Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress

It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones including a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E(1)7), reaching a limiting value at E(1)7> or =-0.1V. The reactivity of quinones towards NQO1 did not depend on their E(1)7. The cytotoxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E(1)7. The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of aziridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitor of NQO1, dicumarol, protected against the toxicity of aziridinyl-benzoquinones except of 2,5-bis-(2'-hydroxyethylamino)-3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate that in addition to the activation by NQO1, the oxidative stress presumably initiated by single-electron transferring enzymes may be an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs.     

Effect of dicumarol,a Nad(P)h: quinone acceptor oxidoreductase 1 (DT-diaphorase) inhibitor on ubiquinone redox cycling in cultured rat hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 μM dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro , was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 μM dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Effect of Dicumarol,a NAD(P)H: Quinone Acceptor Oxidoreductase 1 (DT-diaphorase) Inhibitor on Ubiquinone Redox Cycling in Cultured Rat Hepatocytes

Ubiquinol is considered to serve as an endogenous antioxidant. However, the mechanism by which the redox state of intracellular ubiquinone (UQ) is maintained is not well established. The effect of dicumarol, an inhibitor of NAD(P)H: quinone acceptor oxidoreductase 1 (NQO1=DT-diaphorase, EC 1.6.99.2), on the reduction of UQ in cultured rat hepatocytes was investigated in order to clarify whether or not NQO1 is involved in reducing intracellular UQ. A concentration of 5 &#119 M dicumarol, which does not inhibit cytosolic NADPH-dependent UQ reductase in vitro, was observed to almost completely inhibit NQO1 and thereby to stimulate cytotoxicity of 2-methyl-1,4-naphthoquinone (menadione) in cultured rat hepatocytes. However, 5 &#119 M dicumarol did not inhibit reduction of endogenous UQ-9, as well as exogenous UQ-10 added to the hepatocytes. In addition, it did not stimulate the formation of thiobarbituric acid reactive substances (TBARS) in the hepatocytes. These results suggested that NQO1 is not involved in maintaining UQ in the reduced state in the intact liver cells.     

Redox properties of novel antioxidant 5,8-Dihydroxycoumarin: implications for its prooxidant cytotoxicity

The aim of this work was to characterize the redox properties of the new antioxidant 5,8-dihydroxycoumarin (5,8-DHC), isolated from sweet grass (Hierochlo? odorata L.), and to determine its impact on its cytotoxic action. Reversible electrochemical oxidation of 5,8-DHC at pH 7.0 was characterized by the midpoint potential (E(p/2)) of 0.23 V vs. the normal hydrogen electrode. 5,8-DHC was slowly autoxidized at pH 7.0, and it was active as a substrate for peroxidase (POD, EC 1.11.1.7) and tyrosinase (TYR, EC 1.14.18.1). Oxidation of 5,8-DHC by POD/H202 yielded the product(s) which reacted with reduced glutathione and supported the oxidation of NADPH by ferredoxin:NADP+ reductase (FNR, EC 1.18.1.2) and NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). The concentration of 5,8-DHC for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (60 +/- 5.5) microM. Cytotoxicity of 5,8-DHC was decreased by desferrioxamine, catalase, the antioxidant N,N'-diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea and dicumarol, an inhibitor of NQO1. This shows that 5,8-DHC possesses the oxidative stress-type cytotoxicity, evidently due to the action of quinodal oxidation product(s). The protective effect of isoniazide, an inhibitor of cytochrome P-450 2E1, points to hydroxylation of 5,8-DHC as additional toxification route, whereas the potentiating effect of 3,5-dinitrocatechol, an inhibitor of catechol-o-methyltransferase (COMT, EC 2.1.1.6), points to the o-methylation of hydroxylation products as the detoxification route.     

Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.     

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.     

Role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by aziridinylbenzoquinones RH1 and MeDZQ

We aimed to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1) in apoptosis induction by antitumour quinones RH1 (2,5-diaziridinyl-3-hydroxymethyl-6-methyl-1,4-benzoquinone) and MeDZQ (2,5-dimethyl-3,6-diaziridinyl-1,4-benzoquinone). Digitonin-permeabilized FLK cells catalyzed NADPH-dependent single- and two-electron reduction of RH1 and MeDZQ. At equitoxic concentrations, RH1 and MeDZQ induced apoptosis more efficiently than the nonalkylating duroquinone or H(2)O(2). The antioxidant N,N'-diphenyl-p-phenylene diamine, desferrioxamine, and the inhibitor of NQO1 dicumarol, protected against apoptosis induction by all compounds investigated, but to a different extent. The results of multiparameter regression analysis indicate that RH1 and MeDZQ most likely induce apoptosis via NQO1-linked formation of alkylating species but not via NQO1-linked redox cycling.     

Fungal pathogen-induced changes in the antioxidant systems of leaf peroxisomes from infected tomato plants

Peroxisomes, being one of the main organelles where reactive oxygen species (ROS) are both generated and detoxified, have been suggested to be instrumental in redox-mediated plant cell defence against oxidative stress. We studied the involvement of tomato (Lycopersicon esculentum Mill.) leaf peroxisomes in defence response to oxidative stress generated upon Botrytis cinerea Pers. infection. The peroxisomal antioxidant potential expressed as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione peroxidase (GSH-Px, EC 1.11.1.19) as well as the ascorbate-glutathione (AA-GSH) cycle activities was monitored. The initial infection-induced increase in SOD, CAT and GSH-Px indicating antioxidant defence activation was followed by a progressive inhibition concomitant with disease symptom development. Likewise, the activities of AA-GSH cycle enzymes: ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) as well as ascorbate and glutathione concentrations and redox ratios were significantly decreased. However, the rate and timing of these events differed. Our results indicate that B. cinerea triggers significant changes in the peroxisomal antioxidant system leading to a collapse of the protective mechanism at advanced stage of infection. These changes appear to be partly the effect of pathogen-promoted leaf senescence.     

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.     

Quantitative structure-activity relationships in prooxidant cytotoxicity of polyphenols: role of potential of phenoxyl radical/phenol redox couple

The aim of this work was to characterize the role of the potential of phenoxyl radical/phenol redox couple, E(7)(2), in the cytotoxicity of polyphenols. The cytotoxicity of polyphenols in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK), and human promyelocytic leukemia cells (line HL-60) was partly inhibited by catalase, by the antioxidant N,N'-diphenyl-p-phenylene diamine and desferrioxamine, and potentiated by 1,3-bis-(2-chloro-ethyl)-1-nitrosourea, thus showing its prooxidant character. Dapsone, an inhibitor of myeloperoxidase, did not affect the cytotoxicity of polyphenols in HL-60 cells, whereas dicumarol, an inhibitor of DT-diaphorase, showed controversial effects on their cytotoxicity in FLK cells. Inhibitors of cytochromes P-450, alpha-naphthoflavone and izoniazide, decreased the cytotoxicity of several polyphenols, whereas 3,5-dinitrocatechol, an inhibitor of catechol-o-methyltransferase (COMT), increased it. The cytotoxicity of 13 polyhydroxybenzenes was described by the equations: logcL50 (microM) = -0.67 + 5.46E(7)(2) (V) - 0.16 logD (FLK), and logcL50 (microM) = -1.39 + 6.90E(7)(2) (V) - 0.20logD (HL-60), where cL50 is compound concentration for 50% cell survival, and D is octanol/water distribution coefficient at pH 7.0. The flavonoids comprise a separate series of compounds with lower cytotoxicity. The correlations obtained quantitatively confirm the parallelism between the polyphenol cytotoxicity and the rates of their single-electron oxidation, and point to the leading role of formation of the reactive oxygen species in their cytotoxicity. Depending on the examined system, this parallelism may be distorted due to the cytochrome P-450 and COMT-catalyzed transformation of polyphenols.     

Phenobarbital-induced cytoprotective mechanisms in menadione metabolism: the role of glutathione reductase and DT-diaphorase.

1. Treatment with N,N-bis (2-chloroethyl)-N-nitrosourea (BCNU) (80 microM) led to decreases in cell viability in both naive and sodium phenobarbital (PB) induced hepatocytes. 2. Dicumarol (30 microM) selectively increased the cytotoxicity of menadione in hepatocytes isolated from naive vs PB-pretreated rats. 3. Inclusion of both BCNU and dicumarol to the incubation medium abolished the characteristic concentration-response curves of the hepatocytes for menadione. 4. A greater proportion of menadione was metabolized by DT-diaphorase in the hepatocytes isolated from PB-pretreated rats. 5. The role of glutathione reductase vs DT-diaphorase in mitigating menadione-cytotoxicity in the naive vs PB-induced hepatocyte is discussed.     

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.     

Evaluation of reactive oxygen species involvement in amiodarone pulmonary toxicity in vivo and in vitro

Amiodarone (AM) is an effective antidysrhythmic agent, restricted in use by the development of adverse effects, including potentially fatal AM-induced pulmonary toxicity (AIPT). Although the pathogenesis of AIPT is unknown, an oxidant mechanism has been proposed. The present study evaluated the role of reactive oxygen species (ROS) in AM-induced toxicity. The effect of inhibiting lung antioxidant defense on in vivo development of AIPT was evaluated in hamsters. Lung glutathione reductase activity was inhibited by 66%, 6 hours following administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (20 mg/kg i.p.). When AM (1.83 μmol) was administered intratracheally 6 hours after BCNU, toxicity was enhanced, as indicated by lung hydroxyproline content and histological evaluation 21 days later. However, BCNU treatment did not affect AM-induced alterations in lung glutathione, suggesting that the increased toxicity was not due to decreased antioxidant capacity following BCNU. The effect of BCNU on AM cytotoxicity in vitro was evaluated using rabbit lung alveolar macrophages. Incubation with 5 μM BCNU for 2 hours caused greater than 95% inhibition of glutathione reductase activity. However, BCNU treatment had no effect on 146 μM AM-induced cytotoxicity, as assessed by lactate dehydrogenase latency following 12 hours of incubation. Rabbit macrophages loaded with 2′,7′-dichlorofluorescin, which is oxidized by ROS to fluorescent 2′,7′-dichlorofluorescein (DCF), were used to evaluate ROS generation by AM. Incubation of macrophages with AM (73 or 146 μM) for 1 hour, with or without the catalase inhibitor sodium azide (1 mM), did not result in DCF formation. Overall, these results do not support the hypothesis that AIPT is due to ROS action. © 1997 John Wiley & Sons, Inc.     

Effect of ascorbic acid on DNA damage, cytotoxicity, glutathione reductase, and formation of paramagnetic chromium in Chinese hamster V-79 cells treated with sodium chromate(VI).

The effect of pretreatment with ascorbic acid (vitamin C) on chromate-induced DNA damage, cytotoxicity, and enzyme inhibition as well as on the cellular reduction of chromium(VI) was investigated using Chinese hamster V-79 cells. Cellular pretreatment with nontoxic levels of 1 mM ascorbic acid for 24 h prior to exposure resulted in a significant increase (1.7-fold) in cellular levels of this vitamin. Alkaline elution assays demonstrated that this pretreatment decreased cellular levels of Na2CrO4-induced alkali-labile sites while the numbers of DNA-protein crosslinks produced by chromate increased. In colony-forming assays, pretreatment with ascorbic acid enhanced the cytotoxicity of chromate. However, the inhibition of glutathione reductase attributed to Na2CrO4 was attenuated by this pretreatment. Under the same experimental condition, the uptake of chromate in pretreated cells was found to increase. ESR studies revealed that cellular pretreatment with ascorbic acid reduced the level of chromium(V) intermediate and increased the level of chromium(III) complex, indicating that cellular reduction of chromium(VI) to chromium(III) was accelerated by this vitamin. These results suggest that ascorbic acid decreases chromate-induced alkali-labile sites and chromium inhibition of glutathione reductase, but it enhances DNA-protein cross-links and cytotoxicity caused by this metal through its ability to directly reduce chromium(VI).     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

Modified responses of root growth and reactive oxygen species-scavenging system to combined salt and heat stress in transgenic rice

Root growth and modifications of ROS-scavenging systems were investigated in transgenic rice (Oryza sativa L., cv. Zhonghua no. 11) co-expressing glutathione-S-transferase (GST, EC. 2.5.1.18) and catalase 1 (CAT1, EC 1.11.1.6) and nontransgenic rice exposed to just salt or heat and their combination. The higher number of adventitious roots but the lower root to shoot ratio were observed in the stressed transgenics as compared with nontransgenics. Most antioxidant enzymes, such as CAT, GST, ascorbate peroxidase (APX, EC 1.11.1.11), glutathione reductase (GR, EC.1.6.4.2), dehydroascorbate reductase (DHAR, EC.1.8.5.1), and the redox state of glutathione and ascorbate, measured in the transformant roots, were significantly different from those in nontransformant roots following the three types of stress. The variations of root growth and antioxidant systems in the stressed transgenic rice may be attributed to not only the GST and CAT1 transgenes but also the coordination of the ascorbate-glutathione cycle.     

The role of oxidative processes in the cytotoxicity of substituted 1,4-naphthoquinones in isolated hepatocytes

In order to clarify the role of oxidative processes in cytotoxicity we have studied the metabolism and toxicity of 2-methyl-1,4-naphthoquinone (menadione) and its 2,3 dimethyl (DMNQ) and 2,3 diethyl (DENQ) analogs in isolated rat hepatocytes. The two analogs, unlike menadione, cannot alkylate nucleophiles directly and were considerably less toxic than menadione. This decreased toxicity was consistent with the inability of DMNQ and DENQ to alkylate but we also found them to undergo lower rates of redox cycling in hepatocytes and a higher ratio of two electron as opposed to one electron reduction relative to menadione. Thus, facile analysis of the respective roles of alkylation and oxidation in cytotoxicity was not possible using these compounds. In hepatocytes pretreated with bischloroethyl-nitrosourea (BCNU) to inhibit glutathione reductase, all three naphthoquinones caused a potentiation of reduced glutathione (GSH) removal/oxidized glutathione (GSSG) generation and cytotoxicity relative to that observed in control cells. These data show that inhibition of hepatocyte glutathione reductase by BCNU results in enhanced naphthoquinone-induced oxidative challenge and subsequent cellular toxicity. That DMNQ and DENQ are cytotoxic, albeit at high concentrations, and that this cytotoxicity is potentiated by BCNU pretreatment suggest that oxidative processes alone can be a determinant of cytotoxicity.     

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.