Henry A. Sasame

Cytochrome P-450 and NADPH cytochrome C reductase in rat brain: Formation of catechols and reactive catechol metabolites

Microsomes isolated from whole rat brain were found to contain cytochreme P-450 (0.025 to 0.051 nmoles/mg) and NADPH cytochrome c reductase activity (26.0 to 55.0 nmoles/mg/min). The oxidation of estradiol to a reactive metabolite that became covalently bound to rat brain microsomal protein was inhibited 63% by an atmosphere of CO:O2 (9:1), indicating the involvement of a cytochrome P-450 oxygenase. In contrast, this atmosphere had no effect on the binding of either the catechol estrogen, 2-hydroxyestradiol, or several catecholamines to rat brain microsomes. An antibody prepared against NADPH cytochrome c reductase was found to decrease significantly both the formation of 2-hydroxyestradiol from estradiol by rat brain microsomes and the covalent binding of the catechol estrogen and catecholamines to rat brain microsomal protein.

Studies on the destruction of liver microsomal cytochrome P-450 by carbon tetrachloride administration

Carbon tetrachloride impairs the oxidative enzymes in liver microsomes by decreasing the amount of cytochrome P-450. The destruction is probably not mediated by an increase in lipid peroxidation, since ethanol which promotes lipid peroxidation does not decrease cytochrome P-450 or the metabolism of ethylmorphine. The decrease in cytochrome P-450 is not due to destruction of liver cells or to altered permeability of endoplasmic reticulum because NADPH cytochrome c reductase was not detected in plasma and was not decreased in liver microsomes. The solvent effect of CCl4 probably does not account for the decrease in cytochrome P-450 because CHCl3 and CH2Cl2 did not decrease the cytochrome. Our results are in accord with the view that CCl4 acts through an active metabolite, but they do not necessarily imply that the active metabolite is formed by a cytochrome P-450 enzyme.

Prevention of carbon tetrachloride-induced necrosis by inhibitors of drug metabolism—Further studies on their mechanism of action

Abstract Liver necrosis caused by CCl 4 is known to be decreased by the administration of SKF 525-A (2-diethylaminoethyl 2,2-diphenylvalerate), Sch 5705 (ethyl 2-diethyl-aminoethyl-2-phenyl-2-ethyl malonate), Sch 5706 [ethyl N -(2-diethylaminoethyl) 2-phenyl-2-ethyl malonate], Sch 5712 (ethyl 2-diethylaminoethyl 2-ethyl-2-butyl malonate), CFT 1201 [2-diethylaminoethyl 2-phenyl (2-propene) 4-penten-1-oate], Lilly 18947 (2,4-dichloro-6-phenyl phenoxyethyl diethylamine) and DPEA (2,4-dichloro-6-phenyl phenoxyethylamine). Although these substances are known to inhibit cytochrome P-450 dependent drug-metabolizing enzymes in liver microsomes, they apparently do not evoke their protective effects by slowing the elimination of CCl 4 . In fact, SKF 525-A, but none of the other inhibitors, partially prevents the impairing effects of CCl 4 on cytochrome P-450 in liver. Although SKF 525-A markedly decreased the liver necrosis and the rise in serum isocitrate dehydrogenase (ICD) caused by CCl 4 , it only partially prevented the CCl 4 -induced decrease in body temperature.

Cytochrome P-450-mediated oxidation of 2-hydroxyestrogens to reactive intermediates.

2-Hydroxyestradiol, 2-hydroxyestrone and 2-hydroxy-17α-ethynylestradiol, oxidation products of naturally occurring estrogens and synthetic estrogens in some oral contraceptives were found to be converted by rat liver microsomes to reactive metabolites that become irreversibly bound to microsomal protein. The irreversible binding required microsomes, oxygen and NADPH. The NADPH could be replaced by a xanthine-xanthine oxidase system which is known to generate superoxide anions. The irreversible binding was substantially inhibited by superoxide dismutase, 30% in those incubations containing NADPH and 98% in those incubations containing the xanthine-xanthine oxidase system. Further studies with 2-hydroxyestradiol showed that microsomal cytochrome P-450 was rate limiting in the NADPH-dependent irreversible binding, because the binding was inhibited 62% by an antibody against NADPH-cytochrome c reductase and 70% in an atmosphere of CO:O2 (9:1) when compared to an atmosphere of N2:O2 (9:1). Phenobarbital, a known inducer of cytochrome P-450, had no effect on the irreversible binding of 2-hydroxyestradiol, whereas another inducer of P-450, pregnenolone-16α-carbonitrile, markedly increased the irreversible binding. In contrast, cobaltous chloride, an inhibitor of the synthesis of cytochrome P-450, decreased both P-450 and the irreversible binding. These results are consistent with a mechanism for irreversible binding of estrogens and 2-hydroxyestrogens to microsomes that requires oxidation of the catechol nucleus by cytochrome P-450-generated superoxide anion.

3H-Benzene metabolism in rabbit bone marrow

Abstract An assay for benzene metabolism using 3 H-benzene and high pressure liquid chromatography was developed. 3 H-Benzene metabolism (2 pmoles benzene equivalents/mg protein/min) required the presence of a TPNH generating system and was inhibited 80% in the presence of a CO:O 2 (9:1) atmosphere. The products of 3 H-benzene rabbit bone marrow microsomal metabolism were phenol and an unidentified metabolite. Cytochrome P-450 (26–51 pmoles/mg microsomal protein) and cytochrome c reductase activity (7.8–21.0 nmole/mg microsomal protein/min) were detected in rabbit bone marrow.

Metabolic activation of nitrofurantoin--possible implications for carcinogenesis.

Abstract Although past investigations have indicated that nitrofurantoin is noncarcinogenic, the present studies demonstrate several features of the metabolism of the drug which are similar to those of other nitrofurans that are known carcinogens. Microsomal and soluble fractions from both rat liver and lung mediated the covalent binding of [14C]nitrofurantoin to tissue macromolecules in vitro. Oxygen strongly inhibited the binding in both the microsomal and soluble fractions, and carbon monoxide failed to inhibit binding in microsomal preparations, indicating nitrofurantoin was activated in both systems by nitroreduction and not by oxidation of the furan ring. An antibody against NADPH-cytochrome c reductase inhibited the microsomal nitroreduction and covalent binding of nitrofurantoin, while the addition of a flavin (FAD) markedly enhanced the covalent binding. Maximal rates of covalent binding were obtained in soluble fractions in the presence of NADH or hypoxanthine; covalent binding was inhibited in these fractions by allopurinol. an inhibitor of xanthine oxidase. Nitroreduction of nitrofurantoin was enhanced, but covalent binding was decreased, in liver microsomes from phenobarbital-pretreated rats. Phenobarbital did not alter nitroreduction or covalent binding of nitrofurantoin in lung microsomes or in soluble fractions from lung or liver. Reduced glutathione markedly decreased covalent binding of nitrofurantoin. in both the microsomal and the soluble fractions from liver and lung. but did not alter the rate of nitroreduction in any of the fractions. Radioactivity was covalently bound in several organs of rats given [14C]nitrofurantoin in vivo.

Gastric glutathione depletion and acute ulcerogenesis by diethylmaleate given subcutaneously to rats

Abstract The subcutaneous administration of diethylmaleate (DEM), a drug known previously to deplete liver glutathione (GSH), was shown in rats to cause a severe dose-dependent, rapid and persistent decrease in the glutathione content of the glandular gastric mucosa, a tissue that normally contains extraordinarily high concentrations of GSH. This effect of DEM was accompanied by the occurrence of severe ulcerative lesions of the gastric lining and sometimes also a marked gaseous inflation of the stomach. The acute ulcerative lesions appeared identical to those previously shown to be induced by a variety of physical and/or behavioral stressors in rodents. At least one ulcerogenic experimental stressor (cold-restraint) has been shown previously to lower gastric GSH. Also, a pretreatment (i.e., starvation) that decreases gastric GSH enhances both stress-induced ulcerogenesis and DEM-induced ulcerogenesis. These studies suggest that a possible role for GSH in maintaining the normal homeostasis and integrity of the gastric mucosa should be considered.

Superoxide and hydrogen peroxide production and NADPH oxidation stimulated by nitrofurantoin in lung microsomes: Possible implications for toxicity

Abstract The addition of nitrofurantoin to aerobic incubation mixtures containing rat lung microsomes strongly enhanced the generation of adrenochrome from epinephrine. Adrenochrome formation in this system was blocked by superoxide dismutase, but not by catalase. Hydrogen peroxide production was also strongly enhanced by nitrofurantoin in these preparations; superoxide dismutase did not significantly alter the amount of H 2 O 2 measured, but no H 2 O 2 was detected in incubation mixtures in the presence of catalase. Nitrofurantoin enhanced the oxidation of NADPH in lung microsomal suspensions under aerobic conditions; the enhancement was unaffected by catalase but was partially prevented by superoxide dismutase. Neither adrenochrome formation nor H 2 O 2 production were enhanced by nitrofurantoin under anaerobic (N 2 ) conditions, but NADPH oxidation in the presence of nitrofurantoin was greater under anaerobic conditions than under aerobic conditions. These results are consistent with the view that the redox cycling of nitrofurantoin in lung microsomes in the presence of oxygen results in the consumption of NADPH and the production of activated oxygen species, emphasizing some in vitro metabolic similarities with the lung-toxic herbicide, paraquat.

Effect of adrenalectomy and cortisone administration on components of the liver microsomal mixed function oxygenase system of male rats which catalyzes ethylmorphine metabolism

Abstract The effects of adrenalectomy and cortisone administration on components of the mixed function oxygenase system of liver microsomes from adult male rats were studied. The N -demethylation of ethylmorphine (EM) as measured by formaldehyde production was used as an index of the overall activity of this system. EM demethylase activity in liver microsomes from adrenalectomized rats was 56 per cent of the value obtained with sham-operated controls, while cytochrome P-450 content fell by only 19 per cent. NADPH cytochrome c reductase and cytochrome P-450 reductase activities were decreased 68 and 69 per cent, respectively, in liver microsomes from adrenalectomized animals, while the apparent affinity constant ( K sp ) of EM for cytochrome P-450 spectral change was not altered. The decrease in maximal spectral change caused by adrenalectomy corresponded to the decrease in cytochrome P-450 content. Cortisone acetate administration (5 mg/kg/day for 8 days) to adrenalectomized rats prevented the decrease in EM demethylase activity. Accordingly, the steroid treatment increased the NADPH cytochrome c reductase and cytochrome P-450 reductase activities to 113 and 140 per cent, respectively, of the values obtained with the sham-operated controls, without significantly altering the cytochrome P-450 content or the kinetic constants for the spectral changes. These data suggest that the well known decrease in drug-metabolizing activity seen in liver microsomes of adrenalectomized animals is not related to changes in cytochrome P-450 content or to a decrease in the ability of cytochrome P-450 to bind drug substrates. However, there appears to be a relationship between the reductase activities and the adrenal function, because the changes in activities in NADPH cytochrome c reductase and cytochrome P-450 reductase in liver microsomes from adrenalectomized and cortisone-treated adrenalectomized rats paralleled the changes in EM demethylase activity.

Studies on the inhibitory effects of various substances on drug metabolism by liver microsomes: The effect of nicotinamide in altering the apparent mechanism of inhibition

Abstract Nicotinamide, 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), and amino-ethyldiphenylpropylacetate (SKF 26754A, SKF 525A-PA) inhibit the hydroxylation of aniline, and the N -demethylation of aminopyrine and ethylmorphine by liver microsomes. The mechanism of inhibition, however, depends on the species and the substrate. For example, nicotinamide blocks the aminopyrine demethylating enzyme in rat liver microsomes competitively, and that in mouse liver microsomes by a mixed mechanism. The aniline hydroxylating enzyme in mouse liver microsomes is blocked competitively by nicotinamide, uncompetitively by SKF 525A-PA, and partially uncompetitively by DPEA. The ethylmorphine demethylating enzyme in mouse liver microsomes is inhibited by nicotinamide, SKF 525A-PA and DPEA by dual mechanisms. At low concentrations, the inhibitors block the reaction noncompetitively, but a high concentrations they block it both competitively and non-competitively. Nicotinamide modified the apparent mechanism of inhibition by SKF 525A-PA and DPEA. Thus in the presence of nicotinamide, SKF 525A-PA apparently inhibits the N -demethylation of ethylmorphine by a competitive mechanism.