Interaction of glutathione and sodium selenite in vitro investigated by electrospray ionization tandem mass spectrometry

Selenite has been found to be an active catalyst for the oxidation of sulphhydryl compounds, such as glutathione (GSH). Considering the biological importance of GSH oxidation and the implication of sulphhydryl compounds in selenium poisoning and other biological activities, more information on selenite oxidation of GSH in enzyme-free conditions is desirable. Herein, we describe glutathione and sodium selenite simply mixed in aqueous solutions. The interaction products and transient intermediate are identified and characterized using electrospray ionization (ESI) tandem mass spectrometry. In the first step, GSH directly reacts to form diglutathione (GSSG) and unstable selenodiglutathione (GS-Se-SG). Then selenodiglutathione further reacted with remaining GSH to form diglutathione and elemental selenium, Se(0). As the amount of GSSG significantly increased or acidity of the solution increased, the redox potential of glutathione [E(0')(GSSG/2GSH) approximately -250 mV (NHE)] significantly shifted to the positive direction. This makes the GSSG react with elemental selenium formed in the solution, which can be demonstrated by another unstable intermediate ion identified at m/z 418 by mass spectrometry with the elemental composition of [GSS-Se](-). The reaction mechanism between GSH and sodium selenite has been proposed according to the ESI-MS, NMR and UV-vis spectrometric measurements.     

Oxidation of glutathione and superoxide generation by inorganic and organic selenium compounds

The carcinostatic activities of selenium (Se) compounds have been shown to be composition and concentration dependent. Several studies have indicated that the ratios between glutathione (GSH) and Se may play an important role in Se catalysis and toxicity. The present study examined the catalytic effect of three selenium compounds on GSH oxidation using lucigenin-dependent chemiluminescence (CL) as an indirect measure of superoxide generation. Various GSH:Se ratios were assayed for the glutathione oxidase activity of selenite, selenocystamine and diselenodipropionic acid. CL emitted from the reaction of selenite with GSH increased more rapidly and was greater than those from the diselenides, but the diselenide CL reactions were sustainable. Both selenite- and diselenide-induced CL were markedly suppressed by superoxide dismutase (SOD). Iodoacetic acid (IAc) effectively inhibited CL generated from selenite-, selenocystamine- and diselenodipropionic acid-catalyzed GSH oxidation. These results suggest that GSH oxidation catalyzed by selenite, and the diselenides selenocystamine and diselenodipropionic acid, generated the superoxide radical in which the CL was inhibited by SOD. Furthermore, CL inhibition by IAc suggests that the catalytic species producing superoxide were the GSSe(-) or RSe(-) anion. This redox chemistry may be responsible for selenite and organoselenium toxicity and apoptosis, making possible the design and synthesis of organoselenium-containing pharmaceuticals.     

The investigation of the antioxidative properties of the novel synthetic organoselenium compounds in some rat tissues

DMBA (7,12-dimethylbenz[a]anthracene) is a polycyclic aromatic hydrocarbon (PAH) known to cause tumors in rats. Selenium is an essential element with physiological non-enzymatic antioxidant properties. Because of the health problems induced by many environmental pollutants, many efforts have been undertaken in evaluating the relative antioxidant potential of selenium and synthetic organoselenium compounds. In this study, adult female Wistar rats were treated with DMBA and the novel organoselenium compounds (1-isopropyl-3-methylbenzimidazole-2-selenone [SeI] and 1,3-di-p-methoxybenzylpyrimidine-2-selenone [SeII]) in the determined doses. The protective effects of novel synthetic organoselenium compounds (SeI and SeII) against DMBA-induced changes in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) activities and total glutathione (GSH) and malone-dialdehyde (MDA) levels of rat heart and brain were investigated. It was determined that SeI and SeII fully or partially restored enzyme activity. It was also found that lipid peroxidation was also decreased in SeI and SeII treated groups. Consequently, it was determined that novel synthetic organoselenium compounds (SeI and SeII) provided protection of antioxidant activity, and protection against lipid peroxidation measured as MDA in SeI and SeII treated groups was provided by novel synthesized organoselenium compounds. The ability of the organoselenium compounds to prevent oxidative damage induced by DMBA in rats was rationalized.     

Redox modulation of human prostate carcinoma cells by selenite increases radiation-induced cell killing

Although selenium compounds have been extensively studied as chemopreventative agents for prostate cancer, little is known about the potential use of selenium compounds for chemotherapy. We have shown that selenite inhibits cell growth and induces apoptosis in androgen-dependent LAPC-4 prostate cancer cells. LAPC-4 cells were more sensitive to selenite-induced apoptosis than primary cultures of normal prostate cells. Selenite-induced apoptosis in LAPC-4 cells correlated with a decrease in the Bcl-2:Bax expression ratio. Selenite-induced oxidative stress and apoptosis are dependent upon its reaction with reduced GSH. LAPC-4 cells treated with selenite showed decreased levels of total GSH and increased concentrations of GSSG. Thus, selenite altered the intracellular redox status toward an oxidative state by decreasing the ratio of GSH:GSSG. Because increased levels of Bcl-2 and GSH are associated with radioresistance, we examined the ability of selenite to sensitize prostate cancer cells to gamma-irradiation. Both LAPC-4 and androgen-independent DU 145 cells pretreated with selenite showed increased sensitivity to gamma-irradiation as measured by clonogenic survival assays. Importantly, selenite-induced radiosensitization was observed in combination with a clinically relevant dose of 2 Gy. These data suggest that altering the redox environment of prostate cancer cells with selenite increases the apoptotic potential and sensitizes them to radiation-induced cell killing.     

Catalytic selenols couple the redox cycles of metallothionein and glutathione.

Co-ordination of zinc to the thiol group of cysteine allows mobilization of zinc through oxidation of its ligand. This molecular property links the binding and release of zinc in metallothionein (MT) to the cellular redox state [Maret W. & Vallee B.L. (1998) Proc. Natl Acad. Sci. USA 95, 3483-3488]. Biological disulfides such as glutathione disulfide (GSSG) oxidize MT with concomitant release of zinc, while glutathione (GSH) reduces the oxidized protein to thionein, which then binds to available zinc. Neither of these two redox processes is very efficient, even at high concentrations of GSSG or GSH. However, the GSH/GSSG redox pair can efficiently couple with the MT/thionein system in the presence of a selenium compound that has the capacity to form a catalytic selenol(ate). This coupling provides a very effective means of modulating oxidation and reduction. Remarkably, selenium compounds catalyze the oxidation of MT even under overall reducing conditions such as those prevailing in the cytosol. In this manner, the binding and release of zinc from zinc-thiolate co-ordination sites is linked to redox catalysis by selenium compounds, changes in the glutathione redox state, and the availability of either a zinc donor or a zinc acceptor. The results also suggest that the pharmacological actions of selenium compounds in cancer prevention and other antiviral and anti-inflammatory therapeutic applications, as well as unknown functions of selenium-containing proteins, may relate to coupling between the thiol redox state and the zinc state.     

Role of Synthesized Organoselenium Compounds on Protection of Rat Erythrocytes from DMBA-Induced Oxidative Stress

Formation of free radicals is not limited to normal cellular process but also occur upon exposure to certain chemicals (polycyclic aromatic hydrocarbon, cadmium, lead, etc.), cigarette smoke, radiation, and high-fat diet. Free-radical damage is an important factor in many pathological and toxicological processes. Selenium, an essential micronutrient, is a associated with antioxidant functions, physiological defense mechanisms against different diseases including several types of cancers. Search for new selenium compounds with more chemopreventive activities and less toxicities are in progress. In addition, there has been a growing interest in the synthesis of organoselenium compounds with respect to their use in enzymology and bioorganic chemistry. In the present study, adult female Wistar rats were treated with 7,12-dimethylbenz[a]anthracene (DMBA) and the organoselenium compounds [1-isopropyl-3-methylbenzimidazole-2-selenone (Se I) and 1, 3-di-p-methoxybenzylpyrimidine-2-selenone (Se II)] in determined doses. The protective effects of synthetic organoselenium compounds (Se I and Se II) against DMBA-induced changes in antioxidant enzyme (superoxide dismutase, glutathione peroxidase (GSH-Px), catalase (CAT), glutathione reductase (GR)) activities, total GSH, and malondialdehyde (MDA) levels of rat erythrocyte were investigated. The DMBA-treated group exhibited significant decreases in the levels of erythrocyte GSH-Px, CAT, and GR activities, an increase in MDA levels, and a decrease in total GSH level compared to the control. Se I and Se II fully or partially restored enzyme activity. Lipid peroxidation was also decreased in Se-I- and Se-II-treated groups.     

Methylselenol Formed by Spontaneous Methylation of Selenide Is a Superior Selenium Substrate to the Thioredoxin and Glutaredoxin Systems

Naturally occurring selenium compounds like selenite and selenodiglutathione are metabolized to selenide in plants and animals. This highly reactive form of selenium can undergo methylation and form monomethylated and multimethylated species. These redox active selenium metabolites are of particular biological and pharmacological interest since they are potent inducers of apoptosis in cancer cells. The mammalian thioredoxin and glutaredoxin systems efficiently reduce selenite and selenodiglutathione to selenide. The reactions are non-stoichiometric aerobically due to redox cycling of selenide with oxygen and thiols. Using LDI-MS, we identified that the addition of S-adenosylmethionine (SAM) to the reactions formed methylselenol. This metabolite was a superior substrate to both the thioredoxin and glutaredoxin systems increasing the velocities of the nonstoichiometric redox cycles three-fold. In vitro cell experiments demonstrated that the presence of SAM increased the cytotoxicity of selenite and selenodiglutathione, which could neither be explained by altered selenium uptake nor impaired extra-cellular redox environment, previously shown to be highly important to selenite uptake and cytotoxicity. Our data suggest that selenide and SAM react spontaneously forming methylselenol, a highly nucleophilic and cytotoxic agent, with important physiological and pharmacological implications for the highly interesting anticancer effects of selenium.     

Versatility of selenium catalysis in PHGPx unraveled by LC/ESI-MS/MS

Phospholipid hydroperoxide glutathione peroxidase (PHGPx; EC 1.11.1.12), a broad-spectrum thiol-dependent peroxidase, deserves renewed interest as a regulatory factor in various signaling cascades and as a structural protein in sperm cells. We present a first attempt to identify catalytic intermediates and derivatives of the selenoprotein by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/ESI-MS/MS) and to explain observed specificities by molecular modeling. The ground state enzyme E proved to correspond to position 3-170 of the deduced porcine sequence with selenium being present as selenocysteine at position 46. The selenenic acid form, which is considered to be the first catalytic intermediate F formed by reaction with hydroperoxide, could not be identified. The second catalytic intermediate G was detected as Se-glutathionylated enzyme. This intermediate is generated in the reverse reaction where the active site selenol interacts with glutathione disulfide (GSSG). According to molecular models, specific binding of reduced glutathione (GSH) and of GSSG is inter alia facilitated by electrostatic attraction of Lys-48 and Lys-125. Polymerization of PHGPx is obtained under oxidizing conditions in the absence of low molecular weight thiols. Analysis of MS spectra revealed that the process is due to a selective reaction of Sec-46 with Cys-148' resulting in linear polymers representing dead-end intermediates (G'). FT Docking of PHGPx molecules allowed reactions of Sec-46 with either Cys-66', Cys-107', Cys-168' or Cys-148', the latter option being most likely as judged by the number of proposed intermediates with reasonable hydrogen bonds, interaction energies and interface areas. We conclude that the same catalytic principles, depending on the conditions, can drive the diverse actions of PHGPx, i.e. hydroperoxide reduction, GSSG reduction, S-derivatization and self-incorporation into biological structures.     

Design, synthesis and glutathione peroxidase-like properties of ovothiol-derived diselenides

Eleven imidazole diselenides derived from the naturally occurring family of antioxidants, the ovothiols, were synthetised by cyclisation of selenoamides with trimethylsilyltrifluoromethanesulfonate or refluxing of cyanoamines in a selenium/sodium borohydride mixture. These compounds were assayed for their glutathione peroxidase-like (GSH Px-like) activity and their capacity to be reduced by glutathione. The most active molecules of the series were 4 times more potent in the GSH Px-like test than the widely known reference compound, ebselen. This catalytic activity was mediated by the formation of the antioxidant selenol intermediate upon partial but significant exchange reaction with glutathione.     

Isoselenocyanates derived from amino acid esters: an expedient synthesis and application to the assembly of selenoureidopeptidomimetics,unsymmetrical Selenoureas and selenohydantoins

An important class of organoselenium compounds‐α‐isoselenocyanato esters 4 hasbeen prepared by a reaction of α‐isocyano esters with elemental selenium powder. The reaction issimple, rapid and all the isoselenocyanates havebeen isolated as stable ones after chromatographic purification. These hitherto unreported classes of molecules would be useful building blocks for the preparation of variety of selenium containing peptidomimetics. In this study, the utility of the title molecules in the preparation of selenoureidopeptidomimetics 6, unsymmetrical selenoureas 8 and selenohydantoins 10 isdemonstrated. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Hydrogen selenide ion adsorption to colloidal elemental selenium

Hydrogen selenide ion (HSe^?) has an important role in the metabolism of the essential trace element selenium. Several redox reactions of selenide were found to be dominated by the amount of colloidal elemental selenium (Se°) generated during the reaction. The following reaction of selenide with the disulfide, oxidized glutathione (GSSG), was used as an example: HSe^? + GSSG + H⁺ → Se° + 2 GSH. The resulting thiol is reduced glutathione (GSH; γ-glutamylcysteinylglycine). By following this reaction with polarography, it was seen that the ratio of colloidal selenium produced to selenide unreacted was a constant 2.1 ± 0.1, and was the only factor found to determine the extent of oxidation. This is best explained by the hypothesis that freshly generated colloidal selenium adsorbs selenide readily; no evidence for polyselenide formation was found. Adsorption of selenide should be considered in any reaction involving the oxidation of selenide to colloidal selenium.     

Identification in human urine and blood of a novel selenium metabolite, Se-methylselenoneine, a potential biomarker of metabolization in mammals of the naturally occurring selenoneine, by HPLC coupled to electrospray hybrid linear ion trap-orbital ion trap MS

Speciation analysis of selenium in human urine allowed for the first time the identification of a novel selenium metabolite, Se-methylselenoneine. Despite a concentration at low ppb level, its characterization was achieved after sample purification by solid phase extraction (SPE) followed by the parallel coupling of the bidimensional RP/HILIC chromatography with ICP-MS and ESI-LTQ Orbitrap MS detection. To confirm its biological significance with regards to selenoneine, the recently discovered analog of ergothioneine, and to discard the possibility of sample preparation artifacts, a new method was developed to monitor its actual presence, as well as the occurrence of its sulfur and/or non-methylated analogs, in non-preconcentrated urine and blood samples of non-supplemented humans. It consisted in a HILIC ESI-MS(3) method in high resolution mode (resolution 30?000 at m/z 400) with large isolation width windows for precursor ions. These two particular settings allowed respectively to keep observing the specific mass defect of selenium- and sulfur-containing molecules and to maintain the characteristic selenium pattern in product ions created through MS(n) fragmentations. As a result, all four metabolites were detected in blood and three of them in urine. Moreover, different ratios "methylated/non-methylated" were observed between urine and blood samples, which seemed to indicate their active metabolization. The analytical tool developed here will be of a great importance to further study the occurrence and the potential metabolic role in mammalian organelles, cells and fluids of these very particular and promising redox metabolites.     

Transmembrane glutathione cycling in growing Escherichia coli cells

Glutathione (GSH) plays an important role in bacterial cells, participating in maintenance of redox balance in the cytoplasm and in defense against many toxic compounds and stresses. In this study we demonstrate that in aerobic, exponentially growing Escherichia coli culture endogenous reduced glutathione undergoes continuous transmembrane cycling between the cells and medium. As a result of an establishment of a dynamic balance between GSH efflux and uptake, a constant extracellular concentration of GSH counting per biomass unit is maintained. The magnitude of this concentration strictly depends on external pH. GSH cycling is carried out in respiring cells and disturbed by influences, which change the level of ΔμH(+) and ATP. Export of GSH is modified by phosphate deficiency in the medium.     

Investigation of lipid peroxide and glutathione redox status of chicken concerning on high dietary selenium intake

This study was designed to investigate the effects of excess (24.5 mg Se/kg feed) inorganic and organic dietary selenium supplementation on 3-week-old broilers. The experiments lasted 4 days. Intensity of lipid peroxidation processes (malondialdehyde, MDA) and the amount (reduced glutathione, GSH) and activity (glutathione peroxidase activity, GSHPx) of gluathione redox system were measured in blood plasma, red blood cell hemolysate and liver. Voluntary feed intake in the selenium-treated groups reduced remarkably. Elevated GSH concentration and GSHPx activity were measured in plasma and liver of both selenium-treated groups compared to the untreated control and the 'pair-fed' controls. The lipid peroxidation processes in the liver showed higher intensity than the control due to both selenium treatment. The applied dose of selenite and selenomethionine does not inhibit, but even improves the activity of glutathione redox system in the liver during the early period of selenium exposure.     

Reactions of Pseudomonas aeruginosa pyocyanin with reduced glutathione

Pseudomonas aeruginosa is the most common cause of chronic and recurrent lung infections in patients with cystic fibrosis (CF) whose sputa contain copious quantities of P. aeruginosa toxin, pyocyanin. Pyocyanin triggers tissue damage mainly by its redox cycling and induction of reactive oxygen species (ROS). The reactions between reduced glutathione (GSH) and pyocyanin were observed using absorption spectra from spectrophotometry and the reaction products analysed by nuclear magnetic resonance imaging. Pyocyanin reacted with GSH non-enzymatically at 37 degrees C resulting in the production of red-brown products, spectophotometrically visible as a 480 nm maximum absorption peak after 24 h of incubation. The reaction was concentration-dependent on reduced glutathione but not on pyocyanin. Minimizing the accessibility of oxygen to the reaction decreased its rate. The anti-oxidant enzyme catalase circumvented the reaction. Proton-NMR analysis demonstrated the persistence of the original aromatic ring and the methyl-group of pyocyanin in the red-brown products. Anti-oxidant agents having thiol groups produced similar spectophotometrically visible peaks. The presence of a previously unidentified non-enzymatic GSH-dependent metabolic pathway for pyocyanin has thus been identified. The reaction between pyocyanin and GSH is concentration-, time-, and O(2)-dependent. The formation of H(2)O(2) as an intermediate and the thiol group in GSH seem to be important in this reaction.     

Schisandrin B enhances the glutathione redox cycling and protects against oxidant injury in different types of cultured cells

Tert-butylhydroperoxide (tBHP) challenge caused an initial depletion of cellular reduced glutathione (GSH), which was followed by a gradual restoration of cellular GSH in AML12, H9c2, and differentiated PC12 cells. The time-dependent changes in cellular GSH induced by tBHP were monitored as a measure of GSH recovery capacity (GRC), of which glutathione reductase (GR)-mediated glutathione redox cycling and γ-glutamate cysteine ligase (GCL)-mediated GSH synthesis were found to play an essential role. While glutathione redox cycling sustained the GSH level during the initial tBHP-induced depletion, GSH synthesis restores the GSH level thereafter. The effects of (-)schisandrin B [(-)Sch B] and its analogs (Sch A and Sch C) on GRC were also examined in the cells. (-)Sch B and Sch C, but not Sch A, ameliorated the extent of tBHP-induced GSH depletion, indicative of enhanced glutathione redox cycling. However, the degree of restoration of GSH post-tBHP challenge was not affected or even decreased. Pretreatment with (-)Sch B and Sch C, but not Sch A, protected against oxidant injury in the cells. The (-)Sch B afforded cytoprotection was abolished by N,N'-bis(chloroethyl)-N-nitrosourea pretreatment suggesting the enhancement of glutathione redox cycling is crucially involved in the cytoprotection afforded by (-)Sch B against oxidative stress-induced cell injury.     

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.     

Temporal changes in tissue glutathione in response to chemical form,dose, and duration of selenium treatment

Selenium has been reported to affect glutathione (GSH) concentrations in short-term animal-feeding experiments. Given the central role that this tripeptide plays in maintaining cellular homeostasis, it was hypothesized that perturbations in glutathione metabolism induced by selenium might account for its cancer chemopreventive activity. In the present study, four experiments were conducted in which the effect of acute, short-, or long-term exposure to selenium was assessed. Selenium was provided as either sodium selenite or D,L-selenomethionine. Selenite was observed to induce a biphasic response in total liver GSH. Injected selenium caused an acute reduction in GSH, whereas short-term feeding (up to 8 wk) increased both total GSH and oxidized glutathione (GSSH), an effect that gradually diminished in magnitude with prolonged feeding. Our data suggest that such changes are unlikely to account for the chemopreventive activity of selenium for the following reasons: Perturbations in glutathione metabolism occurred only at doses of selenite that approached toxicity. These doses are higher than what would be required for producing cancer chemoprevention. The transient nature of these changes also contrasts with the need for a continuous supplementation of selenite in suppression of tumorigenesis. Furthermore, selenomethionine was found to have little activity in altering glutathione metabolism, even though it compares favorably with selenite as a cancer chemopreventive agent. Nonetheless, these findings do not discount the possibility that sulfhydryl compounds, such as glutathione, might be used to modify the toxicity and/or enhance the cancer prophylactic activity of selenium compounds.     

Metabolism of selenium compounds catalyzed by the mammalian selenoprotein thioredoxin reductase

The mammalian thioredoxin reductases (TrxR) are selenoproteins with a catalytic selenocysteine residue which in the oxidized enzyme forms a selenenylsulfide and in the reduced enzyme is present as a selenolthiol. Selenium compounds such as selenite, selenodiglutathione and selenocystine are substrates for the enzyme with low K_m-values and the enzyme is implicated in reductive assimilation of selenium by generating selenide for selenoprotein synthesis. Redox cycling of reduced metabolites of these selenium compounds including selenide with oxygen via TrxR and reduced thioredoxin (Trx) will oxidize NADPH and produce reactive oxygen species inducing cell death at high concentrations explaining selenite toxicity. There is no free pool of selenocysteine since this would be toxic in an oxygen environment by redox cycling via thioredoxin systems. The importance of selenium compounds and TrxR in cancer and cardiovascular diseases both for prevention and treatment is discussed. A selenazol drug like ebselen is a direct substrate for mammalian TrxR and dithiol Trx and ebselen selenol is readily reoxidized by hydrogen peroxide and lipid hydroperoxides, acting as an anti-oxidant and anti-inflammatory drug.     

Redox cycling and sulphydryl arylation; their relative importance in the mechanism of quinone cytotoxicity to isolated hepatocytes

Quinones are believed to be toxic by a mechanism involving redox cycling and oxidative stress. In this study, we have used 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ), which redox cycles to the same degree as menadione, but does not react with free thiol groups, to distinguish between the importance of redox cycling and arylation of free thiol groups in the causation of toxicity to isolated hepatocytes. Menadione was significantly more toxic to isolated hepatocytes than 2,3-diOMe-1,4-NQ. Both menadione and 2,3-diOMe-1,4-NQ caused an extensive GSH depletion accompanied by GSSG formation, preceding loss of viability. Both compounds stimulated a similar increase in oxygen uptake in isolated hepatocytes and NADPH oxidation in microsomes suggesting they both redox cycle to similar extents. Further evidence for the redox cycling in intact hepatocytes was the detection of the semiquinone anion radicals with electron spin resonance spectroscopy. In addition we have, using the spin trap DMPO (5,5-dimethyl-1-pyrroline N-oxide), demonstrated for the first time the formation of superoxide anion radicals by intact hepatocytes. These radicals result from oxidation of the semiquinone by oxygen and further prove that both these quinones redox cycle in intact hepatocytes. We conclude that while oxidative processes may cause toxicity, the arylation of intracellular thiols or nucleophiles also contributes significantly to the cytotoxicity of compounds such as menadione.