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.     

Inhibition of peroxynitrite-mediated DNA strand cleavage and hydroxyl radical formation by aspirin at pharmacologically relevant concentrations: Implications for cancer intervention

Epidemiological studies have suggested that the long-term use of aspirin is associated with a decreased incidence of human malignancies, especially colorectal cancer. Since accumulating evidence indicates that peroxynitrite is critically involved in multistage carcinogenesis, this study was undertaken to investigate the ability of aspirin to inhibit peroxynitrite-mediated DNA damage. Peroxynitrite and its generator 3-morpholinosydnonimine (SIN-1) were used to cause DNA strand breaks in φX-174 plasmid DNA. We demonstrated that the presence of aspirin at concentrations (0.25-2 mM) compatible with amounts in plasma during chronic anti-inflammatory therapy resulted in a significant inhibition of DNA cleavage induced by both peroxynitrite and SIN-1. Moreover, the consumption of oxygen caused by 250 μM SIN-1 was found to be decreased in the presence of aspirin, indicating that aspirin might affect the auto-oxidation of SIN-1. Furthermore, EPR spectroscopy using 5,5-dimethylpyrroline-N-oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adduct (DMPO-OH) from authentic peroxynitrite, and that aspirin at 0.25-2 mM potently diminished the radical adduct formation in a concentration-dependent manner. Taken together, these results demonstrate for the first time that aspirin at pharmacologically relevant concentrations can inhibit peroxynitrite-mediated DNA strand breakage and hydroxyl radical formation. These results may have implications for cancer intervention by aspirin.     

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.     

Radiation protection of DNA by ferulic acid under in vitro and in vivo conditions

The effect of ferulic acid was studied on γ-radiation-induced relaxation of plasmid pBR322 DNA and induction of DNA strand breaks in peripheral blood leukocytes and bone marrow cells of mice exposed to whole body γ-radiation. Presence of 0.5 mM ferulic acid significantly inhibited the disappearance of supercoiled (ccc) plasmid pBR322 with a dose modifying factor (DMF) of 2.0. Intraperitoneal administration of different amounts (50, 75 and 100 mg/kg body weight) of ferulic acid 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strands breaks in murine peripheral blood leukocytes and bone marrow cells as evidenced from comet assay. The dose-dependent protection was more pronounced in bone marrow cells than in the blood leukocytes. It was observed that there was a time-dependent disappearance of radiation induced strand breaks in blood leukocytes (as evidenced from comet parameters) following whole body radiation exposure commensuration with DNA repair. Administration of 50 mg/kg body weight of ferulic acid after whole body irradiation of mice resulted disappearance of DNA strand breaks at a faster rate compared to irradiated controls, suggesting enhanced DNA repair in ferulic acid treated animals. (Mol Cell Biochem xxx: 209–217, 2005)     

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.     

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.     

Peroxynitrite increases iNOS through NF-kappa B and decreases prostacyclin synthase in endothelial cells

Peroxynitrite, a marker of oxidativestress, is elevated in conditions associated with vascular endothelialcell dysfunction, such as atherosclerosis, preeclampsia, and diabetes.However, the effects of peroxynitrite on endothelial cell function are not clear. The endothelium-derived enzymes nitric oxide synthase (NOS)and prostaglandin H synthase (PGHS) mediate vascular reactivity andcontain oxidant-sensitive isoforms (iNOS and PGHS-2) that can beinduced by nuclear factor (NF)-B activation. We investigated theeffect(s) of peroxynitrite on NOS and PGHS pathways in endothelial cells. We hypothesized that peroxynitrite will increase levels of iNOSand PGHS-2 through activation of NF-B. Western immunoblots ofendothelial cells show that 3-morpholinosydnonimine (SIN-1; 0.5 mM), aperoxynitrite donor, increased iNOS protein mass, which can beinhibited by pyrroline dithiocarbamate (an NF-B inhibitor) (167 ± 24.2 vs. 78 ± 19%, P < 0.05, n = 6). SIN-1 treatment also significantly increasedNF-B translocation into endothelial cell nuclei (135 ± 10%,P < 0.05). Endothelial NOS, PGHS-1, and PGHS-2 proteinlevels were not altered by SIN-1. However, prostacyclin synthaseprotein mass, but not mRNA, was significantly reduced in SIN-1-treatedendothelial cells (78 ± 8.9%, P < 0.05). Our results illustrate novel mechanisms through which peroxynitrite maymodulate vascular endothelial function.

     

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.     

Protection of cellular DNA from γ-radiation-induced damages and enhancement in DNA repair by troxerutin

The effect of troxerutin on γ-radiation-induced DNA strand breaks in different tissues of mice in vivo and formations of the micronuclei were studied in human peripheral blood lymphocytes ex vivo and mice blood reticulocytes in vivo. Treatments with 1 mM troxerutin significantly inhibited the micronuclei induction in the human lymphocytes. Troxerutin protected the human peripheral blood leucocytes from radiation-induced DNA strand breaks in a concentration dependent manner under ex vivo condition of irradiation (2 Gy). Intraperitoneal administration of troxerutin (175 mg/kg body weight) to mice before and after whole body radiation exposure inhibited micronuclei formation in blood reticulocytes significantly. The administration of different doses (75, 125 and 175 mg/kg body weight) of troxerutin 1 h prior to 4 Gy γ-radiation exposure showed dose-dependent decrease in the yield of DNA strand breaks in murine blood leucocytes and bone marrow cells. The dose-dependent protection was more pronounced in bone marrow cells than in blood leucocytes. Administration of 175 mg/kg body weight of the drug (i.p.) 1 h prior or immediately after whole body irradiation of mice showed that the decrease in strand breaks depended on the post-irradiation interval at which the analysis was done. The observed time-dependent decrease in the DNA strand breaks could be attributed to enhanced DNA repair in troxerutin administered animals. Thus in addition to anti-erythrocytic, anti-thrombic, fibrinolytic and oedema-protective rheological activity, troxerutin offers protection against γ-radiation-induced micronuclei formation and DNA strand breaks and enhances repair of radiation-induced DNA strand breaks. (Mol Cell Biochem xxx: 57–68, 2005)     

A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks

Background  

Formation and repair of DNA single-strand breaks are important parameters in the assessment of DNA damage and repair occurring in live cells. The 'Fluorimetric Detection of Alkaline DNA Unwinding (FADU)' method [Birnboim HC, Jevcak JJ. Cancer Res (1981) 41:1889–1892] is a sensitive procedure to quantify DNA strand breaks, yet it is very tedious to perform.     

DNA protective properties of vanillin against γ-radiation under different conditions: Possible mechanisms

Ionizing radiation is an important genotoxic agent. Protecting against this form of toxicant, especially by a dietary component, has several potential applications. In the present study, we have examined the ability of vanillin (4-hydroxy-3-methoxybenzaldehyde), a naturally occurring food flavouring agent, to inhibit radiation-induced DNA damage measured as strand breaks under in vitro, ex vivo and in vivo conditions besides the possible mechanisms behind the observed protection. Our study showed that there was a concentration-dependent inhibition of the disappearance of super-coiled (ccc) form of plasmid pBR322 (in vitro) upon exposure to 50 Gy of γ-radiation. Presence of 0.5 mM vanillin has a dose-modifying factor (DMF) of 6.75 for 50% inactivation of ccc form. Exposure of human peripheral blood leucocytes (ex vivo) to γ-radiation causes strand breaks in the cellular DNA, as assessed by comet assay. When leucocytes were exposed to 2 Gy of γ-radiation there was an increase in parameters of comet assay such as %DNA in tail, tail length, ‘tail moment’ and ‘Olive tail moment’. The presence of 0.5 mM vanillin during irradiation significantly reduced these parameters. Damage to DNA in mouse peripheral blood leucocytes after whole-body exposure of mice (in vivo) to γ-radiation was studied at 1 and 2 h post-irradiation. There was recovery of DNA damage in terms of the above-mentioned parameters at 2 h post-irradiation. This was more than that observed at 1 h. The recovery was more in vanillin treated mice. Hence our studies showed that vanillin offers protection to DNA against radiation-induced damage possibly imparting a role other than modulation of DNA repair. To examine the possible mechanisms of radioprotection, in terms of radiation-derived radicals, we carried out the reaction of vanillin with ABTS⁺ radical spectrophotometrically besides with DNA peroxyl and carbonyl radicals by using pulse radiolysis. Our present investigations show that vanillin has ability to protect against DNA damage in plasmid pBR322, human and mouse peripheral blood leucocytes and splenic lymphocytes besides enhancing survival in splenic lymphocytes against γ-radiation, and that the possible mechanism may involve scavenging of radicals generated during radiation, apart from modulation of DNA repair observed earlier.     

Formation of plasmid DNA strand breaks induced by low-energy ion beam: indication of nuclear stopping effects

Plasmid pGEM 3zf(+) was irradiated by nitrogen ion beam with energies between 20 and 100 keV and the fluence kept as 1×10¹² ions/cm². The irradiated plasmid was assayed by neutral electrophoresis and quantified by densitometry. The yields of DNA with single-strand and double-strand breaks first increased then decreased with increasing ion energy. There was a maximal yield value in the range of 20–100 keV. The relationship between DNA double-strand breaks (DSB) cross-section and linear energy transfer (LET) also showed a peak-shaped distribution. To understand the physical process during DNA strand breaks, a Monte Carlo calculation code known as TRIM (Transport of Ions in Matter) was used to simulate energy losses due to nuclear stopping and to electronic stopping. It can be assumed that nuclear stopping plays a more important role in DNA strand breaks than electronic stopping in this energy range. The physical mechanisms of DNA strand breaks induced by a low-energy ion beam are also discussed. Received: 30 July 1997 / Accepted in revised form: 18 January 1998     

Two distinct mechanisms of nitric oxide-mediated neuronal cell death show thiol dependency

To better understand the mechanism(s) underlying nitricoxide (· NO)-mediated toxicity, in the presence and absenceof concomitant oxidant exposure, postmitotic terminally differentiatedNT2N cells, which are incapable of producing · NO, wereexposed to PAPA-NONOate (PAPA/NO) and 3-morpholinosydnonimine (SIN-1).Exposure to SIN-1, which generated peroxynitrite in the range of25-750 nM/min, produced a concentration- and time-dependentdelayed cell death. In contrast, a critical threshold concentration(>440 nM/min) was required for · NO to produce significantcell injury. Examination of cells by electron microscopy shows alargely necrotic injury after peroxynitrite exposure but mainlyapoptotic-like morphology after · NO exposure. Cellularlevels of reduced thiols correlated with cell death, and pretreatmentwith N-acetylcysteine (NAC) fully protected from cell death ineither PAPA/NO or SIN-1 exposure. NAC given within the first 3 hposttreatment further delayed cell death and increased theintracellular thiol level in SIN-1 but not · NO-exposedcells. Cell injury from · NO was independent of cGMP,caspases, and superoxide or peroxynitrite formation. Overall, exposureof non-· NO-producing cells to · NO orperoxynitrite results in delayed cell death, which, although occurringby different mechanisms, appears to be mediated by the loss ofintracellular redox balance.

     

Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite

We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified method offers the advantage of providing a straightforward and direct measurement of aldehyde- and ketone-containing strand breaks and abasic sites, with the potential for direct labeling in cells prior to DNA isolation.     

Genotoxicity of chloroquine in rat liver cells: Protective role of free radical scavengers

The genotoxic effect of chloroquine (CQ), a 4-aminoquinoline antimalarial drug was investigated in rat liver cells using the alkaline comet assay. Chloroquine (0–1000 μmol/L) significantly increased DNA strand breaks of rat liver cells dose-dependently. Rat liver cells exposed to CQ (100–500 μmol/L) and treated with endonuclease III and formamidopyrimidine-DNA glycosylase, the bacterial DNA repair enzymes that recognize oxidized pyrimidine and purine, respectively, showed greater DNA damage than those not treated with the enzymes, providing evidence that CQ induced oxidation of purines and pyrimidines. Treatment of cells with 5 mmol/L N-acetylcysteine, an intracellular reactive oxygen species (ROS) scavenger, and 100 μmol/L and 250 μmol/L deferoxamine, an established iron chelator, significantly decreased the CQ-induced strand breaks and base oxidation, respectively. Similarly, the formation of DNA strand breaks and oxidized bases was prevented by vitamin C (10 μmol/L) (a water-soluble antioxidant), quercetin (50 μmol/L) (an antioxidant flavonoid), and kolaviron (30 μmol/L and 90 μmol/L) (an antioxidant and a liver hepatoprotective phytochemical). The results indicate that the genotoxicity of CQ in rat liver cells might involve ROS and that free radical scavengers may elicit protective effects in these cells.     

Effect of peroxynitrite on production of superoxide anion radicals and calcium homeostasis in cells of astroglial origin

We studied the effect of a donor of peroxynitrite, SIN-1, on the morphological characteristics of interweaved rat C6 glioma cells, on menadione-induced production of superoxide anion radicals, and on the concentration of Ca²⁺ in these cells. In concentrations of 1.25·10⁻⁴ to 2.5·10⁻⁷ M, SIN-1 demonstrated cytotoxic and antimitogenic effects. This donor of peroxynitrite caused abnormal modifications of the size of C6 cells and the structure of cellular organelles, intensified in a dose-dependent manner the release of Ca²⁺ from cellular stores into the cytoplasm, and suppressed menadione-induced production of superoxide anion radicals. Therefore, it should be believed that peroxynitrite exerts a modifying effect on the processes of mitotic division and induces apoptosis; it is also involved in the processes of intracellular signalling providing an increase in the concentration of cytosolic Ca²⁺ and a decrease in the redox activity of cells. Neirofiziologiya/Neurophysiology, Vol. 38, Nos. 5/6, pp. 401–406, September–December, 2006.     

Monitoring the (photo)genotoxicity of photosensitizer drugs: direct quantitation of single-strand breaks in deoxyribonucleic acid using an oligonucleotide chip

Oligonucleotide chip-based assays can be a sample-thrifty, time-saving, routine tool for evaluation of chemical-induced DNA strand breaks. This article describes a novel approach using an oligonucleotide chip to determine photosensitizer-induced DNA single-strand breaks. Surface coverage of fluorophore-labeled oligonucleotides on silicon dioxide chip surfaces was determined on alkaline phosphatase digestion. Fluorescence maxima (at 520 nm) of the solutions were converted to molar concentrations of the fluorescein-modified oligonucleotide by interpolation from a predetermined standard linear calibration curve. The photosensitizing activity of chlorpromazine and triflupromazine toward DNA single-strand breaks was then studied at different drug doses and also as a function of photoirradiation time. Photoinduced single-strand breaks calculated using the method described here agreed with values predicted by theoretical extrapolation of the single-strand breaks obtained for plasmid DNAs from agarose gel electrophoresis, and thereby indirectly validated the chip-based assays. Under UV irradiation (93.6 kJ/m²) chlorpromazine (0.08 mM) was found to have significant photogenotoxicity. However, triflupromazine did not exhibit any (photo)genotoxicity over the concentration range studied (0.04–0.20 mM). The method developed will be useful for quantitative screening of drug genotoxicity in terms of induction of breaks in DNA.     

Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Peroxynitrite generated in arteries fromsuperoxide and nitric oxide (NO) may damage their function. Here, wecompare the effects of peroxynitrite and peroxynitrite/NO-generatingagents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP(S-nitroso-N-acetyl-penicillamine), SNP (sodiumnitroprusside), and NONOate (spermine NONOate) on pig coronary artery.Deendothelialized artery rings were pretreated with these agents andthen washed before examining their contractility. Pretreatment with allagents (200 µM) results in a decrease in the force of contraction inresponse to the sarco(endo)plasmic Ca²⁺ (SERCA) pumpinhibitor cyclopiazonic acid (CPA): SNAP > NONOate  peroxynitrite  SIN-1 > SNP. Pretreatment with SNAP,NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plussuperoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca²⁺transients produced in response to CPA. Pretreating isolated membranevesicles with peroxynitrite inhibits the Ca²⁺ uptake due tothe SERCA pump, with all the other agents being less effective. Thusperoxynitrite and NO both inhibit the CPA-induced contractions indeendothelialized artery rings, peroxynitrite by damage to the SERCApump and NO possibly by a step downstream from the increase incytosolic Ca²⁺.

     

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater GWA groundwater aquifer