R. Kuntzman

Lipid peroxidation and the degradation of cytochrome P-450 heme.

Abstract In an in vitro system consisting of rat liver microsomes and NADPH, significant lipid peroxidation was observed along with a concomitant loss of cytochrome P-450. This spectral loss of cytochrome P-450 was shown to be the result of a breakdown of cytochrome P-450 heme. Inhibitors of lipid peroxidation also prevented the loss of cytochrome P-450, demonstrating a direct relationship between lipid peroxidation and breakdown of cytochrome P-450 heme. This breakdown of cytochrome P-450 heme during lipid peroxidation is unrelated to the degradation of cytochrome P-450 heme by an active metabolite of certain allyl-containing barbiturates and barbiturate related compounds. In addition, it appears that different breakdown products of heme are produced by these two mechanisms.

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.

Effects of pesticides on drug and steroid metabolism

Treatment of experimental animals with small amounts of halogenated hydrocarbon insecticides stimulates the activity of oxidative enzymes in liver microsomes that metabolize drugs and steroid hormones. The enhancing effect of these pesticides on drug metabolism alters the duration and intensity of drug action in animals and also markedly influences drug toxicity. The minimum exposure to DDT required to decrease the hypnotic action of pentobarbital in rats gives rise to 10 to 15 μg of DDT per gram of fat, a concentration approaching that reported to occur in the human population. The physiological significance of pesticide‐induced increases in steroid hydroxylases in liver microsomes is not known, and further studies are necessary to elucidate the importance of this effect. In contrast to the stimulatory effect of halogenated hydrocarbon insecticides on hepatic steroid hydroxylases, treatment of rats with organophosphate insecticides, such as chlorthion, inhibits the liver microsomal metabolism of several steroid hormones. Chlorthion has a greater inhibitory effect on the 16α‐hydroxylation of testosterone than on the 6β‐ or 7 α‐hydroxylation of this steroid, suggesting that separate enzyme systems are required for the various hydroxylation reactions. Since the stimulatory effects of several drugs on drug and steroid metabolism in animals is paralleled by enhanced drug and steroid hydroxylation in man, further studies should be carried out to determine whether the chronic environmental exposure of humans to pesticides is sufficient to alter their metabolism of drugs and steroids.

Effect of cigarette smoking on phenacetin metabolism

Nine nonsmokers and nine individuals who smoked more than 15 Cigarettes per day were administered 900 mg of phenacetin orally, and the concentration of phenacetin in plasma was measured 1, 2, 3.5, and 5 hours after the dose. The plasma levels of phenacetin in the smokers were markedly lower than in the nonsmokers. The plasma levels of unconiugated or total N‐acetyl‐p‐aminophenol in the smokers were the same or only slightly lower than in the nonsmokers, and the ratios of the plasma concentration of total N‐acetyl‐p‐aminophenol to phenacetin were increased severalfold in the smokers. These results indicated that cigarette smoking stimulated the metabolism of phenacetin. To learn whether polycyclic hydrocarbons in cigarette smoke could influence the gastrointestinal metaboli5m of phenacetin, we studied the effects of 3,4‐benzpyrene administration on the metabolism of CILphenacetin by enzymes in the intestinal mucosa of the rat. The data indicated that O‐dealkylation of phenacetin did occur—to a small extent—by an enzyme system in the small intestine, and that the activity of this enzyme system was stimulated by treatment of rats with 3,4‐benzpyrene.

Decreased Concentration of Phenacetin in Plasma of Cigarette Smokers

The amount of phenacetin in plasma was determined in nine control subjects (nonsmokers) and nine subjects who smoked at least 15 cigarettes per day. The mean plasma concentration of phenacetin at 1, 2, 3�, and 5 hours after its administration was markedly lower in cigarette smokers than in nonsmokers. At 2 hours after the oral administration of 900 milligrams of phenacetin, the plasma concentration (� standard error) of unchanged drug was 2.24 � 0.73 micrograms per milliliter in the controls and 0.48 � 0.28 micrograms per milliliter in the smokers. The rate of excretion in urine of the major metabolite of phenacetin, N-acetyl-p-aminophenol, was the same in both groups. These results indicate for the first time decreased concentrations of a drug in plasma of persons who smoke cigarettes, and the results suggest that the decrease in the amount of Phenacetin in plasma may result from increased metabolism of phenacetin in cigarette smokers.

Induction of liver microsomal cortisol 6β-hydroxylase by diphenylhydantoin or phenobarbital: An explanation for the increased excretion of 6-hydroxycortisol in humans treated with these drugs

Abstract Treatment of guinea pigs with diphenylhydantoin or phenobarbital for several days causes a marked increase in the activity of an enzyme system in liver microsomes that 6β-hydroxylates cortisol. This effect offers an explanation for the previously reported observation indicating an increased urinary excretion of 6-hydroxycortisol in humans treated with diphenylhydantoin or phenobarbital.

Incorporation of radioactive-δ-aminolevulinic acid into microsomal cytochrome P450: Selective breakdown of the hemoprotein by allylisopropylacetamide and carbon tetrachloride

Abstract Administration of allylisopropylacetamide (AIA) or CCl 4 to rats previously treated with phenobarbital leads to a rapid decrease in cytochrome P 450 within 1 hr. The amount of cytochrome b 5 and NADPH cytochrome c reductase in liver microsomes remains unchanged following AIA treatment. In contrast, CCl 4 administration causes a decrease in total microsomal protein thus leading to a net loss in cytochrome b 5 and NADPH cytochrome c reductase. By using 3 H-δ-aminolevulinic acid to label microsomal cytochrome P 450 heme, the effect of AIA and CCl 4 on this cytochrome was shown to be caused by destruction of preexisting CO-binding pigment and not from inhibition of synthesis. In addition, the breakdown products of cytochrome P 450 heme accumulate in the liver after AIA or CCl 4 treatment.

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates: VII. Stimulation of benzphetamine N-demethylation by lipid and detergent

Abstract The resolved liver microsomal hydroxylation system required lipid for benzphetamine N-demethylation. Certain nonionic detergents, such as Emulgen 911, Triton N-101, and Triton X-100, at appropriate concentrations could substitute for lipid. These results suggest that lipid and detergent activate the cytochrome P-450-containing hydroxylation system by a similar mechanism, probably by enhancing the interaction between cytochrome P-450 and NADPH-cytochrome c reductase.

A reconstituted microsomal enzyme system that converts naphthalene to trans-1,2-dihydroxy-1,2-dihydronaphthalene via naphthalene-1,2-oxide: Presence of epoxide hydrase in cytochrome P-450 and P-448 fractions

Abstract Cytochrome P -450 and P -448 fractions isolated from rat liver microsomes contain high levels of epoxide hydrase activity with styrene oxide or naphthalene-1,2-oxide as substrate. A reconstituted system, which consists of cytochrome P -450 or P -448, reductase, and lipid, converts naphthalene into naphthalene-1,2-oxide, trans -1,2-dihydroxy-1,2-dihydronaphthalene and 1-naphthol. The reconstituted stem with cytochrome P -450 produces mainly 1-naphthol, while with cytochrome P -448, nearly equal amounts of dihydrodiol and 1-naphthol are formed. Naphthalene oxide is formed as the intermediate in both systems, and its conversion to dihydrodiol can be blocked by the epoxide hydrase inhibitor, 3,3,3-trichloropropene oxide.