Russell A. Prough

Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450☆

Abstract The mechanism of NADPH- and cumene hydroperoxide-supported hydroxylation of benzo(a)pyrene as catalyzed by liver microsomes was studied using high pressure liquid chromatography, fluorescence, and spectrophotometric methods. Repetitive scan difference spectral analysis clearly demonstrated that during the steady state of these reactions, different products were formed. While the major products noted with NADPH were phenols, only low concentrations of phenols were observed in the presence of cumene hydroperoxide using all three analytical methods. With the organic hydroperoxide, the metabolite profile was shifted from the preponderate production of phenols and dihydrodiols to the production of the three quinone isomers of the hydrocarbon. At several concentrations of cumene hydroperoxide, epoxide hydrase activity and the stability of the arene oxide substrates tested were unaffected during a 2-min incubation period. The transient nature of benzo(a)pyrene phenol formation was investigated; the 3- and 6-phenols were easily oxidized to quinones by cumene hydroperoxide in a cytochrome P-450-dependent oxidation process which most likely involves the formation of free radicals by a one-electron process. These results indicate that the reaction mechanism operative in the presence of the organic hydroperoxide differs in several regards from that functional in the presence of NADPH and that a common oxidative mechanism may not exist.

Cytosol-mediated reduction of resorufin: A method for measuring quinone oxidoreductase

The reduction of resorufin (7-hydroxyphenoxazone) fluorescence was catalyzed by enzymes present in the hepatic cytosol of rats and hamsters. This reaction was mediated by either NADH or NADPH, was completely inhibited by 10 microM dicumarol, and was not affected by anaerobic conditions (purging the reaction cuvette with nitrogen). The enzyme-mediated decrease in resorufin fluorescence was also associated with the loss of the primary absorbance maximum at 570 nm as well as the shoulders at 530 and 600 nm. Similar spectral changes were observed after resorufin was nonenzymatically reduced by sodium dithionite. The enzymatic activity was induced 20- to 40-fold by animal pretreatment with Aroclor-1254 or methylcholanthrene, but only minimally by phenobarbital. A 2.5-fold increase in the rate of the reaction was noted when the pH of the reaction mixture was lowered from pH 7.5 to 6.0. This pH optimum was not a result of slower rates of reoxidation of the reduced resorufin at lower pH, but was due to increased rates of reduction of the compound. Several of the characteristics of the reaction were congruent with the involvement of DT-diaphorase (quinone oxidoreductase, EC 1.6.99.2), and this newly developed fluorimetric assay would appear to be a rapid, sensitive, and direct method for measurement of DT-diaphorase activity.

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes

Abstract An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b 5 reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b 5 reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b 5 to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b 5 from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b 5 during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b 5 stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b 5 and methemoglobin. An antibody to purified, trypsin-solubilized cytochrome b 5 from rat liver microsomes was shown to inhibit erythrocyte cytochrome b 5 reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b 5 preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b 5 of human erythrocytes are immunochemically similar to NADH-cytochrome b 5 reductase and cytochrome b 5 of liver microsomes, respectively.

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported cytochrome P-450-dependent enzyme activities in liver microsomes

Abstract The role of NADH-cytochrome b 5 reductase and cytochrome b 5 as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P -450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P -450 whereas the third requires the reductase and cytochrome b 5 . Antibody prepared against NADH-cytochrome b 5 reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b 5 and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b 5 in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b 5 reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b 5 to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b 5 involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450 -dependent electron transport reactions, cytochrome b 5 functions as an intermediate electron carrier between NADH-cytochrome b 5 reductase and cytochrome P -450.

The existence of a benzo(a)pyrene-3,6-quinone reductase in rat liver microsomal fractions

Abstract Analysis of repetitive scan difference spectra of incubation mixtures containing liver microsomes from phenobarbital-pretreated rats, benzo(a)pyrene-3,6-quinone, and NADPH reveals the rapid reduction of the quinone to a steady-state level of hydroquinone and the subsequent reoxidation of the hydroquinone. This cyclic process results in NADPH oxidation coupled to oxygen reduction and hydrogen peroxide formation. The reduction of the benzo(a)pyrene-3,6-quinone is not supported by NADH. The initial rate of the NADPH-supported reaction is inhibited by NADP+, metyrapone, and antiNADPH-cytochrome P-450 ( c ) reductase globulin, but not by dicumarol, anaerobiosis, or a gas mixture of carbon monoxide and oxygen (4:1, v v ). These results suggest that cytochrome P-450 and its reductase are involved in this reaction. During the steady-state of metabolism of benzo(a)pyrene by liver microsomes, the 3,6-quinone produced must exist largely as the hydroquinone and may be suitable for disposition as a water-soluble conjugate(s).

Some characteristics of hamster liver and lung microsomal aryl hydrocarbon (biphenyl and benzo(a)pyrene) hydroxylation reactions

Abstract Aryl hydrocarbon hydroxylation (AHH) reactions were compared using liver and lung microsomes of corn oil- and 3-methylcholanthrene (3-MC)-treated hamsters, employing benzo(a)pyrene (BAP) and biphenyl as substrates. The predominant AHH activity of liver and lung microsomes from corn oil- or 3-MC-treated hamsters was biphenyl 4-hydroxylase. Biphenyl 2-hydroxylase and BAP-hydroxylase activities were approximately 50 per cent as active as biphenyl 4-hydroxylase in liver and approximately 1–3 per cent as active as biphenyl 4-hydroxylase in lung microsomes. Biphenyl 4-hydroxylase activity was 70–80 per cent as active in lung as in liver microsomes. Treatment with 3-MC in vivo induced the biphenyl 4-hydroxylation reaction in liver but not in lung microsomes, the biphenyl 2-hydroxylation reaction both in lung and liver microsomes, and the BAP hydroxylation reaction in lung but not in liver microsomes. Biphenyl 2- and 4-hydroxylase activities of liver microsomes displayed similar sensitivities to inhibition by a number of chemical inhibitors in vitro . Inhibition of biphenyl hydroxylation reactions by metyrapone or carbon monoxide did not distinguish between lung or liver microsomal mono-oxygenases of corn oil- or 3-MC-treated hamsters. While small differences were expressed by inhibition with ethylmorphine, large differences became apparent through inhibition studies with BAP or α-naphthoflavone. It is concluded that the major aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the constitutive (uninduced) AHH of the liver microsomes and that the minor aromatic hydroxylase activity of lung microsomes from corn oil- or 3-MC-treated hamsters resembles the induced AHH of the liver microsomes.

Synergistic induction of monooxygenase activity by glucocorticoids and polycyclic aromatic hydrocarbons in human fetal hepatocytes in primary monolayer culture

The ability of polycyclic aromatic hydrocarbons and glucocorticoids to regulate monooxygenase activity of human fetal liver has been studied using hepatocytes prepared by collagenase digestion of liver samples from human abortuses of 13 to 19 weeks of gestational age, and maintained in primary monolayer culture for periods up to 5 days. Addition of 1,2-benzanthracene to the cells caused an increase in monooxygenase activity (3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin) in a time-and concentration-dependent fashion. The concentration of 1,2-benzanthracene required to achieve half-maximal induction was 5 microM. The inductive effect of the polycyclic hydrocarbon was potentiated approximately 2.5-fold when dexamethasone (250 nM) or other glucocorticoids were included in the culture medium. Dexamethasone alone had little or no effect on the induction of monooxygenase activity. The concentration of dexamethasone required for half-maximal stimulation of monooxygenase activity in the presence of 1,2-benzanthracene was 5-10 nM, and the action of dexamethasone was reversed by the addition of cortisol 21-mesylate, consistent with the concept that the action of dexamethasone was mediated by binding to a glucocorticoid receptor. These results are suggestive that glucocorticoids, which are produced by the fetal adrenal and have an important role in the regulation of fetal development, act synergistically with polycyclic aromatic hydrocarbons to induce the activity of liver monooxygenases in the human fetus.

Hydrogen peroxide-supported oxidation of benzo [a]pyrene by rat liver microsomal fractions

Abstract In the presence of liver microsomes from phenobarbital-pretreated rats, hydrogen peroxide oxidized benzo [a]pyrene to a number of biologically significant products at a rate that was approximately 20 per cent as fast as that seen by us and others with NADPH and oxygen. As with NADPH-dependent reactions [J. Capdevila, R. W. Estabrook, and R. A. Prough, Archs. Biochem. Biophys.200, 186 (1980)], the hydrogen peroxide-dependent reactions resulted in the production of relatively large quantities of dihydrodiols as metabolites. This was in marked contrast to the product distribution observed when cumene hydroperoxide was utilized as a cosubstrate (foregoing reference). The formation of the various organic-soluble metabolites was dependent on the presence of functional liver microsomal cytochrome P-450 in the reaction mixture. Approximately 48 per cent of the benzo[a]pyrene metabolites, however, was observed to be bound to microsomal protein, and inhibition of cytochrome P-450 function, by metyrapone or N-octylamine did not affect the extent of covalent binding of the hydrocarbon to the microsomal protein. The differences noted during benzo[a]pyrene metabolism using hydrogen peroxide strongly suggest that at least two distinct mechanisms exist to account for the oxidation of the hydrocarbon, i.e. epoxidation and one-electron oxidation reactions.

The role of NADPH-cytochrome c reductase in microsomal hydroxylation reactions☆

Abstract A specific antibody elicited against NADPH-cytochrome c reductase inhibited cytochrome P-450- and NADPH-dependent hydroxylation of biphenyl by rodent liver and lung microsomal preparations. The inhibition profiles suggested that both the 2- and 4-hydroxylation of biphenyl were mediated by a common NADPH-cytochrome c reductase (NADPH-cytochrome P-450 reductase) and that the same flavoprotein species operated in liver and lung microsomes of corn oil- or 3-methylcholanthrene-pretreated rats and hamsters. An immunochemically identical NADPH-cytochrome c reductase also apparently functioned in the NADPH-supported metabolism of benzo(a)pyrene and ethylmorphine. NADH supported the microsomal metabolism of benzo(a)pyrene and ethylmorphine in liver and biphenyl in liver and lung, but the maximal rates of reaction were slower than when supported with NADPH. The Km of NADH for biphenyl 2- and 4-hydroxylations in control hamster liver microsomes were approximately 5 m m . Anti-NADPH-cytochrome c reductase globulin inhibited NADH-supported biphenyl 2- and 4-hydroxylase activities in corn oil- or 3-methylcholanthrene-pretreated rats and hamsters, even at NADH concentrations as low as 0.25 m m . These results indicate that the same flavoprotein reductase species which mediated NADPH-dependent biphenyl hydroxylase donated at least one electron for the NADH-supported hydroxylation.

The metabolism and toxicity of some organotin compounds in isolated rat hepatocytes

Abstract The metabolism and toxicity of some ethyl-substituted organotin compounds in isolated rat hepatocytes were studied. Tetra- and triethyltin derivatives were metabolized by isolated rat hepatocytes to yield ethane and ethylene. Hydrocarbon formation from tetraethyltin was larger than that obtained with triethyltin bromide, and ethylene was the major product (95%) of tetraethyltin metabolism. At a triethyltin salt concentration of 100 μM, the major product formed by cells from untreated rats was ethane. Pretreatment in vivo by phenobarbital resulted in a marked increase in the overall rate of hydrocarbon production and a change in ethylene to ethane ratio; in this case ethylene was the predominant metabolite produced. 5,6-Benzoflavone pretreatment in vivo resulted in a small depression in overall hydrocarbon production. No metabolism of diethyltin dichloride (100 μM) by isolated rat heptocytes was detected. Triethyltin bromide (100 μM) was a potent inhibitor of both the Phase I (oxidation) and Phase II (conjugation) metabolism of biphenyl in isolated hepatocytes from phenobarbital-pretreated rats, whereas diethyltin dichloride was seen to affect particularly the Phase I metabolism of the aromatic hydrocarbon. Triethyl- and diethyltin salts reduced oxygen consumption and ATP levels in these cells. However, the triethyl derivative was more effective in this respect. Tetraethyltin was not appreciably toxic to hepatocytes. Trypan blue dye exclusion and lactate dehydrogenase loss by the cells isolated from phenobarbital-pretreated rats indicated that triethyltin bromide was more toxic than diethyltin dichloride. In contrast, the diethyl derivative was more potent in stimulating lipid peroxidation as indicated by formation of thiobarbituric acid-reactive products than was triethyltin.