Enzymatic redox properties of novel nitrotriazole explosives implications for their toxicity

The toxicity of conventional nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) is caused by their enzymatic free radical formation with the subsequent oxidative stress, the formation of alkylating nitroso and/or hydroxylamino metabolites, and oxyhemoglobin oxidation into methemoglobin. In order to get an insight into the mechanisms of toxicity of the novel explosives NTO (5-nitro-1,2,4-triazol-3-one) and ANTA (5-nitro-1,2,4-triazol-3-amine), we examined their reactions with the single-electron transferring flavoenzymes NADPH: cytochrome P-450 reductase and ferredoxin:NADP+ reductase, two-electron transferring flavoenzymes mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase), and Enterobacter cloacae NAD(P)H:nitroreductase, and their reactions with oxyhemoglobin. The reactivity of NTO and ANTA in the above reactions was markedly lower than that of TNT. The toxicity of NTO and ANTA in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. This points to the involvement of oxidative stress in their cytotoxicity, presumably to the redox cycling of free radicals. The FLK cell line cytotoxicity and the methemoglobin formation in isolated human erythrocytes of NTO and ANTA were also markedly lower than those of TNT, and similar to those of nitrobenzene. Taken together, our data demonstrate that the low toxicity of nitrotriazole explosives may be attributed to their low electron-accepting properties.     

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.     

Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.     

Flavoenzyme-catalyzed redox cycling of hydroxylamino- and amino metabolites of 2,4,6-trinitrotoluene: implications for their cytotoxicity

The toxicity of 2,4,6-trinitrotoluene (TNT), a widespread environmental contaminant, is exerted through its enzymatic redox cycling and/or covalent binding of its reduction products to proteins and DNA. In this study, we examined the possibility of another cytotoxicity mechanism of the amino- and hydroxylamino metabolites of TNT, their flavoenzyme-catalyzed redox cycling. The above compounds acted as redox-cycling substrates for single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and ferredoxin:NADP(+) reductase (FNR), as well as substrates for the two-electron transferring flavoenzymes rat liver NAD(P)H:quinone oxidoreductase (NQO1) and Enterobacter cloacae NAD(P)H:nitroreductase (NR). Their reactivity in P-450R-, FNR-, and NR-catalyzed reactions increased with an increase in their single-electron reduction potential (E(1)(7)) or the decrease in the enthalpy of free radical formation. The cytotoxicity of the amino- and hydroxylamino metabolites of TNT towards bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was partly prevented by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea, thus pointing to the involvement of oxidative stress. In general, their cytotoxicity increased with an increase in their electron accepting properties, or their reactivity towards the single-electron transferring FNR and P-450R. Thus, our data imply that the flavoenzyme-catalyzed redox cycling of amino and hydroxylamino metabolites of TNT may be an important factor in their cytotoxicity.     

Engineering plants for the phytoremediation of RDX in the presence of the co-contaminating explosive TNT

The explosive compounds hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4,6-trinitrotoluene (TNT) are widespread environmental contaminants commonly found as co-pollutants on military training ranges. TNT is a toxic carcinogen which remains tightly bound to the soil, whereas RDX is highly mobile leaching into groundwater and threatening drinking water supplies. We have engineered Arabidopsis plants that are able to degrade RDX, whilst withstanding the phytotoxicity of TNT. Arabidopsis thaliana (Arabidopsis) was transformed with the bacterial RDX-degrading xplA, and associated reductase xplB, from Rhodococcus rhodochrous strain 11Y, in combination with the TNT-detoxifying nitroreductase (NR), nfsI, from Enterobacter cloacae. Plants expressing XplA, XplB and NR remove RDX from soil leachate and grow on soil contaminated with RDX and TNT at concentrations inhibitory to XplA-only expressing plants. This is the first study to demonstrate the use of transgenic plants to tackle two chemically diverse organic compounds at levels comparable with those found on contaminated training ranges, indicating that this technology is capable of remediating concentrations of RDX found in?situ. In addition, plants expressing XplA and XplB have substantially less RDX available in aerial tissues for herbivory and potential bioaccumulation.     

Nitroreductase II involved in 2,4,6-trinitrotoluene degradation: Purification and characterization from <Emphasis Type="Italic">Klebsiella</Emphasis> sp. Cl

Three 2,4,6-trinitrotoluene (TNT) nitroreductases from Klebsiella sp. CI have different reduction capabilities that can degrade TNT by simultaneous utilization of two initial reduction pathways. Of these, nitroreductase II was purified to homogeneity by sequential chromatographies. Nitroreductase II is an oxygen-insensitive enzyme and reduces both TNT and nitroblue tetrazolium. The N-terminal amino acid sequence of the enzyme did not show any sequence similarity with those of other nitroreductases reported. However, it transformed TNT by the reduction of nitro groups like nitroreductase I. It had a higher substrate affinity and specific activity for TNT reduction than other nitroreductases, and it showed a higher oxidation rate of NADPH with the ortho-substituted isomers of TNT metabolites (2-hydroxylaminodinitrotoluene and 2-aminodinitrotoluene) than with para-substituted compounds (4-hydroxylaminodinitrotoluene and 4-amino-dinitrotoluene).     

Plant processes important for the transformation and degradation of explosives contaminants

Environmental contamination by explosives is a worldwide problem. Of the 20 energetic compounds, 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are the most powerful and commonly used. Nitroamines are toxic and considered as possible carcinogens. The toxicity and persistence of nitroamines requires that their fate in the environment be understood and that contaminated soil and groundwater be remediated. This study, written as a minireview, provides further insights for plant processes important for the transformation and degradation of explosives. Plants metabolize TNT and the distribution of the transformation products, conjugates, and bound residues appears to be consistent with the green liver model concept. Metabolism of TNT in plants occurs by reduction as well as by oxidation. Reduction probably plays an important role in the tolerance of plants towards TNT, and, therefore a high nitroreductase capacity may serve as a biochemical criterion for the selection of plant species to remediate TNT. Because the activities and the inducibilities of the oxidative enzymes are far lower than of nitroreductase, reducing processes may predominate. However, oxidation may initiate the route to conjugation and sequestration leading ultimately to detoxification of TNT, and, therefore, particularly the oxidative pathway deserves more study. It is possible that plants metabolize RDX also according to the green liver concept. In the case of plant metabolism of HMX, a conclusion regarding compliance with the green liver concept was not reached due to the limited number of available data.     

A double mutant of Pseudomonas putida JLR11 deficient in the synthesis of the nitroreductase PnrA and assimilatory nitrite reductase NasB is impaired for growth on 2,4,6-trinitrotoluene (TNT)

Pseudomonas putida JLR11 can grow on 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. We created nasB (nitrite reductase), pnrA (nitroaromatic reductase) and pnrA nasB mutants and tested their growth with TNT as the sole N source. The nasB and pnrA mutants grew at a reduced rate on TNT, whereas the double nasB pnrA mutant did not. This suggests that P. putida JLR11 carries out multiple enzymatic attacks on TNT-releasing nitrite and/or ammonium. The PnrA nitroreductase plays a key role in the reduction of TNT to 2,6-dinitro-4-hydroxylaminotoluene and the subsequent release of ammonium for growth.     

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.     

Transformation of 2,4,6-Trinitrotoluene by Pseudomonas pseudoalcaligenes JS52

Pseudomonas pseudoalcaligenes JS52 grows on nitrobenzene via partial reduction of the nitro group and enzymatic rearrangement of the resultant hydroxylamine. Cells and cell extracts of nitrobenzene-grown JS52 catalyzed the transient formation of 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), 4-amino-2,6-dinitrotoluene (4ADNT), and four previously unidentified metabolites from 2,4,6-trinitrotoluene (TNT). Two of the novel metabolites were identified by liquid chromatography/mass spectrometry and (sup1)H-nuclear magnetic resonance spectroscopy as 2,4-dihydroxylamino-6-nitrotoluene (DHANT) and 2-hydroxylamino-4-amino-6-nitrotoluene (2HA4ANT). A polar yellow metabolite also accumulated during transformation of TNT by cells and cell extracts. Under anaerobic conditions, extracts of strain JS52 did not catalyze the production of the yellow metabolite or release nitrite from TNT; moreover, DHANT and 2HA4ANT accumulated under anaerobic conditions, which indicated that their further metabolism was oxygen dependent. Small amounts of nitrite were released during transformation of TNT by strain JS52. Sustained transformation of TNT by cells required nitrobenzene, which indicated that TNT transformation does not provide energy. Transformation of TNT catalyzed by enzymes in cell extracts required NADPH. Transformation experiments with (sup14)C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with cells. Nitrobenzene nitroreductase purified from strain JS52 transformed TNT to DHANT via 4HADNT, which indicated that the nitroreductase could catalyze the first two steps in the transformation of TNT. The unusual ability of the nitrobenzene nitroreductase to catalyze the stoichiometric reduction of aromatic nitro compounds to the corresponding hydroxylamine provides the basis for the novel pathway for metabolism of TNT.     

DT-diaphorase-catalyzed two-electron reduction of quinone epoxides

DT-diaphorase catalyzes the two-electron reduction of the unsubstituted quinone epoxide, 2,3-epoxy-p-benzoquinone, at expense of NAD(P)H with formation of 2-OH-p-benzohydroquinone as the reaction product. The further conversion reactions of 2-OH-p-benzohydroquinone are influenced by the presence of O2 in the medium. Under aerobic conditions, 2-OH-p-benzohydroquinone undergoes autoxidation--probably with formation of 2-OH-semiquinone intermediates--to 2-OH-p-benzoquinone. The latter product is rapidly reduced by DT-diaphorase and, thus, its accumulation can be only observed upon exhaustion of NADPH. Under anaerobic conditions, 2-OH-p-benzohydroquinone does not undergo autoxidation and its accumulation is stoichiometrically (1:1) related to the amount of NADPH oxidized and epoxide substrate reduced. DT-diaphorase also catalyzes the reduction of the disubstituted quinone epoxide, 2,3-dimethyl-2,3-epoxy-1,4-naphthoquinone. Neither the aliphatic epoxide, trans-stilbene oxide, nor the aromatic epoxide, 4,5-epoxy-benzo[a]pyrene are substrates for DT-diaphorase. The reduction of 2,3-epoxy-p-benzoquinone is also catalyzed by the one-electron transfer enzyme, NADPH-cytochrome P450 reductase at a rate similar to that found with DT-diaphorase. However, this reaction differs from that catalyzed by DT-diaphorase in the distribution of molecular products as well as in the relative contribution of nonenzymatic reactions, i.e. semiquinone disproportionation and autoxidation.     

Prooxidant cytotoxicity of chromate in mammalian cells: the opposite roles of DT-diaphorase and glutathione reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.     

Purification and characterization of the NAD(P)H-nitroreductase for the catabolism of 2,4,6-trinitrotoluene (TNT) in<Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HK-6

The NAD(P)H-nitroreductase of thePseudomonas sp. HK-6 which is capable of catabolizing 2,4,6-trinitrotoluene (TNT), was purified and biochemically characterized. The specific activity of the purified TNT nitroreductase was approximately 1.47 units/mg, and was concentrated to 10.1-fold compared to the crude extract. The optimal temperature and pH of the highest nitroreductase activity was 30°C and 7.5, respectively. The substrate specificity test revealed that the nitroreductase exhibited the highest enzyme activity for the TNT substrate of the nitroaromatic compounds tested in this study. Moreover, the molecular weight of the TNT nitroreductase was approximately 27 kDa on the SDS-PAGE. The N-terminal amino acid sequence of the purified protein was 5′-MDTVSLAKRRYTTKAYDASR, which is identical topnrB ofPseudomonas putida JLR11, and is capable of TNT reduction. The molecular analysis of the approximately 650-bp PCR product, orginating from the HK-6, revealed that the oxygen-insensitive NAD(P)H-nitroreductase gene, which transforms TNT in strain HK-6 with five unique amino acid sequences and diverges from the nitroreductases identified so far inPseudomonas, Burkholderia, andRalstonia, is frequently found amidst the powerful degraders of aromatic compounds.     

Quantitative structure–activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity

The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (ΔHf(ArNO₂^−⋅)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP⁺ reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in ΔHf(ArNO₂^−⋅). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their ΔHf(ArNO₂^−⋅). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.     

Purification and characterization of NAD(P)H-dependent nitroreductase I from Klebsiella sp. C1 and enzymatic transformation of 2,4,6-trinitrotoluene

Three NAD(P)H-dependent nitroreductases that can transform 2,4,6-trinitrotoluene (TNT) by two reduction pathways were detected in Klebsiella sp. C1. Among these enzymes, the protein with the highest reduction activity of TNT (nitroreductase I) was purified to homogeneity using ion-exchange, hydrophobic interaction, and size exclusion chromatographies. Nitroreductase I has a molecular mass of 27 kDa as determined by SDS-PAGE, and exhibits a broad pH optimum between 5.5 and 6.5, with a temperature optimum of 30–40°C. Flavin mononucleotide is most likely the natural flavin cofactor of this enzyme. The N-terminal amino acid sequence of this enzyme does not show a high degree of sequence similarity with nitroreductases from other enteric bacteria. This enzyme catalyzed the two-electron reduction of several nitroaromatic compounds with very high specific activities of NADPH oxidation. In the enzymatic transformation of TNT, 2-amino-4,6-dinitrotoluene and 2,2′,6,6′-tetranitro-4,4′-azoxytoluene were detected as transformation products. Although this bacterium utilizes the direct ring reduction and subsequent denitration pathway together with a nitro group reduction pathway, metabolites in direct ring reduction of TNT could not easily be detected. Unlike other nitroreductases, nitroreductase I was able to transform hydroxylaminodinitrotoluenes (HADNT) into aminodinitrotoluenes (ADNT), and could reduce ortho isomers (2-HADNT and 2-ADNT) more easily than their para isomers (4-HADNT and 4-ADNT). Only the nitro group in the ortho position of 2,4-DNT was reduced to produce 2-hydroxylamino-4-nitrotoluene by nitroreductase I; the nitro group in the para position was not reduced.     

NAD(P)H:flavin mononucleotide oxidoreductase inactivation during 2,4,6-trinitrotoluene reduction

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, K(N), of 394 microM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation.     

Cytotoxicity of nitroaromatic explosives and their biodegradation products in mice splenocytes: implications for their immunotoxicity

Nitroaromatic explosives like 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methyl-nitramine (tetryl) comprise an important group of toxic environmental pollutants, whose toxicity is mainly attributed to the flavoenzyme electrontransferase-catalyzed redox cycling of their free radicals (oxidative stress) and DT-diaphorase [NAD(P)H:quinone oxidoreductase, NQO1, EC 1. 6.99.2]-catalyzed formation of alkylating nitroso and/or hydroxylamine metabolites. Because of the incomprehensive data on the immunotoxic effects of nitroaromatic explosives, we have studied the structure-cytotoxicity relationships in the action of tetryl, TNT as well as its amino and hydroxylamino metabolites, and related nitroaromatic compounds towards mouse splenocyte cells. The protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine against the cytotoxicity of TNT and other nitroaromatics showed that the oxidative stress-type cytotoxicity mechanism takes place. In addition, the cytotoxicity of nitroaromatics is also partly prevented by an inhibitor of NQO1, dicumarol. The cytotoxicity of the amino metabolites of TNT is also partly prevented by alpha-naphthoflavone and isoniazide, which points to the involvement of cytochromes P-450 in their activation. In general the cytotoxicity of nitroaromatics in splenocytes increases with an increase in their single-electron reduction potential, E1(7). This points to the prevailing mechanism of the oxidative stress-type cytotoxicity. The obtained structure-activity relationship and the studies of other mammalian cell lines showed that the immunotoxic potential of nitroaromatic explosives may decrease in the order tetryl > or = TNT > or = hydroxylamino metabolites of TNT > amino and diamino metabolites of TNT.     

Biochemical characterization of trinitrotoluene transforming oxygen-insensitive nitroreductases from <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> ATCC 824

The genes that encode oxygen-insensitive nitroreductases from Clostridium acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized. The gene products NitA (MW 31 kDa) and NitB (MW 23 kDa) were purified to homogeneity. The NitA and NitB are oxygen-insensitive nitroreductases comprised of a single nitroreductase domain. NitA and NitB enzymes show spectral characteristics similar to flavoproteins. The biochemical characteristics of NitA and NitB are highly similar to those of NfsA, the major nitroreductase from E. coli. NitA exhibited broad specificity similar to that of E. coli NfsA and displayed no flavin reductase activity. NitB showed broad substrate specificity toward nitrocompounds in a pattern similar to NfsA and NfsB of Escherichia coli. NitB has high sequence similarity to NAD(P)H nitroreductase from Archaeoglobus fulgidus. NitA could utilize only NADH as an electron donor, whereas NitB utilized both NADH and NADPH as electron donors with a preference for NADH. The activity of both nitroreductases was high toward 2,4-Dinitrotoluene (2,4-DNT) as a substrate. Both the nitroreductases were inhibited by dicoumarol and salicyl hydroxamate. The nitroreductases showed higher relative expression on induction with TNT, nitrofurazone and nitrofurantoin compared to the uninduced control.     

Zeta-crystallin catalyzes the reductive activation of 2,4,6-trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts

Exposure to 2,4,6-trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role. From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species. The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS-PAGE. Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to zeta-crystallin. Superoxide anions were formed during reduction of TNT by zeta-crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens. Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.     

Two-electron reduction of quinones by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships

In order to clarify the poorly understood mechanisms of two-electron reduction of quinones by flavoenzymes, we examined the quinone reductase reactions of a member of a structurally distinct old yellow enzyme family, Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase (PETNR). PETNR catalyzes two-electron reduction of quinones according to a 'ping-pong' scheme. A multiparameter analysis shows that the reactivity of quinones increases with an increase in their single-electron reduction potential and pK(a) of their semiquinones (a three-step (e(-),H(+),e(-)) hydride transfer scheme), or with an increase in their hydride-transfer potential (E(7)(H(-))) (a single-step (H(-)) hydride transfer scheme), and decreases with a decrease in their van der Waals volume. However, the pH-dependence of PETNR reactivity is more consistent with a single-step hydride transfer. A comparison of X-ray data of PETNR, mammalian NAD(P)H : quinone oxidoreductase (NQO1), and Enterobacter cloacae nitroreductase, which reduce quinones in a two-electron way, and their reactivity revealed that PETNR is much less reactive, and much less sensitive to the quinone substrate steric effects than NQO1. This may be attributed to the lack of pi-pi stacking between quinone and the displaced aromatic amino acid in the active center, e.g., with Phe-178' in NQO1.