Antiplasmodial activity of nitroaromatic and quinoidal compounds: redox potential vs. inhibition of erythrocyte glutathione reductase.

Prooxidant nitroaromatic and quinoidal compounds possess antimalarial activity, which might be attributed either to their formation of reactive oxygen species or to their inhibition of antioxidant enzyme glutathione reductase (GR, EC 1.6.4.2). We have examined the activity in vitro against Plasmodium falciparum of 24 prooxidant compounds of different structure (nitrobenzenes, nitrofurans, quinones, 1,1'-dibenzyl-4,4'-bipyridinium, and methylene blue), which possess a broad range of single-electron reduction potentials (E(1)(7)) and erythrocyte glutathione reductase inhibition constants (K(i(GR))). For a series of homologous derivatives of 2-(5'-nitrofurylvinyl)quinoline-4-carbonic acid, the relationship between compound K(i(GR)) and concentration causing 50% parasite growth inhibition (IC(50)) was absent. For all the compounds examined in this study, the dependence of IC(50) on their K(i(GR)) was insignificant. In contrast, IC(50) decreased with an increase in E(1)(7) and positive electrostatic charge of aromatic part of molecule (Z): log IC(50) (microM) = -(0.9846 +/- 0.3525) - (7.2850 +/- 1.2340) E(1)(7) (V) - (1.1034 +/- 0.1832) Z (r(2) = 0.8015). The redox cycling activity of nitroaromatic and quinoidal compounds in ferredoxin:NADP(+) reductase-catalyzed reaction and the rate of oxyhemoglobin oxidation in lysed erythrocytes increased with an increase in their E(1)(7) value. Our findings imply that the antiplasmodial activity of nitroaromatic and quinoidal compounds is mainly influenced by their ability to form reactive oxygen species, and much less significantly by the GR inhibition.

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 (DeltaHf(ArNO(2)(-*)), 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 DeltaHf(ArNO(2)(-*)). 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 DeltaHf(ArNO(2)(-*)). 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.

Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin.

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.

Quantitative structure-activity relationships in two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H:nitroreductase.

Enterobacter cloacae NAD(P)H:nitroreductase (NR; EC 1.6.99.7) catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) to 10(7) M(-1) s(-1). In agreement with a previously proposed scheme of two-step four-electron reduction of nitroaromatics by NR (Koder, R. L., and Miller, A.-F. (1998) Biochim. Biophys. Acta 1387, 395-405), 2 mol NADH per mole mononitrocompound were oxidized. An oxidation of excess NADH by polinitrobenzenes, including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of "redox cycling" was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The initial reduction of tetryl and other polinitrophenyl-N-nitramines by E. cloacae NR was analogous to a two-step four-electron reduction mechanism of TNT and other nitroaromatics. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationship shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Methemoglobin formation in human erythrocytes by nitroaromatic explosives.

We have examined the structure-activity relationships in methemoglobin (MetHb) formation by high explosives 2,4,6-trinitrotoluene (TNT), 2,4,6-trinitrophenyl-N-nitramine (tetryl) and 2,4,6-trinitrophenyl-N-nitraminoethylnitrate (pentryl), and a number of model nitrobenzenes. In lysed human erythrocytes the rate constants of oxyhemoglobin (OxyHb) oxidation increased with an increase in single-electron reduction potential (E(1)7) or with a decrease of the enthalpies of single-electron reduction of nitroaromatics. Tetryl and pentryl oxidized OxyHb almost 3 times faster than TNT. Although the initial rates of MetHb formation in intact erythrocytes by tetryl, pentryl, and TNT matched their order of reactivity in the oxidation of OxyHb in lysed erythrocytes, TNT was a more efficient MetHb forming agent than tetryl and pentryl during a 24-h incubation. The decreased efficiency of tetryl and pentryl was attributed to their reaction with intraerythrocyte reduced glutathione (GSH) producing 2,4,6-trinitrophenyl-Sglutathione, which acted as a less efficient OxyHb oxidizing agent.

Inhibition of phthalocyanine-sensitized photohemolysis of human erythrocytes by polyphenolic antioxidants: description of quantitative structure-activity relationships.

Polyphenolic antioxidants protected against Al-phthalocyanine tetrasulfonate-sensitized photohemolysis of human erythrocytes. A quantitative structure-activity relationship has been obtained to describe the protective effects of di- and trihydroxybenzenes: log cI(50) (microM)=(1.8620+/-1.5565)+(3.6366+/-2.8245) E(1)(7) (V)-(0. 4034+/-0.0765) log P (r(2)=0.8367), where cI(50) represents the concentrations of compounds for the 2-fold increase in the lag-phase of hemolysis, E(1)(7) represents the compound single-electron oxidation potential, and P represents the octanol/water partition coefficient. The cI(50) for quercetin and taxifolin were close, and cI(50) for morin, kaempferol and hesperetin were lower than might be predicted by this equation. The protection from hemolysis by azide, a quencher of singlet oxygen ((1)O(2)) was accompanied by increase in cI(50) of polyphenols, indicating that azide and polyphenols competed for the same damaging species, (1)O(2). These findings point out to two factors, determining the protective efficiency of polyphenols against (1)O(2), namely, ease of electron donation and lipophilicity.

Regioselectivity and reversibility of the glutathione conjugation of quercetin quinone methide.

The chemical reactivity, isomerization, and glutathione conjugation of quercetin o-quinone were investigated. Tyrosinase was used to generate the unstable quercetin o-quinone derivative which could be observed upon its subsequent scavenging by glutathione. Identification of the products revealed formation of 6-glutathionyl-quercetin and 8-glutathionyl-quercetin adducts. Thus, in particular, glutathione adducts in the A ring of quercetin were formed, a result which was not expected a priori. Quantum mechanical calculations support the possibility that the formation of these glutathione adducts can be explained by an isomerization of quercetin o-quinone to p-quinone methides. Surprisingly, additional experiments of this study reveal the adduct formation to be reversible, leading to interconversion between the two quercetin glutathione adducts and possibilities for release and further electrophilic reactions of the quercetin quinone methide at cellular sites different from those of its generation.

Two-electron reduction of nitroaromatic compounds by Enterobacter cloacae NAD(P)H nitroreductase: description of quantitative structure-activity relationships.

Enterobacter cloacae NAD(P)H:nitroreductase catalyzes the reduction of a series of nitroaromatic compounds with steady-state bimolecular rate constants (kcat/Km) ranging from 10(4) M(-1) s(-1) to 10(7) M(-1) s(-1), and oxidizing 2 moles NADH per mole mononitrocompound. Oxidation of excess NADH by polynitrobenzenes including explosives 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), has been observed as a slower secondary process, accompanied by O2 consumption. This type of 'redox cycling' was not related to reactions of nitroaromatic anion-radicals, but was caused by the autoxidation of relatively stable reaction products. The logs kcat/Km of all the compounds examined exhibited parabolic dependence on their enthalpies of single-electron- or two-electron (hydride) reduction, obtained by quantum mechanical calculations. This type of quantitative structure-activity relationships shows that the reactivity of nitroaromatics towards E. cloacae nitroreductase depends mainly on their hydride accepting properties, but not on their particular structure, and does not exclude the possibility of multistep hydride transfer.

Quantitative structure activity relationships for the electron transfer reactions of Anabaena PCC 7119 ferredoxin-NADP+ oxidoreductase with nitrobenzene and nitrobenzimidazolone derivatives: mechanistic implications.

The steady state single electron reduction of polynitroaromatics by ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) from cyanobacterium Anabaena PCC 7119 has been studied and quantitative structure activity relationships are described. The solubility of the polynitroaromatics as well as their reactivity towards ferredoxin-NADP+ oxidoreductase are markedly higher than those for previously studied mononitroaromatics and this enabled the independent measurement of the kinetic parameters-k(cat) and Km. Interestingly, the natural logarithm of the bimolecular rate constant, k(cat)/Km, and also the natural logarithm of k(cat) correlate with the calculated energy of the lowest unoccupied molecular orbital of the polynitroaromatic substrates. The minimal kinetic model in line with these quantitative structure activity relationships is a ping-pong mechanism which includes substrate binding equilibria in the second half reaction.

Enzymatic reduction studies of nitroheterocycles.

The nitroimidazole derivative Megazol is a highly active compound used against several strains of Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanomiasis). With the aim of gaining an insight into the probable mode of action, the interaction of Megazol with different redox enzymes was studied in comparison to that of Nifurtimox and Metronidazole. The three nitroaromatic compounds are reduced by L-lactate cytochrome c-reductase, adrenodoxin reductase, and NADPH:cytochrome P-450 reductase (EC 1.6.2.4), the efficiencies of the enzymatic reductions being roughly related to the reduction potentials of these pseudo-substrates. As the enzyme responsible for the reduction of Megazol within the parasite has not yet been identified, the nitroimidazole was assayed with T. cruzi lipoamide dehydrogenase and trypanothione reductase. Megazol did not inhibit the physiological reactions but proved to be a weak substrate of both flavoenzymes. The single electron reduction of the compound by NADPH:cytochrome P-450 reductase, by rat liver as well as by trypanosome microsomes was confirmed by ESR experiments. As shown here, Megazol interferes with the oxygen metabolism of the parasite, but its extra activity when compared to Nifurtimox may be related to other features not yet identified.

Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product.

In the last ten years, there has been an important increase in interest in quercetin action as a unique antioxidant, but its putative role in numerous prooxidant effects is also being continually updated. The mechanism underlying this undesirable ability seems to involve its metabolic oxidoreductive activation. Based on the structural properties of quercetin, we have investigated whether its catechol moiety may be the potential tool for revealed toxicity. We demonstrated, with an ESR spin-stabilization technique coupled to conventional spectrophotometry, that o-semiquinone and o-quinone are indeed the products of enzymatically catalyzed oxidative degradation of quercetin. The former radical might serve to facilitate the formation of superoxide and depletion of GSH, which could confer a specificity of its prooxidative action in situ. The observed one-electron reduction of o-quinone may enrich the semiquinone pool, thereby magnifying its effect. The two-electron reduction of quinone can result in constant resupply of quercetin in situ, thereby also modulating another pathway of its known biological activities. We have also tried to see whether the intracellular oxidative degradation of quercetin can be confirmed under the controlled conditions of model monolayer cell cultures. The results are indicative of the intracellular metabolic activation of quercetin to o-quinone, the process which can be partially associated with the observed concentration-dependent cytotoxic effect of quercetin.

Role of redox cycling and activation by DT-diaphorase in the cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB-1954) and its analogs.

In tumor cell lines with high content of DT-diaphorase (EC 1.6.99.2), the cytotoxicity of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB-1954) and its derivatives is exerted through DT-diaphorase-catalyzed formation of crosslinking species. However, little is known about other possible mechanisms of CB-1954 action. We have examined the toxicity of CB-1954 and its derivatives to bovine leukemia virus-transformed lamb fibroblasts (line FLK), which possessed moderate DT-diaphorase activity, 260 units/mg protein. The action of these compounds was accompanied by lipid peroxidation, their toxicity was decreased by desferrioxamine and antioxidant N,N'-diphenyl-p-phenylene diamine (DPPD), but, in most cases, not by dicumarol, an inhibitor of DT-diaphorase. Using multiparameter regression analysis, we have found that the toxicity of CB-1954 derivatives as well as that of several non-alkylating nitroaromatics, increased upon the increase in their single-electron reduction potential (E(1)7) and octanol/water partition coefficient (P), and almost did not depend on their reactivity towards DT-diaphorase. It seems that in cell lines with a moderate amount of DT-diaphorase, the toxicity of CB- 1954 and its analogs is exerted through their redox cycling.

Prooxidant toxicity of polyphenolic antioxidants to HL-60 cells: description of quantitative structure-activity relationships.

Polyphenolic antioxidants exhibited a dose-dependent toxicity against human promyelocytic leukemia cells (HL-60). Their action was accompanied by malondialdehyde formation, and was partly prevented by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine. This points to a prooxidant character of their cytotoxicity. A quantitative structure-activity relationship (QSAR) has been obtained to describe the cytotoxicity of 13 polyphenolic antioxidants belonging to three different groups (flavonoids, derivatives of gallic and caffeic acid): log cL50 (microM) = (2.7829+/-0.2339)+(1.2734+/-0.4715) Ep/2 (V)-(0.3438+/-0.0582) log P (r2 = 0.8129), where cL50 represents the concentration for 50% cell survival, Ep/2 represents the voltammetric midpoint potential, and P represents the octanol/water partition coefficient. Analogous QSARs were obtained using enthalpies of single-electron oxidation of these compounds, obtained by quantum-mechanical calculations. These findings clearly point to two important characteristics determining polyphenol cytotoxicity, namely their ease of oxidation and their lipophilicity.

Nitroreductase reactions of Arabidopsis thaliana thioredoxin reductase.

Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) catalyzed redox cycling of aromatic nitrocompounds, including the explosives 2,4,6-trinitrotoluene and tetryl, and the herbicide 3,5-dinitro-o-cresol. The yield of nitro anion radicals was equal to 70-90%. Redox cycling of tetryl was accompanied by formation of N-methylpicramide. Bimolecular rate constants of nitroaromatic reduction (kcat/Km) and reaction catalytic constants (kcat) increased upon an increase in oxidant single-electron reduction potential (E(1)7). Using compounds with an unknown E(1)7 value, the reactivity of TR increased parallelly to the increase in reactivity of ferredoxin:NADP+ reductase of Anabaena PCC 7119 (EC 1.18.1.2). This indicated that the main factor determining reactivity of nitroaromatics towards TR was their energetics of single-electron reduction. Incubation of reduced TR in the presence of tetryl or 2,4-dinitrochlorobenzene resulted in a loss of thioredoxin reductase activity, most probably due to modification of reduced catalytic disulfide, whereas nitroreductase reaction rates were unchanged. This means that on the analogy of quinone reduction by TR (D. Bironaite, Z. Anusevicius, J.-P. Jacquot, N. Cenas, Biochim. Biophys. Acta 1383 (1998) 82-92), FAD and not catalytic disulfide of TR was responsible for the reduction of nitroaromatics. Tetryl, 2,4,6-trinitrotoluene and thioredoxin increased the FAD fluorescence intensity of TR. This finding suggests that nitroaromatics may bind close to the thioredoxin-binding site at the catalytic disulfide domain of TR, and induce a conformational change of enzymes (S.B. Mulrooney, C.H. Williams Jr., Protein Sci. 6 (1997) 2188-2195). Our data indicate that certain nitroaromatic herbicides, explosives and other classes of xenobiotics may interfere with the reduction of thioredoxin by plant TR, and confer prooxidant properties to this antioxidant enzyme.

DT-diaphorase catalyzes N-denitration and redox cycling of tetryl.

Rat liver DT-diaphorase (EC 1.6.99.2) catalyzed reductive N-denitration of tetryl (2,4,6-tri-nitrophenyl-N-methylnitramine) and 2,4-dinitrophenyl-N-methylnitramine, oxidizing the excess of NADPH. The reactions were accompanied by oxygen consumption and superoxide dismutase-sensitive reduction of added cytochrome c and reductive release of Fe2+ from ferritin. Quantitatively, the reactions of DT-diaphorase proceeded like single-electron reductive N-denitration of tetryl by ferredoxin:NADP+ reductase (EC 1.18.1.2) (Shah, M.M. and Spain, J.C. (1996) Biochem. Biophys. Res. Commun. 220, 563-568), which was additionally checked up in this work. Thus, although reductive N-denitration of nitrophenyl-N-nitramines is a net two-electron (hydride) transfer process, DT-diaphorase catalyzed the reaction in a single-electron way. These data point out the possibility of single-electron transfer steps during obligatory two-electron (hydride) reduction of quinones and nitroaromatics by DT-diaphorase.

Prooxidant character of flavonoid cytotoxicity: structure-activity relationships.

The action of flavonoids on bovine leukemia virus-transformed lamb fibroblasts (line FLK) and HL-60 cells was accompanied by lipid peroxidation, their toxicity was partly prevented by iron chelator desferrioxamine and antioxidant N,N'-diphenyl-p-phenylene diamine. This pointed out to the involvement of oxidative stress in flavonoid cytotoxicity. The concentration of compound for 50% survival of FLK cells (cL50) did not show correlation with polarographic oxidation half-peak potential (Ep/2) and/or partition coefficient (log P) of flavonoids; however, their toxicity to HL-60 cells was described by equation log cL50 (microM) = 3.0161 + 1.1099 Ep/2 (V) - 0.3369 log P. The toxicity of quercetin was partly prevented by nontoxic concentrations of other flavonoids examined, thus pointing out to potential neutralization of quercetin cytotoxicity by intake of flavonoid mixtures.

Modulation of erythrocyte photohemolysis rate by glutathione reductase inactivating alkylating agents.

In order to determine the role of glutathione reductase (GR) in protection against Alphtalocyanine tetrasulfonate-sensitized human erythrocyte photolysis, we have studied the effects of antitumour alkylating agents that inactivate GR, on photohemolysis rate. The rates of inactivation of reduced GR decreased in order BCNU > pharanox (N-p-[bis-(2-chloroethyl)-amino]-phenylacetic acid N-oxide) > phenalol (N-p-[bis-(2-chloroethyl)-amino]-phenylacetyl-L- phenylalanine) > o-F- and p-F-lophenal (o- and p-isomers of N-p-[bis-(2-chloroethyl)-amino]-phenylacetyl-D,L-fluorophenylalanine) > D,L-melphalan. As supposed, erythrocyte photolysis was accelerated by BCNU and pharanox, however, it was slowed down by phenylalanine mustards. The latter effect was explained by singlet oxygen quenching and/or photooxidation reactions of these compounds. This points out to a possibility of certain phenylalanine derivatives to neutralize the side-effects of photodynamic therapy.

Quantitative structure activity relationships for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by DT-diaphorase: implications for the kinetic mechanism.

Quantitative structure activity relationships (QSARs) for the conversion of nitrobenzimidazolones and nitrobenzimidazoles by rat liver DT-diaphorase (EC 1.6.99.2) are described. The parameter used for description of the QSARs is the energy of the lowest unoccupied molecular orbital (E(LUMO)) of the nitroaromatic compounds. Interestingly, correlations with E(LUMO) were observed for both the natural logarithm of kcat, but also for the natural logarithm of kcat/Km. The minimal kinetic model in line with these QSARs is a ping-pong mechanism that includes a substrate binding equilibrium in the second half reaction.

Interaction of quinones with Arabidopsis thaliana thioredoxin reductase.

In view of the ubiquitous role of the thioredoxin/thioredoxin reductase (TRX/TR) system in living cells, the interaction of Arabidopsis thaliana NADPH-thioredoxin reductase (EC 1.6.4.5) with quinones, an important class of redox cycling and alkylating xenobiotics, was studied. The steady-state reactions of A. thaliana TR with thioredoxin (TRX) and reaction product NADP+ inhibition patterns were in agreement with a proposed model of E. coli enzyme (B.W. Lennon, C.H. Williams, Jr., Biochemistry, vol. 35 (1996), pp. 4704-4712), that involved enzyme cycling between four- and two-electron reduced forms with FAD being reduced. Quinone reduction by TR proceeded via a mixed single- and two-electron transfer, the percentage of single-electron flux being equal to 12-16%. Bimolecular rate constants of quinone reduction (kcat/km) and reaction catalytic constants (kcat) increased upon an increase in quinone single-electron reduction potential. E(1)7. In several cases, the kcat of quinone reduction exceeded kcat of TRX reduction, suggesting that quinones intercepted electron flux from TR to TRX. Incubation of reduced TR with alkylating quinones resulted in a rapid loss of TRX-reductase activity, while quinone reduction rate was unchanged. In TRX-reductase and quinone reductase reactions of TR, NADP+ exhibited different inhibition patterns. These data point out that FAD and not the catalytic disulfide of TR is responsible for quinone reduction, and that quinones may oxidize FADH2 before it reduces catalytic disulfide. Most probably, quinones may oxidize the two-electron reduced form of TR, and the enzyme may cycle between two-electron reduced and oxidized forms in this reaction. The relatively high rate of quinone reduction by A. thaliana thioredoxin reductase accompanied by their redox cycling, confers pro-oxidant properties to this antioxidant enzyme. These factors make plant TR an attractive target for redox active and alkylating pesticide action.

Nitrobenzimidazoles as substrates for DT-diaphorase and redox cycling compounds: their enzymatic reactions and cytotoxicity.

We have synthesized a number of nitrobenzimidazoles containing nitro groups in the benzene ring and found that they acted as relatively efficient substrates for rat liver DT-diaphorase (EC 1.6.99.2), their reactivity exceeding reactivities of nitrofurans and nitrobenzenes. Nitrobenzimidazoles were competitive with NADPH inhibitors of DT-diaphorase in menadione reductase reactions, their inhibition constant being unchanged in the presence of dicumarol and being increased in the presence of 2',5'-ADP. These data indicate that the poor reactivity of nitrobenzimidazoles and other nitroaromatics in comparison to quinones could be determined by their binding in the adenosine-phosphate binding region of the NADPH-binding site, whereas quinones bind at the nicotinamide-binding pocket at the vicinity of FAD of DT-diaphorase. The reduction of 4,5,6-trinitrobenzimidazol-2-one by DT-diaphorase most probably involves reduction of 5-nitro group to 5-nitroso or 5-hydroxylamine derivative at the initial step. A certain parallelism existed between reactivities of nitrobenzimidazoles toward DT-diaphorase and their reactivities in single-electron reduction by Anabaena ferredoxin:NADP+ reductase (EC 1.18.1.2) and Saccharomyces cerevisiae flavocytochrome b2 (EC 1.1.2.3), the latter being determined by electronic factors. However, we suppose that the relatively high reactivity of polinitrobenzimidazoles toward DT-diaphorase was due not only to electronic effects, but also to a sterical crowding of nitrogroups by each other. The toxicity of nitrobenzimidazoles to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) with a moderate amount of DT-diaphorase (260 U/mg protein) is partly prevented by dicumarol. That points out to partial determination of nitrobenzimidazole cytotoxicity by their reduction by DT-diaphorase. Another important factor of nitrobenzimidazole toxicity to this cell line was oxidative stress, catalyzed by single-electron transferring enzymes.