Selenium and drug metabolism--II. Independence of glutathione peroxidase and reversibility of hepatic enzyme modulations in deficient mice

Male mice were fed a diet containing less than 0.01 ppm selenium (Se-) for 6 months. A control group received the same diet containing 0.5 ppm selenium (Se+). In the livers of the Se- animals a drastic decrease in glutathione peroxidase (GSH-Px) activity was observed. It reached undetectable levels after 17 days of the Se- diet. At that time, GSH-transferase activity began to increase significantly, followed by changes in many other enzyme activities. After the 60th day, these enzyme modulations had reached a plateau with the following percentage changes compared to controls: GSH-transferases: 320% (1,2-dichloro-4-nitrobenzene), 218% (1-chloro-2,4-dinitrobenzene); glutathione reductase: 160%; ethoxycoumarin deethylase: 330%; cytochrome P-450-hydroperoxidase: 230%; heme oxygenase: 240%; UDP-glucuronyltransferase: 200%; GSH-thioltransferase: 64%; sulphotransferase: 62%; NADPH-cytochrome-P-450-reductase: 65%; flavin-containing mono-oxygenase: 57%. No significant changes were observed for GSH-transferase activity assayed with ethacrynic acid or for microsomal H2O2 formation and aniline hydroxylase activity. In single-pulse repletion experiments by injection of 250 micrograms selenium/kg body wt, different individual time constants for the recovery process of the enzymatic perturbations were observed. The half-times for the recovery ranged from 5.7 hr for the microsomal NADPH-cytochrome-P-450 reductase to over 29 hr for GSH-Px up to 44 hr for part of the GSH-transferase activity. 250 micrograms selenium/kg body wt were needed to restore 50% of GSH-Px activity in the long-term Se- mice compared to Se+ controls. All other enzymatic changes in the Se- mice needed a dose of 7 micrograms selenium/kg body wt for 50% restorage . The results demonstrate that processes other than those related to GSH-Px take place in a later phase of selenium deficiency in mouse liver with a chronologically common beginning. The different repletion and depletion kinetics as well as the different need of these processes for the trace element are discussed with respect to the existence of two separate selenium pools.     

Effect of acute and repeated selenium treatment on hepatic monooxygenase enzyme activity in male rats

The effect of sodium selenite administered acutely or repeatedly on the biochemical components of the hepatic microsomal monooxygenase enzyme system was examined in male rats. 72 h following acute administration of selenium (2.4 mg Se/kg, i.p.), there was a significant decrease in ethylmorphine-N-demethylase activity and cytochrome P-450 levels but no change in aniline hydroxylase or NADPH cytochrome c reductase activity. Following repeated administration of selenite in the drinking water (1, 2, or 4 ppm Se) for 30 days, there was no alteration in any of the parameters measured. Following the in vitro additions of selenite to microsomes obtained from untreated rats, ethylmorphine-N-demethylase and aniline hydroxylase activities were inhibited at selenium concentrations of 10(-4) M or greater, but the inhibition achieved was less than 50%. No alterations in cytochrome P-450 levels were observed. These results indicate that selenium is a rather weak, indirect, and substrate-specific inhibitor of the hepatic monooxygenase enzyme system.     

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.     

Hypophysectomy differentially alters P-450 protein levels and enzyme activities in rat liver: pituitary control of hepatic NADPH cytochrome P-450 reductase

Pituitary-determined hormones regulate the expression of hepatic cytochromes P-450 through processes involving both negative and positive controls. Accordingly, protein levels of several P-450 forms are elevated in rat liver following hypophysectomy [P-450 forms designated 2a (gene IIIA2), RLM2 (gene IIA2), and PB-4 (gene IIB1)], whereas protein levels of others are suppressed [e.g., P-450 2c (gene IIC11)]. In the present study, microsomal steroid hydroxylase activities associated with these same P-450 forms were found to be decreased by hypophysectomy, despite elevations in protein levels for several of them. Studies were, therefore, undertaken to determine the biochemical basis for this decrease in microsomal P-450 enzyme specific activity. In vivo treatment of hypophysectomized rats with gonadotropin, under conditions that restore heme to testis P-450, and heme reconstitution experiments carried out with liver homogenates indicated that a deficiency in P-450-associated heme is unlikely to account for the observed decreases in liver P-450 enzyme specific activity. Analysis of the flavoprotein P-450 reductase, however, revealed that the reductase protein and its associated cytochrome c reductase activity are decreased by 50 to 75% in liver microsomes isolated from hypophysectomized rats. Moreover, supplementation of isolated liver microsomes with exogenous purified P-450 reductase stimulated microsomal steroid hydroxylase activity preferentially in the hypophysectomized rats, to levels consistent with the observed changes in P-450 protein levels. Thus, a deficiency in P-450 reductase, which is a rate-limiting component for many P-450-dependent hydroxylation reactions, appears to be responsible for the decrease in steroid hydroxylase specific activity in the hypophysectomized rats. Although growth hormone, adrenocorticotropic hormone, and chorionic gonadotropin were each ineffective at restoring hepatic P-450 reductase when administered to hypophysectomized rats, substantial restoration of P-450 reductase levels could be achieved by treatment of the hypophysectomized rats with thyroxine. Thyroxine treatment of these rats also elevated the microsomal steroid hydroxylase activities associated with the individual hepatic P-450 forms to levels commensurate with their respective P-450 protein levels. These results establish that hepatic P-450 reductase is subject to hormonal controls that are distinct from those governing cytochrome P-450 expression and further demonstrate the complexity of endocrine control of hepatic steroid hormone metabolism.     

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.     

Cytochrome P-450-dependent nicotine oxidation by liver microsomes of guinea pigs. Immunochemical evidence with antibody against phenobarbital-inducible cytochrome P-450

When guinea pigs were treated with phenobarbital (PB), the specific activity of liver microsomal nicotine oxidase increased by 42%. PB-inducible cytochrome P-450 (PB-P-450) was purified to homogeneity from liver microsomes of PB-treated guinea pigs. Purified PB-P-450 catalyzed nicotine oxidation when reconstituted with NADPH-P-450 reductase and phospholipid system. Antibody prepared against the purified PB-P-450 formed single precipitation lines with both purified PB-P-450 and microsomal components in livers of PB-treated guinea pigs, and both precipitation lines fused. The antibody against PB-P-450 strongly inhibited nicotine oxidation in the reconstituted system. The antibody also inhibited liver microsomal nicotine oxidase activities in PB-treated and untreated guinea pigs by about 30% and less than 5% respectively. About 45% of total P-450 in liver microsomes of PB-treated guinea pigs was precipitated by the antibody. These results show that PB-P-450 participates in liver microsomal nicotine oxidation in PB-treated guinea pigs but not in untreated control animals.     

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.     

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1)

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.     

Effects of chronic ethanol administration in the rat: Relative dependency on dietary lipids—I.: Induction of hepatic drug-metabolizing enzymes in vitro

Female Sprague-Dawley rats were fed nutritionally adequate liquid diets with or without ethanol, at two ethanol concentrations, 5 and 6% (w/v). In other animals, various degrees of caloric deficiency were obtained by replacing ethanol by water in one animal of a pair. Ethanol given as a 5% (w/v) solution with high amounts of dietary fat increased cytochrome P-450, the activities of NADPH-cytochrome P-450 reductase, benzphetamine demethylation, aniline hydroxylation and microsomal ethanol-oxidizing system (MEOS). When ethanol was given with a low fat diet as a 5% (w/v) solution, the increase in cytochrome P-450 and P-450 reductase was much less than with a high fat diet; the other enzyme activities, however, were enhanced to a level comparable to that achieved with the high fat diet. When ethanol was administered as a 6% (w/v) solution in presence of a low fat diet, caloric deficiency was observed and no significant induction of any parameter except aniline hydroxylation could be found. When it was given with a high fat diet, in spite of caloric deficiency and lower ethanol ingestion, cytochrome P-450 and P-450 reductase activities were enhanced while that of MEOS was not. Ingestion of ethanol as a 6% (w/v) solution with a high fat diet resulted in a negligible weight gain. Higher basal levels of cytochrome P-450, P-450 reductase and benzphetamine demethylation activities were found in animals rendered caloric-deficient. Ethanol is associated with a greater induction of drug-metabolizing enzyme activities in the high fat model compared to the low fat model. Induction of drug-metabolizing enzymes by ethanol is partly dependent on dietary lipids as well as on the amounts of ethanol ingested and on the caloric status of the animal.     

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.     

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.     

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.     

Cytochrome P-450 isozymes involved in the oxidative metabolism of delta 9-tetrahydrocannabinol by liver microsomes of adult female rats.

Oxidative metabolism of delta 9-tetrahydrocannabinol (THC) by liver microsomes was studied in female rats. Delta 9-THC was mainly biotransformed to 11-hydroxy-delta 9-THC (11-OH-delta 9-THC) and 9 alpha,10 alpha-epoxy-hexahydrocannabinol (EHHC) by liver microsomal fraction of adult female rat. Two isozymes of cytochrome P-450 (P-450) [F-1 (IIC6) and F-2 (IIC12)] were purified from liver microsomes of female rats and oxidation activities toward delta 9-THC were assessed in the reconstituted system containing NADH-P-450 reductase and cytochrome b5. P-450 F-1 showed considerable activity toward 11-OH-delta 9-THC formation (10.62 nmol/min/nmol of P-450), whereas P-450 F-2 did not show any activity toward delta 9-THC oxidation under the conditions used. Preincubation of microsomes with antiserum against P-450 F-1 obtained from rabbits caused a marked decrease in 11-OH-delta 9-THC formation, whereas antiserum against P-450 F-2 did not exhibit any inhibitory effect on the oxidation of delta 9-THC by liver microsomes of adult female rats. Further, antiserum against P-450 F-1 or F-2 did not affect the microsomal formation of 9 alpha,10 alpha-EHHC from delta 9-THC. These results indicate that P-450 F-1 and its immunochemically related P-450 isozyme(s) play important roles in the formation of an active metabolite, 11-OH-delta 9-THC, from delta 9-THC by liver microsomes of adult female rats.     

Altered regulation of cytochrome P-450 enzymes in choline-deficient cirrhotic male rat liver: impaired regulation and activity of the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase, cytochrome P-450UT-A, in hepatic cirrhosis

Total microsomal cytochrome P-450 levels were decreased, to about 50% of control, in liver of male rats made cirrhotic by the prolonged intake of a choline-deficient diet. We have suggested previously that this decrease in cytochrome P-450 levels is not a generalized one, but is selective for certain forms of the enzyme. In the present study, levels of six cytochrome P-450 forms including the sex-specific cytochrome P-450 forms, P-450UT-A, P-450PCN-E, and P-450UT-l, were quantitated immunochemically in hepatic microsomes prepared from control and cirrhotic male rats and were related to changes in the regioselectivity of cytochrome P-450-mediated androst-4-ene-3,17-dione hydroxylation in these fractions. The principal finding of this study was that the male-specific androst-4-ene-3,17-dione 16 alpha-hydroxylase was decreased in cirrhotic microsomes to about 20% of control. The content of P-450UT-A decreased concurrently from about 0.40 to less than 0.01 nmol/mg of microsomal protein. Other pathways of androst-4-ene-3,17-dione hydroxylation were also affected, but to different extents than the 16 alpha-hydroxylase. 6 beta-Hydroxylation decreased in cirrhotic microsomes to about 45% of control, despite a marked decrease in P-450PCN-E from 0.27 to less than 0.002 nmol/mg of microsomal protein. The rate of androst-4-ene-3,17-dione 7 alpha-hydroxylation underwent a less pronounced reduction in cirrhosis to about two-thirds of control microsomal activity, and levels of the cytochrome P-450 associated with this activity, P-450UT-F, were decreased in proportion with the decrease in total microsomal cytochrome P-450. 16 beta-Hydroxylase activity was unaffected by the cirrhotogenic process. From spectral binding studies it was apparent that androst-4-ene-3,17-dione elicited a high affinity type I interaction in control microsomal fractions (Ks = 4.5 microM), whereas no interaction was apparent in cirrhotic liver microsomes. Levels of three other forms of cytochrome P-450--P-450PB-C (a constitutive form inducible by phenobarbital), P-450ISF-G (a major isosafrole-inducible form), and P-450UT-I (the major female sexually-differentiated isozyme)--were apparently unaltered in cirrhosis. These findings are consistent with the assertion that specific forms of cytochrome P-450 are subject to altered regulation in hepatic cirrhosis.     

Enhancement of Hepatic Drug Biotransformation by a Short-term Intermittent Turpentine Exposure in the Rat

Abstract An inhalation exposure of male rats to 300 p.p.m. of a commercial turpentine 6 hrs daily 5 days a week for 8 weeks enhanced the activities of drug biotransformation enzymes of liver microsomes considerably. The activities of NADPH cytochrome c reductase and 7-ethoxycoumarin deethylase, and microsomal content of cytochrome P-450 were increased 35–60 % during the first weeks of the experiment, but had a tendency to return towards the control values later on. A similar enhancement of activities was also found in liver microsomal epoxide hydratase and UDPglucuronosyltransferase, but these enzyme activities tended to adapt less during the experiment. The turpentine treatment increased the affinity of liver microsomal cytochrome P-450 to α-pinene (the main component of the turpentine). The present data suggests that exposure to turpentine is able to modify considerably the biotransformation of drugs.     

Ring- and N-Hydroxylation of 2-acetylaminofluorene by reconstituted mouse liver microsomal cytochrome P-450 enzyme system

The N- and ring-hydroxylation of 2-acetylaminofluorene (AAF) are examined with a reconstituted cytochrome P-450 enzyme system from liver microsomal fractions from both control and 3-methylcholanthrene (MC)-pretreated mice. Partial purification of cytochrome P-450 fraction is achieved by bacterial protease treatment of microsomes followed by Triton X-100 solubilization and ammonium sulfate precipitation. Both cytochrome P-450 and NADPH-cytochrome c reductase fractions are required for optimum oxidative activity. Hydroxylation activity is determined by the source of cytochrome P-450 fraction; cytochrome P-450 fraction from MC-pretreated mice is several fold more active than that from controls.     

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.     

Effects of motorcycle exhaust inhalation exposure on cytochrome P-450 2B1, antioxidant enzymes, and lipid peroxidation in rat liver and lung

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2 h daily for 4 wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7-ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl(3) -ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.     

Erythrocyte and liver characteristics in low and high glutathione Finn sheep

Selenium-uptake, glutathione peroxidase (GSH-Px), hepatic microsomal cytochrome P-450 and hepatic glutathione contents were studied in Finnsheep. The erythrocytes of low-GSH sheep had higher Selenium uptake and GSH-Px activity than did those in high-GSH animals. The low Se uptake in high-GSH sheep could be due to continuous pumping of Se from the cells into the erythrocytes, which reflects the active role of these cells, compared to those in low-GSH sheep. Significant differences in hepatic microsomal cytochrome P-450 content were found in the two groups of sheep. The greater activity among the low-GSH sheep suggests that they may have more active detoxification mechanisms than the high-GSH animals. The glutathione content in the liver was similar in both high and low-GSH sheep.     

Characterization of the cytochrome P-450 monooxygenase system of hamster liver microsomes. Effects of prior treatment with ethanol and other xenobiotics

The cytochrome P-450 monooxygenase system of hamster liver microsomes and its response to prior treatment with ethanol and other xenobiotics have been examined. Male Syrian golden hamsters were administered ethanol (ETOH), phenobarbital (PB), 5,6-benzoflavone (BF) or isoniazid (INH). Each treatment resulted in a moderate increase (20-60%) in the specific content of liver microsomal cytochrome P-450 along with a unique hemeprotein ferrous carbonyl Soret maximum. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of liver microsomes revealed distinctive changes in protein banding patterns in the cytochrome P-450 (45-60 kDa) region with each treatment. NADPH: cytochrome c reductase activity was increased by both PB and INH, whereas cytochrome b5 content was increased by INH only. Microsomal oxidation of ETOH and aniline p-hydroxylation (expressed per nmol cytochrome P-450) were enhanced dramatically by ETOH and INH, whereas PB and BF had no effect on these enzymatic activities. Both ETOH and INH also increased zoxazolamine 6-hydroxylation but, in contrast to other rodent species, this drug-metabolizing activity was decreased in hamster liver microsomes after treatment with either PB or BF. Microsomal benzphetamine N-demethylation was decreased by ETOH, INH and BF administration and was only modestly enhanced after treatment with PB. ETOH and INH had no effect on the O-deethylation of 7-ethoxycoumarin, and enzymatic activity increased by BF but decreased by PB. These results demonstrate that the cytochrome P-450-dependent monooxygenase system of hamster liver microsomes responds to treatment with ETOH and other xenobiotics in a manner that is quantitatively and, in certain respects, qualitatively different from that reported for the rat, rabbit, and mouse.