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Positional Specificity for Methyl-n-amylnitrosamine Hydroxylation by Cytochrome P-450 Isozymes Determined with Monoclonal Antibodies¹

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 [S. S. M., Q. H., C. J., S. W.], and Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, Maryland 20892 [S. S. P., H. V. G.]

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 malespecific 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 Aroclorinduced 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 -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.

¹ This study was supported by NIH Grants R01-CA-35628 and core Grant CA-36727 from the National Cancer Institute and core Grant ACS-Sig-16 from the American Cancer Society. Some of these results were presented at earlier meetings (25, 26).

² To whom requests for reprints should be addressed.

³ Present address: Department of Biochemistry, Vanderbilt University, Nashville, TN 37203.

Received 8/ 9/90. Accepted 11/29/90.

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