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.     

Mechanism of azoreduction of dimethylaminoazobenzene by rat liver NADPH-cytochrome P-450 reductase and partially purified cytochrome P-450. Oxygen and carbon monoxide sensitivity and stimulation by FAD and FMN

We have reported that the hepatocarcinogen dimethylaminoazobenzene (DAB) is reduced by rat liver microsomes in an oxygen- and carbon monoxide-insensitive manner and that activity is induced by clofibrate but no other recognized inducers of cytochrome P-450 activity. In the present study we have shown that the reaction proceeds in a partially purified reconstituted cytochrome P-450 system as well as with purified NADPH-cytochrome P-450 reductase alone. In the latter system, activity is totally inhibited in air whereas the former system is active in air as well as in a carbon monoxide atmosphere. Although clofibrate induces both DAB azoreductase and laurate hydroxylase activities, the suicide substrate 10-undecynoic acid blocks the latter but not the former, implying catalysis by distinct enzymes. FAD and FMN stimulate DAB azoreduction 40-50-fold by both NADPH-cytochrome P-450 reductase alone and by the reconstituted cytochrome P-450 system. However, it was shown that these flavins facilitate electron flow to DAB only from reductase and not from cytochrome P-450. The fact that the reconstituted system, which contains NADPH-cytochrome P-450 reductase, is oxygen insensitive suggests that there is an obligatory electron flow through cytochrome P-450 to DAB, bypassing the oxygen-sensitive step.     

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.     

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.     

Partial purification of pulmonary cytochrome P-448 from 3-methylcholanthrene-treated rats

Pulmonary cytochrome P-448 from 3-methylcholanthrene-pretreated rats was partially purified approximately 20-fold. The purified preparations containing 1.74 nmol per mg protein were essentially free of NADH-cytochrome b5 reductase and NADPH-cytochrome c reductase, and included a small amount of cytochrome b5 spectrophotometrically. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the partially purified cytochrome P-448 gave one major band and several minor bands when stained with Coomassie blue. The major band on which the presence of peroxidase activity could be determined had the apparent molecular weight of 57,000. In the presence of NADPH-cytochrome c reductase, lipid and NADPH, the pulmonary cytochrome P-448 was active in hydroxylation of benzo-[a]pyrene, but catalyzed N-demethylation of benzphetamine in a slow rate.     

Selective inactivation of rat lung and liver microsomal NADPH-cytochrome c reductase by acrolein

Addition of acrolein to rat lung or liver microsomal suspensions resulted in total inactivation of NADPH-cytochrome c reductase and partial conversion of cytochrome P-450 to P-420 in a concentration- and time-dependent fashion. Acrolein also caused total loss of nonprotein sulfhydryl content in both preparations, whereas protein sulfhydryl content was decreased by 40% and 28% in lung and liver preparations, respectively. Maxima of about 60% of the total lung cytochrome P-450 and 50% of the liver cytochrome P-450 in acrolein-treated microsomes did not support the N-demethylation of benzphetamine or ethylmorphine or hydroxylation of aniline because of the total loss of NADPH-cytochrome c reductase. Addition of purified NADPH-cytochrome c reductase to the acrolein-treated lung or liver microsomal suspension largely restored these monooxygenase activities. Addition of glutathione or dithiothreitol to the lung or liver microsomal suspension prior to the addition of acrolein significantly protected cytochrome P-450 from conversion to cytochrome P-420 as well as NADPH-cytochrome c reductase from inactivation. Thus, selective conjugation of acrolein with lung and liver NADPH-cytochrome c reductase but not cytochrome P-450 was responsible for total loss of these lung and liver monooxygenase activities.     

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.     

Purification and properties of cytochrome P-450 and NADPH-cytochrome c (P-450) reductase from human liver microsomes.

Cytochrome P-450 and NADPH-cytochrome c (P-450) reductase were purified to 10.6 nmoles per mg of protein and 19.9 units per mg of protein, respectively, from human liver microsomes. The purified cytochrome was assumed to be in a low spin state as judged by the absolute spectrum. n-Octylamine and aniline produced type II difference spectra and SKF 525-A and benzphetamine type I spectra when bound to the purified cytochrome P-450. The purified human cytochrome P-450 catalyzed laurate oxidation as determined by NADPH oxidation but not aniline hydroxylation, benzphetamine N-demethylation and 7-ethoxycoumarin O-deethylation when reconstituted with the reductases purified from human and rat liver microsomes. The human cytochrome P-450, however, catalyzed drug oxidations when cumene hydroperoxide was used as the oxygen source. The purified human NADPH-cytochrome c (P-450) reductase contained FAD and FMN at a ratio of 1:0.76. The reductase was capable of supporting 7-ethoxycoumarin O-deethylation activity of cytochrome P-448 purified from 3-methylcholanthrene-treated rat liver microsomes.     

Stimulation of microsomal drug oxidation activities by incorporation into microsomes of purified NADPH-cytochrome c (P-450) reductase.

The effects of addition of purified NADPH-cytochrome c (P-450) reductase on microsomal activities of aniline hydroxylation, p-phenetidine O-deethylation and ethylmorphine and aminopyrine N-demethylations were investigated utilizing microsomes from untreated, phenobarbital-treated and 3-methylcholanthrene-treated rats. The purified reductase was incorporated into microsomes. The drug oxidation activities were increased by the fortification of microsomes with the reductase while the extent of increase in the activities varied with the substrate and microsomes employed. The most pronounced enhancement was seen in p-phenetidine O-deethylation, followed by aniline hydroxylation and aminopyrine and ethylmorphine N-demethylations. The enhancement was more remarkable in microsomes from rats treated with 3-methylcholanthrene or phenobarbital. alpha-Naphthoflavone inhibited p-phenetidine O-deethylation activity markedly when the reductase was incorporated into microsomes, indicating that a larger amount of a species of cytochrome P-450 sensitive to the inhibitor was capable of participating in the oxidation of this substrate in the presence of the added reductase. One of the two Km values seen with higher concentrations of aniline or aminopyrine was altered by the fortification of microsomes with the purified NADPH cytochrome c (P-450) reductase. From these results, we propose that NADPH-cytochrome c (P-450) reductase transfers electrons to the selected one or two of multiple species of cytochrome P-450 more preferentially depending upon the substrate and the concentration of the substrate in microsomal membranes.     

Inhibition by SKF 525-A of 7-ethoxycoumarin O-deethylation in microsomal and the reconstituted monooxygenase systems from PCB-treated rat livers

Addition of diethylaminoethyl 2,2-diphenylvalerate-HCl (SKF 525-A) to the incubation mixture containing liver microsomes or purified cytochrome P-450 (PCB P-450) from PCB (KC-500)-treated rats resulted in non-competitive inhibition of 7-ethoxycoumarin O-deethylation activity whereas the addition to the incubation mixture containing purified cytochrome P-448 (PCB P-448) showed a competitive inhibition. Fortification of PCB-induced microsomes with purified NADPH-cytochrome P-450 reductase enhanced the O-deethylation activity. With the reductase-fortified microsomes, SKF 525-A inhibited the O-deethylation in a competitive manner. Based on these results, we confirmed that SKF 525-A inhibits non-competitively and competitively, depending on the species of cytochrome P-450. Our results also support the view that in microsomes from PCB-treated rats, PCB P-450 rather than PCB P-448 is mainly involved in the O-deethylation reaction, presumably due to the presence of a limited amount of NADPH-cytochrome P-450 reductase in microsomes.     

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.     

Drug oxidation activities of horseradish peroxidase, myoglobin and cytochrome P-450cam reconstituted with synthetic hemes

Drug oxidations by horseradish peroxidase (HRP), myoglobin (Mb) and cytochrome P-450cam (P-450cam) reconstituted with synthetic hemes were studied in comparison with a form of cytochrome P-450 purified from liver microsomes of polychlorinated biphenyl (PCB)-treated rats. N,N-Dimethylaniline (DMA) and 7-isopropoxycoumarin were hardly dealkylated by the heme-substituted proteins in the presence of NADPH-cytochrome c (P-450) reductase and NADPH, while substantial activity of this kind was observed in the presence of hydrogen peroxide or cumene hydroperoxide as oxygen donors. Specific activity varied, depending on the substrates, oxygen donors, heme derivatives and apoproteins employed. Very high levels of activity were observed in hydrogen peroxide-dependent DMA N-demethylation with HRP substituted with certain hemes. The highest level of activity was about two hundred times as high as that of rat liver cytochrome P-450. The relationship between such activity and the chemical structure of heme derivatives was discussed.     

The interaction between two forms of cytochrome P-450 during drug oxidation in the reconstituted system containing limited amount of NADPH-cytochrome P-450 reductase

The activities of drug oxidation in a reconstituted system which contains two forms of cytochrome P-450 and a limiting amount of NADPH-cytochrome P-450 reductase were determined. Cytochrome P-450 (termed MC P-4481 and MC P-4482) purified from liver microsomes of 3-methyl-cholanthrene-treated rats was active in both 2- and 4-hydroxylation of biphenyl but cytochrome P-450 (termed PB P-450) purified from liver microsomes of phenobarbital-treated rats was active in 4-hydroxylation of biphenyl only. PB P-450, MC P-4481 and MC P-4482 were most active toward benzphetamine N-demethylation, aniline hydroxylation and 7-ethoxycoumarin O-deethylation, respectively. PB P-450 inhibited the activity of biphenyl 2-hydroxylation supported by MC P-4481 or MC P-4482. On the contrary, no inhibition of PB P-450 supported benzphetamine N-demethylation was observed when MC P-4481 or MC P-4482 was added to the system containing PB P-450 and limited amount of the reductase. The apparent Km of PB P-450 for the reductase obtained from double reciprocal plot of the reductase concentration and the activity of biphenyl hydroxylase or benzphetamine N-demethylation was lower than that of MC P-4481 or MC P-4482. These and other results suggest that there is a certain hierarchy among the cytochrome P-450 species for receiving electrons from reductase.     

Characterization of mixed-function oxidases and purified cytochrome P-450 of livers of house musk shrew, Suncus murinus

Characteristics of mixed-function oxidases in the liver of house musk shrew, Suncus murinus, were studied. The basal level of cytochrome P-450 and the activity of drug-metabolizing enzyme in hepatic microsomes of Suncus murinus is relatively lower than that of rats, while the level of cytochrome b5 and NADPH-cytochrome c reductase is approximately the same as that of rats. The treatment of Suncus murinus with phenobarbital and 3-methylcholanthrene elevated the level of cytochrome P-450 and the activity of drug-metabolizing enzymes, but not the level of cytochrome b5. Two distinct forms of cytochrome P-450 have been purified from hepatic microsomes of 3-methylcholanthrene-treated Suncus murinus. These forms have their absorption maximum at 448.0 nm and 448.5 nm in CO-bound reduced form, and one is in the high-spin state and the other is in the low-spin state. They are different in their molecular weights (53,500 and 55,000) and in their spectral and catalytic properties. Characteristics of these forms were compared with those of the major forms of cytochrome P-450 purified from livers of rats treated with 3-methylcholanthrene.     

Circadian changes of cytochrome P-450-dependent monooxygenase system in the rat liver.

Circadian changes in cytochrome P-450 and cytochrome b5 content and activity of NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases have been studied in rat liver microsomes in season autumn. The obtained results indicate, that cytochrome P-450 in 6-month-old animals shows 12 h rhythm, but in older ones 24 h rhythm. NADPH-cytochrome P-450 reductase activity shows 24 h rhythm in oldest animals only. Cytochrome b5 and its reductase has 24 h rhythm in all examined groups of rats.     

Characteristics of two classes of azo dye reductase activity associated with rat liver microsomal cytochrome P450

Azo dyes are reduced to primary amines by the microsomal enzymes NADPH-cytochrome P450 reductase and cytochrome P450. Amaranth, a highly polar dye, is reduced almost exclusively by rat liver microsomal cytochrome P450 and the reaction is inhibited almost totally by oxygen or CO. Activity is induced by pretreatment with phenobarbital or 3-methylcholanthrene. In contrast, microsomal reduction of the hepatocarcinogen dimethylaminoazobenzene (DAB), a lipid soluble, weakly polar compound, is insensitive to both oxygen and CO. However, reconstitution of activity with purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation indicates that activity is catalyzed almost exclusively by cytochrome P450. Activity is induced by clofibrate but not phenobarbital, beta-naphthoflavone, 3-methylcholanthrene, isosafrol, or pregnenolone-16 alpha-carbonitrile. These observations suggest the existence of at least two classes of azoreductase activity catalyzed by cytochrome P450. To investigate this possibility, the reduction of a number of azo dyes was investigated using microsomal and partially purified systems and the characteristics of the reactions were observed. Microsomal reduction of azo dyes structurally related to DAB required a polar electron-donating substituent on one ring. Activity was insensitive to oxygen and CO if the substrates had no additional substituents on either ring or contained only electron-donating substituents. Introduction of an electron-withdrawing group into the prime ring conferred oxygen and CO sensitivity on the reaction. Substrates in the former group are referred to as insensitive and substrates in the latter group as sensitive. Inhibitors of cytochrome P450 activity depressed reduction of both insensitive and sensitive substrates. In a fully reconstituted system containing lipid, highly purified NADPH-cytochrome P450 reductase and a partially purified cytochrome P450 preparation, rates of reduction of various insensitive substrates varied several-fold, whereas rates of reduction of sensitive substrates varied by three orders of magnitude. Using purified enzymes, each of the insensitive substrates was shown to be reduced by reductase alone, but only at a fraction of the rate seen in the fully reconstituted system, implying that reducing electrons were transferred to the dyes mainly from cytochrome P450. Conversely, there was substantial, in some cases almost exclusive, reduction of sensitive substrates by purified reductase alone and almost no inhibition by CO. Their reduction, however, was inhibited by CO in microsomal systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Suppression of constitutive cytochrome P-450 gene expression in livers of rats undergoing an acute phase response to endotoxin

Administration of purified bacterial lipopolysaccharide (LPS) to male rats suppressed the constitutive hepatic expression of the male-specific cytochrome P-450 [AH, reduced flavoprotein:oxygen oxidoreductase (RH hydroxylating), E.C.1.14.14.1] isozyme P-450h (P450IIC11) to about 35% of control levels within 24 hr. The mRNA for P-450h was more rapidly and more profoundly suppressed than was the protein, indicating (a) that the decrease in the mRNA was responsible for the suppression of the protein and (b) that other mechanisms work to maintain expression of P-450h apoprotein in the face of repression of its mRNA. Suppression of P-450h expression was maximal at an endotoxin dose of 30-100 micrograms/kg, indicating that P-450 suppression is concomitant with the acute-phase response of hepatic secretory proteins. The female-specific cytochrome P-450 isozyme, P-450i (P450IIC12), was suppressed to 17% of control levels by LPS administration in female rats. Suppression of the P-450i apoprotein by LPS, and recovery of its expression, was more rapid than was suppression of P-450h in males. P-450i protein and mRNA levels were concomitantly suppressed by LPS, indicating that although there is a pretranslational component to the suppression, other mechanisms may also contribute. Calculations based on estimations of the microsomal contents of P-450h and P-450i relative to the total cytochrome P-450 in untreated rat livers indicate that suppression of these forms contributes significantly to the decreases in total microsomal P-450 after LPS treatment. In these studies, hepatic microsomal NADPH-cytochrome c reductase (TPNH2-cytochrome c reductase, E.C.1.6.2.4) activities and content of cytochrome b5 were decreased by LPS administration in both male and female rats. Like its effects on cytochrome P-450 expression, endotoxin suppression of NADPH-cytochrome c reductase activities and cytochrome b5 levels was more rapid in female rats than in males. The production of a local inflammatory response in male rats by subcutaneous injection of turpentine caused effects on cytochrome P-450, P-450h expression, and cytochrome b5 that were similar to those of endotoxin but were less rapidly achieved.     

Role of hepatic microsomal and purified cytochrome P-450 in one-electron reduction of two quinone imines and concomitant reduction of molecular oxygen

The possible role of cytochrome P-450 in one-electron reduction of quinoid compounds as well as in the formation of reduced oxygen species was investigated in hepatic microsomal and reconstituted systems of purified cytochrome P-450 and purified NADPH-cytochrome P-450 reductase using electron spin resonance (ESR) methods. Two compounds were selected as model compounds: N-acetyl-parabenzoquinone imine (NAPQI) and 3,5-dimethyl-N-acetyl-para-benzoquinone imine (3,5-dimethyl-NAPQI). Both compounds could be reduced by oxyhaemoglobin, the semiquinones formed were detectable by ESR and did not reduce molecular oxygen. Both NAPQI and 3,5-dimethyl-NAPQI underwent one-electron reduction in microsomal systems and in fully reconstituted systems of cytochrome P-450 and NADPH-cytochrome P-450 reductase under anaerobic and aerobic conditions. In both incubation systems the semiquinone formation was diminished under aerobic circumstances and concomitant reduction of oxygen occurred, leading to the formation of hydrogen peroxide and hydroxyl free radicals. Both the reduction of the quinone imines and the reduction of oxygen were found to be cytochrome P-450 dependent. Both activities of cytochrome P-450 may also be involved in the bioactivation of other compounds with quinoid structural elements, like many chemotherapeutic agents.     

Mechanisms responsible for the thermal sensitivity of adrenal microsomal monooxygenases.

Studies were done to determine the mechanism(s) responsible for the thermal lability of adrenal microsomal monooxygenases. Preincubation of guinea pig adrenal microsomal suspensions at 37 degrees C caused large time-dependent declines in benzo(a)pyrene (BP) hydroxylase and benzphetamine (BZ) demethylase activities. Similar preincubations with hepatic microsomes had little effect on enzyme activities. The decreases in adrenal enzyme activities were completely prevented by co-incubation of microsomes with cytosol, but were not diminished by reduced glutathione, ascorbic acid, or bovine serum albumin. Partial protection was afforded by EDTA, suggesting that lipid peroxidation might be involved, but malonaldehyde production was not demonstrable and MnCl2, a potent inhibitor of lipid peroxidation, did not affect the decline in enzyme activities. The decreases in the rates of BP and BZ metabolism were prevented by including NADPH or NADP+ in the preincubation medium. The preincubation conditions causing losses of adrenal enzyme activities did not affect cytochrome P-450 concentrations or substrate binding to cytochromes P-450, as indicated by type I difference spectra. NADH-cytochrome c reductase activity also was not affected, but there were decreases in NADPH-cytochrome c reductase activity that were proportionately similar to the declines in drug-metabolizing activities. Direct assessment of NADPH-cytochrome P-450 reductase revealed similarly large decreases in enzyme activity resulting from preincubation of adrenal microsomes. The results demonstrate a need for extra caution when doing preincubation experiments with adrenal microsomal preparations, and suggest that the thermal lability of adrenal monooxygenases is attributable to effects at the active site of NADPH-cytochrome P-450 reductase.