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1.
When NADPH-cytochrome P-450 reductase isolated from rat liver microsomes was aerobically incubated with bleomycin, FeCl3, NADPH and DNA parallel NADPH and oxygen were consumed and malondialdehyde was formed. A similar parallelism of NADPH- and oxygen-consumption and malondialdehyde formation was observed when ceil nuclei isolated from rat liver were incubated under the same conditions. The formation of malondialdehyde which was identified by HPLC and which was most likely released from oxidative cleavage of deoxyribose of nuclear DNA required oxygen, bleomycin, FeCl3 and NADPH. This indicates that a nuclear NADPH-enzyme, presumably NADPH-cytochrome P-450 reductase, is able to redox cycle a bleomycin-iron-complex which in the reduced form can activate oxygen to a DNA-damaging reactive species. The data suggest that the activity of this enzyme in the cell nucleus could play an important role in the cytotoxicity of bleomycin in tumor cells.  相似文献   

2.
In the presence of NADPH and O2, NADPH-cytochrome P-450 reductase was found to activate Fe(III)-bleomycin A2 for DNA strand scission. Consistent with observations made previously when cccDNA was incubated in the presence of bleomycin and Fe(II) + O2 or Fe(III) + C6H5IO, degradation of DNA by NADPH-cytochrome P-450 reductase activated Fe(III)-bleomycin A2 produced both single- and double-strand nicks with concomitant formation of malondialdehyde (precursors). Cu(II)-bleomycin A2 also produced nicks in SV40 DNA following activation with NADPH-cytochrome P-450 reductase, but these were not accompanied by the formation of malondialdehyde (precursors). These findings confirm the activity of copper bleomycin in DNA strand scission and indicate that it degrades DNA in a fashion that differs mechanistically from that of iron bleomycin. The present findings also-establish the most facile pathways for enzymatic activation of Fe(III)-bleomycin and Cu(II)-bleomycin, provide data concerning the nature of the activated metallobleomycins, and extend the analogy between the chemistry of cytochrome P-450 and bleomycin.  相似文献   

3.
《Free radical research》2013,47(4-5):261-266
Calf thymus DNA was incubated with bleomycin and FeCl3, in the presence of isolated rat liver microsomal NADH-cytochrome b5 reductase, cytochrome b5 and NADH which catalyze redox cycling of the bleomycin-Fe-complex. Furthermore, isolated rat liver nuclei were incubated with bleomycin, FeCl3 and NADH, a system in which redox cycling of bleomycin-Fe leads to DNA damage. In both systems free bases from DNA were released. Furthermore, 8-hydroxy-guanine was also found in the supernatant. On the other hand, 8-hydroxy-deoxyguanosine was detected in DNA of cell nuclei indicating that hydroxylation of the guanine molecule occurred in intact DNA. The release of bases correlated with the release of malondialydehyde as well as with NADH and oxygen consumption. These results indicate that NADH-cytochrome b5 reductase catalyzes redox cycling of the bleomycin-Fe-complex which results in the formation of reactive oxygen species which oxidize deoxyribose as well as bases of DNA. Both mechanisms may contribute to the cytotoxic and cytostatic effects of bleomycin observed in intact cells.  相似文献   

4.
Highly-purified rat liver microsomal cytochrome P-450 converted cyclohexane to cyclohexanol in the presence of iodosobenzene. Oxygen from 18O-iodosobenzene was not incorporated into cyclohexanol but oxygen from H218O was readily incorporated. Cytochrome P-450 catalyzed the facile exchange of oxygen between iodosobenzene and water but neither cytochrome P-420 nor the apoenzyme did. Under these conditions cytochrome P-450 readily incorporated oxygen from 18O2 into cyclohexanol in the presence of NADPH-cytochrome P-450 reductase and NADPH. The results are interpreted in a mechanism in which cytochrome P-450 forms a common hydroxylating species in the presence of iodosobenzene or O2 plus NADPH.  相似文献   

5.
Preincubation of mouse liver microsomes with NADPH resulted in malondialdehyde formation, destruction of cytochrome P-450, and decreased rates of aniline hydroxylation and N-demethylation of aminopyrine and ethylmorphine. These phenomena were more pronounced in phosphate than in Tris buffer. No reduction in rates of NADPH-linked oxidation of ethanol or in the activities of NADPH oxidase and NADPH-cytochrome c reductase was observed. While addition of EDTA to preincubation mixtures prevented lipid peroxidation, loss of cytochrome P-450, and inactivation of the drug-metabolizing capacity of microsomes, it did not alter ethanol oxidation rates and the activities of NADPH oxidase and NADPH-cytochrome c reductase. These findings argue against the involvement of cytochrome P-450 in the microsomal ethanol-oxidizing system.  相似文献   

6.
Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism.  相似文献   

7.
Rat and rabbit liver microsomes catalyze an NADPH-cytochrome P-450 reductase-dependent peroxidation of endogenous lipid in the presence of the chelate, ADP-Fe3+. Although liver microsomes from both species contain comparable levels of NADPH-cytochrome P-450 reductase and cytochrome P-450, the rate of lipid peroxidation (assayed by malondialdehyde and lipid hydroperoxide formation) catalyzed by rabbit liver microsomes is only about 40% of that catalyzed by rat liver microsomes. Microsomal lipid peroxidation was reconstituted with liposomes made from extracted microsomal lipid and purified protease-solubilized NADPH-cytochrome P-450 reductase from both rat and rabbit liver microsomes. The results demonstrated that the lower rates of lipid peroxidation catalyzed by rabbit liver microsomes could not be attributed to the specific activity of the reductase. Microsomal lipid from rabbit liver was found to be much less susceptible to lipid peroxidation. This was due to the lower polyunsaturated fatty acid content rather than the presence of antioxidants in rabbit liver microsomal lipid. Gas-liquid chromatographic analysis of fatty acids lost during microsomal lipid peroxidation revealed that the degree of fatty acid unsaturation correlated well with rates of lipid peroxidation.  相似文献   

8.
The mechanism by which 2-bromo-4'-nitroacetophenone (BrNAP) inactivates cytochrome P-450c, which involves alkylation primarily at Cys-292, is shown in the present study to involve an uncoupling of NADPH utilization and oxygen consumption from product formation. Alkylation of cytochrome P-450c with BrNAP markedly stimulated (approximately 30-fold) its rate of anaerobic reduction by NADPH-cytochrome P-450 reductase, as determined by stopped flow spectroscopy. This marked stimulation in reduction rate is highly unusual in that Cys-292 is apparently not part of the heme- or substrate-binding site, and its alkylation by BrNAP does not cause a low spin to high spin state transition in cytochrome P-450c. Under aerobic conditions the rapid oxidation of NADPH catalyzed by alkylated cytochrome P-450c was associated with rapid reduction of molecular oxygen to hydrogen peroxide via superoxide anion. The intermediacy of superoxide anion, formed by the one-electron reduction of molecular oxygen, established that alkylation of cytochrome P-450c with BrNAP uncouples the catalytic cycle prior to introduction of the second electron. The generation of superoxide anion by decomposition of the Fe2+ X O2 complex was consistent with the observations that, in contrast to native cytochrome P-450c, alkylated cytochrome P-450c failed to form a 430 nm absorbing chromophore during the metabolism of 7-ethoxycoumarin. Alkylation of cytochrome P-450c with BrNAP did not completely uncouple the catalytic cycle such that 5-20% of the catalytic activity remained for the alkylated cytochrome compared to the native protein depending on the substrate assayed. The uncoupling effect was, however, highly specific for cytochrome P-450c. Alkylation of nine other rat liver microsomal cytochrome P-450 isozymes with BrNAP caused little or no increase in hydrogen peroxide formation in the presence of NADPH-cytochrome P-450 reductase and NADPH.  相似文献   

9.
Liver microsomal steroid 5-alpha-reduction is catalyzed by a NADPH-dependent enzyme system. The requirement of NADPH-cytochrome P-450 reductase to shuttle reduction equivalents from NADPH to steroid 5-alpha-reductase was investigated using an inhibitory antibody against NADPH-cytochrome P-450 reductase. This antibody preparation inhibited cytochrome c reduction in microsomes from female rat liver with an I50 of 0.75 mg antibody/mg of microsomal protein. Benzphetamine N-demethylation and testosterone 6-beta-hydroxylation, two cytochrome P-450-mediated oxidative reactions, were inhibited by the antibody. On the other hand, testosterone 5-alpha-reductase was not affected by the antibody. These results suggest that NADPH-cytochrome P-450 reductase is not an obligatory component of the liver microsomal steroid 5-alpha-reduction.  相似文献   

10.
Indigocarmine, which is widely used as a synthetic colouring agent for foods and cosmetics in many countries, was reduced to its leuco form and decolorized by rat liver microsomes with NADPH under anaerobic conditions. The reductase activity was enhanced in liver microsomes of phenobarbital-treated rats, and inhibited by diphenyliodonium chloride, a NADPH-cytochrome P450 reductase (P450 reductase) inhibitor, but was not inhibited by SKF 525-A or carbon monoxide. Indigocarmine reductase activity was exhibited by purified rat P450 reductase. In contrast, when indigocarmine was incubated with rat liver microsomes and NADPH under aerobic conditions, superoxide radical was produced and its production was inhibited by superoxide dismutase and diphenyliodonium chloride. When indigocarmine was incubated with purified rat P450 reductase in the presence of NADPH, superoxide radical production was enhanced 17.7-fold (similar to the enhancement of indigocarmine-reducing ability) as compared with that of rat liver microsomes. A decrease of one molecule of NADPH was accompanied with formation of about two molecules of superoxide radical. P450 reductase exhibited little reductase activity towards indigo and tetrabromoindigo, which also afforded little superoxide radical under aerobic conditions. These results indicate that indigocarmine is reduced by P450 reductase to its leuco form, and superoxide radical is produced by autoxidation of the leuco form, through a mechanism known as futile redox cycling.  相似文献   

11.
9-Methylfluorene was metabolized by rat liver microsomes to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene. The results were confirmed by using a reconstituted cytochrome P-450 oxygenase system purified from phenobarbital-induced rat liver which established its involvement. SKF-525A strongly inhibited the formation of both oxygenation products. Cytochrome P-450 alone brought about the conversion of the hydroperoxide to its alcohol. NADPH augmented the peroxidative reaction, but the presence of NADPH-cytochrome P-450 reductase was without effect. Certain microsomal preparations and reconstituted enzyme yielded little or no detectable amounts of hydroperoxide. This was due to a too rapid conversion of the hydroperoxide to its alcohol. The addition of metyrapone, a compound that inhibited such conversion, resulted in accumulation of 9-hydroperoxy-9-methylfluorene for positive identification. Incubation of 9-methylfluorene with microsomes and NADPH resulted in covalent binding of its metabolite to microsomal proteins. Incubation of 14C-labeled 9-hydroperoxy-9-methylfluorene caused covalent binding of label to proteins, RNA, and DNA.  相似文献   

12.
The participation of the microsomal electron transport system involving cytochrome P-450 in ω-oxidation of fatty acids by a rat liver preparation was examined since ω-oxidation involves microsomal reactions requiring both NADPH and molecular oxygen.

ω-Oxidation of fatty acids was inhibited by CO and by the antibody against NADPH-cytochrome c reductase. The addition to the reaction mixture of drugs which interact with cytochrome P-450 inhibited ω-oxidation. It is concluded that the microsomal electron transport system involving cytochrome P-450 functions in ω-oxidation of fatty acids.  相似文献   


13.
T D Porter  T W Beck  C B Kasper 《Biochemistry》1990,29(42):9814-9818
cDNA clones to rat liver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 lambda-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.  相似文献   

14.
Using the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) we have demonstrated that hydroxyl radicals are generated indirectly from purified preparations of rat liver microsomal NADPH-cytochrome c (P-450) reductase during NADPH oxidation. Hydroxyl radical formation is completely inhibited by p-chloromercuribenzoate, but not by metyrapone. In addition, hydroxyl radical DMPO adduct formation is blocked by added linolenic acid which, in turn, is peroxidatively degraded into malondialdehyde, suggesting that hydroxyl radicals formed from purified NADPH-cytochrome c (P-450) reductase are capable of initiating lipid peroxidation. A mechanism for the indirect production of hydroxyl radicals from NADPH-cytochrome P-450 reductase is discussed.  相似文献   

15.
A reconstituted lipid peroxidation system consisting of rat liver microsomal NADPH-cytochrome P450 reductase and cytochrome P450 incorporated into phospholipid vesicles was developed and characterized. Peroxidation of the vesicles required NADPH and ADP-Fe3+, just as in the NADPH-dependent peroxidation of microsomes. The peroxidation of the vesicles was inhibited 30-50% by superoxide dismutase, depending upon their cytochrome P450 content: those with higher cytochrome P450 contents exhibited greater rates of malondialdehyde formation which were less sensitive to inhibition by superoxide dismutase. When cytochrome P450 was incorporated into vesicles, EDTA-Fe3+ was not required for lipid peroxidation, distinguishing this system from the one previously described by Pederson and Aust [Biochem. Biophys. Res. Comm. 48, 789; 1972]. Since at least 50% of the malondialdehyde formation in the vesicular system was not inhibited by superoxide dismutase, alternative means of iron reduction (O2-.-independent) were examined. It was found that rat liver microsomes or a reconstituted mixed function oxidase system consisting of NADPH-cytochrome P450 reductase and cytochrome P450 in dilauroylphosphatidylcholine micelles reduced ADP-Fe3+ under anaerobic conditions.  相似文献   

16.
A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4-14C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.  相似文献   

17.
Anthranilamide was slightly hydroxylated by a reconstituted rat liver microsomal monooxygenase system with NADPH, but a large amount of hydrogen peroxide was formed with a consumption of NADPH during the reaction. Superoxide dismutase stimulated the hydroxylation by depressing the hydrogen peroxide formation, in that there was a reverse correlation between the two effects due to the dismutase. In addition, a trace of 3-hydroxyanthranilamide, one of the products, not only stimulated NADPH-dependent hydrogen peroxide formation via NADPH-cytochrome c (P-450) reductase, but also inhibited the reduction of cytochrome P-450 by NADPH in the reconstituted system. These effects of 3-hydroxyanthranilamide were also diminished by superoxide dismutase.  相似文献   

18.
5-(4-Nitrophenyl)penta-2,4-dienal (NPPD) stimulated NADPH-supported oxygen consumption by rat liver microsomes in a concentration-dependent manner. The NPPD stimulation of O2 uptake was not inhibited by metyrapone and was decreased in the presence of NADP+ and p-hydroxymercuribenzoate. These observations suggest that the NPPD initial reduction step is mediated by NADPH-cytochrome P-450 reductase and not by cytochrome P-450. Spin-trapping studies using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) revealed the formation of superoxide anion upon incubation of NPPD, NADPH, DMPO and rat liver microsomes. Hydrogen peroxide generation was also detected in these incubations, thus confirming redox cycling of NPPD under aerobic conditions. NPPD stimulated oxygen consumption, superoxide anion formation and hydrogen peroxide generation by rat kidney, testes and brain microsomes. Other enzymes capable of nitroreduction (NADH dehydrogenase, xanthine oxidase, glutathione reductase, and NADP+ ferredoxin oxidoreductase) were also found to stimulate redox cycling of NPPD. The ability of NPPD to induce superoxide anion and hydrogen peroxide formation might play a role in its reported mutagenicity.  相似文献   

19.
NADPH-dependent oxygen utilization by liver microsomal fractions was stimulated by the addition of increasing concentrations of butylated hydroxyanisole concomitant with the inhibition of benzphetamine N-demethylase activity. The apparent conversion of monooxygenase activity to an oxidase-like activity in the presence of the antioxidant was correlated with the partial recovery of the reducing equivalents from NADPH in the form of increased hydrogen peroxide production. The progress curve of liver microsomal NADPH oxidase activity in the presence of butylated hydroxyanisole displayed a lag phase indicative of the formation of a metabolite capable of uncoupling the monooxygenase activity. Ethyl acetate extracts of microsomal reaction mixtures obtained in the presence of butylated hydroxyanisole, oxygen, and NADPH stimulated the NADPH oxidase activity of either liver microsomes or purified NADPH-cytochrome c (P-450) reductase. Using high performance liquid chromatography, gas chromatography, and mass spectrometry techniques, two metabolites of butylated hydroxyanisole, namely t-butylhydroquinone and t-butylquinone, were identified. The quinone metabolite and/or its 1-electron reduction product interact with the flavoprotein reductase to directly link the enzyme to the reduction of oxygen which results in an inhibition of the catalytic activity of the cytochrome P-450-dependent monooxygenase.  相似文献   

20.
18-Hydroxylation of deoxycorticosterone was studies with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P-450 from rat adrenals and a partially purified NADPH-cytochrome P450 reductase preparation from bovine adrenals. In some experimenta a soluble cytochrome P-450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH-cytochrome P-450 reductase preparation. Optimal assay conditions were determined for 18-hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH-cytochrome P-450 reductase fraction, the rate of 18-hydroxylation was linear with time and with the amount of cytochrome P-450. In incubations with intact rat adrenal mitochondria to which Ca2+ and an excess NADPH had been added, NADPH-cytochrome P-450 reductase increased the rate of 18-hydroxylation about 100%, indicating that NADPH-cytochrome P-45o reductase was to some extent rate-limiting. The rate of 18-hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rat of 18-hydroxylation of corticosterone and progesterone. When the cytochrome P-450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P-450 from bovine adrenals, the rate of 18-hydroxylation decreased considerably. Under all experimental conditions, the 18-hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11beta-hydroxylation. Provided the source of cytochrome P-450 was the same, the ratio between 11beta- and 18hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P-450 are involved in 11beta- and 18-hydroxylation of deoxycorticosterone.  相似文献   

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