Two sites of azo reduction in the monooxygenase system

The mechanism of the azo reduction of sulfonazo III and amaranth by the rat hepatic monooxygenase system was studied. Air strongly inhibited (greater than 95%) the enzymatic reduction of both azo compounds; a 100% CO atmosphere inhibited amaranth reduction (greater than 90%) but only slightly inhibited sulfonazo III reduction (13%). The addition of 50 microM sulfonazo III to microsomal incubations stimulated oxygen consumption, NADPH oxidation, and adrenochrome formation, whereas 100 microM amaranth did not. The reduction potentials of these two azo compounds were also very different (amaranth, E = -0.620 V; sulfonazo III, E = -0.265 V versus normal hydrogen electrode). The organic mercurial mersalyl converted cytochrome P-450 to cytochrome P-420 (68%) and markedly decreased NADPH-cytochrome P-450(c) reductase activity (97%) in microsomal preparations, presumably by inactivating or destroying functional sulfhydryl groups important for the catalytic activity of these enzymes. GSH was used to restore, and NADP+ to protect, the activities of the monooxygenase components from the effects of mersalyl. The data indicate that inactivation of NADPH-cytochrome P-450(c) reductase inhibits sulfonazo III and amaranth reduction, whereas inactivation of cytochrome P-450 inhibits only amaranth reduction. Furthermore, the reduction of sulfonazo III by purified microsomal NADPH-cytochrome P-450(c) reductase was significantly faster than the rate of reduction of amaranth. These studies demonstrate that two distinct sites of azo reduction exist in the monooxygenase system and that not all azo compounds are reduced by cytochrome P-450.     

Immunohistochemical localization and quantitation of NADPH-cytochrome P-450 reductase in human liver

An antibody raised in a goat against the human liver NADPH-cytochrome P-450 reductase (EC 1.6.2.4.) enzyme has been used to: 1) immunoquantify the level of this enzyme in human liver microsomes, and 2) study the distribution of the reductase across the human liver acinus. Employing the Western blot procedure, anti-human reductase IgG recognized a single band in human liver microsomes which corresponded in molecular weight to the purified reductase. The content of the NADPH-cytochrome P-450 reductase in six normal human livers varied from 87 to 121 pmol/mg of microsomal protein. NADPH-cytochrome P-450 reductase activity of the same microsomes ranged from 107 to 222 nmol of cytochrome c reduced per min per mg of protein. The correlation between reductase content and activity (r = 0.54) was not statistically significant (p greater than 0.1). The total cytochrome P-450 content (cytochrome P-450 and P-420) of the same microsomes varied from 423 to 1413 pmol/mg of microsomal protein. The average ratio of cytochrome P-450 to NADPH-cytochrome P-450 reductase was 7.1:1 +/- 3.1 (mean +/- SD) in the human liver microsomal preparations studied. The reductase was found to be nonuniformly distributed across the human liver acinus. Although all hepatocytes stained positively for NADPH-cytochrome P-450 reductase, the staining intensity was highest in zone 3 and in some cases also in zone 1 hepatocytes. These results show that human liver contains a gross excess of cytochrome P-450 molecules to NADPH-cytochrome P-450 reductase molecules. Furthermore, the differential distribution of the reductase within the human liver acinus may lead to a better understanding of the mechanism underlining site-specific drug hepatotoxicity.     

Monooxygenase Activities of Human Liver,Lung, and Kidney Microsomes – A Study of 42 post mortem Cases

Abstract: The cytochrome P-450-dependent monooxygenase system was examined in microsomal fractions prepared from 42 post mortem human livers and 9 lungs and kidneys. Electron microscopy studies indicated that the human liver samples were relatively free of mitochondrial and plasma membrane contamination, but samples of kidney and lung were less pure. The microsomal fractions from all organs were judged to be relatively free of haemoglobin and methaemoglobin. The specific enzyme activities for several drug substrates for the monooxygenase, NADPH-cytochrome c reductase activity and the content of the microsomal cytochromes were measured. The values of the biochemical parameters studied were found to be quite variable and the values for the human liver were appreciably lower than those obtained with liver microsomes from laboratory rodents. The enzyme activities of the human kidney and lung microsomal fractions were 1–10% of those seen for human liver samples, except for NADPH-cytochrome c (P-450) reductase activity. In order to evaluate any post mortem changes in human liver, correlations between drug metabolism activities and either cytochrome P-450 or NADPH-cytochrome c (P-450) reductase content were examined. Strong correlations (r>0.91) were seen only between aminopyrine or ethylmorphine demethylase activity and cytochrome P-450 content in samples obtained within 4 hours of death. Longer post mortem times gave poorer correlation between activity and cytochrome content. These studies document several conditions required in order to obtain human microsomal fractions representative of the activities in fresh, viable tissue.     

Oxidation of thiobenzamide by the FAD-containing and cytochrome P-450-dependent monooxygenases of liver and lung microsomes

Two distinct microsomal pathways involved in the metabolism of thiobenzamide to thiobenzamide S-oxide have been identified and quantitated in the liver and lungs of mice and rats, using a highly inhibitory antibody against NADPH-cytochrome P-450 reductase. Approximately 50 and 65% of the oxidation in mouse and rat liver microsomes, respectively, was due to the FAD-containing monooxygenase, the remainder being catalyzed by cytochrome P-450. In the mouse lung, S-oxidation was predominantly via the FAD-containing monooxygenase while that in the rat lung was about 60% via the FAD-containing enzyme and 40% via cytochrome P-450. Cytochrome P-450-dependent S-oxidation of thiobenzamide was induced in the liver by treatment of mice with phenobarbital and slightly increased by treatment with 3-methylcholanthrene, while in rat liver either of these treatments caused only a small increase in metabolism due to cytochrome P-450. Thermal inactivation of the FAD-containing monooxygenase left the cytochrome P-450 component essentially unchanged. Thermally treated microsomes had a pH activity profile characteristic of cytochrome P-450 and were less inhibited by methimazole and thiourea when compared to untreated microsomes. Female mouse liver microsomes had a much higher, and female rat liver microsomes a lower, ability to S-oxidize thiobenzamide when compared to the males.     

The cytochrome P-450 monooxygenase system of rabbit lung enzyme components, activities, and induction in the nonciliated bronchiolar epithelial (Clara) cell, alveolar type II cell, and alveolar macrophage

Enzyme components and activities of the cytochrome P-450 monooxygenase system in microsomal preparations from the Clara cell, alveolar type II cell, and alveolar macrophage fractions isolated from lungs of untreated rabbits and rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin were examined. Results are compared to those obtained with microsomal preparations from whole lung. Concentrations of cytochrome P-450 isozymes 2 and 5 and NADPH-cytochrome P-450 reductase activities were higher in preparations from Clara cell fractions than in preparations from type II cell fractions or whole lung. For the most part, however, differences among these preparations were 2-fold or less. Microsomal preparations from the macrophage fraction contained low or undetectable levels of cytochrome P-450 isozymes but relatively high levels of cytochrome P-450 reductase activity. The concentration of cytochrome P-450 isozyme 6, in contrast to those of isozymes 2 and 5, was found to be highest in microsomal preparations from whole lung. Treatment of rabbits with 2,3,7,8-tetrachlorodibenzo-p-dioxin increased the concentrations of isozyme 6 in preparations from the Clara and type II cell fractions and from whole lung about 20-fold. In contrast, the content of isozyme 6 in preparations from the macrophage fraction increased greater than 90-fold. In all cases, induction of isozyme 6 resulted in substantial increases in the O-deethylation of 7-ethoxyresorufin and only minor increases in the hydroxylation of benzo(a)pyrene. Activities per unit of isozyme 6, following induction, were similar in all preparations, and we estimate that less than 20% of the potential activity of isozyme 6 is expressed with benzo(a)pyrene and greater than 40% with 7-ethoxyresorufin. These similarities exist in spite of significant differences among the preparations from different fractions in the ratios of isozyme 6 to NADPH-cytochrome P-450 reductase.     

Triphenyltin acetate-mediated in vitro inactivation of rat liver cytochrome P-450

The in vitro effects of the organotin (OT) compound triphenyltin acetate (TPTA) on cytochrome P-450 content and functions were investigated in liver microsomes from untreated, phenobarbital (PB)- or beta-naphthoflavone- (betaNAF) pretreated rats. At a concentration of 0.5 mM, TPTA caused a marked loss in the spectrally detectable content of cytochrome P-450 up to 27% of its original value, along with an increase in the inactive form cytochrome P-420. Both effects were most pronounced in betaNAF-treated microsomes, which showed a shift in the hemoprotein absorption maximum from 448 nm to 451 nm, but in all cases TPTA failed to affect either cytochrome b5 or total heme content, or to increase the production of malondialdehyde. These results suggest that lipid peroxidation of microsomal membranes or damage to the heme moiety should be excluded as contributing factors in the hemoprotein loss. TPTA also produced a concentration-related functional inactivation of cytochrome P-450 that was most pronounced in betaNAF-exposed microsomal preparations, as denoted by a striking reduction in the ethoxyresorufin O-deethylase (EROD) activity (IC50 = 0.088 mM). In contrast, the activities of cytochrome P-450-independent microsomal enzymes such as NADPH cytochrome c reductase and indophenyl acetate esterase (IPA-EST) were not markedly affected even by 0.5 mM TPTA (-30%). As assessed by Lineweaver-Burk plots, the mechanism of inhibition appeared to be noncompetitive for IPA-EST and of mixed type (competitive-noncompetitive) for EROD. Among sulfhydryl-containing compounds, dithiothreitol was considerably more effective than albumin and reduced glutathione in preventing cytochrome P-450 inactivation and even was able to partially reverse the hemoprotein damage when added after TPTA; glycerol, which is known to protect the hydrophobic environment of cytochrome P-450, was as effective as albumin. This study indicates that TPTA behaves as an almost specific and powerful in vitro inhibitor of cytochrome P-450-dependent monooxygenases, apparently through the interaction with critical sulfhydryl groups of the hemoprotein.     

Inhibitory effects of nilutamide, a new androgen receptor antagonist, on mouse and human liver cytochrome P-450

The effects of nilutamide were studied first with human liver microsomes. At concentrations expected in the human liver (110 microM), nilutamide inhibited hexobarbital hydroxylase, benzphetamine N-demethylase, benzo(a)pyrene hydroxylase and 7-ethoxycoumarin O-deethylase activities by 85, 40, 35 and 25%, respectively. There was no in vitro inhibition of NADPH-cytochrome c reductase activity, no in vitro loss of CO-binding cytochrome P-450, and no spectral evidence for the in vitro formation of a possible cytochrome P-450Fe(II)-nitroso metabolite complex. Other studies were performed with mouse liver microsomes. Nilutamide (550 microM) did not significantly increase the consumption of NADPH by aerobic microsomes, and did not modify the kinetics for the reduction of cytochrome P-450 by NADPH-cytochrome P-450 reductase in an anaerobic system. Nilutamide (22 microM) produced either a type I or a type II binding spectrum. Kinetics for the inhibition of hexobarbital hydroxylase were consistent with competitive inhibition. A last series of experiments was performed after administration of nilutamide in mice. Thirty minutes after administration of doses (15 or 30 mumol.kg-1 i.p.) similar to those used in humans, the hexobarbital sleeping time was increased by 40 and 60%, respectively. There was no evidence, however, for the irreversible inactivation of microsomal enzymes since CO-binding cytochrome P-450 and monooxygenase activities remained unchanged in liver microsomes from mice killed 1 or 6 hr after administration of nilutamide (30 mumol.kg-1 i.p.). These results show that nilutamide inhibits hepatic cytochrome P-450 activity, and suggest that inhibition may actually occur after therapeutic doses of nilutamide in humans.     

Solubilisation, purification and reconstitution of hepatic microsomal azoreductase activity

Microsomal NADPH-cytochrome c (P-450) reductase and cytochrome P-450 were purified from the livers of phenobarbitone-treated rats. Purified NADPH-cytochrome c (P-450) reductase effected the NADPH-dependent reduction of FMN and FAD under anaerobic conditions in a non-enzymic manner, but was unable to reduce directly the azo dye, amaranth. In the presence of FMN, the purified reductase effected reduction of amaranth through the production of reduced FMN. Incorporation of NADPH-cytochrome c (P-450) reductase into the microsomal fraction increased the azoreductase activity of liver preparations from phenobarbitone-treated rats, but had no effect on azoreductase activity in preparations from control animals. Azoreductase activity was reconstituted into dilauroyl phosphatidylcholine vesicles containing purified cytochrome P-450 and purified NADPH-cytochrome c (P-450) reductase. In the absence of supplementary FMN, amaranth reduction was completely dependent upon all three components, but in the presence of FMN, the omission of any one component failed to abolish completely azoreductase activity.     

Inhibition of microsomal cytochrome c reductase activity by a series of alpha, beta-unsaturated aldehydes

alpha, beta-Unsaturated aldehydes are reactive and cytotoxic compounds which occur in the environment and are also formed in vivo. Many of these aldehydes have been reported to inhibit hepatic cytochrome P-450. Our laboratory has shown that trans,trans-muconaldehyde (a possible metabolite of benzene) as well as acrolein and crotonaldehyde, when added to hepatic microsomes, decreased cytochrome P-450 (measured spectrophotometrically). Additional studies showed that several alpha, beta-unsaturated aldehydes also inhibited hepatic microsomal NADPH-cytochrome c reductase. Acrolein, crotonaldehyde and trans,trans-muconaldehyde all decreased NADPH-cytochrome c reductase activity in vitro. Concentrations of 0.5, 1.0 and 1.5 mM acrolein decreased activity to 60, 26 and 11% of control respectively. Similar concentrations of trans,trans-muconaldehyde inhibited NADPH-cytochrome c reductase. Crotonaldehyde was a less effective inhibitor of this enzyme. Propionaldehyde, a saturated aldehyde, had no effect on NADPH-cytochrome c reductase activity. Time course experiments with acrolein over a period of 5-45 min suggest that the loss of NADPH-cytochrome c reductase activity is non-linear. The addition of reduced glutathione protected against the inhibition of reductase activity by acrolein. Formation of these aldehydes and their subsequent inhibition of these enzymes may have important consequences in xenobiotic metabolism.     

Perinatal development of hepatic microsomal mixed function oxidase activity in swine

Hepatic microsomal cytochrome P-450, cytochrome b₅, NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase levels were measured in fetal (107-days gestation), newborn and 1-, 2-, 3-, 4- and 6-week-old swine. Cytochrome P-450 levels and NADPH-cytochrome c reductase and NADPH-cytochrome P-450 reductase activities increased in near parallel with ethylmorphine demethylase (V_max) activity between the first and the sixth postnatal week. The activities or levels of all parameters measured appeared to plateau between the fourth and sixth week post-partum. The only qualitative change observed after 1 week of age was a slight increase in the K_m for ethylmorphine demethylation. NADPH-cytochrome c reductase activity of fetal liver was relatively high, being approximately 40 per cent of the values attained at 6 weeks of age. This was in contrast to very low levels of NADPH-cytochrome P-450 reductase activity and cytochrome P-450 content of fetal liver. Clearly the activity of the flavoprotein NADPH-cytochrome c reductase does not limit the rate of reduction of cytochrome P-450 in the microsomal fraction of fetal liver. The possibility that cytochrome P-450 exists in a different form, or ratio of forms, in fetal liver could not be ascertained from carbon monoxide (CO) or ethylisocyanide (EtCN) difference spectra of fetal microsomal preparations. However, the dithionite difference CO spectra of cytochrome P-450 did not change with age.     

The increasing and decreasing effects of aromatic hydrocarbon solvents on pulmonary and hepatic cytochrome P-450 in the rat

The effects of pretreatment with benzene and various methylbenzenes, ethyl- and propylbenzene, cumene and styrene on hepatic and pulmonary microsomal enzymes were studied in male rats. In the lungs, all the substituted benzenes, but not benzene itself, decreased cytochrome P-450 concentration, and most of them also decreased 7-ethoxycoumarin O-deethylase activity, whereas 7-ethoxyresorufin O-deethylase activity was increased by the same treatment. The change in aryl hydrocarbon hydroxylase activity was negligible. Neither NADPH-cytochrome c reductase activity, nor cytochrome b5 concentration were changed after hydrocarbon treatment. In the liver, all the compounds studied, except for benzene, increased 7-ethoxycoumarin O-deethylase and 7-ethoxyresorufin O-deethylase, and most of them also cytochrome P-450, aryl hydrocarbon hydroxylate and NADPH-cytochrome c reductase. The effect on cytochrome b5 in the liver was less marked. In the liver, all the monooxygenases studied seemed to be inducible by alkylbenzenes and styrene, whereas the effect was selective in the lung; depending on the monooxygenase, the activity can increase, decrease or remain unchanged.     

One-electron reduction of mitomycin c by rat liver: role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450.     

Development of liver microsomal oxidations in the chick.

1. Liver microsomal preparations from chick embryos (1 day before hatching) and from 1-7 day old chicks were assayed for oxidative drug-metabolizing activity with aminopyrine, aniline and naphthalene as substrates. 2. Activities for all three substrates were highest in preparations from 1 day-old chicks. These were more than twice as active as the 7 day-old preparations and about three times as active as those from the embryos. 3. The increase in drug-metabolizing activities in newly-hatched chicks was the same for either sex and persisted for 3 days before declining towards the 7 day-old levels. 4. The developmental time-course fo the liver microsomal drug-metabolizing activities was independent of any factor in the 105 000 g supernatant fractions and of such microsomal parameters as cytochrome b5 and cytochrome P-450 content, and NADPH-cytochrome c reductase activity, but was related to changes in NADPH-cytochrome P-450 reductase levels. 5. Treatment of 7 day-old chicks with exogenous inducers, 3-methylcholanthrene or phenobarbital sodium (100 mg/kg, intraperitoneally) brought about maximal stimulation of microsomal activity as 18-24 h. The time-course of this induction was reflected by changes in microsomal cytochrome P-450 content and NADPH-cytochrome P=450 reductase activities. 6. Some induction of liver microsomal drug metabolism in 7 day-old chicks could also be brought about by injecting certain lipid-soluble egg yolk extracts.     

Studies on the mechanisms of thallium-mediated inhibition of hepatic mixed function oxidase activity. Correlation with inhibition of NADPH-cytochrome c (P-450) reductase

Thallium (TlCl3) administration to rats produced a dose-dependent loss of hepatic NADPH-cytochrome c (P-450) reductase and microsomal mixed function oxidase activities within 2-4 hr following treatment. These changes occurred independently of apparent effects on microsomal heme or cytochrome P-450 content, both of which remained unchanged with respect to control levels despite transient inhibition of delta-aminolevulinic acid (ALA) synthetase and induction of heme oxygenase. These results are consistent with the recognized properties of thallium as both a flavoprotein antagonist and sulfhydryl inhibitor and differ uniquely from the action of other metals which impair mixed function oxidase activity through compromise of heme biosynthesis and heme depletion. The potential utility of thallium compounds in further evaluating the functional characteristics of NADPH-cytochrome c (P-450) reductase and its role in microsomal oxidative processes is suggested from these observations.     

Nitrite binding to cytochrome P-450 from liver microsomes of trout (Salmo gairdneri Rich.) and effects on two microsomal enzymes

Addition of nitrite to dithionite-reduced trout liver microsomes leads to the conversion of cytochrome P-450 into a cytochrome P-420-NO complex, as it does in mammalian microsomes. A loss in cytochrome P-450 and an inhibition of aminopyrine demethylase (AP) activity were observed in vitro at nitrite-concentrations found in the liver of trout exposed in vivo to this toxin. Nitrite had no effect on dimethylaniline monooxygenase (DMA), a cytochrome P-450-independent enzyme.     

Loss of rat liver microsomal cytochrome P-450 during methimazole metabolism. Role of flavin-containing monooxygenase

The role of flavin-containing monooxygenase (FMO) in the decrease in cytochrome P-450 content during the microsomal metabolism of methimazole (N-methyl-2-mercaptoimidazole) was investigated by heat inactivation of FMO. Incubation of liver microsomes from untreated Fischer 344 rats with NADPH and methimazole resulted in a 25% loss of cytochrome P-450 detectable as its ferrous-carbon monoxide complex. The same extent of cytochrome P-450 loss was observed with 1 and 20 mM methimazole, suggesting saturation of the process. There was no significant loss of cytochrome P-450 when microsomal FMO was heat-inactivated prior to incubation with NADPH and methimazole. Heat pretreatment of the microsomes did not affect cytochrome P-450 concentrations and cytochrome P-420 was not observed. These results indicate that FMO-catalyzed metabolism of methimazole is necessary for the loss of cytochrome P-450 in microsomes from untreated rats. Sulfite and N-methylimidazole, the ultimate products of methimazole metabolism, did not cause a significant loss of cytochrome P-450. There was no loss of cytochrome P-450 when glutathione was included in the incubation with methimazole, suggesting that cytochrome P-450 loss was due to an interaction with oxygenated metabolites of methimazole formed by FMO. Losses of cytochrome P-450 were also observed after incubation of microsomes from phenobarbital- (31%) of beta-naphthoflavone-pretreated rats (44%) with NADPH and methimazole. In contrast to microsomes from untreated rats, heat inactivation of FMO did not prevent the loss of cytochrome P-450 in microsomes from the pretreated rats. These results indicate that both phenobarbital and beta-naphthoflavone induce isozymes of cytochrome P-450 capable of directly activating methimazole.     

Sites of action of cadmium in vitro on hepatic,adrenal, and pulmonary microsomal monooxygenases in guinea pigs

Preincubation of hepatic, adrenal, or pulmonary microsomal preparations with cadmium produced time-dependent decreases in monooxygenase (benzphetamine demethylase, benzo(a)pyrene hydroxylase) activities. Addition of cadmium after the preincubation period had little or no effect on microsomal metabolism. As a result of preincubation with cadmium, hepatic cytochrome P-450 levels declined and the magnitude of the benzphetamine-induced type I spectral change in hepatic microsomes decreased. Cadmium also decreased hepatic NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities but had no effect on NADH-cytochrome c reductase activity. Cadmium similarly decreased cytochrome P-450 concentrations and NADPH-cytochrome c reductase activity in lung microsomes without affecting NADH-cytochrome c reductase activity. Preincubation of adrenal microsomes with cadmium had no effects on cytochrome P-450 levels, on the benzphetamine-induced type I spectrum, or on NADH-cytochrome c reductase activity. However, decreases in adrenal NADPH-cytochrome c and NADPH-cytochrome P-450 reductase activities resulted which closely paralleled the decline in adrenal monooxygenase activities. EDTA extraction of hepatic, adrenal, or pulmonary microsomes after the preincubation exposure removed about 95% of the cadmium but did not diminish the effects of the metal on microsomal monooxygenases. The results indicate that cadmium has somewhat varying sites of action on hepatic, adrenal, and pulmonary monooxygenases, but in all three tissues electron transfer to cytochrome P-450 is compromised. In addition, the effects of cadmium on microsomal metabolism persist fully even after removal of approximately 95% of the metal.     

One-electron reduction of mitomycin c by rat liver: Role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H₂O₂ from redox cycling of the reduced mitomycin c in the presence of O₂ and the alkylation of ρ-nitrobenzylpyridine (NBP) in the absence of O₂ were taken as parameters.

2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems.

3. Under aerobic conditions the H₂O₂ production in the microsomal systems was dependent on NADPH, O₂ and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A.

4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O₂, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient.

5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450.

6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase.

7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O₂ tension is more likely the haem site of cytochrome P-450.     

Differences in the mechanism of functional interaction between NADPH-cytochrome P-450 reductase and its redox partners

Selective methylamidation of NADPH-cytochrome P-450 reductase (EC 1.6.2.4) carboxyl groups was used to assess the relative importance of these groups in the enzyme-catalyzed reduction of cytochromes c, b5, and P-450. Methylamidation of as few as 7 mol of carboxyl groups per mol of reductase caused 80% inhibition of cytochrome c reduction, 50% inhibition of rat liver microsomal RLM3 reduction, and up to 90% inhibition in the capacity of the reductase to support reconstituted monooxygenase activities of RLM3, RLM5, and LM2. In marked contrast, cytochrome b5 reduction measured under comparable conditions was stimulated by 50%. The impaired interactions between the reductase and cytochromes P-450 LM2 and RLM5 were shown not to arise from an impaired capacity for the proteins to bind each other but more likely to be due to an inhibition of a step(s) subsequent to complex formation between the oxidized proteins. These results show that the reductase interacts functionally with cytochrome c and cytochromes P-450 on the one hand and cytochrome b5 on the other through different mechanisms.     

EFFECTS OF MOTORCYCLE EXHAUST ON CYTOCHROME P-450-DEPENDENT MONOOXYGENASES AND GLUTATHIONE S-TRANSFERASE IN RAT TISSUES

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450) -dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/ kg markedly elevated benzo[a]pyrene hydroxylation and 7- ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/ kg/d for 4d resulted in significant increases of P-450 and cytochrome b contents and NADPH-cytochrome c reductase 5 activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S -transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.