Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene.
Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.
D016604:Aflatoxin B1; D000348:Aflatoxins / Q000378:metabolism*; D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism*; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D004128:Dimethylnitrosamine / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014316:Tritium
D000348:Aflatoxins; D002273:Carcinogens; D014316:Tritium; D001564:Benzo(a)pyrene; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D004128:Dimethylnitrosamine