Photoinactivation of Salmonella enterica exposed to 5-aminolevulinic acid: Impact of sensitization conditions and irradiation time.

The photodynamic inactivation (PDI) represents the potential alternative to traditional antibiotic therapy, and can be applied to treat various bacterial infections, including those caused by Gram-negative bacterial strains. One of the treatment modalities is based on the capacity of bacterial cells to synthesize the excess amounts of porphyrins after exposure to an externally applied 5-aminolevulinic acid (5-ALA), which makes them photosensitive and leads to reduced survival after irradiation with an appropriately selected light source. This study focuses on the sensitization and the photoinduced inactivation of Salmonella enterica cells in PBS containing 0.5 mM 5-ALA, incubated at 37 °C for 4 h or for 20 h and afterwards irradiated with violet LED light (11.1 mW/cm2, a peak at 400 nm). It has been found that both amounts and composition of endogenous porphyrins not only depended on the incubation duration, but also were affected by externally induced photo- and chemo-oxidation reactions. The application of different sensitization conditions has revealed that the increasing amounts of endogenously produced porphyrins do not ensure the proportional reduction of bacterial cell survival numbers. The comparative investigations also demonstrated that the presence of endogenously produced porphyrins in the medium results in secondary sensitization of bacterial cells and causes a notably stronger photoinactivation effect in comparison to their externally applied standards.

Sensitization of Salmonella enterica with 5-aminolevulinic acid-induced endogenous porphyrins: a spectroscopic study.

Photodynamic therapy (PDT) of bacterial strains presents an attractive potential alternative to antibiotic therapies in search of the solution for the chemoresistance problem. The efficacy of the treatment is dependent on the interaction of photochemically active substances called photosensitizers (PSs) with the bacterial cell wall or their intracellular accumulation. In addition to exogenous PSs, other molecules such as 5-aminolevulinic acid (5-ALA), a natural precursor of heme, are gaining interest. When provided exogenously to cells, 5-ALA uptake results in the overproduction of various photoactive porphyrins. The pattern of their intracellular accumulation and release to the surroundings depends on incubation conditions such as the applied 5-ALA concentration, cell density and incubation duration. The detection of endogenously synthesized porphyrins in samples of Salmonella enterica cells and supernatants was accomplished after 4 h and 20 h incubation periods by means of fluorescence spectroscopy. The relative proportions of different types of porphyrins were assessed by modeling the registered spectra with the fluorescence spectra of standard porphyrins. After the shorter incubation period, the dominant porphyrins in the supernatant medium were coproporphyrins. The longer incubation period shifted the relative proportion of intracellular porphyrins from protoporphyrin IX towards water-soluble porphyrins such as uroporphyrin I, which interfered with additional by-products. The time-dependent changes in compositions of both intracellular and extracellular porphyrins imply that 5-ALA-induced sensitization might have triggered a complex protective mechanism of bacterial cells. Thus, identification and evaluation of the relative amounts of porphyrins, which accumulate in bacterial cells and are extruded outside after different time periods, could provide access to valuable information, working towards more efficient applications of 5-ALA-based antibacterial PDT.

Quinones and nitroaromatic compounds as subversive substrates of Staphylococcus aureus flavohemoglobin.

In microorganisms, flavohemoglobins (FHbs) containing FAD and heme (Fe3+, metHb) convert NO. into nitrate at the expense of NADH and O2. FHbs contribute to bacterial resistance to nitrosative stress. Therefore, inhibition of FHbs functions may decrease the pathogen virulence. We report here a kinetic study of the reduction of quinones and nitroaromatic compounds by S. aureus FHb. We show that this enzyme rapidly reduces quinones and nitroaromatic compounds in a mixed single- and two-electron pathway. The reactivity of nitroaromatics increased upon an increase in their single-electron reduction potential (E17), whereas the reactivity of quinones poorly depended on their E17 with a strong preference for a 2-hydroxy-1,4-naphthoquinone structure. The reaction followed a 'ping-pong' mechanism. In general, the maximal reaction rates were found lower than the maximal presteady-state rate of FAD reduction by NADH and/or of oxyhemoglobin (HbFe2+O2) formation (~130 s-1, pH 7.0, 25 °C), indicating that the enzyme turnover is limited by the oxidative half-reaction. The turnover studies showed that quinones prefreqently accept electrons from reduced FAD, and not from HbFe2+O2. These results suggest that quinones and nitroaromatics act as 'subversive substrates' for FHb, and may enhance the cytotoxicity of NO. by formation of superoxide and by diverting the electron flux coming from reduced FAD. Because quinone reduction rate was increased by FHb inhibitors such as econazole, ketoconazole, and miconazole, their combined use may represent a novel chemotherapeutical approach.

Flavoenzyme-mediated reduction reactions and antitumor activity of nitrogen-containing tetracyclic ortho-quinone compounds and their nitrated derivatives.

Nitrogen-based tetracyclic ortho-quinones (naphtho[1'2':4.5]imidazo[1,2-a]pyridine-5,6-diones, NPDOs) and their nitro-substituted derivatives (nitro-(P)NPDOs) were obtained by condensation of substituted 2,3-dichloro-1,4-naphthoquinones with 2-amino-pyridine and -pyrimidine and nitration at an elevated temperature. The structural features of the compounds as well as their global and regional electrophilic potency were characterized by means of DFT computation. The compounds were highly reactive substrates of single- and two-electron (hydride) - transferring P-450R (CPR; EC 1.6.2.4) and NQO-1 (DTD; EC 1.6.99.2), respectively, concomitantly producing reactive oxygen species. Their catalytic efficiency defined in terms of the apparent second-order rate constant (kcat/KM (Q)) values in P-450R- and NQO-1-mediated reactions varied in the range of 3-6 × 107 M-1 s-1 and 1.6-7.4 × 108 M-1 s-1, respectively. The cytotoxic activities of the compounds on tumor cell lines followed the concentration-dependent manner exhibiting relatively high cytotoxic potency against breast cancer MCF-7, with CL50 values of 0.08-2.02 µM L-1 and lower potency against lung cancer A-549 (CL50 = 0.28-7.66 µM L-1). 3-nitro-pyrimidino-NPDO quinone was the most active compound against MCF-7 with CL50 of 0.08 ± 0.01 µM L-1 (0.02 µg mL-1)) which was followed by 3-nitro-NPDO with CL50 of 0.12 ± 0.03 µM L-1 (0.035 µg mL-1)) and 0.28 ± 0.08 µM L-1 (0.08 µg mL-1) on A-549 and MCF-7 cells, respectively, while 1- and 4-nitro-quinoidals produced the least cytotoxic effects. Tumor cells quantified by AO/EB staining showed that the cell death induced by the compounds occurs primarily through apoptosis.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones: Synthesis, enzymatic reduction and cytotoxic activity.

Naphtho[1',2':4,5]imidazo[1,2-a]pyridine-5,6-diones (NPDOs), a new type of N-heterocycle-fused o-quinones, have been synthesized. They have been found to be efficient electron-accepting substrates of NADPH-dependent single-electron-transferring P-450R and two-electron transferring NQO1, generating reactive oxygen species (ROS) with a concomitant decrease in NADPH, which is consistent with redox-cycling. The reactivity of NPDOs toward P-450R (in terms of kcat/Km) varied in the range of 10(6)-10(7)M(-1)s(-1), while their reduction by NQO1 proceeded much faster, approaching the diffusion control limit (kcat/Km∼10(8)-10(9)M(-1)s(-1)). NPDOs exhibited relatively high cytotoxic activity against human lung carcinoma (A-549) and breast tumor (MCF-7) cell lines (LC50=0.1-8.3µM), while promyelocytic leukemia cells (HL-60) were less sensitive to NPDOs (LC50⩾10µM). 3-Nitro-substituted NPDO (11) revealed the highest potency against both A-549 and MCF-7 cell lines, with LC50 of 0.12±0.03µM and 0.28±0.08µM, respectively. Dicoumarol partly suppressed the activity of the compounds against A-594 and MCF-7 cell lines, suggesting that their cytotoxic action might be partially influenced by NQO1-mediated bioreductive activation.

New 1-(3-nitrophenyl)-5,6-dihydro-4H-[1,2,4]triazolo[4,3-a][1,5]benzodiazepines: synthesis and computational study.

Triazole derivatives constitute an important group of heterocyclic compounds have have been the subject of extensive study in the recent past. These compounds have shown a wide range of biological and pharmacological activities. In this work, new fused tricyclic 1-(3-nitrophenyl)-5,6-dihydro-4H-[1,2,4]triazolo[4,3-a][1,5]-benzodiazepines have been synthesized by the thermal cyclization of N'-(2,3-dihydro-1H-1,5-benzodiazepin-4-yl)-3-nitrobenzohydrazides. After screening ethanol, toluene and 1-butanol as solvents, butanol-1 was found to be the best choice for the cyclization reaction in order to obtain the highest yields of tricyclic derivatives. The chemical structures of the synthesized compounds were elucidated by the analysis of their IR, 1H- and 13C-NMR spectral data. For tentative rationalization of the reaction processes, the global and local reactivity indices of certain compounds, taking part in the reaction pathway, were assessed by means of quantum mechanical calculations using the conceptual density functional theory (DFT) approach. This work could be useful for the synthesis of new heterocyclic compounds bearing a fused triazole ring.

The study of NADPH-dependent flavoenzyme-catalyzed reduction of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans).

The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs) towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R) and two-electron (hydride) transferring NAD(P)H: quinone oxidoreductase (NQO1) was examined in this work. Since the =N+ (→O)O- moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs) whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs) of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity indices were assessed by quantum mechanical methods. In P-450R-catalyzed reactions, both BFXs and NACs showed the same reactivity dependence on their electron-accepting potency which might be consistent with an "outer sphere" electron transfer mechanism. In NQO1-catalyzed two-electron (hydride) transferring reactions, BFXs acted as more efficient substrates than NACs, and the reduction efficacy of BFXs by NQO1 was in general higher than by single-electron transferring P-450R. In NQO1-catalyzed reactions, QSARs obtained showed that the reduction efficacy of BFXs, as well as that of NACs, was determined by their electron-accepting potency and could be influenced by their binding mode in the active center of NQO1 and by their global softness as their electronic characteristic. The reductive conversion of benzofuroxan by both flavoenzymes yielded the same reduction product of benzofuroxan, 2,3-diaminophenazine, with the formation of o-benzoquinone dioxime as a putative primary reductive intermediate, which undergoes a further reduction process. Overall, the data obtained show that by contrast to NACs, the flavoenzyme-catalyzed reduction of BFXs is unlikely to initiate their redox-cycling, which may argue for a minor role of the redox-cycling-type action in the cytotoxicity of BFXs.

Single-electron reduction of quinone and nitroaromatic xenobiotics by recombinant rat neuronal nitric oxide synthase.

We examined the kinetics of single-electron reduction of a large number of structurally diverse quinones and nitroaromatic compounds, including a number of antitumour and antiparasitic drugs, and nitroaromatic explosives by recombinant rat neuronal nitric oxide synthase (nNOS, EC 1.14.13.39), aiming to characterize the role of nNOS in the oxidative stress-type cytotoxicity of the above compounds. The steady-state second-order rate constants (kcat/Km) of reduction of the quinones and nitroaromatics varied from 10² M⁻¹s⁻¹ to 106 M⁻¹s⁻¹, and increased with an increase in their single-electron reduction potentials (E¹7). The presence of Ca²âº/calmodulin enhanced the reactivity of nNOS. These reactions were consistent with an 'outer sphere' electron-transfer mechanism, considering the FMNH∙/FMNH2 couple of nNOS as the most reactive reduced enzyme form. An analysis of the reactions of nNOS within the 'outer sphere' electron-transfer mechanism gave the approximate values of the distance of electron transfer, 0.39-0.47 nm, which are consistent with the crystal structure of the reductase domain of nNOS. On the other hand, at low oxygen concentrations ([O2] = 40-50 µM), nNOS performs a net two-electron reduction of quinones and nitroaromatics. This implies that NOS may in part be responsible for the bioreductive alkylation by two-electron reduced forms of antitumour aziridinyl-substituted quinones under a modest hypoxia.

Redox properties and prooxidant cytotoxicity of a neuroleptic agent 6,7-dinitrodihydroquinoxaline-2,3-dione (DNQX).

In order to characterize the possible mechanism(s) of cytotoxicity of a neuroleptic agent 6,7-dinitrodihydroquinoxaline-2,3-dione (DNQX) we examined the redox properties of DNQX, and its mononitro- (NQX) and denitro- (QX) derivatives. The irreversible electrochemical reduction of the nitro groups of DNQX was characterized by the reduction peak potentials (Ep,7) of -0.43 V and -0.72 V vs. Ag/AgCl at pH 7.0, whereas NQX was reduced at Ep,7 = -0.67 V. The reactivities of DNQX and NQX towards the single-electron transferring enzymes NADPH:cytochrome P-450 reductase and NADPH:adrenodoxin reductase/adrenodoxin complex were similar to those of model nitrobenzenes with the single-electron reduction potential (E¹7) values of -0.29 V - -0.42 V. DNQX and NQX also acted as substrates for two-electron transferring mammalian NAD(P)H:quinone oxidoreductase (DT-diaphorase). The cytotoxicity of DNQX in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) was prevented by antioxidants and an inhibitor of NQO1, dicoumarol, and was enhanced by the prooxidant alkylating agent 1,3-bis(2-chloromethyl)-1-nitrosourea. A comparison with model nitrobenzene compounds shows that the cytotoxicity of DNQX and NQX reasonably agrees with the ease of their electrochemical reduction, and/or their reactivities towards the used enzymatic single-electron reducing systems. Thus, our data imply that the cytotoxicity of DNQX in FLK cells is exerted mainly through oxidative stress.

Quinone- and nitroreductase reactions of Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme.

Thermotoga maritima peroxiredoxin-nitroreductase hybrid enzyme (Prx-NR) consists of a FMN-containing nitroreductase (NR) domain fused to a peroxiredoxin (Prx) domain. These domains seem to function independently as no electron transfer occurs between them. The reduction of quinones and nitroaromatics by NR proceeded in a two-electron manner, and follows a 'ping-pong' scheme with sometimes pronounced inhibition by quinone substrate. The comparison of steady- and presteady-state kinetic data shows that in most cases, the oxidative half-reaction may be rate-limiting in the catalytic cycle of NR. The enzyme was inhibited by dicumarol, a classical inhibitor of oxygen-insensitive nitroreductases. The reduction of quinones and nitroaromatic compounds by Prx-NR was characterized by the linear dependence of their reactivity (logk(cat)/K(m)) on their single-electron reduction potentials E(7)(1), while the reactivity of quinones markedly exceeded the one with nitroaromatics. It shows that NR lacks the specificity for the particular structure of these oxidants, except their single-electron accepting potency and the rate of electron self-exchange. It points to the possibility of a single-electron transfer step in a net two-electron reduction of quinones and nitroaromatics by T. maritima Prx-NR, and to a significant diversity of the structures of flavoenzymes which may perform the two-electron reduction of quinones and nitroaromatics.

Redox reactions of the FAD-containing apoptosis-inducing factor (AIF) with quinoidal xenobiotics: a mechanistic study.

Mitochondrial apoptosis-inducing factor (AIF) is a FAD-containing protein that under certain conditions translocates to the nucleus and causes a programmed cell death, apoptosis. The apoptogenic action of AIF is redox controlled as the NADH-reduced AIF dimer has lower affinity for DNA than the oxidized monomer. To gain further insights into the mechanism of AIF, we investigated its interaction with a series of quinone oxidants, including a number of anticancer quinones. Our data indicate that the NADH:quinone oxidoreduction catalyzed by AIF follows a "ping-pong" scheme, with the reductive half-reaction being rate-limiting and the FADH(-)-NAD(+) charge-transfer complex serving as an electron donor. AIF is equally reactive toward benzo- and naphthoquinones, but may discriminate structures with a higher number of aromatic rings. The reactivity of quinones is mainly defined by their one-electron reduction potential, whereas the size and nature of the substituents play a minor role. AIF is unlikely to significantly contribute to bioreductive activation of low-potential quinoidal anticancer quinones. However, high-potential quinones, e.g. a toxic natural compound naphthazarin, maintain AIF in the oxidized state when a significant excess of NADH is present. Thus, these compounds may prevent the accumulation of the reduced form of AIF in vivo, and enhance AIF-mediated apoptosis.

Reduction of aliphatic nitroesters and N-nitramines by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships.

Enterobacter cloacae PB2 NADPH:pentaerythritol tetranitrate reductase (PETNR) performs the biodegradation of explosive organic nitrate esters via their reductive denitration. In order to understand the enzyme substrate specificity, we have examined the reactions of PETNR with organic nitrates (n = 15) and their nitrogen analogues, N-nitramines (n = 4). The reactions of these compounds with PETNR were accompanied by the release of 1-2 mol of nitrite per mole of compound, but were not accompanied by their redox cycling and superoxide formation. The reduction rate constants (k(cat)/K(m)) of inositol hexanitrate, diglycerol tetranitrate, erythritol tetranitrate, mannitol hexanitrate and xylitol pentanitrate were similar to those of the established PETNR substrates, PETN and glycerol trinitrate, whereas the reactivities of hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine were three orders of magnitude lower. The log k(cat)/K(m) value of the compounds increased with a decrease in the enthalpy of formation of the hydride adducts [DeltaH(f)(R-O-N(OH)O(-)) or DeltaH(f)(R(1),R(2) > N-N(OH)O(-))], and with an increase in their lipophilicity (octanol/water partition coefficient, log P(ow)), and did not depend on their van der Waals' volumes. Hydrophobic organic nitroesters and hydrophilic N-nitramines compete for the same binding site in the reduced enzyme form. The role of the hydrophobic interaction of PETNR with glycerol trinitrate was supported by the positive dependence of glycerol trinitrate reactivity on the solution ionic strength. The discrimination of nitroesters and N-nitramines according to their log P(ow) values seems to be a specific feature of the Old Yellow Enzyme family of flavoenzymes.

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.

New approach to the fungal decontamination of wheat used for wheat sprouts: effects of aminolevulinic acid.

Nowadays, there is a growing interest in natural, minimally processed, nutritional and healthy foods. Sprouted seeds can be offered as natural nutritive products. Regrettably, existing seed decontamination technologies are limited and have specific disadvantages. 5-aminolevulinic acid (5-ALA) as a novel and effective tool for wheat decontamination from microfungi is proposed in this work. Inhibition of wheat with 5-ALA revealed a drastically suppressed development of microfungi. Studies of wheat germination characteristics showed that 5-ALA stimulates the growth of wheat seedlings and roots without impairing the vigor of germination and the viability of seeds. 5-ALA also induces either marginal or significant activities of antioxidant enzymes which can be associated with enhanced cellular capacity to detoxify reactive oxygen species. The results indicate that 5-ALA application may be an effective, environmentally friendly and inexpensive technology to be used in producing sprouts for human consumption.

Reduction of nitroaromatic compounds by NAD(P)H:quinone oxidoreductase (NQO1): the role of electron-accepting potency and structural parameters in the substrate specificity.

We aimed to elucidate the role of electronic and structural parameters of nitroaromatic compounds in their two-electron reduction by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The multiparameter regression analysis shows that the reactivity of nitroaromatic compounds (n=38) increases with an increase in their single-electron reduction potential and the torsion angle between nitrogroup(s) and the aromatic ring. The binding efficiency of nitroaromatics in the active center of NQO1 exerted a less evident role in their reactivity. The reduction of nitroaromatics is characterized by more positive entropies of activation than the reduction of quinones. This points to a less efficient electronic coupling of nitroaromatics with the reduced isoalloxazine ring of FAD, and may explain their lower reactivity as compared to quinones. Another important but poorly understood factor enhancing the reactivity of nitroaromatics is their ability to bind at the dicumarol/quinone binding site in the active center of NQO1.

FAD semiquinone stability regulates single- and two-electron reduction of quinones by Anabaena PCC7119 ferredoxin:NADP+ reductase and its Glu301Ala mutant.

Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.

Enzymatic redox reactions of the explosive 4,6-dinitrobenzofuroxan (DNBF): implications for its toxic action.

With an aim to understand the toxicity mechanisms of the explosive 4,6-dinitro- benzofuroxan (DNBF), we studied its single-electron reduction by NADPH:cytochrome P450 reductase and ferredoxin:NADP(+) reductase, and two- electron reduction by DT-diaphorase and Enterobacter cloacae nitroreductase. The enzymatic reactivities of DNBF and another explosive 2,4,6-trinitrotoluene (TNT) were similar, except for the much lower reactivity of DNBF towards nitroreductase. DNBF was less cytotoxic in FLK cells than TNT. However, their action shared the same mechanisms, oxidative stress and activation by DT-diaphorase. The lower cytotoxicity of DNBF may be explained by the negative electrostatic charge of its adduct with water which may impede cellular membrane penetration, and by the formation of its less reactive adducts with intracellular reduced glutathione.

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.

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.