Cytotoxicity, redox cycling and photodynamic action of two naturally occurring quinones

Two naturally occurring anthraquinones, barleriaquinone-I (BQ-I) and barleriaquinone-II (BQ-II), extracted from Barleria buxifolia, are tested for their cytotoxic action by aerobic incubation with human breast adenocarcinoma cells (MCF7). Cytotoxicities, measured as LD(50) (50% inhibition of colony formation) values, show BQ-II to be more active than BQ-I. Electron paramagnetic resonance studies confirm that BQ-II is reductively activated by NADH:cytochrome c reductase to superoxide anion radical. Cyclic voltammetric studies show one quasi-reversible redox couple for both BQ-I and BQ-II. Also, aerobic solutions of both BQ-I and BQ-II on visible illumination generate reactive oxygen species. Formation of O*-2 is studied by both EPR spin trapping and SOD-inhibitable cytochrome c reduction techniques. BQ-I generates more singlet oxygen as evidenced from the photobleaching of N,N-dimethyl-4-nitrosoaniline.     

Adriamycin-catalyzed aerobic photooxidation of NAD dimers to NAD+

The photooxidation of the dimers of nicotinamide adenine dinucleotide, (NAD)2, is catalyzed by adriamycin under aerobic conditions. (NAD)2 and O2 react in 1:1 molar ratio to yield 2 mol of NAD+. Experiments carried out by irradiating at 340 and 485 nm, corresponding to the absorption maxima of (NAD)2 and adriamycin, respectively, clearly indicate that the process is primed by photoexcitation of adriamycin. The key step of the process is the redox reaction between (NAD)2 and adriamycin with formation of the semiquinone radical anion, identified by the EPR spectrum. The semiquinone is then oxidized back to adriamycin by oxygen with formation of the superoxide radical.     

Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate.

The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated rats. In the present study, 2,3,5,6-tetramethylbenzoquinone (TMQ) was chosen as a model quinone. Anaerobic one-electron reduction of TMQ by PB-microsomes showed relatively strong electron spin resonance (ESR) signals of the oxygen-centered semiquinone free radical (TMSQ), whereas these signals were hardly detectable with beta NF-microsomes. Under aerobic conditions TMSQ formation was diminished and concomitant reduction of molecular oxygen occurred in PB-microsomes. Interestingly, TMQ-induced superoxide anion radicals, measured by ESR (using the spin trap 5,5'-dimethyl-1-pyrroline-N-oxide), and hydrogen peroxide generation was found to occur with beta NF-microsomes as well. Furthermore, SK&F 525-A (a type I ligand inhibitor of cytochrome P450) inhibited TMQ-induced hydrogen peroxide formation in both PB- and beta NF-microsomes. However, metyrapone and imidazole (type II ligand inhibitors of cytochrome P450) inhibited molecular oxygen reduction in beta NF-microsomes and not in PB-microsomes. The present study indicates that cytochrome P450-mediated one-electron reduction of TMQ to TMSQ and subsequent redox cycling of TMSQ with molecular oxygen constitutes the major source for superoxide anion radical and hydrogen peroxide generation in PB-microsomes (i.e. from the reductase activity of cytochrome P450). However, most of the superoxide anion radical formed upon aerobic incubation of TMQ with beta NF-microsomes originates directly from the dioxyanion-ferri-cytochrome P450 complex (i.e. from the oxidase activity of cytochrome P450). In conclusion, both the one-electron reduction of TMQ and molecular oxygen were found to be cytochrome P450 dependent. Apparently, both the reductase and oxidase activities of cytochrome P450 may be involved in the reductive cytotoxicity of chemotherapeutic agents containing the quinoid moiety.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Glutathionyl- and hydroxyl radical formation coupled to the redox transitions of 1,4-naphthoquinone bioreductive alkylating agents during glutathione two-electron reductive addition.

The kinetic parameters of the redox transitions subsequent to the two-electron transfer implied in the glutathione (GSH) reductive addition to 2- and 6-hydroxymethyl-1,4-naphthoquinone bioalkylating agents were examined in terms of autoxidation, GSH consumption in the arylation reaction, oxidation of the thiol to glutathione disulfide (GSSG), and free radical formation detected by the spin-trapping electron spin resonance method. The position of the hydroxymethyl substituent in either the benzenoid or the quinonoid ring differentially influenced the initial rates of hydroquinone autoxidation as well as thiol oxidation. Thus, GSSG- and hydrogen peroxide formation during the GSH reductive addition to 6-hydroxymethyl-1,4-naphthoquinone proceeded at rates substantially higher than those observed with the 2-hydroxymethyl derivative. The distribution and concentration of molecular end products, however, was the same for both quinones, regardless of the position of the hydroxymethyl substituent. The [O2]consumed/[GSSG]formed ratio was above unity in both cases, thus indicating the occurrence of autoxidation reactions other than those involved during GSSG formation. EPR studies using the spin probe 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) suggested that the oxidation of GSH coupled to the above redox transitions involved the formation of radicals of differing structure, such as hydroxyl and thiyl radicals. These were identified as the corresponding DMPO adducts. The detection of either DMPO adduct depended on the concentration of GSH in the reaction mixture: the hydroxyl radical adduct of DMPO prevailed at low GSH concentrations, whereas the thiyl radical adduct of DMPO prevailed at high GSH concentrations. The production of the former adduct was sensitive to catalase, whereas that of the latter was sensitive to superoxide dismutase as well as to catalase. The relevance of free radical formation coupled to thiol oxidation is discussed in terms of the thermodynamic and kinetic properties of the reactions involved as well as in terms of potential implications in quinone cytotoxicity.     

Generation of superoxide radicals by heart mitochondria: study by spin trapping under continuous oxygenation

The generation of superoxide radicals by isolated rat heart mitochondria was studied by the spin trapping technique. The sample was placed into the cavity of an EPR spectrometer in a thin-wall teflon capillary tube, which made it possible to maintain the partial oxygen pressure in the mitochondrial suspension at a constant level. Tiron was used as a spin trap, and the intensity of its EPR signal corresponded to the rate of O2-. formation in the sample. The addition of oxidation substrates (succinate, glutamate, and malate) into the incubation mixture caused the appearance of the Tiron EPR signal. The rate of superoxide radical generation by heart mitochondria strongly increased in the presence of antimycin A, an inhibitor of the Q-cycle in complex III of the respiratory chain, but it was completely depressed by another inhibitor of Q-cycle myxothiazol. The inhibition of the reverse electron transport in complex I of the respiratory chain by rotenone (oxidation substrate--succinate) caused a substantial decrease in the rate of O2-. formation by mitochondria.     

Polycyclic aromatic hydrocarbon quinones may be either substrates for or irreversible inhibitors of the human placental NAD-linked 15-hydroxyprostaglandin dehydrogenase.

Under aerobic conditions, 9,10-phenanthrenequinone and 5,6-chyrsenequinone undergo oxidation-reduction cycling in the presence of NADH and the NAD-linked 15-hydroxyprostaglandin dehydrogenase. This results in the formation of potentially hazardous semiquinones, the superoxide anion, and H2O2. Superoxide dismutase inhibits this cycling by destroying the free radical chain propagator, the superoxide anion. Four other polycyclic aromatic hydrocarbon quinones are not substrates of the enzyme and they cause it to undergo a time-dependent inactivation. This presumably results from alkylation of the enzyme. Glutathione fully protects the enzyme against inactivation by 1,2-naphthoquinone but is only partially effective against 7,8-benzo[a]pyrenequinone. These results suggest that in tissues which contain the NAD-linked 15-hydroxyprostaglandin dehydrogenase some polycyclic aromatic hydrocarbon quinones might produce deleterious effects by undergoing redox cycling. Others might cause such effects by irreversibly inhibiting the enzyme which catalyzes the first step in prostaglandin catabolism.     

Electron paramagnetic resonance study of the generation of reactive oxygen species catalysed by transition metals and quinoid redox cycling by inhalable ambient particulate matter

A range of epidemiological studies in the 1990s showed that exposure to ambient particulate matter (PM) is associated with adverse health effects in the respiratory system and increased morbidity and mortality rates. Oxidative stress has emerged as a pivotal mechanism that underlies the toxic pulmonary effects of PM. A key question from a variety of studies was whether the adverse health effects of PM are mediated by the carbonaceous particles of their reactive chemical compounds adsorbed into the particles. Experimental evidence showed that PM contains redox-active transition metals, redox cycling quinoids and polycyclic aromatic hydrocarbons (PAHs) which act synergistically to produce reactive oxygen species (ROS). Fine PM has the ability to penetrate deep into the respiratory tree where it overcomes the antioxidant defences in the fluid lining of the lungs by the oxidative action of ROS. From a previous study [Valavanidis A, Salika A, Theodoropoulou A. Generation of hydroxyl radicals by urban suspended particulate air matter. The role of iron ions. Atmospher Environ 2000; 34 : 2379-2386], we established that ferrous ions in PM play an important role in the generation of hydroxyl radicals in the presence of hydrogen peroxide (H2O2). In the present study, we investigated the synergistic effect of transition metals and persistent quinoid and semiquinone radicals for the generation of ROS without the presence of H2O2. We experimented with airborne particulate matter, such as TSPs (total suspended particulates), fresh automobile exhaust particles (diesel, DEP and gasoline, GEP) and fresh wood smoke soot. Using electron paramagnetic resonance (EPR), we examined the quantities of persistent free radicals, characteristic of a mixture of quinoid radicals with different structures and a carbonaceous core of carbon-centred radicals. We extracted, separated and analysed the quinoid compounds by EPR at alkaline solution (pH 9.5) and by TLC. Also, we studied the direct production of superoxide anion and the damaging hydroxyl radical in aqueous and in DMSO suspensions of PM without H2O2. From these results, it is suggested that the cytotoxic and carcinogenic potential of PM can be partly the result of redox cycling of persistent quinoid radicals, which generate large amounts of ROS. In the second phase, the water-soluble fraction of PM elicits DNA damage via reactive transition metal-dependent formation of hydroxyl radicals, implicating an important role for hydrogen peroxide. Together, these data indicate the importance of mechanisms involving redox cycling of quinones and Fenton-type reactions by transition metals in the generation of ROS. These results are supported by recent studies indicating cytotoxic effects, especially mitochondrial damage, by PM extracts and differential mechanisms of cell killing by redox cycling quinones.     

Metabolic activation of sulfur mustard leads to oxygen free radical formation

We recently published electron paramagnetic resonance (EPR) spin trapping results that demonstrated the enzymatic reduction of sulfur mustard sulfonium ions to carbon-based free radicals using an in vitro system containing sulfur mustard, cytochrome P450 reductase, NADPH, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in buffer (A.A. Brimfield et al., 2009, Toxicol. Appl. Pharmacol. 234:128-134). Carbon-based radicals have been shown to reduce molecular oxygen to form superoxide and, subsequently, peroxyl and hydroxyl radicals. In some cases, such as with the herbicide paraquat, a cyclic redox system results, leading to magnified oxygen free radical concentration and sustained tissue damage. Low mustard carbon radical concentrations recorded by EPR in our in vitro system, despite a robust (4.0mM) sulfur mustard starting concentration, led us to believe a similar oxygen reduction and redox cycling process might be involved with sulfur mustard. A comparison of the rate of mustard radical-POBN adduct formation in our in vitro system by EPR at atmospheric and reduced oxygen levels indicated a sixfold increase in 4-POBN adduct formation (0.5 to 3.0 μM) at the reduced oxygen concentration. That result suggested competition between oxygen and POBN for the available carbon-based mustard radicals. In parallel experiments we found that the oxygen radical-specific spin trap 5-tert-butoxycarbonyl-5-methylpyrroline-N-oxide (BMPO) detected peroxyl and hydroxyl radicals directly when it was used in place of POBN in the in vitro system. Presumably these radicals originated from O(2) reduced by carbon-based mustard radicals. We also showed that reactive oxygen species (ROS)-BMPO EPR signals were reduced or eliminated when mustard carbon radical production was impeded by systematically removing system components, indicating that carbon radicals were a necessary precursor to ROS production. ROS EPR signals were completely eliminated when superoxide dismutase and catalase were included in the complete in vitro enzymatic system, providing additional proof of oxygen radical participation. The redox cycling hypothesis was supported by density functional theory calculations and frontier molecular orbital analysis.     

One-electron reduction of quinones by the neuronal nitric-oxide synthase reductase domain

Flavin electron transferases can catalyze one- or two-electron reduction of quinones including bioreductive antitumor quinones. The recombinant neuronal nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FMN prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADPH-oxidation in the presence of the model quinone compound menadione (MD), including antitumor mitomycin C (Mit C) and adriamycin (Adr). Calcium/calmodulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-mediated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxidoreductase (QR), but Mit C- and Adr-mediated NADPH oxidations were not. In anaerobic conditions, cytochrome b5 as a scavenger for the menasemiquinone radical (MD*-) was stoichiometrically reduced by the nNOS reductase domain in the presence of MD, but not of QR. These results indicate that the nNOS reductase domain can catalyze a only one-electron reduction of bivalent quinones. In the presence or absence of Ca2+/CaM, the semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone did not react rapidly with MD, but the fully reduced species of both flavins, FAD and FMN, could donate one electron to MD. The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsuda, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data suggest that the enzyme functions between the 1e- <==> 3e- level during one-electron reduction of MD, and that the rates of quinone reductions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM.     

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O2*-, *OH, and GHB*-) and singlet oxygen (1O2).

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB*-) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB*-) and hydroquinone (GHBH2) of GHB, respectively. GHBH2 is readily formed via the decay of GHB*- in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O2*-) is formed, via the electron transfer from GHB*-, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O2* and *OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen (1O2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.     

Superoxide dismutase-like activities of copper(II) complexes tested in serum

The superoxide dismutase-like activities of a series of coordination complexes of copper were evaluated and compared to the activities of bovine erythrocyte superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) in serum using the nitroblue tetrazolium chloride (NBT)-reduction assay and electron paramagnetic resonance (EPR) spectroscopy. A 40% inhibition was observed for the initial rate of the NBT reduction by superoxide dismutase in serum, but more than 40% inhibition was achieved with CuSO4, Cu(II)-dimethylglyoxime, Cu(II)-3,8-dimethyl-4,7-diazadeca-3,7-dienediamide, Cu2[N,N'-(2-(O-hydroxy-benzhydrylidene)amino)ethyl]2-1,2-ethane dia mine), Cu(II)-(diisopropylsalicylate)2, Cu(II)-(p-bromo-benzoate)2, Cu(II)-(nicotinate)2 and Cu(II)-(1,2-diamino-2-methylpropane)2. The electron paramagnetic resonance technique of spin trapping was used to detect the formation of superoxide (O2-.) and other free radicals in the xanthine-xanthine oxidase system under a variety of conditions. Addition of the spin trapping agent 5,5-dimethylpyrroline 1-oxide (DMPO) to the xanthine-xanthine oxidase system in fetal bovine serum produced the O2-.-spin adduct of DMPO (herein referred to as superoxide spin adduct, DMPO-OOH) as the well known short-lived nitroxyl whose characteristic EPR spectrum was recorded before its rapid decay to undetectable levels. The hydroxyl radical (HO.) adduct of the spin trap DMPO (herein referred to as DMPO-OH) was detected to a very small extent. When CuSO4, or the test complexes of copper, were added to the xanthine-xanthine oxidase system in serum containing the spin trap, the yield of DMPO-OOH was negligible. In addition to their superoxide dismutase-like activity, CuSO4 and the copper complexes also behaved as Fenton-type catalysts as seen by the accumulation of varying amounts of the hydroxyl spin adduct DMPO-OH. Both the Fenton-type catalysis and the superoxide dismutase-like action of these compounds were lost when a chelator such as EDTA was included in the xanthine-xanthine oxidase incubation mixture. Addition of superoxide dismutase instead of the copper compounds to this enzyme system abolished the formation of superoxide adduct DMPO-OOH, and no hydroxyl adduct DMPO-OH was detected. This effect of superoxide dismutase remained unaltered by EDTA.     

5,5-dimethyl-1-pyrroline-N-oxide alone enhances the spontaneous superoxide generation by primaquine.

Primaquine (PQ), a well-known antimalarial drug, has been reported to generate superoxide (O2-) in the presence of reducing agents such as NADPH. In the present study, chemiluminescence was detected by adding only PQ to aqueous 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo-[1,2-alpha] pyrazin-3-one (MCLA), which is a specific chemiluminescent probe for O2-, and was quenched by superoxide dismutase (SOD), indicating that PQ alone can generate O2- in aerobic conditions. Furthermore, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) enhanced the O2- generation by PQ. Superoxide spin adduct, DMPO-OOH, was also detected by ESR both in aqueous solutions and in dimethyl sulfoxide with DMPO. The level of O2- generation showed a linear correlation with the DMPO concentration, and SOD competitively inhibited the DMPO-OOH formation. The results suggested that in aerobic conditions PQ is autoxidized to 5-hydroxy-PQ, which generates O2-, and DMPO accelerates the autoxidation process by trapping O2-. DMPO or M4PO alone enhances the spontaneous O2- generation by PQ, therefore cautious evaluation is necessary in all studies using the ESR/spin trapping technique to elucidate the mechanism of PQ-related radical generation.     

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.     

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.     

Cytotoxicity of the redox cycling compound diquat in isolated hepatocytes: involvement of hydrogen peroxide and transition metals

Diquat is a hepatotoxin whose toxicity in vivo and in vitro is mediated by redox cycling and greatly enhanced by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase. The mechanism by which redox cycling mediates diquat cytotoxicity is unclear, however. Here, we have attempted to examine the roles of three potential products of redox cycling, namely superoxide anion radical (O2-.), hydrogen peroxide (H2O2), and hydroxyl radical (.OH), in the toxicity of diquat to BCNU-treated isolated hepatocytes. Addition of high concentrations of catalase, but not superoxide dismutase, to the incubations provided some protection against the toxic effect of diquat, but much better protection was observed when catalase was added in combination with the iron chelator desferrioxamine. Addition of desferrioxamine alone also provided considerable protection, whereas the addition of copper ions enhanced diquat cytotoxicity. Taken together, these results indicate that both H2O2 and the transition metals iron and copper could play major roles in the cytotoxicity of diquat. The role of O2-. remains less clear, however, but studies with diethylenetriaminepentaacetic acid indicate that O2-. is unlikely to significantly contribute to the reduction of Fe3+ to Fe2+. The hydroxyl radical or a related species seems the most likely ultimate toxic product of the H2O2/Fe2+ interaction, but hydroxyl radical scavengers afforded only minimal protection.     

Histidinyl radical formation in the self-peroxidation reaction of bovine copper-zinc superoxide dismutase.

In the absence of suitable oxidizable substrates, the peroxidase reaction of copper-zinc superoxide dismutase (SOD) oxidizes SOD itself, ultimately resulting in its inactivation. A SOD-centered free radical adduct of 2-methyl-2-nitrosopropane (MNP) was detected upon incubation of SOD with the spin trap and a hydroperoxide (either H(2)O(2) or peracetic acid). Proteolysis by Pronase converted the anisotropic electron paramagnetic resonance (EPR) spectrum of MNP/(center dot)SOD to a nearly isotropic spectrum with resolved hyperfine couplings to several atoms with non-zero nuclear spin. Authentic histidinyl radical (from histidine + HO(center dot)) formed a MNP adduct with a very similar EPR spectrum to that of the Pronase-treated MNP/(center dot)SOD, suggesting that the latter was centered on a histidine residue. An additional hyperfine coupling was detected when histidine specifically (13)C-labeled at C-2 of the imidazole ring was used, providing evidence for trapping at that atom. All of the experimental spectra were convincingly simulated assuming hyperfine couplings to 2 nearly equivalent nitrogen atoms and 2 different protons, also consistent with trapping at C-2 of the imidazole ring. Free histidinyl radical consumed oxygen, implying peroxyl radical formation. MNP-inhibitable oxygen consumption was also observed when cuprous SOD but not cupric SOD was added to a H(2)O(2) solution. Formation of 2-oxohistidine, the stable product of the SOD-hydroperoxide reaction, required oxygen and was inhibited by MNP. These results support formation of a transient SOD-peroxyl radical.     

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.     

Electron paramagnetic resonance decay constant and oxidative stresses in liver microsomes of the selenium-deficient rat

The free radical-reducing activity and the membrane fluidity of liver microsomes from selenium-deficient (SeD) rats were examined by means of electron paramagnetic resonance (EPR) spin label method using nitroxyl-labeled stearic acids. Our findings show that the membrane fluidity and lipid peroxidation levels in SeD rat liver microsome were relatively unchanged compared with normal rat. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity. The nitroxyl spin probes are substrates for reduction-relating cytochrome P-450. Previous in vivo studies suggested that the total liver free radical reduction activity in SeD rat was decreased. In contrast, SeD caused the induction of liver microsomal cytochrome P-450 activity, and the reduction rate of nitroxyl radical existing at shallow depth in membrane was increased. Selenium-deficient rats experienced an increase in hydrogen peroxide (H2O2) due to a pronounced loss of glutathione peroxidase (GSH-Px) activity. This masked the overall reduction rate of the nitroxyl spin probe by reoxidation of the hydroxylamine form. Although the SeD condition caused induction of liver cytochrome P-450 and chronic increased H2O2, this did not result in oxidative liver damage. An increased level of glutathione in SeD liver was also evident, likely due to the absence of GSH-Px activity. Using the EPR spin label method, we have shown that SeD causes complicated redox changes in the liver, notably, alterations in the levels of cytochrome P-450 and GSH-Px systems.     

Activation of cytochrome c to a peroxidase compound I-type intermediate by H2O2: relevance to redox signalling in apoptosis

The release of cytochrome c from mitochondria during apoptosis results in the enhanced production of superoxide radicals, which are converted to H2O2 by Mn-superoxide dismutase. We have been concerned with the role of cytochrome c/H2O2 in the induction of oxidative stress during apoptosis. Our initial studies showed that cytochrome c is a potent catalyst of 2',7'-dichlorofluorescin oxidation, thereby explaining the increased rate of production of the fluorophore 2',7'-dichlorofluorescein in apoptotic cells. Although it has been speculated that the oxidizing species may be a ferryl-haem intermediate, no definitive evidence for the formation of such a species has been reported. Alternatively, it is possible that the hydroxyl radical may be generated, as seen in the reaction of certain iron chelates with H2O2. By examining the effects of radical scavengers on 2',7'-dichlorofluorescin oxidation by cytochrome c/H2O2, together with complementary EPR studies, we have demonstrated that the hydroxyl radical is not generated. Our findings point, instead, to the formation of a peroxidase compound I species, with one oxidizing equivalent present as an oxo-ferryl haem intermediate and the other as the tyrosyl radical identified by Barr and colleagues [Barr, Gunther, Deterding, Tomer and Mason (1996) J. Biol. Chem. 271, 15498-15503]. Studies with spin traps indicated that the oxo-ferryl haem is the active oxidant. These findings provide a physico-chemical basis for the redox changes that occur during apoptosis. Excessive changes (possibly catalysed by cytochrome c) may have implications for the redox regulation of cell death, including the sensitivity of tumour cells to chemotherapeutic agents.