Positional specificity for methyl-n-amylnitrosamine hydroxylation by cytochrome P-450 isozymes determined with monoclonal antibodies.
Inhibitory monoclonal antibodies (MAbs) were used to determine the contribution of epitope-specific cytochrome P-450 isozymes in rat liver microsomes to hydroxylation of the esophageal carcinogen methyl-n-amylnitrosamine. These P-450-catalyzed reactions form 2-, 3-, 4-, and 5-hydroxymethyl-n-amylnitrosamine, formaldehyde (demethylation), and pentaldehyde (depentylation). With uninduced microsomes from male rats, MAb 1-68-11 inhibited 4-hydroxylation by 73% and demethylation by 46%. This indicated the major contribution of constitutive male-specific P-450 IIC11 to the metabolism. Inhibition studies with MAbs 2-66-3 and 1-91-3 indicated that P-450 IIB1 contributed 19% and IIE1 35% to demethylation. With uninduced microsomes from females, MAb 1-68-11 produced similar inhibitions to those in male rats, indicating that female-specific P-450 IIC12 (which is closely related to IIC11) also catalyzed 4-hydroxylation and demethylation. With microsomes from 3-methylcholanthrene-induced male rats, P-450 IA1 and/or IA2 were responsible for 60% of 3-hydroxylation and 40% of depentylation. With microsomes from phenobarbital-treated rats, P-450 IIB1 and IIB2 catalyzed all 6 reactions but especially 4-hydroxylation and depentylation, which were 50-75% inhibited by MAb 2-66-3. Microsomes from Aroclor-induced males behaved as if they were induced by both 3-methylcholanthrene and phenobarbital. After treatment with isoniazid (a P-450 IIE1 inducer), inhibition by MAb 1-91-3 indicated a 45% contribution of P-450 IIE1 to demethylation, and both P-450 IIE1 and IIB1 (or IIB2) appear to have been induced. A major finding with uninduced microsomes was the high specificity of MAb 1-68-11 for inhibiting 4-hydroxylation, indicating that P-450 IIC11 and IIC12 catalyzed most of this omega-1-hydroxylation. In microsomes from induced rats, the MAb inhibitions showed the role of the induced P-450 IA1 (or IA2), IIB1 (or IIB2), and IIE1 in methyl-n-amylnitrosamine hydroxylation at different positions, as well as the presence of P-450 IIC11. This study illustrates the usefulness of inhibitory MAbs for defining the contribution of individual P-450s to position-specific metabolism.
Mirvish
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SS
Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha 68105.
Huang
Q
Q
Ji
C
C
Wang
S
S
Park
S S
SS
Gelboin
H V
HV
D000818:Animals; D000911:Antibodies, Monoclonal; D001140:Aroclors / Q000494:pharmacology; D002273:Carcinogens / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006900:Hydroxylation; D007527:Isoenzymes / Q000378:metabolism*; D007538:Isoniazid / Q000494:pharmacology; D008099:Liver / Q000201:enzymology; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008861:Microsomes / Q000502:physiology; D009602:Nitrosamines / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D013379:Substrate Specificity / Q000187:drug effects
D000911:Antibodies, Monoclonal; D001140:Aroclors; D002273:Carcinogens; D007527:Isoenzymes; D009602:Nitrosamines; C025033:N-amyl-N-methylnitrosamine; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D007538:Isoniazid; D010634:Phenobarbital