Hepatic mitochondrial cytochrome P-450 system. Identification and characterization of a precursor form of mitochondrial cytochrome P-450 induced by 3-methylcholanthrene.

Hepatic mitoplasts from 3-methylcholanthrene-treated rats contain cytochrome P-450 which can metabolize polycyclic aromatic hydrocarbons like benzo(a)pyrene. Mitochondrial cytochrome P-450 was partially purified and reconstituted in vitro using adrenodoxin and the adrenodoxin reductase electron transfer system and [3H]benzo(a)pyrene as the substrate. A polyclonal antibody to purified microsomal P-450c (a major 3-methylcholanthrene-inducible form) inhibited the activity of mitochondrial enzyme in a concentration-dependent manner and also reacted with a 54-kDa protein on the immunoblots. A monoclonal antibody having exclusive specificity for P-450c, on the other hand, did not inhibit the aryl hydrocarbon hydroxylase activity of the mitochondrial enzyme and showed no detectable cross-reaction with the 54-kDa mitochondrial protein. Similarly, two-dimensional analysis and immunodetection using the polyclonal antibody showed distinct molecular properties of the mitochondrial enzyme different from the similarly induced microsomal P-450c with respect to the isoelectric pH. In vitro translation of free polysomes from 3-methylcholanthrene-induced liver, transport of precursor proteins by isolated mitochondria in vitro, and immunoprecipitation with the polyclonal antibody showed the presence of a 57-kDa putative precursor which is transported and processed into mature 54-kDa species. These results present evidence for the true intramitochondrial location of the P-450c-antibody reactive isoform detected in 3-methylcholanthrene-induced rat liver mitochondria.

Stimulation of transcription and translation by aurin tricarboxylic acid in mitochondrial lysates from Ehrlich ascites cells.

We have prepared a submitochondrial fraction from Ehrlich ascites tumor cell mitochondria which shows transcription and translation activities. The antibiotic aurin tricarboxylic acid (ATA) at low concentrations induces both RNA and protein synthetic activities of the mitochondrial lysate by several fold. At high concentrations, however, ATA inhibits the translation activity but continues to stimulate the transcription activity in a dose dependent manner up to 0.5 mM concentration tested. The lysate system transcribes endogenous DNA yielding RNA species resembling control mitochondrial RNA and synthesizes authentic cytochrome oxydase I and cytochrome oxidase II subunits.

Protection of mitochondrial genetic system against aflatoxin B1 binding in animals resistant to aflatoxicosis.

Administration of a single dose of aflatoxin B1 (AFB1) (6 mg/kg) to Sprague-Dawley rats results in a high level of modification of hepatic mitochondrial DNA (2.1 nmol of AFB1 adducts per mumol DNA-phosphate) and long-term inhibition of mitochondrial transcription and translation activities (N. Bhat et al., Cancer Res., 42: 1876-1880, 1982). Similar doses of AFB1 given to ICR mice and Syrian golden hamsters result in negligible to very low levels (0-06 nmol) of adducts in hepatic mitochondrial DNA. Intact mitochondria from rat liver can metabolize significant amounts of AFB1 (0.29 nmol/mg of protein) without externally added reduced nicotinamide adenine dinucleotide phosphate, and the metabolic activity is stimulated nearly 3-fold by Kreb's cycle intermediates (glutamate and malate), which support intramitochondrial reduced nicotinamide adenine dinucleotide phosphate production. Intact mitochondria from mice and hamsters, on the other hand, metabolize negligible or very low levels of AFB1 (0-0.1 nmol of AFB1 per mg of protein) even when intramitochondrial reduced nicotinamide adenine dinucleotide phosphate production is stimulated by the addition of Kreb's acids. Detergent-solubilized mitoplasts containing less than 1% microsome contamination from all three sources can catalyze the metabolic activation of AFB1 to electrophilic reactive forms as determined in an in vitro DNA binding assay at comparable levels (1.2-2.2 nmol of AFB1 bound per mumol of cytochrome P-450), suggesting that the low levels of AFB1 metabolism by intact mouse and hamster mitochondria and the relative resistance of macromolecular synthesis in these particles to added AFB1 may be due to mitochondrial membrane impermeability. In support of this possibility, AFB1 transported into mouse liver mitochondria through a liposome delivery system causes about 80% inhibition of protein synthesis.

Formation of benzo(alpha)pyrene metabolites and DNA adducts catalyzed by a rat liver mitochondrial monooxygenase system.

Sonic disrupted mitoplasts from 3-methylcholanthrene (MCA) treated rats can catalyze the formation of benzo(a)pyrene (BaP) adducts with calf thymus DNA in the presence of an NADPH generating system. The mitoplasts used in this study contained less than 1% microsomal marker enzymes: rotenone insensitive NADPH cytochrome c reductase and glucose-6-phosphatase. The rates of BaP metabolism and DNA adduct formation per nanomole cytochrome P-450 were different for MCA induced mitochondrial and microsomal enzymes. The major B(a)P DNA adducts formed in incubations with lysed mitoplasts were derived from reaction of 9-OH-B(a)P-4,5 oxide with deoxyguanosine. The results suggest a potential role of mitochondrial monooxygenase activity in the covalent binding of B(a)P to mitochondrial DNA.

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.

Qualitative and comparative nature of mitochondrial translation products in mammalian cells.

A method has been described for the efficient incorporation of [35S]methionine into isolated mitochondrial particles from various mammalian tissues. The method involves the incubation of digitonin-treated mitochondrial particles (mitoplasts) in a low sucrose medium. Electrophoretic analysis of 35S-labeled products on sodium dodecyl sulfate-polyacrylamide gels under reducing conditions shows that mitoplasts from Ehrlich ascites cells, mouse liver, and rat liver synthesize 19-24 polypeptide species including some high molecular weight components in the size range of 1.0 X 10(5). The polypeptide species synthesized in the mitoplast system resemble the cycloheximide-resistant products synthesized in the intact cells with respect to size distribution and total number, although significant quantitative differences between the two systems are observed. Experiments on pulse--chase analysis of 35S-labeled mitochondrial products and the effects of protease inhibitors on the electrophoretic profiles suggest no significant proteolytic degradation during the incubation or analysis. Further, control experiments with nuclease-treated mitoplasts and use of specific protein synthesis inhibitors show that all of the labeled polypeptides are the intramitochondrial translation products. Extensive comparison between the products synthesized in Ehrlich ascites and mouse and rat liver mitochondria, using one- and two-dimensional gels under denaturing conditions, shows striking variations, suggesting possible heterogeneity.

Inhibition of mitochondrial protein synthesis during early stages of aflatoxin B1-induced hepatocarcinogenesis.

Experiments were designed to determine the in vivo effects of a single 6-mg/kg dose aflatoxin B1 on rat liver mitochondrial transcription and translation processes. With the use of intact hepatocytes and also a highly active mitoplast system for incorporation, it was observed that both mitochondrial transcription and translation activities are inhibited progressively even after 24 hr of carcinogen administration. Electrophoretic patterns of mitochondrial translation products show some qualitative changes during early periods of carcinogen administration. At later stages (greater than 12 hr), however, there is a general inhibition of many of the products, although by this time there is a qualitative and quantitative recovery in the synthesis of mitochondrial proteins imported from the cytoplasm. These results, along with the data showing considerably high levels of aflatoxin B1 binding to mitochondrial DNA suggest that mitochondrial genetic system is one of the direct targets during experimental carcinogenesis.

Preferential attack of mitochondrial DNA by aflatoxin B1 during hepatocarcinogenesis.

Administration of the hepatic carcinogen aflatoxin B1 to experimental animals results in covalent binding to liver mitochondrial DNA at concentrations three to four times higher than nuclear DNA. The concentration of carcinogen adducts in mitochondrial DNA remains unchanged even after 24 hours, possible because of lack of excision repair. Similarly, mitochondrial transcription and translation remain inhibited up to 24 hours suggesting long-term effects of aflatoxin B1 on the mitochondrial genetic system.

Modulation of hepatic transcription and translation during early stages of aflatoxin B1 carcinogenesis.

Isolated rat hepatocytes have been used to study the transcription and translation processes at varied time intervals after administration of a single dose (6 mg/kg) of hepatocarcinogen aflatoxin B1 (AFB1). The effects of AFB1 during the first 24 h of drug treatment show a characteristic inhibition followed by periods of rapid recovery and also a hyperactive state when both heterogeneous nuclear RNA (HnRNA) transcription and cytoplasmic translation processes reach about 200% of control. Analysis of [3H]orotic acid pulse-labeled HnRNA and cytoplasmic polyadenylic acid(poly (A)) containing mRNA on sucrose gradients under conditions which prevent aggregation show a stepwise reduction in the total isotopic incorporation as well as in the size distribution during the first 9 h of AFB1 treatment. Between 9 and 24 h after AFB1 treatment, there is a recovery in the total incorporation as well as in the size of HnRNA and poly (A) containing mRNA. Analysis of translation products by a two-dimensional procedure shows loss of some high molecular weight products in AFB1-treated cells. At 24 h after AFB1 treatment (hyperactive stage) there is a total recovery of activity. Also, several new translation products not detected in the control hepatocytes are synthesized at this stage. The hyperactive stage might represent gene-derepression or reprogramming of gene expression.

Polysome-dependent in vitro translation system capable of peptide chain reinitiation.

A sensitive in vitro translation system has been developed which makes use of cellular polysomes as the source of mRNA and ribosomes. The soluble factors are derived from the preincubated S-30 fraction by centrifugation through a discontinuous sucrose gradient. Of the four fractions tested, fraction 1 (topmost fraction in the gradient) and fraction 2 (fraction sedimenting in 0.5 M sucrose) were stimulatory. These two fractions together yield the highest activity, corresponding to about 125 times the background incorporation. The polysome-directed system exhibits optimal activity in the range 1.8-2 mM Mg2+ and 125-175 mM KCl. The polysome-directed in vitro products exhibit a complexity comparable to the in vivo products resolved on the two-dimensional polyacrylamide gels of O'Farrell [O'Farrell, P. (1975) J. Biol. Chem. 250, 4007-4021]. The system is capable of active chain reinitiation as indicated by partial inhibition by 7-methylguanosine 5'-monophosphate and pactomycin and N-terminal end analysis of in vitro products. This system can also translate polysomes from diverse tissues such as mouse liver, rat liver, and rat brain. The levels and also the authenticity of translation of rat liver albumin and mouse liver carbamoyl phosphate synthetase I were tested by immunoprecipitation with monospecific antibodies. The results show that the major as well as the minor translation products are synthesized in this system at levels comparable to the physiological levels.

Activation of aflatoxin B1 by a mono-oxygenase system localized in rat liver mitochondria.

Structurally intact rat liver mitoplasts free of detectable microsomal contamination contain enzymatic activity to metabolize aflatoxin B1 (AFB1). The activated component(s) bind to mitochondrial macromolecules and also inhibit mitochondrial protein synthesis. The activity of intact mitoplasts or sonicated particles is partly dependent on the addition of NADPH-generating system. Under optimal conditions, the mitochondrial enzyme has specific activity of 60 to 65 pmol/mg and represents about 15 to 18% of total cytoplasmic activity for AFB1 activation. The enzyme is localized in the soluble fraction of mitochondrial matrix and appears to be distinctly different from the microsomal activity.