Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene.

B G Niranjan,N M Wilson,C R Jefcoate,N G Avadhani

doi:10.1016/s0021-9258(18)90774-x

Abstract

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.

Highlights

From the $Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and the §Department of Phrmncology, University of Wisconsin Medical Center, Madison, Wisconsin53706
Rat liver mitoplasts containing less than 1% micro- There is compelling evidence for the existence of somalcontamination contain cytochromeP-450 at 25%multiple forms of cytochrome P-450 in mammalian liver
In thispaper we report further characteristics of this raltiver mitochondrial system which can activate structurally different carcinogens such as DMN and BaP.Studieson the differential inducibility of mitochondrial cytochrome P-450 by PB and3MC indicate that two different types of cytochrome P-450 may be involved in the activation of the carcinogens AFB, and BaP

Summary

EXPERIMENTAL PROCEDURES

Materials-NADP', isocitric acid (trisodium salt), isocitrate dehydrogenase type IV, digitonin, D-mannitol, Hepes, bovine serum albumin (Fraction V), Triton N-101, sodium phenobarbital, and 3MC were obtained from Sigma. The mitoplasts isolated from livers of PB-induced and 3-MC-induced rats were suspended in 100mM potassium phosphate buffer (pH 7.4)containing 20% glycerol, 0.4% sodium cholate, and 1 mM dithiothreitol a t a concentration of 10 mg protein/mI. Calf thymus DNA (1 mg/ml), labeled carcinogens (13H]AFB1,13H]BaP,or ["CIDMN), and mitochondrial or microsomal proteins as the enzyme source were added. Metabolism of Carcinogens by Intact Mitoplusts-Mitoplasts were suspended at 2 to 5 mg/ml in a buffer containing 4 mM Hepes (pH 7.4), 3 mM potassium phosphate (pH 7.4), 1 mM EDTA, 5 mM 2mercaptoethanol, and 0.25 M sucrose, and incubated a t 30 "C for the required time periods with l3H]AFB1(5 nmol/mg protein) or [3H] BaP (10 nmol/mg protein). Other Procedures-Protein was estimated by the method of Lowry et al [34] or by the differential absorption at 280 and 310nm as described before [21]

Two Distinct Forms of HepaMtiictochondrCiayltochromes

Source of enzyme

Two Distinct Forms of HepaMtiictochondCriayltochromes

Adrenodoxin adrenodoxin reductase

Partially purified Partially purified

DISCUSSION