Nitric oxide promotes strong cytotoxicity of phenolic compounds against Escherichia coli: the influence of antioxidant defenses

The induction of mutagenic and cytotoxic effects by simple phenolics, including catechol (CAT), 3,4-dihydroxyphenylacetic acid (DOPAC), hydroquinone (HQ), and 2,5-dihydroxyphenylacetic (homogentisic) acid (HGA), appears to occur through an oxidative mechanism based on the ability of these compounds to undergo autoxidation, leading to quinone formation with the production of reactive oxygen species. This is supported by the detection of such adverse effects in plate assays using Escherichia coli tester strains deficient in the OxyR function, but not in OxyR(+) strains. The OxyR protein is a redox-sensitive regulator of genes encoding antioxidant enzymes including catalase and alkyl hydroperoxide reductase, which would eliminate hydrogen peroxide. Methyl-substituted phenolics such as 4-methylcatechol (MCAT) and methylhydroquinone (MHQ) produced, in addition to oxidative toxicity, marked cytotoxic effects against OxyR(+) cells, thus revealing a mechanism of toxicity not mediated by hydrogen peroxide that could involve quinones and quinone methides arising from MCAT and MHQ oxidation. Quinone compounds could also be responsible for the enhanced cytotoxicity of certain phenolics when combined with a nitric oxide (NO(*)) donor such as diethylamine/NO (DEA/NO). Phenolics scavenge NO(*) and, in turn, NO(*) oxidizes phenolics to form their quinone derivatives. In OxyR(+) cells, where the oxidative toxicity is inhibited, DEA/NO promoted exceptional increases in the cytotoxicity of CAT and 3,4-dihydroxycinnamic (caffeic) acid (CAF), which both exhibited very low oxidative cytotoxicity, as well as in that of MCAT, HQ, and MHQ. In contrast, DEA/NO failed to promote toxicity by DOPAC and HGA, probably due to their ability to undergo oxidative polymerization, leading to the formation of melanins. Spectroscopic studies demonstrated quinone generation from the oxidation of CAF, HQ, and MHQ by DEA/NO. The o-quinone derived from CAF was rather unstable and decomposed during its isolation. For the generation of toxic quinones, e.g., to be used as therapeutic agents producing antitumor or antibacterial effects, the isolation step could be avoided with the method proposed. It combines quinone precursors, i.e. phenolic compounds, with an oxidant such as NO(*).     

Toxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to cultured cancer cells

Toxicity of eight 22,23-dihydroxystigmastane derivatives (four pairs of (22R,23R)- and (22S,23S)-isomers differing in steroid backbone structure) to human breast carcinoma MCF-7 cells was compared. For every pair of structurally related compounds, (22R,23R) isomer was found to be significantly more toxic than (22S,23S) isomer. Computational analysis showed that side chain of (22R,23R)-22,23-dihydroxystigmastane derivatives is rigid, whereas that of (22S,23S)-isomers is rather flexible. Structure of steroid backbone significantly affects cytotoxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to human breast carcinoma MCF-7 cells, human ovary carcinoma CaOv cells, and human prostate carcinoma LnCaP cells. (22R,23R)-3β,22,23-trihydroxystigmast-5-ene and (22R,23R)-3β,22,23-trihydroxystigmast-5-en-7-one, both comprising equatorial 3β-hydroxyl group, exhibited the highest cytotoxicity, while the most polar 28-homobrassinolide and 28-homocastasterone, both comprising 2α,3α-dihydroxy groups, exhibited the lowest toxicity. Binding of (22R,23R)-22,23-dihydroxystigmastane derivatives to plasmatic membrane was suggested to be important for cytotoxicity.     

Quinone toxicity in hepatocytes without oxidative stress

The toxicity of quinones is believed to be mediated via redox cycling involving formation of semiquinone radicals which autoxidize to form active oxygen species. However, when the cytotoxicity of benzoquinones was compared using freshly isolated rat hepatocytes, benzoquinones which did not mediate oxidative stress were highly toxic. Thus, the benzoquinone analogs in decreasing order of cytotoxicity were 2-CH3-, 2-Br-, unsubstituted, 2,6-(CH3)2-, 2,5-(CH3)2-, and 2,3,5-(CH3)3-benzoquinone. Cellular thiols were rapidly depleted and glutathione (GSH) was converted to a quinone conjugate without oxidation to glutathione disulfide. No increase in cyanide-resistant respiration was observed and benzoquinone-induced cytotoxicity was not enhanced by inactivation of catalase or glutathione reductase. In contrast, duroquinone [2,3,5,6-(CH3)4-benzoquinone], which stimulated cyanide-resistant respiration and GSH oxidation, was only cytotoxic when catalase or glutathione reductase was inactivated. These results suggest that alkylation and/or oxidative stress may be important mechanisms in the cytotoxicity of benzoquinone derivatives.     

Taiwaniaquinoid and abietane quinone derivatives with trypanocidal activity against T. cruzi and Leishmania spp

The in vitro leishmanicidal (Leishmania infantum and Leishmania braziliensis) and trypanocidal (Trypanosoma cruzi) activities of different compounds were evaluated. These compounds, of vegetal origin but synthesised in our laboratory, included five taiwaniaquinoid derivatives (S-567; S-569; S-589; S-602 and A-246) and one abietane quinone (P-1). The in vitro activity of the compounds on extracellular and intracellular forms of the two Leishmania species and T. cruzi was assayed. Infectivity and cytotoxicity tests for the Leishmania species were conducted on J774.2 macrophage cells using Glucantime as the reference drug. From all the compounds assayed, the derivatives P-1>S-567 were more active and less toxic than Glucantime. Infection rates and amastigote means indicated that these two compounds were the most active in both Leishmania species. In the case of T. cruzi, the best derivatives were P-1 and S-567, at the same levels as for the Leishmania species. These compounds exhibited the most potent anti-proliferative activity against the extracellular vector form (the epimastigote), the extracellular host form (the trypomastigote), and the intracellular host form (the amastigote), with lower toxicity than that of the reference drug Benznidazole. Metabolite excretion studies showed that alterations mainly at the level of the mitochondria may explain observed metabolic changes in succinate and acetate production, perhaps due to the disturbance of enzymes involved in sugar metabolism within the mitochondrion. The in vivo studies for T. cruzi provided results consistent with those found in vitro. No signs of toxicity were detected in mice treated with the compounds tested, and the parasitic charge was slightly lower than in the control. The effects of these two compounds were also demonstrated with the change in the anti-T. cruzi antibody levels during the chronic stage.     

Cytotoxicity of quinone drugs on highly proliferative human leukemia T cells: reactive oxygen species generation and inactive shortened SOD1 isoform implications

Drugs containing the quinone group were tested on hyperproliferative leukemia T cells (HLTC: Jhp and Jws) and parental Jurkat cells. Doxorubicin, menadione and adaphostin produced different effects on these cell lines. Rapid doxorubicin-induced cell death in Jurkat cells was mediated by caspase activation. Doxorubicin-induced cell death of HLTCs was delayed due to the absence of caspase-3 and -8 expression. Delayed HLTC cell death was mediated and triggered by the generation of reactive oxygen species (ROS). Other drugs containing quinone groups, such as menadione and adaphostin, were also tested on HLTC and both were toxic by a caspase-independent mechanism. The toxicity of these drugs correlated with the generation of the superoxide anion, which increased and was more effective in HLTCs than in parental Jurkat cells. Accordingly, SOD1 activity was much lower in HLTCs than in Jurkat cells. This lower SOD1 activity in HLTCs was associated not only with the absence of the wild-type (16 kDa) SOD1 monomer but also with the presence of a shortened (14 kDa) SOD1 monomer isoform. Moreover, the cytotoxicity of drugs containing the quinone group was prevented by incubation with manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable superoxide dismutase mimetic and a potent inhibitor of oxidation. These findings could explain the sensitivity of HLTCs to drugs containing the quinone group using a mechanism dependent on oxidative stress. These observations can also be useful to target hyperproliferative leukemias that are resistant to the classical caspase-dependent apoptotic pathway.     

Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

Molecular mechanisms of quinone cytotoxicity

Quinones are probably found in all respiring animal and plant cells. They are widely used as anticancer, antibacterial or antimalarial drugs and as fungicides. Toxicity can arise as a result of their use as well as by the metabolism of other drugs and various environmental toxins or dietary constituents. In rapidly dividing cells such as tumor cells, cytotoxicity has been attributed to DNA modification. However the molecular basis for the initiation of quinone cytotoxicity in resting or non-dividing cells has been attributed to the alkylation of essential protein thiol or amine groups and/or the oxidation of essential protein thiols by activated oxygen species and/or GSSG. Oxidative stress arises when the quinone is reduced by reductases to a semiquinone radical which reduces oxygen to superoxide radicals and reforms the quinone. This futile redox cycling and oxygen activation forms cytotoxic levels of hydrogen peroxide and GSSG is retained by the cell and causes cytotoxic mixed protein disulfide formation. Most quinones form GSH conjugates which also undergo futile redox cycling and oxygen activation. Prior depletion of cell GSH markedly increases the cell's susceptibility to alkylating quinones but can protect the cell against certain redox cycling quinones. Cytotoxicity induced by hydroquinones in isolated hepatocytes can be attributed to quinones formed by autoxidation. The higher redox potential benzoquinones and naphthoquinones are the most cytotoxic presumably because of their higher electrophilicty and thiol reactivity and/or because the quinones or GSH conjugates are more readily reduced to semiquinones which activate oxygen.     

Quinoprotein Adducts Accumulate in the Substantia Nigra of Aged Rats and Correlate with Dopamine-Induced Toxicity in SH-SY5Y Cells

Parkinson’s disease (PD) is an age-dependent neurodegenerative disorder characterized by dopaminergic neuron loss in substantia nigra. Previous studies have implicated a role of dopamine oxidation in PD. Dopamine oxidation leads to the formation of dopamine quinone, which generates reactive oxygen species and covalently modifies cysteinyl proteins to form quinoprotein adduct. We compared quinoprotein adduct formation and lipid peroxidation in different brain regions of young and old rats. We found a prominent age-dependent accumulation of quinoprotein adducts in the substantia nigra, while no significant change of lipid peroxidation was detected in any brain regions of 2- to 15-month old rats. To determine whether quinoprotein adduct formation correlates with dopamine-induced cytotoxicity, we analyzed dopamine treated SH-SY5Y cells and found a strong correlation between quinoprotein adduct formation and cytotoxicity. Together, our results indicate that quinoprotein adduct formation may play a role in the age-dependent selective vulnerability of dopaminergic neurons in PD.     

The potent pro‐oxidant activity of rhododendrol–eumelanin induces cysteine depletion in B16 melanoma cells

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD), a skin‐whitening agent, is known to induce leukoderma in some people. To explore the mechanism underlying this effect, we previously showed that the oxidation of RD with mushroom or human tyrosinase produces cytotoxic quinone oxidation products. We then examined the metabolism of RD in B16F1 melanoma cells in vitro and detected RD‐pheomelanin and RD‐quinone bound to non‐protein and protein thiols. In this study, we examined the changes in glutathione (GSH) and cysteine in B16 cells exposed to RD for up to 24 h. We find that the levels of cysteine, but not those of GSH, decrease during 0.5‐ to 3‐h exposure, due to oxidation to cystine. This pro‐oxidant activity was then examined using synthetic melanins. Indeed, we find that RD‐eumelanin exerts a pro‐oxidant activity as potent as Dopa‐pheomelanin. GSH, cysteine, ascorbic acid, and NADH were oxidized by RD‐eumelanin with a concomitant production of H₂O₂. We propose that RD‐eumelanin induces cytotoxicity through its potent pro‐oxidant activity.     

Synthesis and protective effects of coumarin derivatives against oxidative stress induced by doxorubicin

The use of doxorubicin (DOX) in the treatment of solid tumors is limited by cardiotoxicity essentially due to oxidative stress generation. The aim of this study was to identify coumarin derivatives displaying a protective antioxidant activity without affecting DOX antitumoral efficiency. A set of eighteen coumarinic derivatives was synthesized. Their antioxidant power was evaluated in vitro with the FRAP (ferric reducing ability of plasma) method and in human breast adenocarcinoma MCF7 cells using H(2)DCFDA (2',7'-dichlorodihydrofluorescein diacetate) in a cytometric analysis. 4-Methyl-7,8-dihydroxycoumarin was found to exhibit an important antioxidant strength, a low cytotoxicity, and could decrease ROS (reactive oxygen species) production generated by DOX treatment without affecting DOX cytotoxicity in MCF7 cells.     

Metabolic activation of 3-hydroxyanisole by isolated rat hepatocytes

A tyrosinase-directed therapeutic approach for malignant melanoma therapy uses the depigmenting phenolic agents such as 4-hydroxyanisole (4-HA) to form cytotoxic o-quinones. However, renal and hepatic toxicity was reported as side effects in a recent 4-HA clinical trial. In search of novel therapeutics, the cytotoxicity of the isomers 4-HA, 3-HA and 2-HA were investigated. In the following, the order of the HAs induced hepatotoxicity in mice, as measured by increased in vivo plasma transaminase activity, or in isolated rat hepatocytes, as measured by trypan blue exclusion, was 3-HA > 2-HA > 4-HA. Hepatocyte GSH depletion preceded HA induced cytotoxicity and a 4-MC-SG conjugate was identified by LC/MS/MS mass spectrometry analysis when 3-HA was incubated with NADPH/microsomes/GSH. 3-HA induced hepatocyte GSH depletion or GSH depletion when 3-HA was incubated with NADPH/microsomes was prevented by CYP 2E1 inhibitors. Dicumarol (an NAD(P)H: quinone oxidoreductase inhibitor) potentiated 3-HA- or 4-methoxycatechol (4-MC) induced toxicity whereas sorbitol (an NADH generating nutrient) greatly prevented cytotoxicity indicating a quinone-mediated cytotoxic mechanism. Ethylendiamine (an o-quinone trap) largely prevented 3-HA and 4-MC-induced cytotoxicity indicating that o-quinone was involved in cytotoxicity. Dithiothreitol (DTT) greatly reduced 3-HA and 4-MC induced toxicity. The ferric chelator deferoxamine slightly decreased 3-HA and 4-MC induced cytotoxicity whereas the antioxidants pyrogallol or TEMPOL greatly prevented the toxicity suggesting that oxidative stress contributed to 3-HA induced cytotoxicity. In summary, ring hydroxylation but not O-demethylation/epoxidation seems to be the bioactivation pathway for 3-HA in rat liver. The cytotoxic mechanism for 3-HA and its metabolite 4-MC likely consists cellular protein alkylation and oxidative stress. These results suggest that 3-HA is not suitable for treatment of melanoma.     

Tyrosinase‐catalyzed metabolism of rhododendrol (RD) in B16 melanoma cells: production of RD‐pheomelanin and covalent binding with thiol proteins

RS‐4‐(4‐Hydroxyphenyl)‐2‐butanol (rhododendrol, RD) was reported to induce leukoderma of the skin. To explore the mechanism underlying that effect, we previously showed that oxidation of RD with mushroom tyrosinase produces RD‐quinone, which is converted to secondary quinone products, and we suggested that those quinones are cytotoxic because they bind to cellular proteins and produce reactive oxygen species. We then confirmed that human tyrosinase can oxidize both enantiomers of RD. In this study, we examined the metabolism of RD in B16F1 melanoma cells in vitro. Using 4‐amino‐3‐hydroxy‐n‐butylbenzene as a specific indicator, we detected moderate levels of RD‐pheomelanin in B16F1 cells exposed to 0.3 to 0.5 mM RD for 72 h. We also confirmed the covalent binding of RD‐quinone to non‐protein thiols and proteins through cysteinyl residues. The covalent binding of RD‐quinone to proteins was 20‐ to 30‐fold greater than dopaquinone. These results suggest that the tyrosinase‐induced metabolism of RD causes melanocyte toxicity.     

An approach to evaluate two-electron reduction of 9,10-phenanthraquinone and redox activity of the hydroquinone associated with oxidative stress

Quinones are widely used as medicines or redox agents. The chemical properties are based on the reactions against an electron donor. 9,10-Phenanthraquinone (PQ), which is a quinone contaminated in airborne particulate matters, forms redox cycling, not Michael addition, with electron donors. Redox cycling of PQ contributes to its toxicity, following generation of reactive oxygen species (ROS). Detoxification of quinones is generally thought to be two-electron reduction forming hydroquinones. However, a hydroquinone of PQ, 9,10-dihydroxyphenanthrene (PQH(2)), has been never detected itself, because it is quite unstable. In this paper, we succeeded in detecting PQH(2) as its stable derivative, 9,10-diacetoxyphenanthrene (DAP). However, higher concentrations of PQ (>4 microM) form disproportionately with PQH(2), producing the 9,10-phenanthraquinone radical (PQ(-)) which is a one-electron reducing product of PQ. In cellular experiments using DAP as a precursor of PQH(2), it was shown that PQH(2) plays a critical role in the oxidative protein damage and cellular toxicity of PQ, showing that two-electron reduction of PQ can also initiate redox cycling to cause oxidative stress-dependent cytotoxicity.     

Oxidation of 3,4-dihydroxyphenylacetaldehyde, a toxic dopaminergic metabolite, to a semiquinone radical and an ortho-quinone

The oxidation and toxicity of dopamine is believed to contribute to the selective neurodegeneration associated with Parkinson disease. The formation of reactive radicals and quinones greatly contributes to dopaminergic toxicity through a variety of mechanisms. The physiological metabolism of dopamine to 3,4-dihydroxyphenylacetaldehyde (DOPAL) via monoamine oxidase significantly increases its toxicity. To more adequately explain this enhanced toxicity, we hypothesized that DOPAL is capable of forming radical and quinone species upon oxidation. Here, two unique oxidation products of DOPAL are identified. Several different oxidation methods gave rise to a transient DOPAL semiquinone radical, which was characterized by electron paramagnetic resonance spectroscopy. NMR identified the second oxidation product of DOPAL as the ortho-quinone. Also, carbonyl hydration of DOPAL in aqueous media was evident via NMR. Interestingly, the DOPAL quinone exists exclusively in the hydrated form. Furthermore, the enzymatic and chemical oxidation of DOPAL greatly enhance protein cross-linking, whereas auto-oxidation results in the production of superoxide. Also, DOPAL was shown to be susceptible to oxidation by cyclooxygenase-2 (COX-2). The involvement of this physiologically relevant enzyme in both oxidative stress and Parkinson disease underscores the potential importance of DOPAL in the pathogenesis of this condition.     

Synthesis and assessment of the relative toxicity of the oxidised derivatives of campesterol and dihydrobrassicasterol in U937 and HepG2 cells

The cytotoxic effects of the oxidised derivatives of the phytosterols, stigmasterol and β-sitosterol, have previously been shown to be similar but less potent than those of the equivalent cholesterol oxides in the U937 cell line. The objective of the present study was to compare the cytotoxic effects of the oxidised derivatives of synthetic mixtures of campesterol and dihydrobrassicasterol in both the U937 and HepG2 cell lines. The parent compounds consisted of a campesterol: dihydrobrassicasterol mix at a ratio of 2:1 (2CMP:1DHB) and a dihydrobrassicasterol:campesterol mix at a ratio of 3:1 (3DHB:1CMP). The 2CMP:1DBH oxides were more cytotoxic in the U937 cells than the 3DBH:1CMP oxides but the difference in cytotoxicity was less marked in the HepG2 cells. The order of toxicity of the individual oxidation products was found to be similar to that previously observed for cholesterol, β-sitosterol and stigmasterol oxidation products in the U937 cell line. There was an increase in apoptotic nuclei in U937 cells incubated with the 7-keto and 7β-OH derivatives of both 2CMP:1DHB and 3DHB:1CMP and also in the presence of 3DHB:1CMP-3β,5α,6β-triol and 2CMP:1DHB-5β,6β-epoxide. An additional oxidation product synthesised from 2CMP:1DHB, 5,6,22,23-diepoxycampestane, was cytotoxic but did not induce apoptosis. These results signify the importance of campesterol oxides in the overall paradigm of phytosterol oxide cytotoxicity.     

Menadione stress in Saccharomyces cerevisiae strains deficient in the glutathione transferases

Using S. cerevisiae as a eukaryotic cell model we have analyzed the involvement of both glutathione transferase isoforms, Gtt1 and Gtt2, in constitutive resistance and adaptive response to menadione, a quinone which can exert its toxicity as redox cycling and/or electrophiles. The detoxification properties, of these enzymes, have also been analyzed by the appearance of S-conjugates in the media. Direct exposure to menadione (20 mM/60 min) showed to be lethal for cells deficient on both Gtt1 and Gtt2 isoforms. However, after pre-treatment with a low menadione concentration, cells deficient in Gtt2 displayed reduced ability to acquire tolerance when compared with the control and the Gtt1 deficient strains. Analyzing the toxic effects of menadione we observed that the gtt2 mutant showed no reduction in lipid peroxidation levels. Moreover, measuring the levels of intracellular oxidation during menadione stress we have shown that the increase of this oxidative stress parameter was due to the capacity menadione possesses in generating reactive oxygen species (ROS) and that both GSH and Gtt2 isoform were required to enhance ROS production. Furthermore, the efflux of the menadione-GSH conjugate, which is related with detoxification of xenobiotic pathways, was not detected in the gtt2 mutant. Taken together, these results suggest that acquisition of tolerance against stress generated by menadione and the process of detoxification through S-conjugates are dependent upon Gtt2 activity. This assessment was corroborated by the increase of GTT2 expression, and not of GTT1, after menadione treatment.     

Autoxidation of extracellular hydroquinones is a causative event for the cytotoxicity of menadione and DMNQ in A549-S cells

Cytotoxicity of 1,4-naphthoquinones has been attributed to intracellular reactive oxygen species (ROS) generation through one-electron-reductase-mediated redox cycling and to arylation of cellular nucleophiles. Here, however, we report that in a subclone of lung epithelial A549 cells (A549-S previously called A549-G4S (Watanabe, et al., Am. J. Physiol. 283 (2002) L726-736), the mechanism of ROS generation by menadione and by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and therefore that of cytotoxicity, differs from the paradigm. Ninety percent of H(2)O(2) generation by both the quinones can be prevented by dicumarol, an inhibitor of NAD(P)H quinone oxidoreductase (NQO1), at the submicromolar level, regardless of the quinone concentrations. Exogenous SOD also inhibits H(2)O(2) production at low but not high concentrations of the quinones, especially DMNQ. Thus, at low quinone concentrations, superoxide-driven hydroquinone autoxidation accounts for more than half of H(2)O(2) generation by both quinones, whereas at high quinone concentrations, especially for DMNQ, comproportionation-driven hydroquinone autoxidation becomes the predominant mechanism. Hydroquinone autoxidation appears to occur predominantly in the extracellular environment than in the cytosol as extracellular catalase can dramatically attenuate quinone-induced cytotoxicity throughout the range of quinone concentrations, whereas complete inactivation of endogenous catalase or complete depletion of intracellular glutathione has only a marginal effect on their cytotoxicity. Finally, we show evidence that ROS production is a consequence of the compensatory defensive role of NQO1 against quinone arylation.     

Synthesis and cytotoxicity of pyranonaphthoquinone natural product analogues under bioreductive conditions

We have synthesised a focused library of derivatives of natural products containing the pyranonaphthoquinone moiety including the first report of such a scaffold with an appended tetrazole functionality. Examples include kalafungin derivatives as well as analogues of nanaomycin and eleutherin. These compounds were assessed for cytotoxic activation by breast cancer cell lines engineered to express the prototypic human one- and two-electron quinone bioreductive enzymes, NADPH: cytochrome P450 oxidoreductase (POR) and NAD(P)H: quinoneoxidoreductase 1 (NQO1; DT-diaphorase), respectively. Several compounds were observed to be cytotoxic at sub-micromolar level and a pattern of increased aerobic potency was observed in cells over expressing POR. A subset of analogues was assessed under anoxic conditions, where cytotoxicity was reduced, implicating redox cycling as a major mechanism of toxicity. The substrate specificity for reductive enzymes is relevant to the future design of bioreductive prodrugs to treat cancer.     

The aromatic amine carcinogens o-toluidine and o-anisidine induce free radicals and intrachromosomal recombination in Saccharomyces cerevisiae

Aniline-based aromatic amine carcinogens are poorly detected in short-term mutagenicity assays such as the Salmonella reverse mutation (Ames) assay. More information on the mechanism of toxicity of such Salmonella-negative carcinogens is needed. Aniline and o-toluidine are negative in the Ames assay, but induce deletions (DEL) due to intrachromosomal recombination in Saccharomyces cerevisiae with an apparent threshold. We show here that the DEL assay also detects the genotoxic activity of another aromatic amine carcinogen, o-anisidine, which is also negative in the Salmonella assay. We also show that the DEL assay distinguishes between o-anisidine and its non-carcinogenic structural analog 2, 4-dimethoxyaniline. We have investigated whether the ability of the DEL assay to detect the carcinogens and to distinguish between the carcinogen/non-carcinogen pair is linked to rises in intracellular free radical species following exposure to the carcinogens. Toxicity induced by all three compounds was reduced in the presence of the free radical scavenger and antioxidant N-acetyl cysteine, recombination induced by o-anisidine and o-toluidine was also reduced by N-acetyl cysteine. All three compounds induced oxidation of the free radical-sensitive reporter compound dichlorofluorescin diacetate. Superoxide dismutase-deficient strains, however, were hypersensitive to cytotoxicity induced by o-toluidine and o-anisidine but not by the non-carcinogen 2,4-dimethoxyaniline, indicating a different potential for generating superoxide radical between the carcinogens and the non-carcinogen analog. The results indicate that the yeast DEL assay is a useful tool for investigating the genotoxic activity of aromatic amine carcinogens.     

DNA strand scission and free radical production in menadione-treated cells. Correlation with cytotoxicity and role of NADPH quinone acceptor oxidoreductase.

Menadione (MD; 2-methyl-1,4-naphthoquinone), a redox cycling quinone was shown to induce single (ss)- and double (ds)-strand DNA breaks in human MCF-7 cells. This DNA damage was mediated via the hydroxyl radical as evidenced by electron spin resonance spectroscopy (ESR) studies utilizing the spin trap, 5,5-dimethyl-1-pyrroline-1-oxide. The free radical production and DNA damage were shown to play a role in MD cytotoxicity as revealed by the reversal of MD toxicity and inhibition of hydroxyl radical production by exogenously added catalase. The role of NADPH quinone acceptor oxidoreductase in the metabolism of MD was evaluated. Purified quinone acceptor oxidoreductase in combination with MD resulted in the production of significant levels of the hydroxyl radical as measured by ESR. Dicumarol, an inhibitor of quinone acceptor oxidoreductase, decreased the production of the hydroxyl radical and attenuated DNA strand breaks in MCF-7 cells treated with MD.