Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.5.2 (NQO1)
6,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ellagic acid (EA), a naturally occurring plant polyphenol possesses broad chemoprotective properties. Dietary EA has been shown to reduce the incidence of N-2-fluorenylacetamide-induced hepatocarcinogenesis in rats and N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumors. In this study changes in the expression and activities of specific rat hepatic and esophageal mucosal cytochromes P450 (P450) and phase II enzymes following dietary EA treatment were investigated. Liver and esophageal mucosal microsomes and cytosol were prepared from three groups of Fisher 344 rats which were fed an AIN-76 diet containing no EA or 0.4 or 4.0 g/kg EA for 23 days. In the liver total P450 content decreased by up to 25% and P450 2E1-catalyzed p-nitrophenol hydroxylation decreased by 15%. No changes were observed in P450 1A1, 2B1 or 3A1/2 expression or activities or cytochrome b5 activity. P450 reductase activity decreased by up to 28%. Microsomal epoxide hydrolase (mEH) expression decreased by up to 85% after EA treatment, but mEH activities did not change. The hepatic phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone reductase [NAD-(P)H:QR] and UDP glucuronosyltransferase (UDPGT) activities increased by up to 26, 17 and 75% respectively. Assays for specific forms of GST indicated marked increases in the activities of isozymes 2-2 (190%), 4-4 (150%) and 5-5 (82%). In the rat esophageal mucosa only P450 1A1 could be detected by Western blot analysis and androstendione was the only P450 metabolite of testosterone detectable. However, there were no differences in the expression of P450 1A1, the formation of androstendione or NAD(P)H:QR activities between control and EA-fed rats in the esophagus. Although there was no significant decrease in overall GST activity, as measured with 1-chloro-2,4-dinitrobenzene (CDNB), there was a significant decrease in the activity of the 2-2 isozyme (66% of control). In vitro incubations showed that EA at a concentration of 100 microM inhibited P450 2E1, 1A1 and 2B1 activities by 87, 55 and 18% respectively, but did not affect 3A1/2 activity. Using standard steady-state kinetic analyses, EA was shown to be a potent non-competitive inhibitor of both liver microsomal ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activities, with apparent Ki values of approximately 55 and 14 microM respectively. In conclusion, these results demonstrate that EA causes a decrease in total hepatic P450 with a significant effect on hepatic P450 2E1, increases some hepatic phase II enzyme activities [GST, NAD-(P)H:QR and UDPGT] and decreases hepatic mEH expression. It also inhibits the catalytic activity of some P450 isozymes in vitro. Thus the chemoprotective effect of EA against various chemically induced cancers may involve decreases in the rates of metabolism of these carcinogens by phase I enzymes, due to both direct inhibition of catalytic activity and modulation of gene expression, in addition to effects on the expression of phase II enzymes, thereby enhancing the ability of the target tissues to detoxify the reactive intermediates.
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PMID:The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes. 862 97

The pathways participating in the metabolism of the nitrofuran antimicrobial drug N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone (furazolidone) in intact cells were investigated in the human intestinal cell line Caco-2. One-electron reduction of furazolidone led to the formation of a free radical intermediate that could be monitored in dense cell suspensions by noninvasive electron spin resonance spectroscopy. The effects of enzyme inhibitors on the kinetics of radical production and decay were used to estimate the relative contribution of different enzymes to the reductive activation of the drug. Although many enzymes are known to reduce nitrofurans in vitro (e.g., xanthine oxidase, aldehyde oxidase, DT-diaphorase, mitochondrial redox chain components), their contributions were insignificant in living Caco-2 cells. The first reducing equivalent required for the formation of the nitroanion derivative of furazolidone appeared to be provided essentially by the microsomal cytochrome P450 reductase. This was confirmed through studies of the NADPH-dependent radical formation by microsomes. Differentiated Caco-2 cells, an established enterocyte model, showed only modestly increased radical formation and the same enzyme-specificity pattern as undifferentiated cells. Consistently, only a small increase in P450 reductase activity was found in differentiated cells, in contrast to the 10-fold increase seen in typical differentiation marker enzymes. With the electron spin resonance method that we describe, it is possible to distinguish between sites of bioactivation of redox active drugs in intact cells.
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PMID:N-[5-nitro-2-furfurylidene]-3-amino-2-oxazolidinone activation by the human intestinal cell line Caco-2 monitored through noninvasive electron spin resonance spectroscopy. 864 95

We have purified membrane-bound fatty acid (omega-1-omega-3) hydroxylase of the fungus Fusarium oxysporum MT-811 and found that the activity depends on a single polypeptide with an apparent M(r) value of 118,000. The purified hydroxylase exhibited spectral characteristics of cytochrome P450 (P450), and could catalyze the hydroxylation without the aid of any other proteinaceous components, such as NADPH-P450 reductase. These properties of the fungal hydroxylase are the same as those of bacterial P450BM3 of Bacillus megaterium, a catalytically self-sufficient fused protein of P450 and its reductase. Other properties of the two enzymes, such as molecular weight, high catalytic turnover, and the regiospecificity of the hydroxylating position, were also almost identical. Further, the fungal hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase reacted with the antibody to P450BM3. It was thus shown that the fungal fatty acid hydroxylase structurally and functionally bears a close resemblance to P450BM3, although it is membrane-bound, unlike the bacterial counterpart. On the other hand, a unique phenomenon was found with the fungal hydroxylase: its NADPH-cytochrome c- or NADPH-menadione reductase activity was enhanced enormously upon binding of its substrate (fatty acid). This appears to be the first instance in which the reactivity of P450 reductase against an artificial electron acceptor was enhanced by the binding of the substrate (to be hydroxylated) to P450. These results raise interesting questions about the molecular evolution of P450. Here we term the fungal hydroxylase cytochrome P450foxy.
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PMID:Cytochrome P450foxy, a catalytically self-sufficient fatty acid hydroxylase of the fungus Fusarium oxysporum. 883 36

1. The catalytic activities of several phase I and II xenobiotic-metabolizing enzymes and the immunochemical detection of P4501A and 2B have been investigated in liver microsomes and cytosol of male rats fed for 15 days with diets containing canthaxanthin, astaxanthin, lycopene or lutein (as lutein esters) (300 mg/kg diet) and in rats fed increasing levels (10, 30, 100 and 300 ppm) of canthaxanthin or astaxanthin in the diet. 2. Canthaxanthin increased the liver content of P450, the activities of NADH- and NADPH-cytochrome c reductase, and produced a substantial increase of some P450-dependent activities, especially ethoxyresorufin O-deethylase (EROD) (x 139) and methoxyresorufin O-demethylase (MROD) (x 26). Canthaxanthin also increased pentoxy-(PROD) and benzoxyresorufin O-dealkylases (BROD), but did not affect. NADPH-cytochrome c reductase and erythromycin N-demethylase (ERDM) activities and decreased nitrosodimethylamine N-demethylase (NDMAD) activity. Phase II p-nitrophenol UDP-glucuronosyl transferase (4NP-UGT) and quinone reductase (QR) activities were also increased by canthaxanthin treatment. These enhancing effects on EROD, MROD and 4NP-UGT were clearly detectable at a dose as low as 10 ppm of canthaxanthin in the diet; the induction of QR was only observed in rats fed > or = 100 ppm. Astaxanthin induced the same pattern of enzymes activities as canthaxanthin, but to a lesser extent: its effects on phase I enzymes and 4NP-UGT were observed in rats fed > or = 100 ppm, and QR was not increased. Western blots of microsomal proteins clearly showed the induction of P4501A1 and 1A2 by canthaxanthin and astaxanthin. By contrast, lutein had no effect on the phase I and II xenobiotic-metabolizing enzymes activities measured. Lycopene only decreased NDMAD activity. 3. The two 4-oxocarotenoids canthaxanthin and astaxanthin are substantial inducers of liver P4501A1 and 1A2 in the rat, and coinduce 4NP-UGT and QR, just like polycyclic aromatic hydrocarbon, beta-naphtoflavone or dioxin (TCDD). However, these latter classical P4501A inducers also induce aldehyde dehydrogenase class 3 (ALDH3); this enzyme is not increased, or only marginally, by canthaxanthin and astaxanthin. These two oxocarotenoids form a new class of inducers of P4501A, are structurally very different from the classical inducers quoted above, which are ligands of the AH receptor.
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PMID:Effects of canthaxanthin, astaxanthin, lycopene and lutein on liver xenobiotic-metabolizing enzymes in the rat. 885 21

Inactivation of the human DNA repair protein, O6-alkylguanine-DNA-alkyltransferase (AGT), by exposure to O6-benzylguanine leads to a dramatic enhancement in the cytotoxic response of cells to chemotherapeutic alkylnitrosoureas. Benzylated pyrimidines identified as more potent inactivators than O6-benzylguanine in vitro include 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (5-nitroso-BP) and 2,4-diamino-6-benzyloxy-5-nitropyrimidine (5-nitro-BP). In efforts to determine the clinical usefulness of these benzylated pyrimidines, we examined the metabolism and pharmacokinetics of 5-nitroso-BP in Sprague-Dawley rats, together with its potency as an AGT inactivator in mice. The mean plasma half-life, clearance, and volume of distribution of 5-nitroso-BP in rats were, respectively, 3.8 min, 22 liters/hr/kg, and 2.1 liters/kg. Two metabolites were identified in rat plasma (i.e. 5-nitro-BP and 2,4,5-triamino-6-benzyloxypyrimidine) after intravenous administration of 5-nitroso-BP in rat. Reduction of 5-nitroso-BP (100 microM) occurred primarily in cytosol and was inhibited (> 95%) by 1 mM menadione. Dicumarol (100 microM), a DT-diaphorase inhibitor, did not significantly inhibit this reaction. This indicated a possible role of a dicumarol-resistant quinone reductase. At higher substrate and protein concentration, NADPH-dependent oxidation of 5-nitroso-BP to 5-nitro-BP primarily occurred in microsomes and was completely inhibited by 1-aminobenzotriazole (1 mM), a P450 inhibitor. Unfortunately, neither 5-nitroso-BP nor 5-nitro-BP was as effective as O6-benzylguanine at depleting AGT activity in mouse liver or spleen. At 1 hr after injection of 15 mg/kg O6-benzylguanine, 5-nitroso-BP, or 5-nitro-BP, AGT levels in liver fell to 1%, 66%, and 71% basal activity, respectively. Rapid cytosolic reduction of 5-nitroso-BP may explain the lack of potency of the pyrimidines in vivo.
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PMID:Pharmacokinetics and metabolism in rats of 2,4-diamino-6-benzyloxy-5- nitrosopyrimidine, an inactivator of O6-alkylguanine-DNA alkyltransferase. 893 54

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 micrograms) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide.
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PMID:Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage. 898 Oct 40

There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure, as may be facilitated by early menses, late menopause and long-term estrogen replacement therapy. Equilenin (1,3,5(10),6,8-estrapentaen-3-ol-17-one) or its 17-hydroxylated analogs make up 15% of the most widely prescribed estrogen replacement formulation, Premarin, and yet there is very little information on the human metabolism of these estrogens. In this study, we synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin, and examined how aromatization of the B ring affects the formation and reactivity of the o-quinone (3,5-cyclohexadien-1,2-dione). 4-Hydroxyequilenin-o-quinone is much more redox-active and longer-lived than the endogenous catechol estrone-o-quinones, which suggests that the mechanism(s) of toxicity of the former could be quite different. Interestingly, the rate of reduction of the 4-hydroxyequilenin-o-quinone is increased at least 13-fold in the presence of NAD(P)H:quinone oxidoreductase (DT-diaphorase). Once NADH is consumed however, the catechol auto-oxidized rapidly to the o-quinone. NADH consumption was accompanied by dicumarol-sensitive oxygen uptake both with the purified enzyme and with cytosol from human melanoma cells with high levels of DT-diaphorase activity. P450 reductase and rat liver microsomes also catalyzed NADPH consumption and oxygen uptake. 4-Hydroxyestrone-o-quinone was also rapidly reduced by NAD(P)H; however, this o-quinone does not auto-oxidize and once the o-quinone is reduced the reaction terminates. Including oxidative enzymes in the incubation completes the redox couple and 4-hydroxyestrone-o-quinone behaves like 4-hydroxyequilenin-o-quinone. These data suggest that reduction of estrogen-o-quinones may not result in detoxification. Instead this could represent a cytotoxic mechanism involving consumption of reducing equivalents (NADH/NADPH) as well as formation of superoxide and other reactive oxygen species leading to oxidative stress. Finally, we have compared the cytotoxicity of 4-hydroxyequilenin with that of the estrone catechols in human melanoma cells. 4-Hydroxyequilenin is 5-fold more toxic in these cells compared with 4-hydroxyestrone (ED50 = 7.8 versus 38 microM, respectively) suggesting that formation of the longer-lived redox-active 4-hydroxyequilenin-o-quinone was responsible for the cytotoxic differences. These results substantiate the conclusion that the involvement of quinoids in catechol estrogen toxicity depends on a combination of the rate of formation of the o-quinone, the lifetime of the o-quinone, and the electrophilic/redox reactivity of the quinoids.
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PMID:Bioreductive activation of catechol estrogen-ortho-quinones: aromatization of the B ring in 4-hydroxyequilenin markedly alters quinoid formation and reactivity. 916 1

The role of microsomal NADPH:cytochrome P450 reductase (P450 reductase) and cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase) in the mutagenicity of benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was studied using supF tRNA gene as the mutational target. pUB3 carrying the supF tRNA gene upon transformation into the Escherichia coli ES87 cells exhibited a spontaneous mutation frequency of 0.62 x 10(-6). Chemical modification of the pUB3 DNA with BP-3,6-Q caused a fourfold increase in the mutation frequency, compared with the spontaneous mutations. P450 reductase catalysed metabolic activation of BP-3,6-Q into reactive products (semiquinone and reactive oxygen species), which caused a further increase in the mutation frequency to eightfold over spontaneous mutations. Oxygen radical scavengers (SOD and catalase) blocked the P450 reductase-activated BP-3,6-Q-induced stimulation of mutations. This indicates that redox cycling of the semiquinone leading to the generation of reactive oxygen species (ROS) was directly responsible for the increased mutation frequency of P450 reductase-activated BP-3,6-Q. Analysis of the mutation spectra revealed that P450 reductase-activated BP-3,6-Q showed a significantly higher preference for frameshift mutations, particularly deletions, compared with the spontaneous mutations and the mutations generated by benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE). The single most frequently observed mutation by P450 reductase-activated quinone (semiquinone + ROS) was deletion of a single guanosine. Among the base substitutions, G:C --> T:A, G:C --> A:T and G:C --> C:G were also noticed. Interestingly, NQO1 competed with P450 reductase and specifically prevented the P450 reductase-activated BP-3,6-Q-induced mutations. However, BP-hydroquinone (BP-3,6-HQ) generated during the metabolic reduction of BP-3,6-Q catalysed by NQO1 caused specific mutations involving the deletion of a single cytosine from the DNA sequence 5'-CCCCC-3' in supF tRNA gene at a significantly high frequency. A similar cytosine deletion was also observed with benzoquinone hydroquinone (HQ), indicating that the deletion of cytosine is associated with a hydroquinone class of compounds. These results suggest that: (1) quinones and P450 reductase-activated products of quinones (semiquinones and ROS) are mutagenic compounds; (2) the mutational spectra of quinones, semiquinones and hydroquinones differ from each other with respect to their mutational frequency and specificity; (3) NQO1 competes with P450 reductase and protects the cells from quinone mutagenicity; and (4) the NQO1 -metabolized quinones (hydroquinones), if not eliminated, cause specific mutations that are not observed with quinones and P450 reductase-activated quinones (semiquinones and ROS).
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PMID:NAD(P)H:quinone oxidoreductase 1 reduces the mutagenicity of DNA caused by NADPH:P450 reductase-activated metabolites of benzo(a)pyrene quinones. 951 48

Benzo(a)pyrene and benzene are human carcinogens. The metabolic activation of these compounds into ultimate mutagenic and carcinogenic metabolites is prerequisite for their carcinogenic effects. In this report, the mutagenicity and carcinogenicity of hydroquinones of benzo(a)pyrene and benzene was investigated to address two important questions: (1) do hydroquinones contribute to benzo(a)pyrene and benzene carcinogenicity; and (2) how safe is it to increase the levels of NAD(P)H:quinone oxidoreductase 1 (NQO1), a key enzyme in the generation of hydroquinone. The supF tRNA of the plasmid pSP189 was used as the mutational target in a cell-free and Chinese hamster ovary (CHO) cell system to study hydroquinone mutagenicity. RNA and protein-free pSP189 DNA was incubated in a cell-free system with benzo(a)pyrene-3,6-quinone and purified NQO1 or with benzoquinone hydroquinone to generate adducted pSP189 DNA. The adducted pSP189 DNA was transfected in human embryonic kidney cells Ad293. In the CHO cell system, monolayer cultures of CHO cells and CHO cells overexpressing NQO1 or P450 reductase were transfected with pSP189 vector DNA, treated with benzo(a)pyrene-3,6-quinone. The adducted and replicated pSP189 DNA was rescued from transfected Ad293 (cell-free system) and CHO cells (CHO cell system), digested with the restriction enzyme Dpn1 to remove unreplicated DNA followed by transformation in Escherichia coli MBM7070. The mutant colonies [white/pale blue on 5-bromo-4-chloro-3-indolyl beta-D-galactoside/isopropyl beta-D-thiogalactoside (X-gal/IPTG) plates] were selected, regrown and analysed by DNA sequencing. Mutagenesis experiments demonstrated that hydroquinones cause sequence-specific frameshift mutations involving deletion of a single cytosine from the DNA sequence 5'-172-CCCCC176-3' or a single guanosine from the complementary strand sequence 5'-GGGGG-3' in the supF tRNA gene. This mutation was specific to the hydroquinones, as it was not observed with quinones and other components of the redox cycling (semiquinones and reactive oxygen species). Exposure of BALBc/3T3 cells to hydroquinones resulted in cellular transformation leading to the loss of contact inhibition and regulation of cell growth. The transformation efficiency of BALBc/3T3 cells exposed to hydroquinones was significantly increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), indicating that hydroquinones are excellent initiators that require additional co-carcinogens or promoters to exert an effect. The hydroquinone + TPA as well as hydroquinone-transformed BALBc/3T3 cells, when injected s.c. in severe combined immunodeficient (SCID) mice, produced tumours at 100% frequency. These results establish that hydroquinones lead to mutagenicity and carcinogenicity.
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PMID:Hydroquinones cause specific mutations and lead to cellular transformation and in vivo tumorigenesis. 970 76

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in North America. In addition to endogenous estrogens, Premarin contains unsaturated equine estrogens, including equilenin [1,3,5(10),6,8-estrapentaen-3-ol-17-one]. In previous work, we showed that the equilenin metabolite 4-hydroxyequilenin (4-OHEN) can be autoxidized to 4-OHEN-o-quinone which readily entered into a redox couple with the semiquinone radical catalyzed by NAD(P)H, P450 reductase, or quinone reductase, resulting in generation of reactive oxygen species [Shen, L., Pisha, E., Huang, Z., Pezzuto, J. M., Krol, E., Alam, Z., van Breemen, R. B., and Bolton, J. L. (1997) Carcinogenesis 18, 1093-1101]. As oxidative damage to DNA by reactive oxygen species generated by redox active compounds has been proposed to lead to tumor formation, we investigated whether 4-OHEN could cause DNA damage. We treated lambda phage DNA with 4-OHEN and found that extensive single-strand breaks could be obtained with increasing concentrations of 4-OHEN as well as increasing incubation times. If scavengers of reactive oxygen species are included in the incubations, DNA could be completely protected from 4-OHEN-mediated damage. In contrast, NADH and CuCl2 enhanced the ability of 4-OHEN to cause DNA single-strand breaks presumably due to redox cycling between 4-OHEN and the semiquinone radical generating hydrogen peroxide and ultimately copper peroxide complexes. We also confirmed that 4-OHEN could oxidize DNA bases since hydrolysis of 4-OHEN-treated calf thymus DNA and HPLC separation with electrospray MS detection revealed oxidized deoxynucleosides, including 8-oxodeoxyguanosine and 8-oxodeoxyadenosine. Our data suggest that DNA single-strand breaks and oxidation of DNA bases by 4-OHEN could contribute to the carcinogenic mechanism(s) of equine estrogens.
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PMID:The equine estrogen metabolite 4-hydroxyequilenin causes DNA single-strand breaks and oxidation of DNA bases in vitro. 976 Feb 86


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