lung, and adrenal microsomes. The regioselectivity of the anti-P450b/e-sensitive metabolism (predominantly 12-methyl hydroxylation), however, indicated a much greater contribution from P450e than P450b in every tissue examined despite a several fold higher expression of P450b than of P450e. P450b was expressed constitutively in lung microsomes from both strains but again failed to exhibit appreciable DMBA metabolizing activity. Based on these activities and microsomal P450b contents, P450b consistently exhibited turnover numbers (0.02-0.15 nmol/nmol P450b/min) that were at least 10-fold lower than those of pure P450b. In contrast, the calculated turnover numbers for microsomal P450e were consistently comparable to those of pure P450e (approximately 1 nmol/nmol P450e/min).
Christou
M
M
Environmental Toxicology Center, University of Wisconsin, Madison 53706.
Mitchell
M J
MJ
Jovanovich
M C
MC
Wilson
N M
NM
Jefcoate
C R
CR
D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism*; D000311:Adrenal Glands / Q000201:enzymology / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D007074:Immunoglobulin G / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008168:Lung / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D011922:Rats, Mutant Strains
D065607:Cytochrome P-450 Enzyme Inhibitors; D007074:Immunoglobulin G; D007527:Isoenzymes; D015127:9,10-Dimethyl-1,2-benzanthracene; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital