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At least 19 recordsLinked to original sources

PubMed1986

Metabolic activation of 1-nitropyrene and 1,6-dinitropyrene by nitroreductases from Bacteroides fragilis and distribution of nitroreductase activity in rats.

Nitrated polycyclic aromatic compounds, 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-diNP), are environmental mutagens and carcinogens. Nitroreductases purified from an anaerobic bacterium, Bacteroides fragilis, catalyzed the metabolic activation of these compounds to produce DNA- and tRNA-bound adducts in vitro. Formation of the adducts was inhibited by p-chloromercuribenzoic acid, which is an inhibitor of nitroreductases from B. fragilis. The enzyme and coenzyme (NADPH) were essential for the adduct formation. These results suggest that nitroreduction is a necessary step in the metabolic activation of nitropyrenes. 1-NP bound specifically to poly(G) and poly(dG), and 1,6-diNP bound to poly(G), poly(dG), and poly(X). The other purine polynucleotides were weak acceptors. However, the reactive products of nitropyrenes formed by nitroreductases could not bind to pyrimidine polynucleotides. Enzymatic hydrolysis of 1-NP-bound DNA and subsequent analysis by high-performance liquid chromatography showed one major and two minor adducts in the hydrolysate. The peak of the major adduct corresponded to that of N-(deoxyguanosin-8-y1)-1-aminopyrene, which is the same as an adduct formed by xanthine oxidase, a mammalian nitroreductase. Nitroreductase activity in the various organs and intestinal contents of Sprague-Dawley rats was assayed in the presence of NADPH or NADH under nitrogen gas. Nitroreductase activity was widely distributed in the organs of the rats; in particular, that of the liver and of the small intestine was relatively high, but that of the respiratory organs such as lung and alveolar macrophages was very low. Intestinal contents had high nitroreductase activity, which was proportional to the number of bacteria, especially anaerobic bacteria, in the intestine. These results suggest that the nitroreductase activity of the normal bacterial flora is very high in rats and that the intestinal bacteria play a major role in the metabolism of nitropyrenes in vivo.

Animals↗

PubMed2001

Retro-nitroreductase, a putative evolutionary precursor to Enterobacter cloacae strain 96-3 nitroreductase.

Enterobacter cloacae strain 96-3 nitroreductase (NR) is a homodimeric flavoenzyme that catalyzes the pyridine nucleotide-dependent four-electron reduction of a variety of nitroaromatic compounds, including the explosives TNT (2,4,6-trinitrotoluene), RDX (1,3,5-trinitro-1,3,5-triazine), tetryl (2,4,6-trinitrophenyl-N-methylnitramine), and pentryl (2,4,6-trinitrophenyl-N-nitroaminoethylnitrate). The enzyme was initially characterized by Bryant et al. from a strain of Enterobacter that had been isolated from a weapons dump in La Jolla, CA. The enzyme displays a catalytic efficiency for nitroreduction at least 10-fold higher than that of several highly homologous bacterial nitroreductases and has long been thought to have evolved to be a more efficient nitroreductase due to the high nitroaromatic compound concentrations in its environment. We report the cloning and biochemical characterization of a nitroreductase gene from a clinical isolate of Enterobacter cloacae, a strain that presumably had not encountered high concentrations of nitroaromatics. The new enzyme, which we term retro-nitroreductase, had an amino acid sequence 96.7% identical to NR, and most differences are relatively conservative. The catalytic efficiency of the new enzyme is twofold less than that of NR for the oxidation of NADH and is not significantly different from the value observed for NR for the reduction of dinitrobenzyl alcohol. We conclude that NR has not significantly evolved to be a more efficient nitroreductase as a result of its environment, and the relatively high catalytic activity of the enzyme is a general property of Enterobacter cloacae nitroreductases.

Amino Acid Sequence↗

PubMed1998

Purification and characterization of wild-type and mutant "classical" nitroreductases of Salmonella typhimurium. L33R mutation greatly diminishes binding of FMN to the nitroreductase of S. typhimurium.

"Classical" nitroreductase of Salmonella typhimurium is a flavoprotein that catalyzes the reduction of nitroaromatics to metabolites that are toxic, mutagenic, or carcinogenic. This enzyme represents a new class of flavin-dependent enzymes, which includes nitroreductases of Enterobacter cloacae and Escherichia coli, flavin oxidoreductase of Vibrio fischeri, and NADH oxidase of Thermus thermophilus. To investigate the structure-function relation of this class of enzymes, the gene encoding a mutant nitroreductase was cloned from S. typhimurium strain TA1538NR, and the enzymatic properties were compared with those of the wild-type. DNA sequence analysis revealed a T to G mutation in the mutant nitroreductase gene, predicting a replacement of leucine 33 with arginine. In contrast to the wild-type enzyme, the purified protein with a mutation of leucine 33 to arginine has no detectable nitroreductase activities in the standard assay conditions and easily lost FMN by dialysis or ultrafiltration. In the presence of an excess amount of FMN, however, the mutant protein exhibited a weak but measurable enzyme activity, and the substrate specificity was similar to that of the wild-type enzyme. Possible mechanisms by which the mutation greatly diminishes binding of FMN to the nitroreductase are discussed.

Amino Acid Sequence↗

PubMed1983

Purification and characterization of 1-nitropyrene nitroreductases from Bacteroides fragilis.

We isolated four nitroreductases from Bacteroides fragilis GAI0624 and examined their physicochemical and functional properties. Two major enzyme activities were found in the adsorbed and unadsorbed fractions from DEAE-cellulose column chromatography. The adsorbed fraction was subjected to Sephadex G-200 column chromatography, and two further activities were separated. One has high nitroreductase activity (nitroreductase I), and the other has low activity and relatively high molecular weight (nitroreductase III). The nitroreductase I fraction was subjected to hydroxylapatite and chromatofocusing column chromatography, and nitroreductase I was purified about 416-fold with a yield of 6.77%. The unadsorbed fraction from DEAE-cellulose column chromatography was subjected to Sepharose 2B and Sepharose 6B column chromatography. Two enzyme activities were obtained by the Sepharose 6B column chromatography. One has high activity (nitroreductase II), and the other has low activity (nitroreductase IV). Nitroreductase II was rechromatographed by Sepharose 6B gel filtration and purified about 178-fold with a yield of 9.65%. The four enzymes (nitroreductases I, II, III, and IV) were shown to be different by several criteria. Their molecular weights, determined by gel filtration, were 52,000, 320,000, 180,000, and 680,000, respectively. The substrate specificity, the effect on mutagenicity of mutagenic nitro compounds, of nitroreductases I, III, and IV was relatively high for 1-nitropyrene, dinitropyrenes, and 4-nitroquinoline 1-oxide, respectively, but nitroreductase II had broad specificity. Nitroreductase activity required a coenzyme; nitroreductases II, III, and IV were NADPH linked, but nitroreductase I was NADH linked. All enzyme activity was enhanced by addition of flavin mononucleotide and inhibited significantly by dicumarol, p-chloromercuribenzoic acid, o-iodosobenzoic acid, sodium azide, and Cu2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteroides fragilis↗

PubMed2000

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine).

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.

Enterobacter cloacae↗

PubMed1997

Targeted disruption of the gene encoding the classical nitroreductase enzyme in Salmonella typhimurium Ames test strains TA1535 and TA1538.

The gene encoding the 'classical nitroreductase' (CNR) of Salmonella typhimurium was disrupted. In this manner, cnr null mutant derivatives of strains TA1535 and TA1538 were constructed, and named YG7131 and YG7127, respectively. In both strain backgrounds, cnr gene disruption reduced nitrofurazone-reductase activity. This reduction almost completely eliminated the nitroreductase activity of strain TA1538. In contrast, the nitroreductase activity of strain TA1535 was much higher than that in TA1538. In this background, cnr gene disruption resulted in a reduction in nitroreductase activity by a similar absolute amount as in TA1538, but representing only about one-quarter of the original activity of TA1535. The results suggest that S. typhimurium has originally at least two distinct nitroreductases, one of which is already deficient in strain TA1538; the CNR is present in both TA1535 and TA1538. Also, these two strains (including their derivatives, TA98 and TA100) are not isogenic with regard to nitroreductase activity. After the introduction of plasmid pKM101, the sensitivities of the strains YG7132 and YG7128, the cnr-null mutants of TA98 and TA100, respectively, against several nitro compounds were compared with those of the conventional cnr-deficient strains TA98NR and TA100NR and the wild-type strains TA98 and TA100. The mutagenicities of 2-nitrofluorene and 1-nitropyrene in YG7132 or TA98NR were ten-fold lower than those of the compounds in TA98. Similarly, the mutagenicity of 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide in strain YG7128 or TA100NR was substantially lower than that of the compound in TA100. However, the mutagenicity of 2-nitronaphthalene in YG7128 was between those observed with TA100 and TA100NR, suggesting that a nitroreductase in S. typhimurium other than CNR is involved in the metabolic activation of this compound. The cnr gene of S. typhimurium positively hybridized with DNA at 13 min on the E. coli chromosome where the nfsB and nfnB genes of E. coli are mapped. These results suggest that the cnr gene of S. typhimurium is a counterpart of the nfsB and nfnB genes of E. coli, and that the newly constructed cnr-deletion strains are useful to assess the role of nitroreductases in the metabolic activation of mutagenic nitro compounds.

Blotting, Southern↗

PubMed2002

Identification and characterization of SnrA, an inducible oxygen-insensitive nitroreductase in Salmonella enterica serovar Typhimurium TA1535.

The biological activity of many nitrosubstituted compounds, many of which are produced commercially or have been identified as environmental contaminants, is dependent on metabolic activation catalyzed by nitroreductases. In the current study, we have cloned a nitroreductase gene, Salmonella typhimurium nitroreductase A (snrA), from S. enterica serovar Typhimurium strain TA1535, and characterized the purified gene product. SnrA is 240 amino acids in length and shares 87% sequence identity to the Escherichia coli homolog, E. coli nitroreductase A (NfsA). SnrA is the major nitroreductase in S. enterica serovar Typhimurium strain TA1535 and catalyzes nitroreduction through a ping-pong bi-bi mechanism in a NADPH and flavine mononucleotide (FMN) dependent manner. SnrA exhibits extremely low levels of FMN reductase activity but the nitroreductase activity of SnrA is competitively inhibited by exogenously added FMN. Treatment of TA1535 with paraquat resulted in induction of nitroreductase activity, suggesting that SnrA is a member of the S. enterica serovar Typhimurium SoxRS regulon associated with cellular defense against oxidative damage. Examination of the microbial genomes databases shows that SnrA homologs are widely distributed in the microbial world, being present in isolates of both Archea and Eubacteria. Southern hybridization and PCR failed to detect the snrA gene in the closely related S. enterica serovar Typhimurium strain TA1538. S. enterica serovar Typhimurium strains TA1535 and TA1538 and their derivatives are commonly used in mutagenicity testing. Differences in metabolic capacity between these two strains may have implications for the interpretation of mutagenicity data.

Amino Acid Sequence↗

PubMedDate not supplied

Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene.

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines. A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells. Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase. Significant bystander cell killing was dependent on a high cell density. In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.

Antineoplastic Agents↗

PubMed1998

Activation of 1-nitropyrene by nitroreductase increases the DNA adduct level and mutagenicity.

1-Nitropyrene (1-NP) is a mutagenic nitro compound in the environment. We studied correlations between the mutagenicity of 1-NP for three strains of Salmonella typhimurium, the activity of bacterial nitroreductases and the amount of 1-NP-derived DNA adducts. Bacterial strains used in this study were S. typhimurium strains TA98, nitroreductase-less mutant TA98NR and YG1021 carrying a nitroreductase-producing plasmid. The mutagenicity of 1-NP was measured using the Ames assay, and the nitroreductase activities of these strains were assayed by quantification of 1-aminopyrene produced from 1-NP. The DNA adducts were measured by the 32P-postlabeling method. Among the three bacterial strains, strain YG1021 was the highest in mutagenicity of 1-NP, the nitroreductase activity and the DNA adduct level. However, S. typhimurium strain TA98NR had the lowest values of these three parameters. Nitroreductase activity, DNA adduct level and mutagenicity were strongly correlated with each other. These results indicate that bacterial nitroreductase plays an important role in forming the DNA adducts, and that the higher the adduct level the higher the level of mutagenicity.

DNA Adducts↗

PubMed1995

Virtual cofactors for an Escherichia coli nitroreductase enzyme: relevance to reductively activated prodrugs in antibody directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli that has the unusual property of being equally capable of using either NADH or NADPH as a cofactor for the reduction of its substrates which include menadione as well as 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954). This property is shared with the mammalian enzyme, DT diaphorase. The nitroreductase can, like DT diaphorase, also use simple reduced pyridinium compounds as virtual cofactors. The intact NAD(P)H molecule is not required and the simplest quaternary (and therefore reducible) derivative of nicotinamide, 1-methylnicotinamide (reduced), is as effective as NAD(P)H in its ability to act as an electron donor for the nitroreductase. The structure-activity relationship is not identical to that of DT diaphorase and nicotinic acid riboside (reduced) is selective, being active only for the nitroreductase. Irrespective of the virtual cofactor used, the nitroreductase formed the same reduction products of CB 1954 (the 2- and 4-hydroxylamino derivatives in equal proportions). Nicotinic acid riboside (reduced), unlike NADH, was stable to metabolism by serum enzymes and had a plasma half-life of seven minutes in the mouse after an i.v. bolus administration. NADH had an unmeasurably short half-life. Nicotinic acid riboside (reduced) could also be produced in vivo by administration of nicotinic acid 5'-O-benzoyl riboside (reduced). These results demonstrate that the requirement for a cofactor need not be a limitation in the use of reductive enzymes in antibody directed enzyme prodrug therapy (ADEPT). It is proposed that the E. coli nitroreductase would be a suitable enzyme for ADEPT in combination with CB 1954 and a synthetic, enzyme-selective, virtual cofactor such as nicotinic acid riboside (reduced).

Animals↗

PubMed1991

Purification and characterization of an oxygen-insensitive NAD(P)H nitroreductase from Enterobacter cloacae.

The reductive products of several nitroaromatic compounds have been found to be toxic, mutagenic, and carcinogenic. The nitroreductases present in intestinal microflora have been implicated in the biotransformation of these compounds to their deleterious metabolites. A "classical" nitroreductase has been purified from Enterobacter cloacae 587-fold using a protocol which yields approximately 1 mg of purified nitroreductase from 10 liters of cell culture. An analysis of the physical properties of the nitroreductase indicates that the enzyme is active as a monomer with a calculated molecular mass of 27 kDa. FMN has been identified as a required flavin cofactor and is present at a stoichiometry of 0.88 mol of FMN bound/mol of active enzyme. The enzyme was found capable of reducing nitrofurazone under aerobic conditions indicating that the mechanism involves an obligatory two-electron transfer. Thus, this enzyme can be classified as an oxygen-insensitive nitroreductase. The purified nitroreductase can utilize either NADH or NADPH as a source of reducing equivalents and can reduce a variety of nitroaromatic compounds including nitrofurans and nitrobenzenes as well as quinones. Studies in which the rates of nitroreduction for a series of para substituted nitrobenzene derivatives were determined suggest that a linear free energy relationship exists between the rate and the redox midpoint potential of the substrate.

Bacterial Proteins↗

PubMed2000

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase.

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001).

Adenoviridae↗

PubMed1997

Involvement of nitroreductase and O-acetyltransferase on the mutagenicity of plant-activated benzidine and 4-aminobiphenyl.

Benzidine and 4-aminobiphenyl (4-ABP) are activated by intact plant cells and cell free TX1MX into mutagenic metabolites that induce frameshift and base pair substitution mutations in Salmonella typhimurium. The plant activation of these agents is plant peroxidase-mediated and bacterial O-acetyltransferase (OAT) dependent. TX1MX-activated benzidine and 4-ABP were analyzed with S. typhimurium frameshift tester strains, YG1021, YG1024, TA98, TA98NR, TA98/1,8-DNP6, MP219, and base pair substitution tester strains, YG1026, YG1029, TA100, TA100NR, TA100TN:OAT, and MP208. Concentration ranges for benzidine and 4-ABP were 1-50 microM and 0.1-1 mM, respectively. This study was conducted to determine if the plant-activation of benzidine and 4-ABP follows the prostaglandin H synthase-mediated activation pathway in mammals [Smith et al. (1992): Chem Res Toxicol 5;431-439]. In this model, benzidine is N-acetylated by S. typhimurium OAT. This acetylated product is a substrate for PHS and is converted into a 4-nitro product which is catalyzed by nitroreductase into a N-hydroxy intermediate. The pathway assigns a specific role for nitroreductase in the activation of benzidine. By employing S. typhimurium strains that express different levels of OAT and/or nitroreductase, we determined that the plant-activation of benzidine and 4-ABP has an absolute requirement of bacterial OAT activity for the induction of frameshift mutations at hisD3052 and is required for the optimal mutagenic response at hisG46. Nitroreductase also plays a role in the plant activation of these agents. The data suggest that the plant-activation of benzidine and 4-ABP generates at least two classes of proximal mutagenic intermediates. One class requires S. typhimurium OAT alone to be transformed into the ultimate mutagen and a second class requires both OAT and nitroreductase.

Acetyltransferases↗

PubMed1992

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--I. Purification and properties of a nitroreductase enzyme from Escherichia coli--a potential enzyme for antibody-directed enzyme prodrug therapy (ADEPT).

A nitroreductase enzyme has been isolated from Escherichia coli B. This enzyme is an FMN-containing flavoprotein with a molecular mass of 24 kDa and requires either NADH or NADPH as a cofactor. Partial protein sequence analysis showed extensive homology with the "classical nitroreductase" of Salmonella typhimurium and a nitroreductase induced in Enterobacter cloacae. In common with the Salmonella enzyme, the E. coli B enzyme is capable of reducing nitrofurazone. The E. coli nitroreductase is also capable of reducing the anti-tumour agent CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide], a property shared with the mammalian enzyme DT diaphorase [NAD(P)H dehydrogenase (quinone)] as isolated from Walker cells. The reduction of CB1954 by the E. coli enzyme results in the generation of cytotoxic species. Both enzymes also share the properties of being able to reduce quinones and are both inhibited by dicoumarol. The nitroreductase is a more active enzyme against CB1954 (kcat = 360 min-1) than Walker DT diaphorase (kcat = 4 min-1) and also has a lower Km for NADH (6 vs 75 microM).

Amino Acid Sequence↗

PubMed1992

The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954)--II. A comparison of an Escherichia coli nitroreductase and Walker DT diaphorase.

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.

Antibodies↗

PubMed1992

A sensitive umu test system for the detection of mutagenic nitroarenes in Salmonella typhimurium NM1011 having a high nitroreductase activity.

A sensitive umu test system for the detection of mutagenic nitroarenes has been developed using a new tester strain Salmonella typhimurium NM1011 having a high nitroreductase activity. The new strain was constructed by subcloning the bacterial nitroreductase gene into a plasmid pACYC184 and introducing the plasmid into the original strain S. typhimurium TA1535/pSK1002 harboring a fusion gene umuC'-'lacZ (pSK1002). Thus, the tester strain enabled us to monitor the genotoxic activities of various nitroarene compounds by measuring the beta-galactosidase activity in the cells. The sensitivity of strain NM1011 was compared with that of the parent tester strain S. typhimurium TA1535/pSK1002 or a nitroreductase-deficient strain S. typhimurium NM1000 with respect to the induction of umuC gene expression by 17 mutagenic nitroarenes. The newly developed strain with high nitroreductase activity had about 3 times higher nitrofurazone-reductase activity than the parent strain and was highly sensitive to the compounds 2-nitrofluorene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, m-dinitrobenzene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 3,9-dinitrofluoranthene, 5-nitroacenaphthene and 2,4-dinitrotoluene. By contrast, the enzyme-deficient strain did not show any considerable response to 2-nitrofluorene, m-dinitrobenzene, 1-nitronaphthalene, 2-nitronaphthalene, 1-nitropyrene, 4,4'-dinitrobiphenyl, 3-nitrofluoranthene, 3,7-dinitrofluoranthene, 2,4-dinitrotoluene and 5-nitroacenaphthene. These results suggest that the newly developed tester strain with high nitroreductase activity is very useful for the detection of potent mutagenic nitroarene compounds.

Cloning, Molecular↗

PubMed1990

Effect of lindane on nitroreductase and dechlorinase enzyme activity in the gastrointestinal tract.

A previously reported acceleration of parathion metabolism in the gastrointestinal (GI) tract of lindane-pretreated rats could have been due to either a prolonged residence time of parathion or increased GI nitroreductase activity or both. Thus to determine the effect on GI nitroreductase and dechlorinase activity, 20 mg/kg lindane or 535 mg/kg neomycin were administered daily, by gavage, to weanling F-344 rats. Enzyme activity in the small intestine and cecum were assayed after 2 weeks and 5 weeks of treatment. Neomycin treatment inhibited the activity of both enzymes in the cecum but had no significant effect on enzyme activity in the small intestine, suggesting the presence of mucosal nitroreductase and dechlorinase in the small intestine. In contrast, lindane, which had no effect on enzyme activity in the cecum, significantly increased nitroreductase activity in the small intestine after treatment for 5 weeks. This increased nitroreductase may account for the previously reported lindane-parathion interaction and could influence the metabolism, toxicity, and risk assessment of many other environmental nitro-compounds that become toxic, mutagenic or carcinogenic upon reduction of their nitro-groups.

Animals↗

PubMed1999

Nitroreductase A is regulated as a member of the soxRS regulon of Escherichia coli.

Nitroreductase A catalyzes the divalent reduction of nitro compounds, quinones, and dyes by NADPH. In this paper, nitroreductase A is induced in Escherichia coli by exposure to paraquat in a manner that depends on the expression of soxR. Nitroreductase activity was only slightly induced by paraquat in a strain bearing a mutational defect in the gene encoding nitroreductase A, but it was approximately 3-fold induced in the parental strain. Nitroreductase A thus appears to be a member of the soxRS regulon and probably contributes to the defenses against oxidative stress by minimizing the redox cycling attendant upon the univalent reduction of nitro compounds, quinones, and dyes.

Drug Resistance, Neoplasm↗