Protection against peroxynitrite-induced DNA damage by mesalamine: implications for anti-inflammation and anti-cancer activity

Mesalamine (5-aminosalicylic acid, 5-ASA) is known to be the first-line medication for treatment of patients with ulcerative colitis. Studies have demonstrated that ulcerative colitis patients treated with 5-ASA have an overall decrease in the risk of developing colorectal carcinoma. However, the mechanisms underlying 5-ASA-mediated anti-inflammatory and anti-cancer effects are yet to be elucidated. Because peroxynitrite has been critically involved in inflammatory stress and carcinogenesis, this study was undertaken to investigate the effects of 5-ASA in peroxynitrite-induced DNA strand breaks, an important event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA with the peroxynitrite generator 3-morpholinosydnonimine (SIN-1) led to the formation of both single- and double-stranded DNA breaks in a concentration-dependent manner. The presence of 5-ASA at 0.1 and 1.0 mM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by SIN-1 was found to not be affected by 5-ASA at 0.1–50 mM, indicating that 5-ASA at these concentrations is not involved in the auto-oxidation of SIN-1 to form peroxynitrite. It is observed that 5-ASA at 0.1–1 mM showed considerable inhibition of peroxynitrite-mediated luminol chemiluminescence in a dose-dependent fashion, suggesting that 5-ASA is able to directly scavenge the peroxynitrite. Electron paramagnetic resonance (EPR) spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap resulting in the formation of DMPO-hydroxyl radical adduct from peroxynitrite, and 5-ASA only at higher concentration (1 mM) inhibited the hydroxyl radical adduct while shifting EPR spectra, indicating that 5-ASA at higher concentrations may generate a more stable free radical species rather than acting purely as a hydroxyl radical scavenger. Taken together, these studies demonstrate for the first time that 5-ASA can potently inhibit peroxynitrite-mediated DNA strand breakage, scavenge peroxynitrite, and affect peroxynitrite-mediated radical formation, which may be responsible, at least partially, for its anti-inflammatory and anti-cancer effects.     

Ethyl Pyruvate Inhibits Peroxynitrite-induced DNA Damage and Hydroxyl Radical Generation: Implications for Neuroprotection

Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite elicited cytotoxicity. Incubation of φX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time- dependent manner. The presence of EP (0.5–10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent fashion. The consumption of oxygen induced by 250 μM SIN-1 was found to be decreased in the presence of EP (0.5–10 mM), indicating that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5–10 mM). EPR spectroscopy in combination with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite, and that EP at 0.5–10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation.     

Up regulation of the GRP-78 and GADD-153 and down regulation of Bcl-2 proteins in primary glomerular diseases: a possible involvement of the ER stress pathway in glomerulonephritis

Alpha-lipoic acid (LA) has recently been reported to afford protection against neurodegenerative disorders in humans and experimental animals. However, the mechanisms underlying LA-mediated neuroprotection remain an enigma. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders, this study was undertaken to investigate the effects of LA in peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite-elicited cytotoxicity. Incubation of φX-174 plasmid DNA with the 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time-dependent fashion. The presence of LA at 100–1,600 μM was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent manner. The consumption of oxygen induced by 250 μM SIN-1 was found to be decreased in the presence of high concentrations of LA (400–1,600 μM), indicating that LA at these concentrations may affect the generation of peroxynitrite from auto-oxidation of SIN-1. It is observed that incubation of the plasmid DNA with authentic peroxynitrite resulted in a significant formation of DNA strand breaks, which could also be dramatically inhibited by the presence of LA (100–1,600 μM). EPR spectroscopy in combination with spin-trapping experiments, using 5,5-dimethylpyrroline-N-oxide (DMPO) as spin trap, resulted in the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite and LA at 50–1,600 μM inhibited the adduct signal. Taken together, these studies demonstrate for the first time that LA can potently inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. In view of the critical involvement of peroxynitrite in the pathogenesis of various neurodegenerative diseases, the inhibition of peroxynitrite-mediated DNA damage by LA may be responsible, at least partially, for its neuroprotective activities.     

Taurine is a weak scavenger of peroxynitrite and does not attenuate sodium nitroprusside toxicity to cells in culture

Summary. Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (↓75%) and SNP (↓50%) at 10 mM. Other taurine analogs (homotaurine, β-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1–2 mM) or taurine (20–40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite. Received May 9, 2000 Accepted June 13, 2000     

Human sensitive to apoptosis gene protein inhibits peroxynitrite-induced DNA damage

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein, which is redox responsive and protects mammalian cells from apoptosis, is a metal chelator and a potential reactive oxygen species scavenger, but its antioxidant properties have not been completely defined. The present study was undertaken to test the hypothesis that human SAG protects from DNA damage induced by peroxynitrite, a potent physiological inorganic toxin. The present study has shown that SAG significantly inhibits single strand breaks in supercoiled plasmid DNA induced by synthesized peroxynitrite (ONOO(-)) and 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion. The formation of 8-hydroxy-2(')-deoxyguanosine in calf thymus DNA by peroxynitrite and SIN-1 was also significantly inhibited by SAG. The protective effect on peroxynitrite-mediated DNA damage was completely abolished by the reaction of SAG with N-ethylmaleimide, a chemical modification agent for the sulfhydryl group of proteins. These observations suggested that the sulfhydryl group of cysteines in SAG could react directly with peroxynitrite to prevent DNA damage.     

Hispidin produced from Phellinus linteus protects against peroxynitrite- mediated DNA damage and hydroxyl radical generation

Oxidative stress plays an important role in the progression of many chronic diseases including cardiovascular diseases, diabetes, cancer and neurodegenerative disorders. One such mediator of oxidative stress is peroxynitrite, which is highly toxic to cultured neurons and astrocytes, and has been reported to be involved in the pathogenesis of various types of neuronal diseases. Therefore, searching for natural compounds with peroxynitrite-scavenging activity might be an effective therapy for peroxynitrite-mediated cytotoxicity. Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong antioxidant, anticancer, and antidiabetic properties. However, the astrocyte protective efficacy of hispidin has been not examined. This study was undertaken to investigate whether the astrocyte protective effect of hispidin is associated with inhibition of peroxynitrite-induced DNA damage, a critical event leading to peroxynitrite-mediated cytotoxicity. Our results showed that peroxynitrite can cause DNA damage in φX-174 plasmid DNA and rat primary astrocytes. The presence of hispidin (10-20μg/ml) was found to significantly inhibit peroxynitrite-induced DNA damage and cytotoxicity. EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from peroxynitrite, and that hispidin potently diminished the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that hispidin can protect against peroxynitrite-mediated cytotoxicity, DNA damage and hydroxyl radical formation.     

Induction of SOS response in Salmonella typhimurium TA4107/pSK1002 by peroxynitrite-generating agent, N-morpholino sydnonimine

Salmonella typhimurium TA4107/pSK1002 strain was used to measure the SOS response induced by peroxynitrite. The parent strain TA4107 (oxydelta1[oxydelta(oxyR argH)1]) is sensitive to oxidative stress and the plasmid of pSK1002 carries a fused gene umuC'-'lacZ, in which umu and lacZ genes are involved in the induction of mutagenesis and beta-galactosidase activity, respectively. Therefore, the level of SOS response was monitored via beta-galactosidase activity. A bolus addition of authentic peroxynitrite (0.3-0.6 mM) increased about eight times the enzyme activity. In N-morpholino sydnonimine (SIN-1), which produces peroxynitrite from superoxide and nitric oxide generated through hydrolysis, addition of over 1mM SIN-1 induced four-five-fold activity. The SIN-1-induced SOS response was scarcely influenced by superoxide dismutase (SOD), catalase or a combination of both, removing the possibility of induction by superoxide, hydrogen peroxide and hydroxyl radical. Two types of peroxynitrite scavengers, mannitol (type I) and glutathione (type II), decreased the response. Mannitol showed a constant inhibition (70%) at a concentration up to 20 mM, exhibiting kinetics that are zero-order in mannitol and first-order in peroxynitrite. On the other hand, glutathione sharply reduced the response dependent on concentration up to 2 mM (90%), indicating second-order kinetics, first-order in both glutathione and peroxynitrite. Dihydrorhodamine (DHR)123, which traps peroxynitrite in a molar ratio of 1:1, efficiently inhibited the SOS response. These effects suggest that peroxynitrite, generated gradually from SIN-1, penetrates through the cell membrane, damages the DNA and induces the SOS response. This strain can thus, be used in screening of antioxidants against peroxynitrite-induced DNA damage in cells.     

Reaction of peroxynitrite with reduced nicotinamide nucleotides, the formation of hydrogen peroxide.

NAD(P)H acts as a two-electron reductant in physiological, enzyme-controlled processes. Under nonenzymatic conditions, a couple of one-electron oxidants easily oxidize NADH to the NAD(.) radical. This radical reduces molecular oxygen to the superoxide radical (O-(2)) at a near to the diffusion-controlled rate, thereby subsequently forming hydrogen peroxide (H(2)O(2)). Because peroxynitrite can act as a one-electron oxidant, the reaction of NAD(P)H with both authentic peroxynitrite and the nitric oxide ((. )NO) and O-(2) releasing compound 3-morpholinosydnonimine N-ethylcarbamide (SIN-1) was studied. Authentic peroxynitrite oxidized NADH with an efficiency of approximately 25 and 8% in the absence and presence of bicarbonate/carbon dioxide (HCO(3)(-)/CO(2)), respectively. NADH reacted 5-100 times faster with peroxynitrite than do the known peroxynitrite scavengers glutathione, cysteine, and tryptophan. Furthermore, NADH was found to be highly effective in suppressing peroxynitrite-mediated nitration reactions even in the presence of HCO(3)(-)/CO(2). Reaction of NADH with authentic peroxynitrite resulted in the formation of NAD(+) and O-(2) and, thus, of H(2)O(2) with yields of about 3 and 10% relative to the added amounts of peroxynitrite and NADH, respectively. Peroxynitrite generated in situ from SIN-1 gave virtually the same results; however, two remarkable exceptions were recognized. First, the efficiency of NADH oxidation increased to 60-90% regardless of the presence of HCO(3)(-)/CO(2), along with an increase of H(2)O(2) formation to about 23 and 35% relative to the amounts of added SIN-1 and NADH. Second, and more interesting, the peroxynitrite scavenger glutathione (GSH) was needed in a 75-fold surplus to inhibit the SIN-1-dependent oxidation of NADH half-maximal in the presence of HCO(3)(-)/CO(2). Similar results were obtained with NADPH. Hence, peroxynitrite or radicals derived from it (such as, e.g. the bicarbonate radical or nitrogen dioxide) indeed oxidize NADH, leading to the formation of NAD(+) and, via O-(2), of H(2)O(2). When peroxynitrite is generated in situ in the presence of HCO(3)(-)/CO(2), i.e. under conditions mimicking the in vivo situation, NAD(P)H effectively competes with other known scavengers of peroxynitrite.     

Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation

Uric acid has been considered to be an efficient scavenger of peroxynitrite but the reaction between urate and peroxynitrite has been only partially characterized. Also, previous studies have indicated that urate may increase peroxynitrite-mediated oxidation of low density lipoprotein (LDL). Here, we examined the reaction between urate and peroxynitrite by combining kinetic, oxygen consumption, spin trapping, and product identification studies; in parallel, we tested the effect of urate upon peroxynitrite-mediated lipid oxidation. Our results demonstrated that urate reacts with peroxynitrite with an apparent second order rate constant of 4.8 x 10(2) M(-1). s(-1) in a complex process, which is accompanied by oxygen consumption and formation of allantoin, alloxan, and urate-derived radicals. The main radical was identified as the aminocarbonyl radical by the electrospray mass spectra of its 5, 5-dimethyl-l-pyrroline N-oxide adduct. Mechanistic studies suggested that urate reacts with peroxynitrous acid and with the radicals generated from its decomposition to form products that can further react with peroxynitrite anion. These many reactions may explain the reported efficiency of urate in inhibiting some peroxynitrite-mediated processes. Production of the aminocarbonyl radical, however, may propagate oxidative reactions. We demonstrated that this radical is likely to be the species responsible for the effects of urate in amplifying peroxynitrite-mediated oxidation of liposomes and LDL, which was monitored by the formation of lipid peroxides and thiobarbituric acid-reactive substances. The aminocarbonyl radical was not detectable during urate attack by other oxidants and consequently it is unlikely to be responsible for all previously described prooxidant effects of uric acid.     

Nitroxidative chemistry interferes with fluorescent probe chemistry: Implications for nitric oxide detection using 2,3-diaminonaphthalene

Simultaneous production of nitric oxide (NO) and superoxide generates peroxynitrite and causes nitroxidative stress. The fluorometric method for NO detection is based on the formation of a fluorescent product from the reaction of a nonfluorescent probe molecule with NO-derived nitrosating species. Here, we present an example of how nitroxidative chemistry could interact with fluorescent probe chemistry. 2,3-Naphthotriazole (NAT) is the NO-derived fluorescent product of 2,3-diaminonaphthalene (DAN), a commonly used NO-detecting molecule. We show that NO/superoxide cogeneration, and particularly peroxynitrite, mediates the chemical decomposition of NAT. Moreover, the extent of NAT decomposition depends on the relative fluxes of NO and superoxide; the maximum effect being reached at almost equivalent generation rates for both radicals. The rate constant for the reaction of NAT with peroxynitrite was determined to be 2.2 × 10³ M⁻¹ s⁻¹. Further, various peroxynitrite scavengers were shown to effectively inhibit NO/superoxide- and peroxynitrite-mediated decomposition of NAT. Taken together, the present study suggests that the interference of a fluorometric NO assay can be originated from the interaction between the final fluorescent product and the formed reactive nitrogen and oxygen species.     

Oxidation of 2',7'-Dichlorofluorescin by Peroxynitrite

The simultaneous production of nitric oxide and superoxide anion leads to the formation of peroxynitrite, a potent oxidant which may be an important mediator of cellular injury. Oxidation of dichlorofluorescin to the fluorescent dichlorofluorescein has been used as a marker for cellular oxidant production. The mechanisms of peroxynitrite-mediated oxidation of dichlorofluorescin to dichlorofluorescein were investigated. Chemically synthesized peroxynitrite (50-500 nM) induced the oxidation of dichlorofluorescin to dichlorofluorescein in a linear fashion. In addition, the simultaneous generation of nitric oxide and superoxide anion induced the oxidation of dichlorofluorescin to dichlorofluorescein, while nitric oxide (1-10 μM) alone under aerobic conditions did not. Peroxynitrite-mediated oxidation of dichlorofluorescin was not inhibited by the hydroxyl radical scavengers mannitol (100 mM) or dimethylsulfoxide (100 mM). Moreover, peroxynitrite-mediated oxidation of dichlorofluorescin was not dependent upon metal ion-catalyzed reactions. Furthermore, dichlorofluorescein formation was diminished at alkaline pH. These findings suggest that peroxynitrite-mediated dichlorofluorescein formation results directly from the protonation of peroxynitrite to form the conjugate peroxynitrous acid. L-cysteine was an efficient inhibitor (K₁ = 25 μM) of dichlorofluorescin oxidation through competitive oxidation of free sulfhydryls. Urate was a less efficient with a maximum inhibition of only 49%. These results demonstrate that dichlorofluorescin is efficiently oxidized by peroxynitrite.

Therefore, under conditions where nitric oxide and superoxide are produced simultaneously, oxidation of dichlorofluorescin may be mediated by the formation of peroxynitrite.     

Kinetic and mechanistic considerations to assess the biological fate of peroxynitrite

Background

Peroxynitrite, the product of the reaction between superoxide radicals and nitric oxide, is an elusive oxidant with a short half-life and a low steady-state concentration in biological systems; it promotes nitroxidative damage.

Scope of review

We will consider kinetic and mechanistic aspects that allow rationalizing the biological fate of peroxynitrite from data obtained by a combination of methods that include fast kinetic techniques, electron paramagnetic resonance and kinetic simulations. In addition, we provide a quantitative analysis of peroxynitrite production rates and conceivable steady–state levels in living systems.

Major conclusions

The preferential reactions of peroxynitrite in vivo include those with carbon dioxide, thiols and metalloproteins; its homolysis represents only < 1% of its fate. To note, carbon dioxide accounts for a significant fraction of peroxynitrite consumption leading to the formation of strong one-electron oxidants, carbonate radicals and nitrogen dioxide. On the other hand, peroxynitrite is rapidly reduced by peroxiredoxins, which represent efficient thiol-based peroxynitrite detoxification systems. Glutathione, present at mM concentration in cells and frequently considered a direct scavenger of peroxynitrite, does not react sufficiently fast with it in vivo; glutathione mainly inhibits peroxynitrite-dependent processes by reactions with secondary radicals. The detection of protein 3-nitrotyrosine, a molecular footprint, can demonstrate peroxynitrite formation in vivo. Basal peroxynitrite formation rates in cells can be estimated in the order of 0.1 to 0.5 μM s^− 1 and its steady-state concentration at ~ 1 nM.

General significance

The analysis provides a handle to predict the preferential fate and steady-state levels of peroxynitrite in living systems. This is useful to understand pathophysiological aspects and pharmacological prospects connected to peroxynitrite. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Peroxynitrite induces F-actin depolymerization and blockade of myosin ATPase stimulation

Treatment of F-actin with the peroxynitrite-releasing agent 3-morpholinosydnonimine (SIN-1) produced a dose-dependent F-actin depolymerization. This is due to released peroxynitrite because it is not produced by 'decomposed SIN-1', and it is prevented by superoxide dismutase concentrations efficiently preventing peroxynitrite formation. F-actin depolymerization has been found to be very sensitive to peroxynitrite, as exposure to fluxes as low as 50-100nM peroxynitrite leads to nearly 50% depolymerization in about 1h. G-actin polymerization is also impaired by peroxynitrite although with nearly 2-fold lower sensitivity. Exposure of F-actin to submicromolar fluxes of peroxynitrite produced cysteine oxidation and also a blockade of the ability of actin to stimulate myosin ATPase activity. Our results suggest that an imbalance of the F-actin/G-actin equilibrium can account for the observed structural and functional impairment of myofibrils under the peroxynitrite-mediated oxidative stress reported for some pathophysiological conditions.     

Aspirin potently inhibits oxidative DNA strand breaks: implications for cancer chemoprevention

Epidemiological studies have suggested that the use of aspirin is associated with a decreased incidence of human malignancies, particularly colorectal cancer. Since reactive oxygen species (ROS) are critically involved in multistage carcinogenesis, this study was undertaken to examine the ability of aspirin to inhibit ROS-mediated DNA damage. Hydrogen peroxide (H2O2)+Cu(II) and hydroquinone (HQ) + Cu(II) were used to cause oxidative DNA strand breaks in phiX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.5-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a marked inhibition of oxidative DNA damage induced by either H2O2/Cu(II) or HQ/Cu(II). The inhibition of oxidative DNA damage by aspirin was exhibited in a concentration-dependent manner. Moreover, aspirin was found to be much more potent than the hydroxyl radical scavengers, mannitol and dimethyl sulfoxide, in protecting against the H2O2/Cu(II)-mediated DNA strand breaks. Since the reduction of Cu(II) to Cu(I) is crucially involved in both H2O2/Cu(II)- and HQ/Cu(II)-mediated formation of hydroxyl radical or its equivalent, and the subsequent oxidative DNA damage, we examined whether aspirin could inhibit this Cu(II)/Cu(I) redox cycle. It was observed that aspirin at concentrations that showed the inhibitory effect on oxidative DNA damage did not alter the Cu(II)/Cu(I) redox cycle in either H2O2/Cu(II) or HQ/Cu(II) system. In addition, aspirin was not found to significantly scavenge H2O2. This study demonstrates for the first time that aspirin potently inhibits both H2O2/Cu(II)- and HQ/Cu(II)-mediated oxidative DNA strand breaks most likely through scavenging the hydroxyl radical or its equivalent derived from these two systems. The potent inhibition of oxidative DNA damage by aspirin may thus partially contribute to its anticancer activities observed in humans.     

Biotransformation and cytotoxic effects of hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes

The biotransformation and cytotoxic effects of hydroxychavicol (HC; 1-allyl-3,4-dihydroxybenzene), which is a catecholic component in piper betel leaf and a major intermediary metabolite of safrole in rats and humans, was studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to HC caused not only concentration (0.25-1.0 mM)- and time (0-3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. At a concentration of 1 mM, the cytotoxic effects of safrole were less than those of HC. The loss of mitochondrial membrane potential and generation of oxygen radical species assayed using 2′,7′-dichlorodihydrofluoresein diacetate (DCFH-DA) in hepatocytes treated with HC were greater than those with safrole. HC at a weakly toxic level (0.25 and/or 0.50 mM) was metabolized to monoglucuronide, monosulfate, and monoglutathione conjugates, which were identified by mass spectra and/or ¹H nuclear magnetic resonance spectra. The amounts of sulfate rather than glucuronide or glutathione conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In hepatocytes pretreated with either diethyl maleate or salicylamide, HC-induced cytotoxicity was enhanced, accompanied by a decrease in the formation of these conjugates and by the inhibition of HC loss. Taken collectively, our results indicate that (a) mitochondria are target organelles for HC, which elicits cytotoxicity through mitochondrial failure related to mitochondrial membrane potential at an early stage and subsequently lipid peroxidation through oxidative stress at a later stage; (b) the onset of cytotoxicity depends on the initial and residual concentrations of HC rather than those of its metabolites; (c) the toxicity of HC is greater than that of safrole, suggesting the participation of a catecholic intermediate in safrole cytotoxicity in rat hepatocytes.     

A reevaluation of the peroxynitrite scavenging activity of some dietary phenolics

Peroxynitrite is implicated in many diseases. Hence, there is considerable interest in potential therapeutic peroxynitrite scavengers. Diet-derived phenolics have been claimed to be powerful peroxynitrite scavengers. However, the reactivity of peroxynitrite can be significantly modified by bicarbonate and this has not been considered in evaluations of the scavenging activity of phenols. Bicarbonate (25 mM) significantly decreased the ability of several phenolic compounds (caffeic acid, o- and p-coumaric acid, gallic acid, ferulic acid) but not others (catechin and epicatechin) to inhibit peroxynitrite-mediated tyrosine nitration. Bicarbonate (25 mM) also decreased the ability of catechin, epicatechin, quercetin and ferulic acid but not chlorogenic acid, gallic acid, caffeic acid and o-coumaric acid to inhibit peroxynitrite-mediated alpha(1)-antiproteinase inactivation. These results show that physiological concentrations of bicarbonate substantially modify the ability of dietary phenolics to prevent peroxynitrite-mediated reactions. When assessing compounds for peroxynitrite scavenging, experiments should be conducted in the presence of bicarbonate to avoid misleading results.     

Induction of endogenous glutathione by the chemoprotective agent, 3H-1,2-dithiole-3-thione, in human neuroblastoma SH-SY5Y cells affords protection against peroxynitrite-induced cytotoxicity

Substantial evidence suggests that peroxynitrite generated from the bi-radical reaction of nitric oxide and superoxide is critically involved in the pathogenesis of neurodegenerative disorders, such as Parkinson's disease. Reaction with sulfhydryl (SH)-containing molecules has been proposed to be a major detoxification pathway of peroxynitrite in biological systems. This study was undertaken to determine if chemically elevated intracellular reduced glutathione (GSH), a major SH-containing biomolecule, affords protection against peroxynitrite-mediated toxicity in cultured neuronal cells. Incubation of human neuroblastoma SH-SY5Y cells with the unique chemoprotectant, 3H-1,2-dithiole-3-thione (D3T), led to a significant elevation of cellular GSH in a concentration-dependent fashion. To examine the protective effects of D3T-induced GSH on peroxynitrite-mediated toxicity, SH-SY5Y cells were pretreated with D3T and then exposed to either the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), or the authentic peroxynitrite. We observed that D3T-pretreated cells showed a markedly increased resistance to SIN-1- or authentic peroxynitrite-induced cytotoxicity, as assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium reduction assay. Conversely, depletion of cellular GSH by buthionine sulfoximine (BSO) caused a marked potentiation of SIN-1- or authentic peroxynitrite-mediated cytotoxicity. To further demonstrate the causal role for GSH induction in D3T-mediated cytoprotection, SH-SY5Y cells were co-treated with BSO to abolish D3T-induced GSH elevation. Co-treatment of the cells with BSO was found to significantly reverse the protective effects of D3T on SIN-1- or authentic peroxynitrite-elicited cytotoxicity. Taken together, this study demonstrates for the first time that D3T can induce GSH in cultured SH-SY5Y cells, and that the D3T-augmented cellular GSH defense affords a marked protection against peroxynitrite-induced toxicity in cultured human neuronal cells.     

Sensitivity of different endpoints for in vitro measurement of genotoxicity of extractable organic matter associated with ambient airborne particles (PM10)

Sensitivity and correlations among three endpoints were evaluated to assess the genotoxic potential of organic complex mixtures in vitro. This study was focused on DNA adduct formation, DNA single strand break induction and tumour suppressor p53 protein up-regulation produced by extractable organic matter (EOM) absorbed on respirable particulate matter PM₁₀ (particulate matter < 10 μm) collected in three European cities (Prague, Sofia, Košice) during winter and summer period. To compare the sensitivity of particular endpoints for in vitro measurement of complex mixture genotoxicity, the metabolically competent human hepatoma cell line Hep G2 was treated with equivalent EOM concentration of 50 μg/ml. Cell exposure to EOMs resulted in significant DNA adduct formation and DNA strand break induction, however, a lack of protein p53 up-regulation over the steady-state level was found. While the maximum of DNA strand breaks was determined after 2 h cell exposure to EOMs, 24 h treatment interval was optimal for DNA adduct determination.No substantial location- and season-related differences in EOM genotoxicity were detected using DNA strand break assessment. In agreement with these results no significant variation in DNA adduct levels were found in relation to the locality and season except for the monitoring site in Prague. The Prague EOM sample collected during summer period produced nearly three-fold lower DNA adduct level in comparison to the winter EOM sample.Comparable results were obtained when the ambient air genotoxicity, based on the concentration of carcinogenic PAHs in cubic meter of air (ng c-PAHs/m³), was elicited using either DNA adduct or strand break determination. In general, at least six-fold higher genotoxicity of the winter air in comparison to the summer air was estimated by each particular endpoint. Moreover, the genotoxic potential of winter air revealed by DNA adduct assessment and DNA strand break measurement increased in the same order: Košice  Prague < Sofia.Based on these data we suppose that two endpoints DNA breakage and DNA adduction are sensitive in vitro biomarkers for estimation of genotoxic activity of organic complex mixture associated with airborne particles. On the other hand, the measurement of protein p53 up-regulation manifested some limitations; therefore it cannot be used as a reliable endpoint for in vitro genotoxicity assessment.     

Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration

Background

Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported.

Methods

We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches.

Results

Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H₂O₂, NO, GSH and peroxynitrite showed that the ONOO⁻ molecule caused the highest inhibition of activity (51% at 5 mM SIN-1), with 5 mM H₂O₂ having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.

Conclusion

These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.

General significance

This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.     

The effect of alpha-tocopherol on the nitration of gamma-tocopherol by peroxynitrite

It has been proposed (S. Christen et al. Proc. Natl. Acad. Sci. USA 94, 3217-3222, 1997) that although alpha-tocopherol (alpha-TH) is an efficient antioxidant, the presence of gamma-tocopherol (gamma-TH) may be required to scavenge peroxynitrite-derived reactive nitrogen species. To investigate the reactions between alpha-TH, gamma-TH, and peroxynitrite, endogenous levels of both alpha-TH and gamma-TH were monitored when low-density lipoprotein was oxidized in the presence of the peroxynitrite generator 5-amino-3-(4-morpholinyl)-1, 2,3-oxadiazolium (SIN-1). SIN-1 oxidized alpha-TH while gamma-TH levels remained constant. The sparing of gamma-TH was also demonstrated when 1,2-dilauroyl-sn-glycero-3-phosphocholine liposomes containing alpha-TH and gamma-TH were incubated with either SIN-1 or peroxynitrite. Our data show that alpha-TH inhibits peroxynitrite-mediated gamma-TH nitration, i.e., 5-NO2-gamma-tocopherol formation. The rate constants for the reactions between both alpha-TH and gamma-TH with peroxynitrite suggest that the sparing of gamma-TH by alpha-TH does not occur by competitive scavenging, but may be due to the formation of a transient gamma-TH intermediate. Nitration of gamma-TH becomes significant only after alpha-TH levels have been depleted. We conclude alpha-TH alone is sufficient to remove any peroxynitrite-derived reactive nitrogen species, as the presence of alpha-TH attenuates nitration of both gamma-TH and tyrosine. The present results also indicate that a bolus addition of peroxynitrite or SIN-1 to liposomes containing gamma-TH forms 5-NO2-gamma-tocopherol in similar yields. This is in contrast to their reaction profile with tyrosine in aqueous solution. Under these conditions, SIN-1 does not form nitrotyrosine at detectable yields.