Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.6.99.3 (diaphorase)
 5,903 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We investigated the requirement of ascorbic acid for the induction by polychlorinated biphenyls (PCB) of hepatic drug-metabolizing enzymes in ODS-od/od rat (OD rat) which is a rat mutant unable to synthesize ascorbic acid. ODS- +/+ rats (+/+ rat), which can synthesize ascorbic acid, were used as controls. In OD rats, the dietary requirement of ascorbic acid to maintain normal growth and prevent any signs of scurvy is about 300 mg of ascorbic acid per kilogram diet. In this study, dietary levels of ascorbic acid tested were 0, 50, 300, 1000 and 3000 mg ascorbic acid per kilogram diet with or without 200 mg of PCB per kilogram diet. Feeding PCB did not affect growth in rats of either genotype. When statistical analysis was done within groups fed diets without PCB, ascorbic acid deficiency caused significant decreases in body weight gain, hepatic activities of drug-metabolizing enzymes and level of hepatic cytochrome P-450. When OD rats were fed a diet without PCB, the supplementation of about 300 mg ascorbic per kilogram diet was sufficient to maintain normal activities of hepatic aminopyrine N-demethylase, aniline hydroxylase, cytochrome c reductase and reduction of cytochrome P-450 and a normal level of hepatic cytochrome P-450. However, when OD rats were fed a diet supplemented with 200 mg PCB per kilogram of diet, significantly higher activities of hepatic aminopyrine N-demethylase and aniline hydroxylase and significantly higher level of hepatic cytochrome P-450 were observed in OD rats fed a diet supplemented with 1000 mg or 3000 mg ascorbic acid per kilogram of diet than in rats fed a diet supplemented with 300 mg of ascorbic acid. It is concluded that the dietary requirement of ascorbic acid is increased severalfold by the administration of xenobiotics, such as PCB, for the maximum induction of hepatic drug metabolism.
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PMID:Ascorbic acid requirement for the induction of microsomal drug-metabolizing enzymes in a rat mutant unable to synthesize ascorbic acid. 309 36

The ability of hepatic microsomes from senescent rats to metabolize the two potent hepatocarcinogens dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) was investigated. Seven and 24-month-old male Sprague-Dawley rats were used. Liver weights, and microsomal protein per gram tissue weight were higher, whereas cytochrome P-450 and cytochrome b5 were significantly lower in older rats. Glutathione S-transferases and NADPH cytochrome c reductase activities were dramatically reduced in senescent rats. There was no difference in the formation of formaldehyde from DMN in vitro (31 vs. 34 pmol/nmol P-450) between the young and old rats. In contrast, increased microsome mediated binding of AFB1 to DNA was observed in older rats (116 vs. 228 pmol/nmol P-450) suggesting the possibility of either quantitative or qualitative changes in P-450 species. Additionally the cytoplasmic GSH S-transferases from older rats affected lower inhibition of binding of AFB1 to DNA. These results indicated differential abilities in the hepatic microsomal metabolism of these two carcinogens which may cause differential effects of these carcinogens in senescent rats.
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PMID:Differential effects on the metabolism of dimethylnitrosamine and aflatoxin B1 by hepatic microsomes from senescent rats. 310 18

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.
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PMID:Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid. 310 2

The presence of a very active cytochrome P-450-dependent drug-metabolizing system in the olfactory epithelium has been confirmed by using 7-ethoxycoumarin, 7-ethoxyresorufin, hexobarbitone and aniline as substrates, and the reasons for the marked activity of the cytochrome P-450 in this tissue have been investigated. The spectral interaction of hexobarbitone and aniline with hepatic and olfactory microsomes has been examined. By this criterion there was no evidence for marked differences in the spin state of the cytochromes of the two tissues, or for the olfactory epithelium containing a greater amount of cytochrome capable of binding hexobarbitone, a very actively metabolized substrate. Rates of NADPH and NADH: cytochrome c reductase activity were found to be higher in the olfactory epithelium than in the liver, and direct evidence was obtained for a greater amount of the NADPH-dependent flavoprotein in the olfactory microsomes. Investigation of male rats and male and female mice, as well as male hamsters, demonstrated that, in all cases, the cytochrome P-450 levels of the olfactory epithelium were lower than those of the liver, while the 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities were higher. A correlation was found between 7-ethoxycoumarin de-ethylase and NADPH:cytochrome c reductase activities for both tissues in all species examined. The ratio of reductase to cytochrome P-450 was found to be considerably higher in the olfactory epithelium (1:2-1:3) than in the liver (1:11-1:15), regardless of the species examined, suggesting that facilitated electron flow may contribute significantly to the cytochrome P-450 catalytic turnover in the olfactory tissue.
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PMID:NADPH: cytochrome P-450 reductase in olfactory epithelium. Relevance to cytochrome P-450-dependent reactions. 310 74

The mixed-function-oxidase (MFO) activities, ethoxyresorufin and pentoxyphenoxazone O-dealkylase, of cultured Hooded-Lister(HL)-rat hepatocytes declined rapidly during 72 h of culture, whereas in Sprague-Dawley(SD)-rat hepatocytes the MFO activities increased between 24 and 72 h in culture. Cytochrome P-450 content declined at the same rate in both HL- and SD-rat hepatocyte cultures. NADPH:cytochrome c reductase and NADH:cytochrome b5 reductase were more stable in SD- than in HL-rat hepatocyte cultures. 16,16-Dimethylprostaglandins E2 and F2 alpha improved the maintenance of cytochrome P-450 content, MFO activity and NADPH:cytochrome c reductase in the HL-rat hepatocyte cultures. In SD-rat hepatocytes, the prostaglandins had no effect on cytochrome P-450 content or NADPH:cytochrome c reductase activity, whereas they prevented the increase observed in MFO activities between 24 and 72 h after culture.
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PMID:Strain differences in the maintenance of cytochrome P-450 and mixed-function-oxidase activities in cultured rat hepatocytes. Effect of prostaglandins. 310 7

The effect of aluminum injection on the hepatic mixed function oxidase was examined in male Wistar rats. A cannula was surgically implanted in both the control and aluminum treated animals to provide a common port for aluminum injection. In addition, the control animals were pair-fed to the aluminum treated animals. The treated animals accumulated aluminum at about 0.1 mg/gm dry weight of liver/day. At 14 days, the cytochrome P-450 was decreased 20%, but the other components, cytochrome b5 and cytochrome reductases, were unchanged. By day 21 both cytochrome P-450 and cytochrome b5 were reduced 25%. Although NADPH cytochrome c reductase was not affected, the other flavoprotein, NADH cytochrome c reductase, was reduced. Drug metabolism, O-demethylation of p-nitroanisole and p-hydroxylation of aniline, was not affected at 14 days. However, at 21 days O-demethylation was not affected, but aniline hydroxylation was decreased, indicating an affect of aluminum on a specific isoenzyme of cytochrome P-450. Uniquely, the nonactivated glucuronyl transferase activity was fourfold greater in the aluminum treated animals. The increase was greater than cation activation and was similar to the detergent activated activity. Thus, aluminum infusion does produce specific alterations in microsomal function, including drug metabolism and conjugation.
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PMID:Effect of aluminum on the hepatic mixed function oxidase and drug metabolism. 310 94

A cDNA containing the complete coding nucleotide sequence for rat liver NADPH-cytochrome P-450 oxidoreductase was constructed from two overlapping cDNA clones. This full-length cDNA was inserted into the plasmid expression vector pCQV2, transfected into Escherichia coli, and expressed reductase was identified in cell lysates by electrophoresis followed by electrophoretic transfer to nitrocellulose and immunodetection. Various strains were screened for maximal expression and minimal intracellular degradation of the expressed protein, and strain C-1A was selected for preparation of the expressed enzyme. Induced cells from 12-liter cultures were pelleted, lysed in a French press, and the 50,000g supernate was fractionated by DEAE-cellulose and 2'5'-ADP agarose chromatography. Thirty-five grams of packed cells yielded approximately 2 mg of affinity-purified protein that was essentially free of E. coli proteins. The final preparation exhibited considerable proteolytic degradation and only an estimated 5-10% of the immunoreactive protein was undegraded. Four principal forms could be distinguished upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with molecular weights of 65,000, 66,000, 74,000, and 78,000, the latter being equivalent to that of intact reductase. High-performance liquid chromatography with a Spherogel-DEAE column resolved these forms but resulted in the loss of the 78-kDa form; three peaks eluted with molecular weights of 65,000. Several of the HPLC fractions exhibited cytochrome c reductase activity, indicating correct incorporation of both flavin prosthetic groups, with the 66-kDa form showing the highest specific activity (44 mumol of cytochrome c reduced/mg reductase/min at 22 degrees C). HPLC assay of flavin content demonstrated equimolar FMN and FAD concentrations, and spectrophotometric analysis of the 66-kDa form revealed a spectrum essentially identical to that of reductase purified from rat liver. When the affinity-purified preparation was reconstituted with cytochrome P-450c, rates of benzo[a]pyrene metabolism approaching rates observed with liver reductase were obtained, indicating that the undegraded component in the affinity-purified preparation was able to interact with cytochrome P-450 and catalyze electron transfer from NADPH.
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PMID:Expression of a functional 78,000 dalton mammalian flavoprotein, NADPH-cytochrome P-450 oxidoreductase, in Escherichia coli. 310 72

Gelatin-sorbitol microcapsules containing 44.1% trichloroethylene (TCE) were prepared and mixed in NIH-07 rodent meal diet and provided at microcapsule concentrations of 0 (untreated control group), 1.25, 2.5, 5.0, or 10% (equivalent to 0, 0.55, 1.10, 2.21, or 4.41% TCE, respectively) to groups of 10 male F344 rats for 14 days. An additional control group received diets containing 5% empty capsules. For comparisons, TCE dissolved in corn oil was administered by gavage to different groups of 10 male rats for 14 consecutive days at dose levels adjusted to correspond to those in the feed study. Treatment-related deaths occurred only in the highest dose group of the gavage study. Body weight gain and feed consumption were reduced in high-dose groups of both the feed and gavage studies. There was no measurable loss of TCE in feed sampled from the cages during the study. Dose-related increases in organ (liver and kidney) weight/body weight ratios, individual cell necrosis in the liver, and hepatic microsomal NADPH cytochrome c reductase and peroxisomal palmitoyl-CoA oxidase and catalase activities were found in both the dosed-fed and gavage groups. Induction of cytochrome P-450 occurred only in the dosed-feed study. There were no significant compound-related pathologic lesions observed in the kidneys, the only other organ examined microscopically. Differences in lethality, cytochrome P-450 levels, and induction of microsomal or peroxisomal enzyme activities were attributed to differences in the method of dosing (gavage versus dosed-feed). The demonstration of no significant loss of TCE from the feed and of similar toxic effects produced by microencapsulated TCE via feed and TCE in corn oil via gavage indicate that microencapsulation can provide an excellent alternative exposure route for studying the oral toxicological properties of volatile chemicals, such as TCE, in rats.
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PMID:Application of microencapsulation for toxicology studies. II. Toxicity of microencapsulated trichloroethylene in Fischer 344 rats. 311 23

NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2',5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80,000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demethylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymorphonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.
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PMID:Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes. 312 59

The hydroxylation of 6-deoxyerythronolide B (6D) to erythronolide B, a step in the biosynthesis of the 14-membered macrolide antibiotic erythromycin A by Saccharopolyspora erythraea, is catalyzed by a cytochrome P-450 monooxygenase that requires two electron transport proteins for the function of this terminal hydroxylase (A. Shafiee and C. R. Hutchinson, Biochemistry 26:6204-6210, 1987). Two flavoproteins and an iron-sulfur protein (erythrodoxin) were purified from S. erythraea CA340 and shown to act with 6D hydroxylase to catalyze the hydroxylation of (9R)-[9-3H]9-deoxo-9-hydroxy-6D in vitro in a suitably reconstituted system. The flavoproteins contained flavin adenine dinucleotide and exhibited characteristic absorption maxima at 356 and 456 nm. The one with an Mr of 47,000 showed NADPH-dependent diaphorase and cytochrome c reductase activity, and the other, with an Mr of 53,000 showed NADH-dependent activities of the same two types. Erythrodoxin contained acid-labile sulfur and iron, had an Mr of 27,500, and showed a broad absorption maximum between 394 and 404 nm. The sequence of its first 15 amino acids, except for position 12, was the same as that of the ferredoxin from Mycobacterium smegmatis.
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PMID:Purification and reconstitution of the electron transport components for 6-deoxyerythronolide B hydroxylase, a cytochrome P-450 enzyme of macrolide antibiotic (erythromycin) biosynthesis. 312 76


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