Susan B. West

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. I. Determination of substrate specificity by the cytochrome P-450 and P-448 fractions.

The reconstituted liver microsomal hydroxylation system was used to study the formation of a metabolite-cytochrome P-450 complex absorbing maximally at 455 nm, with benzphetamine and N-hydroxyamphetamine as substrates. Complex formation required the presence of NADPH, substrate, NADPH-cytochrome c reductase, lipid, and cytochrome P-450, indicating that metabolism of the substrate is essential. In the presence of fixed amounts of lipid and NADPH-cytochrome c reductase, the rate of complex formation with cytochrome P-450 isolated from phenobarbital-treated rats was much greater than that observed with cytochrome P-48 from 3-methylcholanthrene-treated rats or rabbits. These results are consistent with recent studies indicating that different forms of cytochrome P-450 with distinct spectral, catalytic, and immunological properties exist in liver microsomes.Abstract The reconstituted microsomal hydroxylation enzyme system from rats treated with phenobarbital exhibited high activity for benzphetamine N-demethylation, but very low activity for 3,4-benzpyrene hydroxylation. However, when the cytochrome P-450 fraction from phenobarbital-treated rats was replaced by the cytochrome P-448 fraction from rats treated with 3-methylcholanthrene, the N-demethylation of benz-phetamine was decreased while the hydroxylation of 3,4-benzpyrene was greatly increased. On the other hand, the reconstituted system from 3-methylcholanthrene-treated rats showed good benzpyrene hydroxylase activity which could be greatly decreased if the cytochrome P-448 fraction was replaced by the P-450 fraction from rats treated with phenobarbital. These and other experiments indicate that the substrate specificity of the hydroxylation system resides in the cytochrome fraction rather than in the reductase or lipid fraction, and the data suggest that cytochromes P-450 and P-448 have different catalytic activities.

Pregnenolone-16α-carbonitrile: A new type of inducer of drug-metabolizing enzymes

Pretreatment of female rats with pregnenolone-16α-carbonitrile (PCN) for 212 days resulted in a significant increase in cytochrome P-450 content and NADPH-cytochrome c reductase activity in hepatic microsomes. The spectral characteristics of the microsomal hemoprotein from PCN-treated rats were similar to those obtained from untreated and phenobarbital (PB)-treated rats but different from 3-methylcholanthrene (3-MC)-treated animals. However, the specificity of the induction effect of PCN on the oxidative metabolism of benzphetamine, ethylmorphine, and 3,4-benzpyrene differed from the specificity of either PB or 3-MC. Studies on the induction of enzymes that metabolize the above three substrates revealed that PB, PCN, and 3-MC exerted their greatest stimulatory effect on benzphetamine N-demethylation, ethylmorphine N-demethylation, and 3,4-benzpyrene hydroxylation, respectively.

Highly purified cytochrome P-448 and P-450 from rat liver microsomes

Abstract Cytochrome P-448 from 3-methylcholanthrene-treated rats has been purified to a specific content of greater than 20 nmoles/mg protein, and cytochrome P-450 from phenobarbital-treated rats to greater than 17 nmoles/mg protein. Both cytochromes are catalytically active when reconstituted with lipid and NADPH-cytochrome c reductase and exhibit differential substrate specificities for benzphetamine and benzo[a]pyrene. Cytochrome P-448 has a minimum molecular weight of approximately 53,000, and cytochrome P-450, 48,000 by SDS polyacrylamide gel electrophoresis.

A simple and rapid procedure for the purification of phenobarbitalinducible cytochrome P-450 from rat liver microsomes

Abstract A rapid and simple procedure has been developed for the purification of a phenobarbital-inducible form of cytochrome P-450 from the liver microsomes of phenobarbitalpretreated rats. Within 2 days approximately 1000–1500 nmol of highly purified cytochrome P-450 with a specific content of 16 nmol/mg protein can be recovered from 4 g of microsomal protein. The procedure consists of solubilization of microsomal protein with sodium cholate, fractionation with polyethylene glycol, and column chromatography at room temperature on DEAE-cellulose. The resulting DEAE-cellulose fraction electrophoreses on polyacrylamide gels in the presence of sodium dodecyl sulfate as a major protein band with a minimum molecular weight of 52,000 and a few faint bands. Further chromatography on QAE Sephadex A-25 essentially removes these faint bands and increases the specific content slightly to 17 nmol/mg protein. Relatively low amounts of this form of cytochrome P-450 appear to be present in microsomes of untreated rats since less than 1% can be recovered as the DEAE-cellulose fraction by this procedure. An identical form is inducible by phenobarbital in rats of different ages and sex. In a reconstituted system under optimal assay conditions, this form of cytochrome P-450 catalyses the N-demethylation of benzphetamine with a turnover number greater than 100 and hydroxylates testosterone at the 16α position but not at the 6β or 7α position.

Mutagenicity of metabolically activated benzo[a]anthracene 3,4-dihydrodiol: evidence for bay region activation of carcinogenic polycyclic hydrocarbons.

Summary Benzo[a]anthracena and the 5 metabolically possible trans dihydrodiols of benzo[a]anthracene were metabolized, in the presence of S. thyphimurium strain TA100, by a highly purified hepatic microsomal monooxygenase system. The metabolic product(s) of benzo[a]anthracene 3,4-dihydrodiol was nearly 10 times as mutagenic to the bacteria as were the metabolites of benzo[a]anthracene and the other four dihydrodiols. The marked activation of benzo[a]anthracene 3,4-dihydrodiol, presumably to the 3,4-diol-1,2-epoxide is consistent with and supports the hypothesis that bay region epoxides of unsubstituted polycyclic hydrocarbons are ultimately reactive forms of these carcinogenic compounds.

Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes

Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b5, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens.

Liver microsomal electron transport systems: II. The involvement of cytochrome b5 in the NADH-dependent hydroxylation of 3,4-benzpyrene by a reconstituted cytochrome P-448-containing system

Abstract An enzyme system in rat liver microsomes which catalyzes the NADH-dependent hydroxylation of 3,4-benzpyrene has been reconstituted. The essential components of this NADH-mediated electron transport chain are cytochrome b5, NADH-cytochrome b5 reductase, lipid, and cytochrome P-448.

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates: V. Competition between cytochromes P-450 and P-448 for reductase in 3,4-benzpyrene hydroxylation

Abstract The competition between cytochromes P-450 (from rats treated with phenobarbital) and P-448 (from rats treated with 3-methylcholanthrene) for reductase in the 3,4-benzpyrene hydroxylation reaction was studied using the reconstituted microsomal hydroxylation system. Cytochrome P-450 stimulated the rate of reaction at low concentrations of cytochrome P-448 but inhibited the reaction at higher concentrations of cytochrome P-448. The inhibition of cytochrome P-448-supported 3,4-benzpyrene hydroxylation by cytochrome P-450 could be reversed by increasing the concentration of reductase. The kinetic data were consistent with the view that cytochromes P-450 and P-448 compete for the reductase in the reaction mixture.

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds, and endogenous substrates. IV. Hydroxylation of aniline.

Abstract The hydroxylation of aniline required the three components of the reconstituted liver microsomal system—cytochrome P-450 or P-448, reductase, and lipid fractions—for maximal activity. The eytochrome P-450 fractions prepared from untreated and phenobarbital-treated rats and the cytochrome P-448 fraction from rats treated with 3-methylcholanthrene were all active for aniline hydroxylation. Benzphetamine inhibited the cytochrome P-450-dependent hydroxylation of aniline to a greater extent than it inhibited the cytochrome P-448-dependent hydroxylation. In contrast, 3,4-benzpyrene was a more potent inhibitor of the cytochrome P-448-dependent aniline hydroxylation than of the cytochrome P-450-dependent reaction.Abstract The hydroxylation of aniline required the three components of the reconstituted liver microsomal system—cytochrome P-450 or P-448, reductase, and lipid fractions—for maximal activity. The eytochrome P-450 fractions prepared from untreated and phenobarbital-treated rats and the cytochrome P-448 fraction from rats treated with 3-methylcholanthrene were all active for aniline hydroxylation. Benzphetamine inhibited the cytochrome P-450-dependent hydroxylation of aniline to a greater extent than it inhibited the cytochrome P-448-dependent hydroxylation. In contrast, 3,4-benzpyrene was a more potent inhibitor of the cytochrome P-448-dependent aniline hydroxylation than of the cytochrome P-450-dependent reaction.

Liver microsomal electron transport systems: Properties of a reconstituted, NADH-mediated benzo[a]pyrene hydroxylation system☆

Abstract An enzyme system from rat liver microsomes which catalyzes the NADH-mediated hydroxylation of benzo[ a ]pyrene has been reconstituted. The essential microsomal components of this NADH-dependent pathway were NADH-cytochrome b 5 reductase, cytochrome b 5 , cytochrome P -448 and, phosphatidyl choline. Highly purified NADPH-cytochrome c reductase containing small amounts of deoxycholate stimulated this NADH-mediated pathway supported by 0.2 m m NADH whereas boiled reductase had little effect. Part of this stimulation could be attributed to hydroxylation of benzo[ a ]pyrene via a second pathway; i.e., NADPH-cytochrome c reductase in combination with cytochrome P -448 and phosphatidylcholine also supported a low rate of NADH-dependent hydroxylation. The mechanism of the remaining stimulation is not known. However, the effect of NADPH-cytochrome c reductase on the reconstituted cytochrome b 5 -dependent pathway was not unique; high concentrations of deoxycholate also stimulated this pathway, perhaps by facilitating the transfer of electrons from NADH-cytochrome b 5 reductase to cytochrome b 5 . The addition of NADPH-cytochrome c reductase to the cytochrome b 5 -dependent reconstituted system also affected the apparent K m of NADH for benzo[ a ]pyrene hydroxylation. In the absence of NADPH-cytochrome c reductase, the apparent K m of NADH was 1.3 μ m while in its presence a low (1.3 μ m ) and a high (1700 μ m ) K m were observed, consistent with the affinities of the two flavoproteins for NADH. Our results also indicate that the relative contribution of the pathway due to NADPH-cytochrome c reductase in combination with phosphatidyl choline and cytochrome P -448 to the overall rate of NADH-supported benzo[ a ]pyrene hydroxylation in microsomes would be greatly dependent on the concentration of NADH chosen. The rate of benzo[ a ]pyrene hydroxylation by these reconstituted components was almost 10-fold greater with 10 m m NADH than with 0.2 m m NADH, a result consistent with the reduction of NADPH-cytochrome c reductase by high concentrations of NADH.