A convenient approach for evaluating the toxicity profiles of in vitro neuroprotective alkylaminophenol derivatives

The cytotoxicity profiles of a series of quinol-type derivatives were examined through simple Escherichia coli plate assays discriminating the two main cytotoxicity mechanisms associated with polyphenol oxidation to quinone. Toxicity mediated by reactive oxygen species (ROS-TOX) was detected in the OxyR(-) assay using cells sensitive to oxidative stress due to a deficiency in the OxyR function. Toxicity arising from the high susceptibility of quinone toward endogenous nucleophiles (Q-TOX) was detected using OxyR(+) cells, in the presence of a nitric oxide donor to promote the quinol oxidation to the corresponding quinone. The toxicity profile markedly depended on structural features. Strong ROS-TOX required a pyrogallol arrangement (exifone; 2,3,4-trihydroxybenzophenone, 1; baicalein) or a 2-aminoresorcinol sequence (3-amino-2,4-dihydroxybenzophenone, 4). The pyrogallol moiety determined a low Q-TOX, suggesting the conversion of quinones into oxidation products of low toxicity. Compounds lacking a 2-hydroxyl substituent (derivatives 2 and 5, related to 1 and 4, respectively) induced a weak ROS-TOX, but a significant Q-TOX. The electrochemical oxidation of the studied compounds corroborated the crucial role of the 2-hydroxyl group, which had two effects: to protect the quinonoid species from Michael addition, the reaction at the origin of Q-TOX, and, due to the contraction of hydrogen bonding, to stabilize every intermediary oxidation product, very likely involved in ROS-TOX.     

Oxidation of DOPAC by nitric oxide: effect of superoxide dismutase

This study aimed to characterize the redox interaction between 3,4-dihydroxyphenylacetic acid (DOPAC) and nitric oxide (.NO), and to assess the reductive and oxidative decay pathways of the DOPAC semiquinone originating from this interaction. The reaction between DOPAC and.NO led to the formation of the DOPAC semiquinone radical, detected by electron paramagnetic resonance (EPR) and stabilized by Mg(2+), and the nitrosyl anion detected as nitrosylmyoglobin. The EPR signal corresponding to the DOPAC semiquinone was modulated as follows: (i) it was suppressed by glutathione and ascorbic acid with the formation of new EPR spectra corresponding to the glutathionyl and ascorbyl radical, respectively; (ii) it was enhanced by Cu,Zn-superoxide dismutase; the enzyme also accelerated the decay of the semiquinone species to DOPAC quinone. These results are interpreted as a one-electron oxidation of DOPAC by.NO; the reductive decay of the semiquinone back to DOPAC was facilitated by reducing agents, such as glutathione and ascorbate, whereas the oxidative decay to DOPAC quinone was facilitated by superoxide dismutase. The latter effect is understood in terms of a reversible conversion of nitrosyl anion to.NO by the enzyme. The biological relevance of these reactions is also discussed in terms of the reactivity of peroxynitrite towards DOPAC as a model with implications for aerobic conditions.     

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.     

Characterization and partial purification of microsomal NAD(P)H:quinone oxidoreductases

Quinone oxidoreductases are flavoproteins that catalyze two-electron reduction and detoxification of quinones. This leads to the protection of cells against toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] were previously identified, purified, and cloned. A role of cytosolic NQO1 in protection of cells from oxidative stress, cytotoxicity, and mutagenicity of quinones was established. Currently, we have characterized and partially purified the NQO activity from rat liver microsomes. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donors and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. The mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytosolic NQO1. Western analysis of microsomal proteins revealed 29- and 18-kDa bands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of microsomes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephacel column chromatography. The microsomal mNQO proteins are expected to provide additional protection after cytosolic NQOs against quinone toxicity and mutagenicity.     

Alterations in hepatocyte cytoskeleton caused by redox cycling and alkylating quinones

Quinones may induce toxicity by a number of mechanisms, including alkylation and oxidative stress following redox cycling. The metabolism of quinones by isolated rat hepatocytes is associated with cytoskeletal alterations, plasma membrane blebbing, and subsequent cytotoxicity. The different mechanisms underlying the effects of alkylating (p-benzoquinone), redox cycling (2,3-dimethoxy-1,4-naphthoquinone), and mixed redox cycling/alkylating (2-methyl-1,4-naphthoquinone) quinones on hepatocyte cytoskeleton have been investigated in detail in this study. Analysis of the cytoskeletal fraction extracted from quinone-treated cells revealed a concentration-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols, irrespective of the quinone employed. In the case of redox cycling quinones, these alterations were associated with an oxidation-dependent actin crosslinking (sensitive to the thiol reductant dithiothreitol). In contrast, with alkylating quinones an oxidation-independent cytoskeletal protein crosslinking (insensitive to thiol reductants) was observed. In addition to these changes, a dose-dependent increase in the relative abundance of F-actin was detected as a consequence of the metabolism of oxidizing quinones in hepatocytes. Addition of dithiothreitol solubilized a considerable amount of polypeptides from the cytoskeletal fraction isolated from hepatocytes exposed to redox cycling but not alkylating quinones. Our findings indicate that the hepatocyte cytoskeleton is an important target for the toxic effects of different quinones. However, the mechanisms underlying cytoskeletal damage differ depending on whether the quinone acts primarily by oxidative stress or alkylation.     

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.     

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.     

Enhancement of quinone redox cycling by ascorbate induces a caspase-3 independent cell death in human leukaemia cells. An in vitro comparative study

Since the higher redox potential of quinone molecules has been correlated with enhanced cellular deleterious effects, we studied the ability of the association of ascorbate with several quinones derivatives (having different redox potentials) to cause cell death in K562 human leukaemia cell line. The rationale is that the reduction of quinone by ascorbate should be dependent of the quinone half-redox potential thus determining if reactive oxygen species (ROS) are formed or not, leading ultimately to cell death or cell survival. Among different ROS that may be formed during redox cycling between ascorbate and the quinone, the use of different antioxidant compounds (mannitol, desferal, N-acetylcysteine, catalase and superoxide dismutase) led to support H2O2 as the main oxidizing agent. We observed that standard redox potentials, oxygen uptake, free ascorbyl radical formation and cell survival were linked. The oxidative stress induced by the mixture of ascorbate and the different quinones decreases cellular contents of ATP and GSH while caspase-3-like activity remains unchanged. Again, we observed that quinones having higher values of half-redox potential provoke a severe depletion of ATP and GSH when they were associated with ascorbate. Such a drop in ATP content may explain the lack of activation of caspase-3. In conclusion, our results indicate that the cytotoxicity of the association quinone/ascorbate on K562 cancer cells may be predicted on the basis of half-redox potentials of quinones.     

Increased nitric oxide-dependent nitrosylation of 4,5-diaminofluorescein by oxidants: implications for the measurement of intracellular nitric oxide

4,5 diaminofluorescein (DAF-2) is increasingly utilized as a fluorescent detector for nitric oxide (*NO) in cells and tissues. In oxygenated solutions, reactive nitrogen species derived from (*) NO autoxidation nitrosate DAF-2 to yield the highly fluorescent DAF-2 triazole. In the present study, we investigated the nitrosation of DAF-2 at a neutral pH by absorption and fluorescence spectroscopy using NONOates as chemical sources of (*) NO. We found that both chemically synthesized peroxynitrite and horseradish peroxidase in the presence of hydrogen peroxide (H(2)O(2)) oxidized DAF-2 to a relatively stable nonfluorescent intermediate (t(1/2) approximately 90 s). Oxidation of DAF-2 prior to the addition of the z.rad;NO donor DEA/NO resulted in an increase in fluorescence that was approximately 7-fold higher than treatment with DEA/NO alone. The increase in DAF-2 triazole formation upon oxidation of DAF-2 was confirmed by high performance liquid chromatography. Peroxynitrite generated in situ from the equimolar production of (*) NO and superoxide (O(2)(*-)) also increased the yields of DAF-2 triazole formation, which was completely inhibited when O(2)(*-) was in excess of (*) NO. We propose that DAF-2 is oxidized to a free radical intermediate that directly reacts with (*) NO, thereby bypassing the requirement for (*)NO autoxidation for the formation of DAF-2 triazole. Our findings indicate that DAF-2 fluorometric assays are quantitatively difficult to interpret in cells and in solution when oxidants and (*) NO are co-generated.     

Transformations of 2,6-diisopropylphenol by NO-derived nitrogen oxides, particularly peroxynitrite.

To investigate the protective effect of the anesthetic 2, 6-diisopropylphenol, or propofol, in oxidative processes in which (*)NO and peroxynitrite are involved, direct interactions were explored. The reactions of the highly lipophilic propofol with (*)NO in methanolic or aqueous buffered solutions under air were shown to produce the same compounds as those detected with peroxynitrite, but with very low yields and slow rates. In aqueous neutral medium, peroxynitrite (ONOO(-), ONOOCO(-)(2), ONOOH) was able to nitrate and oxidize propofol: In addition to oxidation products, quinone and quinone dimer, the formation of the 4-nitropropofol derivative was detected, increasing with peroxynitrite or CO(2) concentrations. Nitration reached 20% after the addition of 25 mM bicarbonate to an equimolecular mixture of peroxynitrite and propofol in methanol/phosphate-buffered solution (1/4,v/v) at pH 7.4. However, peroxynitrite either in methanol or in alkaline-buffered mixture (optimum pH 10-12) resulted in the rapid and almost complete transformation of propofol to an intermediate compound 1, which further decomposed to 4-nitrosopropofol. The transient compound 1 was obtained from either peroxynitrite or (*)NO in the presence of oxygen. From mass spectrometry determination of compound 1 we propose the involvement of the nitrosodioxyl radical ONOO(*), forming an adduct with the propofoxyl radical, to yield 4-nitrosodioxypropofol and finally 4-nitrosopropofol.     

Structures of mammalian cytosolic quinone reductases

The metabolism of quinone compounds presents one source of oxidative stress in mammals, as many pathways proceed by mechanisms that generate reactive oxygen species as by-products. One defense against quinone toxicity is the enzyme NAD(P)H:quinone oxidoreductase type 1 (QR1), which metabolizes quinones by a two-electron reduction mechanism, thus averting production of radicals. QR1 is expressed in the cytoplasm of many tissues, and is highly inducible. A closely related homologue, quinone reductase type 2 (QR2), has been identified in several mammalian species. QR2 is also capable of reducing quinones to hydroquinones, but unlike QR1, cannot use NAD(P)H. X-ray crystallographic studies of QR1 and QR2 illustrate that despite their different biochemical properties, these enzymes have very similar three-dimensional structures. In particular, conserved features of the active sites point to the close relationship between these two enzymes.     

The biological significance of non-enzymatic reaction of menadione with plasma thiols: enhancement of menadione-induced cytotoxicity to platelets by the presence of blood plasma.

To test the hypothesis that the non-enzymatic reaction of quinones with thiols in plasma can generate reactive oxygens (ROS), thereby leading to potentiated cellular toxicity, we have studied the effect of a representative quinone compound, menadione, on plasma isolated from rats. The experimental results are as follows: (1) menadione generated ROS via non-enzymatic reaction with protein thiols in plasma; (2) the presence of plasma increased menadione-induced cytotoxicity to platelets; (3) pretreatment of plasma with a thiol-depleting agent significantly suppressed menadione-induced ROS and cytotoxicity. These results suggest that the non-enzymatic reaction of menadione with plasma thiols could be an important process in quinone-induced cellular toxicity.     

Cytotoxicity of natural hydroxyanthraquinones: role of oxidative stress

In order to assess the role of oxidative stress in the cytotoxicity of natural hydroxyanthraquinones, we compared rhein, emodin, danthron, chrysophanol, and carminic acid, and a series of model quinones with available values of single-electron reduction midpoint potential at pH 7.0 (E(1)7), with respect to their reactivity in the single-electron enzymatic reduction, and their mammalian cell toxicity. The toxicity of model quinones to the bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK), and HL-60, a human promyelocytic leukemia cell line, increased with an increase in their E(1)7. A close parallelism was found between the reactivity of hydroxyanthraquinones and model quinones with single-electron transferring flavoenzymes ferredoxin: NADP+ reductase and NADPH:cytochrome P450 reductase, and their cytotoxicity. This points to the importance of oxidative stress in the toxicity of hydroxyanthraquinones in these cell lines, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N'-diphenyl-p-phenylene diamine, by the potentiating effects of 1,3-bis-(2-chloroethyl)-1-nitrosourea, and an increase in lipid peroxidation.     

Comparative toxicities of putative phagocyte-generated oxidizing radicals toward a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae)

Toxicities of the radiolytically generated oxidizing radicals HO(*), CO(3)(-)(*), and NO(2)(*) toward suspension cultures of a bacterium (Escherichia coli) and a yeast (Saccharomyces cerevisiae) were examined. As demonstrated by the absence of protection from the membrane-impermeable HO(*) scavenger polyethylene glycol (PEG), externally generated HO(*) was not bactericidal under these conditions; however, partial protection by PEG was observed for S. cerevisiae, indicating the presence of a fungicidal pathway involving external HO(*). For both organisms, conversion of external HO(*) to the secondary radical, CO(3)(-)(*), by reaction with HCO(3)(-) increases their susceptibility to radiolytic killing. In contrast, externally generated NO(2)(*) exhibited toxicity comparable to that of CO(3)(-)(*) toward E. coli, but completely blocked the extracellular toxicity of HO(*) toward S. cerevisiae. Cogeneration of equal fluxes of NO(2)(-)(*) and either HO(*) or CO(3)(-)(*) also essentially eliminated the extracellular microbicidal reactions. This behavior is consistent with expectations based upon relative rates of radical-radical self-coupling and cross-coupling reactions. The different patterns of toxicity observed imply fundamentally different microbicidal mechanisms for the two organisms, wherein the bacterium is susceptible to killing by oxidation of highly reactive targets on its cellular envelope but, despite undergoing similar oxidative insult, the fungus is not.     

Enhanced NAD(P)H:Quinone Reductase Activity Prevents Glutamate Toxicity Produced by Oxidative Stress

Glutamate toxicity in the N18-RE-105 neuronal cell line results from the inhibition of high-affinity cystine uptake, which leads to a depletion of glutathione and the accumulation of oxidants. Production of superoxides by one-electron oxidation/reduction of quinones is decreased by NAD(P)H:quinone reductase, an enzyme with DT-diaphorase activity. Using glutamate toxicity in N18-RE-105 cells as a model of neuronal oxidative stress, we report that the degree of glutamate toxicity observed is inversely proportional to quinone reductase activity. Induction of quinone reductase activity by treatment with t-butylhydroquinone reduced glutamate toxicity by up to 80%. In contrast, treatment with the quinone reductase inhibitor dicumarol potentiated the toxic effect of glutamate. Measurement of cellular glutathione indicates that increases in its levels are not responsible for the protective effect of t-butylhydroquinone treatment. Because many types of cell death may involve the formation of oxidants, induction of quinone reductase may be a new strategy to combat neurodegenerative disease.     

Calculating molar absorptivities for quinones: application to the measurement of tyrosinase activity

The molar absorptivities of the quinones produced from different o-diphenols, triphenols, and flavonoids were calculated by generating the respective quinones through oxidation with an excess of periodate. Oxidation of these substrates by this reagent was analogous to oxidation by tyrosinase with molecular oxygen, although the procedure showed several advantages over the enzymatic method in that oxidation took place almost immediately and quinone stability was favored because no substrate remained. The o-diphenols studied were pyrocatechol, 4-methylcatechol, 4-tert-butylcatechol, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyphenylethylamine, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxyphenylpropionic acid, and caffeic acid; the triphenols studied were pyrogallol, 1,2,4-benzenetriol, 6-hydroxydopa, and 6-hydroxydopamine; and the flavonoids studied were (+)catechin, (-)epicatechin, and quercetin. In addition, the stability of the quinones generated by oxidation of the compounds by [periodate]0/[substrate]0 < 1 was studied. Taking the findings into account, tyrosinase could be measured by following o-quinone formation in rapid kinetic studies using the stopped-flow method. However, measuring o-quinone formation could not be useful for steady-state studies. Therefore, several methods for following tyrosinase activity are proposed, and a kinetic characterization of the enzyme's action on these substrates is made.     

An Improved HPLC-Electrochemical Detection Method for Measuring Brain Levels of 5-S-Cysteinyldopamine, 5-S-Cysteinyl-3,4-Dihydroxyphenylalanine, and 5-S-Cysteinyl-3,4-Dihydroxyphenylacetic Acid

Brain levels of the 5-S-cysteinyl adducts of 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenylacetic acid (DOPAC), and dopamine were determined in several mammalian species. The low levels of the compounds and the risk of artifacts during sample preparation necessitated rather profound modifications of the assaying method. The refined method has made it possible to present more accurate data than those previously reported from this laboratory. The occurrence of low levels of the 5-S-cysteinyl adducts in dopamine-rich brain areas, but not in cerebellum, is indirect evidence of in vivo autoxidation of DOPA, DOPAC, and dopamine. The products generated during catechol autoxidation, including quinones and reduced forms of oxygen, are known to be potentially cytotoxic.     

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.     

Biological response of human diploid keratinocytes to quinone-producing compounds: role of NAD(P)H:quinone oxidoreductase 1

Reactive oxygen species (ROS) and quinones are known to determine redox balance alteration, oxidative stress and carcinogenicity. Keratinocytes of the human epidermis, a tissue particularly exposed to oxidant stimuli, possess a wide range of antioxidant and detoxifying mechanisms aimed to avoid oxidative damage of the tissue. In the present study, we evaluate the response of diploid and transformed human keratinocytes to exposure to L-dopa and tetrahydropapaveroline (THP), catechol compounds susceptible to undergo oxidation to form quinones with concomitant production of reactive oxygen species. We demonstrated that these compounds elicit up-regulation of intracellular antioxidant enzymes, in a different degree in normal cells with respect to transformed ones. Normal diploid keratinocytes adequately scavenge toxic substances through the activation of several, concurrent pathways. Conversely, in transformed cells, the whole oxidative burden must be detoxified by the limited set of conserved pathways that, accordingly, have to be highly activated. The biological response to catechol toxicity appears to rely on the pathway of NAD(P)H:quinone oxidoreductase 1 (NQO1). In conclusion, NAD(P)H:quinone oxidoreductase 1 confirms its antioxidant and detoxifying role contributing to the capacity of keratinocytes to protect epidermis against oxidative stress. Being retained in almost any cell, it represents a mechanism of general relevance in cell physiology.