Nitroreductase activity

Saito et al. (134) found that the cytosolic nitroreductase activity was due to DT-diaphorase, aldehyde oxidase, xanthine oxidase plus other unidentified nitroreductases. As anticipated, the microsomal reduction of 1-nitropyrene was inhibited by 0 and stimulated by FMN which was attributed to this cofactor acting as an electron shuttle between NADPH-cytochrome P-450 reductase and cytochrome P-450. Carbon monoxide and type II cytochrome P-450 inhibitors decreased the rate of nitroreduction which was consistent with the involvement of cytochrome P-450. Induction of cytochromes P-450 increased rates of 1-aminopyrene formation and nitroreduction was demonstrated in a reconstituted cytochrome P-450 system, with isozyme P-448-IId catalyzing the reduction most efficiently. 

The enzymatic system participating in degradation of TNT or other nitroaromatics in plants has not yet been sufficiently characterised. Based on recent results, TNT is not metabolised by highly specific nitroreductases, which would be purposefully synthesized by plants, but by constitutive enzymes with nitroreductase activity (Nepovim 2005b). This assumption is in agreement with results presented by Ekman (2005) showed an increase of the expression of a couple of enzymes in root mRNA due to the induction caused by explosives. 

The metabolic transformation of many drugs is catalysed by various enzyme of the intestinal microflora. The anaerobic microflora and colon are rich in reductases which may be responsible for a significant proportion of the azoreductase and nitroreductase activity. The enzymes and other factors that may produce change in the nature of intestinal microflora might also produce changes in the metabolism pattern of the drugs. 

Reduction. The activity of azo- and nitroreductase varies between different species, as shown by the in vitro data in Table 5.11. Thus, azoreductase activity is particularly high in the guinea pig, relative to the other species studied, whereas nitroreductase activity is greatest in the mouse liver. 

Table 5.11 Hepatic Azoreductase and Nitroreductase Activities of Various Species...

The enteric bacterium Enterobacter cloacae produces a nitroreductase that reduces nitrofurans, nitroimidazoles, nitrobenzene derivatives, and quinones (Bryant DeLuca, 1991). This oxygen-insensitive enzyme has been purified and is known to require FMN to transfer reducing equivalents from NAD(P)H to the nitroaromatic compounds, TNT being the preferred substrate. Aerobically, this enzyme reduces nitrofurazone through the hydroxylamine intermediate, which then tautomerizes to yield an oxime end-product. Anaerobically, however, the reduction proceeds to the fully reduced amine adduct. When E. cloacae was grown in the presence of TNT, the nitroreductase activity increased five- to tenfold. 

Reductive reactions, like oxidation, are carried out at different rates by enzyme preparations from different species. Microsomes from mammalian liver are 18 times or more higher in azoreductase activity and more than 20 times higher in nitroreductase activity than those from fish liver. Although relatively inactive in nitroreductase, fish can reduce the nitro group of parathion, suggesting multiple forms of reductase enzymes. 

The increased CVB in pectin-fed and cereal-based diet-fed animals (compared to purified diet) was correlated with an increase in microfloral enzyme activity. Animals fed the pectin-containing purified diets, NIH-07, or Purina 5002 had significantly higher (two- to threefold) cecal 3-glucuronidase and nitroreductase activities than animals fed the purified diet (Table II). The mean specific activities of both enzymes were similar in the pectin-fed groups and cereal-based diet fed groups. 

Table II. Effect of Diet on the Weight of Cecal Contents and Microfloral 8-Glucuronidase and Nitroreductase Activities...

A plot of this sort is useful for qualitative analysis, especially of outliers. A study of the test conditions (e.g., the activating systems used) the particular strain of test organisms (e.g., its sensitivity to frame shifts vs. base substitutions), or the chemical nature of the mutagen may reveal the basis for the discrepancy and ultimately yield deeper insights. For example, the discrepancy in the two tests for furylfuramide (the extreme outlier in Figure 9-2) could be due to different nitroreductase activities in the two systems, which would lead to differences in capacity to convert the chemical into an active form. 

Nitroreductase activity has been demonstrated in liver homogenates as well as in the soluble fraction, whereas other studies have reported that nitroreductase activity has been found in all liver fractions evaluated. The reductase appears to be distributed in liver, kidney, lung, heart, and brain. The reaction utilizes both NADPH and NADH and requires anaerobic conditions. The reaction can be inhibited by the addition of oxygen. The reaction is stimulated by FMN and FAD, and at high flavin concentrations they can act simply as nonenzymatic electron donors. The reduction... 

TABLE 5.11 Hepatic azoreductase and nitroreductase activities of various species... 

The activation of DNT has been shown to be a multistep process involving metabolism in the liver, excretion into the bile, deconjugation of metabolites and further metabolism by the intestinal flora, re-uptake (enterohepatic transport) of metabolites into liver, and finally activation and binding to cellular macromolecules in the liver [56], More recent studies [57] involving rats pretreated with coal tar creosote, which potentiates the genotoxicity of 2,6-DNT, elucidated a complex interaction that balances metabolic activation, uptake, and detoxification. The study monitored intestinal flora enzyme activities, bacterial analysis, mutagenicity of urine samples, HPLC analysis, and hepatic DNA adducts over a five-week exposure period. The location of nitroreductase activity was an... 

Azoreductase activity (substrate l,2-dimethyl-4-(/ -carboxyphenylazo)-5-hydroxybenzene (CPA) (Hanzel and Carlson 1974) and nitroreductase activity (substrate. 77-nitrobenzoic acid) (Carlson 1972) have been detected in digestive gland of M. mercenaria (Table 12). Their properties were similar to those of mammalian enzyme activities and considered indicative of the existence of two separate enzyme systems. Nitroreductase activity was maximal at 35 to 45 C and pH 6.0, Stimulated by flavin mononucleotide, and inhibited by potassium cyanide and air, but not by SKF-525A or carbon monoxide (cytochrome P-450 inhibitors). Azoreductase activity was similarly stimulated by flavin mononucleotide and inhibited by air, but had a pH optimum of 8.0 activity was higher at 37 C than at 22 °C. Both enzyme activities had cytosolic and microsomal subcellular localizations, viz. activity in nmol min g wet weight (% distribution in brackets)... 

Nitroreductase activity (substrate / -nitrobenzoic acid) was present in the hepatopancreas of H. americanus (Table 20), mainly in the cytosol (79% of total activity), but also in the microsomes (11%) and mitochondria (10%) (Elmamlouk and Gessner 1976). The mitochondrial activity was more active with NADH than with NADPH, whereas the reverse was seen for the cytosolic enzyme. Differential inhibitory and stimulatory effects of FAD and FMN indicated the existence of distinct NADH- and NADPH-dependent cytosolic reductases. The activity of the cytosolic NADPH-dependent reductase was increased under anaerobic conditions. 

Early observations of bacterial nitroreductase activity were made with cells capable of reducing chloramphenicol and/or / -nitrobenzoic acid to the corresponding aryl amines (78, 78, 80, 81, 82). From this body of work there emerged an early image of a bacterial nitroreductase as a soluble metalloflavoprotein that uses reduced pyridine nucleotide as an electron donor and catalyzes at least one two-electron reduction (Fig. 4). 

Yamashina and co-workers (94, 95) showed that azoxy-, azo- and hydrazo-com-pounds were not reduced by bacterial nitroreductase. Their results supported the observations of Saz and coworkers (78, 78, 80, 81) on the occurrence of individual nitro andnitroso reductase activities, and inhibitor studies indicated the involvement of metals in enzymatic activity. Furthermore, these studies contradicted the earlier work of Egami et al. (24) which indicated that nitroreductase activity could be attributed to nitrite reductase. 

Cartwright and Cain (11) studied reduction of the nitro group of nitrobenzoic acids by enzymes in cell-free extracts of Nocardia erythropolis. Detection of the nitroso and hydroxylamino derivatives of nitrobenzoate indicated that they were intermediates in reduction to aminobenzoate. This study attributed nitroreductase activity to a single soluble metalloflavoprotein that catalyzes the entire reduction from the nitro to the amino group. Glucose grown cells reduced nitrobenzoate to aminobenzoate, which suggested that the reaction was not catalyzed by a specific inducible nitroreductase. 

Several studies have shown that bacteria possess multiple nitroreductase activities (2, 8,21,43,61, 84). Although results vary from strain to strain, the typical Type I enzyme was found to be an FMN-containing flavoprotein that uses NAD(P)H as an electron donor and is markedly inhibited by / -chloromercuribenzoate, o-iodosobenzoic acid, and dicuma-rol. Dicumarol is a specific inhibitor of menadione reductase and DT diaphorase, suggesting that the Type I enzymes serve as quinone reductases in vivo (1,21, 84). These studies gave no evidence for the involvement of a metal cofactor in the reduction process. 

Kinouchi, T., and Y. Ohnishi. 1986. Metabolic activation of 1-nitropyrene and 1,6-dinitropyrene by nitroreductases from Bacteroides fragilis and distribution of nitroreductase activity in rats. Microbiol. Immunol. 30 979-992. 

McCoy, E. C., H. S. Rosenkranz, and P. C. Howard. 1990. Salmonella typhimurium TA100Tn5-1012, a strain deficient in arylhydroxylamine O-esterificase, exhibits a reduced nitroreductase activity. Mutat. Res. 243 141-144. 

Zcnno, S., S. Inouye, and H. Kanoh. 1993. Gene encoding enzyme having flavin reducing activity and nitroreductase activity. European Patent Application no. 0 547 876 Al. 

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