title: Monoclonal antibodies to ethanol-induced rat liver cytochrome P-450 that metabolizes aniline and nitrosamines.
abstract: Hybridomas were prepared from mouse myeloma cells and spleen cells derived from female BALB/c mice that had been immunized with a partially purified ethanol-induced rat liver cytochrome P-450 (P-450et). Monoclonal antibodies (MAbs) produced by the hybridomas were screened for binding to P-450et with a radioimmunoassay. Thirty-one independent hybrid clones produced MAbs that had a high affinity for P-450et. Each clone produced MAbs of a single subclass of the mouse immunoglobulins IgG1, IgG2a, IgM, or IgA. Ten of the 31 MAbs also immunoprecipitated P-450et as determined by Ouchterlony double-immunodiffusion analyses. One of the MAbs was tested for cross-reactivity with other rabbit and rat liver cytochromes P-450 and was found not to cross-react with rat liver P-450 induced by either phenobarbital, beta-naphthoflavone, or rabbit liver P-450LM2 or P-450LM4. Nine of the MAbs were tested for cross-reactivity with rat liver clofibrate-induced P-450, rat liver pregnenolone-16-alpha-carbonitrile-induced P-450, and a human liver P-450. All the MAbs showed no cross-reactivity except for one MAb which cross-reacted with both pregnenolone-16-alpha-carbonitrile and human P-450 and three MAbs which cross-reacted with human P-450. Three antigen-precipitating MAbs and four nonprecipitating MAbs were tested for their effects on the aniline p-hydroxylase activity of liver microsomes of untreated rats and from rats treated with acetone, pyrazole, methylpyrazole, or imidazole. One of the seven MAbs tested, 1-91-3, inhibited enzyme activity of acetone-, pyrazole-, or methylpyrazole-induced microsomes by 54, 47, and 48%, respectively. This indicates that at least 50% of microsomal cytochrome P-450 aniline p-hydroxylase activity in the latter is a function of a P-450 enzyme that contained the epitope to which the MAb 1-91-3 is directed. With untreated and imidazole-induced microsomes, 32 and 21% inhibition of the enzyme activity was observed. In reconstituted systems containing phospholipid and NADPH-cytochrome P-450 reductase, MAb 1-91-3 inhibited aniline p-hydroxylase activity of purified ethanol-induced P-450et and acetone-induced P-450 by more than 90%. Nitrosodimethylamine demethylase activity of acetone-induced rat microsomes was inhibited by the various MAbs up to 77% and the activity of the purified acetone-induced P-450 was inhibited up to 92%.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000096:Acetone / Q000494:pharmacology; D000814:Aniline Compounds / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology*; D000431:Ethanol / Q000494:pharmacology*; D005260:Female; D005779:Immunodiffusion; D007527:Isoenzymes / Q000276:immunology; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000201:enzymology; D009602:Nitrosamines / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D000814:Aniline Compounds; D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D009602:Nitrosamines; D000096:Acetone; D000431:Ethanol; D003577:Cytochrome P-450 Enzyme System; C023650:aniline