UNIPROT:Q8NEX9 - FACTA Search

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of highly purified rat liver cytosolic NAD(P)H-quinone oxidoreductase [EC 1.6.99.2] on the mutagenicity of 1,3- 1,6- and 1,8-dinitropyrene (DNP) was studied in the Ames Salmonella typhimurium mutagenicity assay. NAD(P)H-quinone oxidoreductase over the range of 0.02-0.8 micrograms/plate (38-1500) units increased up to threefold the mutagenicity of all three DNPs in S. typhimurium TA 98. In TA98NR, a strain deficient in "classical" nitro-reductase, the mutagenicity of 1,6- and 1,8-DNP was essentially unchanged, whereas that of 1,3-DNP was markedly reduced. NAD(P)H-quinone oxidoreductase enhanced the mutagenicity of 1,6- and 1,8-DNP to approximately equivalent extents in TA98NR and TA98. The mutagenicity of 1,3-DNP in TA98NR was potently enhanced by the addition of NAD(P)H-quinone oxidoreductase in a dose-responsive manner. In the presence of 0.8 micrograms NAD(P)H-quinone oxidoreductase, 1,3-DNP displayed a mutagenic response in TA98NR that was comparable to that obtained in TA98. NAD(P)H-quinone oxidoreductase was found to increase the mutagenicity of 1,6- but not 1,3- or 1,8-DNP to mutagenic intermediates in TA98/1,8-DNP6, a strain deficient in O-acetyltransferase activity. The results suggest that NAD(P)H-quinone oxidoreductase not only catalyzes reduction of the parent DNP but also that of partially reduced metabolites generated from that DNP. Such reductive metabolism may lead to increased formation of the penultimate mutagenic species.
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PMID:Purified NAD(P)H-quinone oxidoreductase enhances the mutagenicity of dinitropyrenes in vitro. 177 70

The metabolism of low-density lipoproteins (LDL) in vitro in the presence of insulin was studied in freshly isolated human peripheral-blood lymphocytes. Insulin appeared to decrease the binding affinity of 125I-LDL to its cell-surface receptor, without any change in apparent Vmax or in the number of LDL receptors. As a consequence, the absolute amounts of 125I-LDL internalized and degraded were lower in the presence of insulin than in its abscence, although the fraction of internalized 125I-LDL degraded in either instance was quite similar. 3-Hydroxy-3-methylglutaryl-CoA reductase activity, and hence cholesterol synthesis, were stimulated by insulin. This effect of insulin was independent of the inhibitory effect of LDL on cholesterol synthesis. At the same time, acid cholesterol esterase and acyl-CoA: cholesterol O-acetyltransferase activities were lower in cells incubated with insulin than in controls. The net effect of these metabolic alterations seems to be that cells accumulate greater quantities of free and esterified cholesterol when treated with insulin.
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PMID:Effect of insulin on low-density-lipoprotein metabolism in human lymphocytes in vitro. 351 64

Most promutagens and procarcinogens exert their genotoxicity after undergoing metabolic activation. Metabolism of chemicals is an important factor in limiting the extent of the action of a chemical. In this study, we established cell lines which carried cDNAs coding for human CYP1A2 and N-acetyltransferase (NAT); the latter functions as O-acetyltransferase for N-hydroxyarylamines formed by CYP1A2. A cell line which expresses CYP1A2 together with P450 reductase activated aflatoxin B1, but not heterocyclic amines. A cell line which carries CYP1A2 and polymorphic NAT (NAT2) in addition to P450 reductase efficiently activated IQ and some other heterocyclic amines. However, a cell line which carries CYP1A2 and monomorphic NAT (NAT1) had only low activity toward the same heterocyclic amines. In order to determine the presence of and frequency of genetic polymorphisms of CYP1A2 and NAT2 in humans, we performed in caffeine phenotyping test on 205 Japanese volunteers. Analyses of metabolic ratios of urinary metabolites showed a bimodal distribution, indicating that about 86% and 91% of Japanese were extensive metabolizers (EM) of CYP1A2 and NAT2, respectively. The genotype NAT2 determined by the PCR-RFLP method agreed completely with the phenotype. To determine the mechanism of the differences in CYP1A2 activity, genomic DNA from peripheral lymphocytes of poor metabolizers (PM) and EM was subjected to DNA sequencing. No differences in nucleotide sequence were observed between PMs and EMs in the exons, exon-intron junctions and 5'-flanking region of the CYP1A2 gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Polymorphic drug metabolism: studies with recombinant Chinese hamster cells and analyses in human populations. 758 92

5-nitro-3-thiophenecarboxanilide (NTCA3) was clearly mutagenic in Salmonella typhimurium strains TA98, YG1021 (the strain with elevated nitroreductase) and YG1024 (the strain with elevated O-acetyltransferase) and only slightly mutagenic at the gpt locus in AS52 cells. Clastogenic activity in human lymphocytes was dependent on the length of exposure: detectable chromosome aberrations were observed following a 24 h treatment period, but not after 3 h exposure. S9 increased genotoxicity in both mammalian cells and human lymphocytes. Metabolites formed by incubation of NTCA3 with the different cell systems were examined. A time-course study in cell whole extracts showed that bacterial and mammalian cells can acetylate NTCA3 forming 5-acetylamino-3-thiophene-carboxanilide. The formation of this metabolite in human lymphocyte extracts was not confirmed. These data support the conclusions that: (i) both bacterial and mammalian activation pathways play a role in mutations by NTCA3; (ii) the N-acetylated derivative is generated by acyl-transferase after reduction and is the end product of the metabolism in both bacterial and mammalian cells; and (iii) different levels of reductase and acetyltransferase activity may contribute to the differential sensitivity of the different cellular species to the genotoxicity of NTCA3. The fact that NTCA3 serves as substrate for enzymatic activities of importance also in human metabolism needs consideration in assessing the potential risk posed by NTCAs.
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PMID:Analysis of metabolism and genotoxicity of 5-nitro-3-thiophenecarboxanilides in bacterial, mammalian and human cells. 766 67

Epidemiologic studies have suggested that aromatic amines (and nitroaromatic hydrocarbons) may be carcinogenic for human pancreas. Pancreatic tissues from 29 organ donors (13 smokers, 16 non-smokers) were examined for their ability to metabolize aromatic amines and other carcinogens. Microsomes showed no activity for cytochrome P450 (P450) 1A2-dependent N-oxidation of 4-aminobiphenyl (ABP) or for the following activities (and associated P450s): aminopyrine N-demethylation and ethylmorphine N-demethylation (P450 3A4); ethoxyresorufin O-deethylation (P450 1A1) and pentoxyresorufin O-dealkylation (P450 2B6); p-nitrophenol hydroxylation and N-nitrosodimethyl-amine N-demethylation (P450 2E1); lauric acid omega-hydroxylation (P450 4A1); and 4-(methylnitrosamino)-1-(3-pyridyl-1-butanol) (NNAL) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-oxidation (P450 1A2, 2A6, 2D6). Antibodies were used to examine microsomal levels of P450 1A2, 2A6, 2C8/9/18/19, 2E1, 2D6, and 3A3/4/5/7 and epoxide hydrolase. Immunoblots detected only epoxide hydrolase at low levels; P450 levels were <1% of liver. Microsomal benzidine/prostaglandin hydroperoxidation activity was low. In pancreatic cytosols and microsomes, 4-nitrobiphenyl reductase activities were present at levels comparable to human liver. The O-acetyltransferase activity (AcCoA-dependent DNA-binding of [3H]N-hydroxy-ABP) of pancreatic cytosols was high, about twothirds the levels measured in human colon. Cytosols showed high activity for N-acetylation of p-aminobenzoic acid, but not of sulfamethazine, indicating that acetyltransferase-1 (NAT1) is predominantly expressed in this tissue. Cytosolic sulfotransferase was detected at low levels. Using 32P-post-labeling enhanced by butanol extraction, putative arylamine-DNA adducts were detected in most samples. Moreover, in eight of 29 DNA samples, a major adduct was observed that was chromatographically identical to the predominant ABP-DNA adduct, N-(deoxyguanosin-8-yl)-ABP. These results are consistent with a hypothesis that aromatic amines and nitroaromatic hydrocarbons may be involved in the etiology of human pancreatic cancer.
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PMID:Metabolic activation of aromatic amines by human pancreas. 916

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the human urothelium. Since preliminary studies indicated that even higher levels of ABP-DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by 32P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N-(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP-DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N-oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the primary involvement of myeloperoxidase rather than prostaglandin H synthase or lipoxygenase. This was confirmed by immunoinhibition and immunoprecipitation studies using solubilized human lung microsomes and antisera specific for myeloperoxidase. These data suggest that ABP-DNA adducts in human lung result from some environmental exposure to 4-nitrobiphenyl. The bioactivation pathways appear to involve: (1) metabolic reduction to N-hydroxy-ABP and subsequent O-acetylation by NAT1 and/or NAT2; and (2) metabolic reduction to ABP and subsequent peroxidation by myeloperoxidase. The myeloperoxidase activity appears to be the highest peroxidase activity measured in mammalian tissue and is consistent with the presence of neutrophils and polymorphonuclear leukocytes surrounding particulate matter derived from cigarette smoking.
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PMID:Immunochemical, 32P-postlabeling, and GC/MS detection of 4-aminobiphenyl-DNA adducts in human peripheral lung in relation to metabolic activation pathways involving pulmonary N-oxidation, conjugation, and peroxidation. 928 89

Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity. They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT). To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain. The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase. The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine compared with the parent Ames tester strain TA1538. The minimum concentrations of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline, IQ, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively. When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO. These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.
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PMID:Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines. 958 21

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.
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PMID:Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay. 1101 10

We investigated roles of different forms of cytochrome P450 (P450 or CYP) in the metabolic activation of heterocyclic amines (HCAs) and other procarcinogens to genotoxic metabolite(s) in the newly developed umu tester strains Salmonella typhimurium (S. typhimurium) OY1002/1A1, OY1002/1A2, OY1002/1B1, OY1002/2C9, OY1002/2D6, OY1002/2E1 and OY1002/3A4, which express respective human P450 enzymes and NADPH-cytochrome P450 reductase (reductase) and bacterial O-acetyltransferase (O-AT). These strains were established by introducing two plasmids into S. typhimurium TA1535, one carrying both P450 and the reductase cDNA in a bicistronic construct under control of an IPTG-inducible double tac promoter and the other, pOA102, carrying O-AT and umuC"lacZ fusion genes. Expression levels of CYP were found to range between 35 to 550 nmol/l cell culture in the strains tested. O-AT activities in different strains ranged from 52 to 125 nmol isoniazid acetylated/min/mg protein. All HCAs tested, and 2-aminoanthracene and 2-aminofluorene exhibited high genotoxicity in the OY1002/1A2 strain, and genotoxicity of 2-amino-3-methylimidazo [4,5-f]quinoline was detected in both the OY1002/1A1 and OY1002/1A2 strains. 3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]-indole and 3-amino-1-methyl-5H-pyrido[4,3-b]-indole were activated in the OY1002/1A1, OY1002/1B1, OY1002/1A2, and OY1002/3A4 strains. Aflatoxin B(1) exhibited genotoxicity in the OY1002/1A2, OY1002/1A1, and OY1002/3A4 strains. beta-Naphthylamine and benzo[a]pyrene did not exhibit genotoxicity in any of the strains. These results suggest that CYP1A2 is the major cytochrome P450 enzyme involved in bioactivation of HCAs.
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PMID:Metabolic activation of heterocyclic amines and other procarcinogens in Salmonella typhimurium umu tester strains expressing human cytochrome P4501A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4 and human NADPH-P450 reductase and bacterial O-acetyltransferase. 1137 47

Norharman (9H-pyrido[3,4-b]indole) and harman (1-methyl-9H-pyrido[3,4-b]indole) contained in cigarette smoke and cooked foodstuffs, are non-mutagenic to Salmonella strains, but show co-mutagenicity with S9 mixture in the presence of aniline or o-toluidine. The resulting 9-(4'-aminophenyl)-9H-pyrido[3,4-b]indole (aminophenylnorharman, APNH), 9-(4'-amino-3'-methylphenyl)-9H-pyrido[3,4-b]indole (aminomethylphenylnorharman, AMPNH) and 9-(4'-aminophenyl)-1-methyl-9H-pyrido[3,4-b]indole (aminophenylharman, APH) are produced by coupling of norharman and aniline, norharman and o-toluidine, and harman and aniline in the presence of S9 mixture, respectively. To clarify the role of human cytochrome P450 (P450) and N-acetyltransferase (NAT) enzymes in the metabolic activation of APNH, AMPNH and APH, we determined the genotoxicity of these coupling chemicals using a variety of umu tester strains established in our laboratories. APNH, AMPNH and APH induced umuC gene expression more strongly in a bacterial O-acetyltransferase-overproducing strain than the parent strain. These chemicals were also found to induce umuC gene expression in NAT2-overexpressing strain at much higher rate than the NAT1-overexpressing strain. Among seven OY strains expressing human P450s and NADPH-P450 reductase used, the genotoxicity of APNH, AMPNH and APH was detected in OY1002/1A2 strain, OY1002/1A1 and OY1002/1A2 strains, and in OY1002/1A2 strain, respectively. From these results, it is concluded that APNH, AMPNH and APH are mainly bioactivated by P450 1A2 and NAT2, followed by NAT1 enzymes. P450 1A1 was also found to activate AMPNH at relatively slower rates.
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PMID:Activation of aminophenylnorharman, aminomethylphenylnorharman and aminophenylharman to genotoxic metabolites by human N-acetyltransferases and cytochrome P450 enzymes expressed in Salmonella typhimurium umu tester strains. 1706 97

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