Oestrogenic compounds and oxidative stress (in human sperm and lymphocytes in the Comet assay)

Reactive oxygen species (ROS) are produced by a wide variety of chemicals and physiological processes in which enzymes catalyse the transfer of electrons from a substrate to molecular oxygen. The immediate products of such reactions, superoxide anion radicals and hydrogen peroxide can be metabolised by enzymes such as superoxide dismutase (SOD) and catalase (CAT), respectively, and depending on its concentration by Vitamin C (Vit C). Under certain circumstances the ROS form highly reactive hydroxyl radicals. We examined human sperm and lymphocytes after treatment with six oestrogenic compounds in the Comet assay, which measures DNA damage, and observed that all caused damage in both cell types. The damage was diminished in nearly all cases by catalase, and in some instances by SOD and Vit C. This response pattern was also seen with hydrogen peroxide. This similarity suggests that the oestrogen-mediated effects could be acting via the production of hydrogen peroxide since catalase always markedly reduced the response. The variable responses with SOD indicate a lesser involvement of superoxide anion radicals due to SOD-mediated conversion of superoxide to hydrogen peroxide generally causing a lower level of DNA damage than other ROS. The variable Vit C responses are explained by a reduction of hydrogen peroxide at low Vit C concentrations and a pro-oxidant activity at higher concentrations. Together these data provide evidence that inappropriate exposure to oestrogenic compounds could lead to free-radical mediated damage. It is believed that the observed activities were not generated by cell free cell culture conditions because increased responses were observed over and above control values when the compounds were added, and also increasing dose-response relationships have been found after treatment with such oestrogenic compounds in previously reported studies.     

Simultaneous protective and damaging effects of cysteamine on intracellular DNA of leukocytes

Incubation of human leukocytes with cysteamine can lead to the induction of DNA strand breaks. The induction of breaks is biphasic with increasing concentration of scavenger. The number of breaks increases in a dose-dependent manner to a maximum and then decreases at higher concentrations. Catalase has been shown to prevent the production of breaks, indicating an involvement of hydrogen peroxide. Cysteamine reacts with oxygen to generate hydrogen peroxide but at higher concentrations it also reacts with hydrogen peroxide. Thus, the biphasic effect of cysteamine on leukocyte DNA may be due to the sum of two separate reaction pathways. (i) Cysteamine reacts with oxygen to generate hydrogen peroxide which leads to DNA strand breakage. (ii) At higher concentrations, it eliminates hydrogen peroxide by reacting with it, thereby protecting the cellular DNA. Other antioxidant scavengers such as WR2721, acetylcysteine and ascorbate can also autooxidize to produce strand breaks. Thiourea and tetramethylurea do not. When tested for their ability to protect cells against DNA damage from added H2O2, the agent which most damaging by itself, cysteamine, was also the most protective.     

Reactive oxygen species and DNA damage after ultrasound exposure

The aim of this work was to detect the formation of hydrogen peroxide and hydroxyl radicals after ultrasound (US) exposure and test the hypothesis that reactive oxygen species induced by ultrasound can contribute to DNA damage. Formation of reactive oxygen species was observed in incubated medium after sonication with 1 MHz continuous ultrasound at the intensities of 0.61-2.44 W/cm2. Free radicals and hydrogen peroxide produced by ultrasound exposure of cells can lead to DNA damage. Comet assay was used to assess the effect of ultrasound on the level of nuclear DNA damage. The nucleated erythrocytes from fish were exposed in vitro to ultrasound at the same intensities and frequency. It was noticed that ultrasound in all used intensities induced DNA damage. The effect was not eliminated by the addition of catalase, which indicates that DNA damage was not caused by hydrogen peroxide only. The results showed that the DNA damage can be repair and this mechanism was the most effective after 30 and 60 min after sonication. Furthermore, the ultrasound-induced DNA damage in the presence of sonosensitizer (Zn- and AlCl-phthalocyanine) was studied. It was noticed that phthalocyaniens (Pcs) alone or with ultrasound did not induce significant changes in the level of DNA damage.     

Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions

Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress. We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase. The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway. Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide. Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide.     

Carcinogenic semicarbazide induces sequence-specific DNA damage through the generation of reactive oxygen species and the derived organic radicals

Semicarbazide, a hydrazine derivative, is carcinogenic to mice but shows no or little mutagenicity in the Salmonella-microsome test. To clarify whether or not the genotoxic mechanism contributes to the non-mutagenic carcinogenicity of semicarbazide, we investigated DNA damage induced by semicarbazide using 32P-5'-end-labeled DNA fragments obtained from the c-Ha-ras-1 protooncogene and the p53 tumor suppressor gene. Semicarbazide caused DNA damage frequently at the thymine and cytosine residues in the presence of Cu(II). Catalase and bathocuproine partially inhibited DNA damage, suggesting that hydrogen peroxide plus Cu(I) participates in DNA damage. When a high concentration of semicarbazide was used in the presence of catalase, DNA damage was induced, especially at G in 5'-AG and slightly at 5'-G in GG and GGG sequences. An electron paramagnetic resonance (EPR) spectroscopic study has confirmed that the reaction of semicarbazide with Cu(II) produces carbamoyl radicals (z.rad;CONH(2)), possibly generated via the nitrogen-centered radicals of semicarbazide. Azodicarbonamide also produced carbamoyl radicals and induced DNA damage frequently at 5'-G in GG and GGG sequences, suggesting that carbamoyl radicals participate in this sequence-specific DNA damage by semicarbazide. On the basis of our previous reports, we consider that the sequence-specific DNA damage at G in 5'-AG in the present study is due to the nitrogen-centered radicals. This study has shown that semicarbazide induces DNA damage in the presence of Cu(II) through the formation of hydrogen peroxide and Cu(I). In addition, semicarbazide-derived free radicals participate in DNA damage. DNA damage induced by these reactive species may be relevant to the carcinogenicity of semicarbazide.     

Mapping of peroxyl radical induced damage on genomic DNA

We have examined the DNA damage produced by reaction of peroxyl radicals with human fibroblast DNA. DNA damage consisted of both strand breaks and base modifications. The extent of strand breaks and base modifications induced as a function of peroxyl radical concentration was determined by quantitation of fragment size distributions using denaturing glyoxal-agarose gel electrophoresis. Both strand breaks and base modifications increased in a log linear fashion with respect to peroxyl radical concentration. Oxidative base modifications were observed to occur to a greater extent than strand breaks at every concentration measured. The sequence-specific distribution of peroxyl radical induced base damage was mapped for 803 nucleotide positions using the method of ligation mediated PCR. A total of 87% of all guanine positions in the examined sequences was found to be significantly oxidized. The order of reactivity of DNA bases toward oxidation by peroxyl radicals was found to be G > C > T. Adenine is essentially unreactive. The yield of oxidative base modifications at guanines and cytosines by peroxyl radicals depends on the exact specification of 5' and 3' flanking bases in a polarity dependent manner. Every guanine in the 5'XGC3' motif was found to be oxidized, where X is any 5' neighbor. In contrast, 5' and 3' purine flanks drastically reduced the extent of peroxyl radical G oxidation. The pattern of base modification and the influence of nearest neighbors differs substantially from that previously reported for hydrogen peroxide damage mediated by low valent transition metal ions for the identical DNA sequences.     

Ganoderma lucidum total triterpenes prevent radiation-induced DNA damage and apoptosis in splenic lymphocytes in vitro

The development of radioprotective agents has been the subject of intense research, especially in the field of radiotherapy. In this study, we examined the radioprotective activity of the total triterpenes isolated from Ganoderma lucidum (Fr.) P. Karst in mouse splenic lymphocytes in vitro. Using the MTT assay, Ganoderma triterpenes were found to have no effect on cell viability, indicating that they are non-toxic to splenic lymphocytes. The effect of the total triterpenes on DNA damage and apoptosis induced by radiation was analyzed using the comet assay, DNA ladder assay and flow cytometric analysis. Total triterpenes were found to be highly effective in preventing DNA laddering, even at low concentrations (25μg/ml). The comet assay demonstrated that the G. triterpenes effectively prevented DNA damage, and flow cytometry revealed a reduction in apoptotic cells. The effect of the total triterpenes on intracellular reactive oxygen species (ROS) level and endogenous antioxidant enzyme activity in splenic lymphocytes were determined to elucidate possible radioprotective mechanisms. Total triterpenes successfully reduced the formation of intracellular ROS and enhanced endogenous antioxidant enzyme activity in splenic lymphocytes following irradiation. Thus, these findings indicate that the total triterpenes isolated from G. lucidum have a remarkable ability to protect normal cells from radiation-induced damage, which suggests therapeutic potential.     

黄芪总黄酮对DNA损伤防护作用的研究

用DNA解旋荧光检测法(FADU)研究了黄芪总黄酮(TFA)对γ射线和H₂O₂所致V79细胞DNA链断裂的防护作用. 结果表明TFA对这两种损伤因子所致的DNA损伤均有不同程度的防护作用, 当TFA浓度达到0.4g/L和0.6g/L时, 分别对H₂O₂和γ射线所致的损伤有保护作用(P<0.05), 而浓度增至0.8g/L和1.2g/L时, 分别对两种因素所致的DNA链断裂损伤有非常显著的防护效果(P<0.01), 对H₂O₂的防护效果优于对γ射线.     

Repair response of Escherichia coli to hydrogen peroxide DNA damage.

The repair response of Escherichia coli to hydrogen peroxide-induced DNA damage was investigated in intact and toluene-treated cells. Cellular DNA was cleaved after treatment by hydrogen peroxide as analyzed by alkaline sucrose sedimentation. The incision step did not require ATP or magnesium and was not inhibited by N-ethylmaleimide (NEM). An ATP-independent, magnesium-dependent incorporation of nucleotides was seen after the exposure of cells to hydrogen peroxide. This DNA repair synthesis was not inhibited by the addition of NEM or dithiothreitol. In dnaB(Ts) strain CRT266, which is thermolabile for DNA replication, normal levels of DNA synthesis were found at the restrictive temperature (43 degrees C), showing that DNA replication was not necessary for this DNA synthesis. Density gradient analysis also indicated that hydrogen peroxide inhibited DNA replication and stimulated repair synthesis. The subsequent reformation step required magnesium, did not require ATP, and was not inhibited by NEM, in agreement with the synthesis requirements. This suggests that DNA polymerase I was involved in the repair step. Furthermore, a strain defective in DNA polymerase I was unable to reform its DNA after peroxide treatment. Chemical cleavage of the DNA was shown by incision of supercoiled DNA with hydrogen peroxide in the presence of a low concentration of ferric chloride. These findings suggest that hydrogen peroxide directly incises DNA, causing damage which is repaired by an incision repair pathway that requires DNA polymerase I.     

Hydrogen peroxide-induced base damage in deoxyribonucleic acid

Aqueous solutions of calf thymus deoxyribonucleic acid (DNA) were exposed to hydrogen peroxide in the presence of air. Base products formed in DNA were identified and quantitated following acid hydrolysis and trimethylsilylation using gas chromatography-mass spectrometry. The yields of these products were dependent upon the hydrogen peroxide concentration, and increased in the following order: 8-hydroxyadenine, cytosine glycol, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 8-hydroxyguanine, thymine glycol, and 4,6-diamino-5-formamidopyrimidine. Previous studies have shown that these compounds are typically formed in DNA in aqueous solution by hydroxyl radicals generated by ionizing radiation. Hydrogen peroxide is thought to participate in a Fenton-like reaction with transition metals, which are readily bound to DNA in trace quantities, resulting in the production of hydroxyl radicals close to the DNA. This proposed mechanism was examined by exposing DNA to hydrogen peroxide either in the presence of a hydroxyl radical scavenger or following pretreatment of DNA with metal-ion chelators. The results indicate that trace quantities of transition metal ions can react readily with hydrogen peroxide to produce radical species. The production of radical species was monitored by determining the altered bases that resulted from the reaction between radicals and DNA. The yields of the base products were reduced by 40 to 60% with 10 mmol dm-3 of dimethyl sulfoxide. A 100-fold increase in the concentration of dimethyl sulfoxide did not result in a further reduction in hydrogen peroxide-induced base damage. DNA which was freed from bound metal ions by pretreatment with metal ion chelators followed by exhaustive dialysis was found to be an ineffective substrate for hydrogen peroxide. The yields of base products measured in this DNA were at background levels. These results support the role of metal ions bound to DNA in the site-specific formation of highly reactive radical species, most likely hydroxyl radicals, in hydrogen peroxide-induced damage to the bases in DNA.     

Hydrogen peroxide: a potent activator of dioxygenase activity of soybean lipoxygenase

Hydrogen peroxide, an ubiquitous biologically occurring peroxide, was found to stimulate the dioxygenase activity of soybean lipoxygenase at the physiologically attainable concentration. The increase in enzyme specific activity was directly proportional to hydrogen peroxide concentration up to 0.5 nM. A decrease in the stimulation of dioxygenase activity was observed at higher concentrations. At low enzyme concentration up to 28-fold stimulation was noted when the formation of lipid hydroperoxide was monitored spectrophotometrically. The stimulation was further confirmed by increased oxygen uptake. It is proposed that the mechanism for in vivo activation involves hydrogen peroxide.     

Effects of polyphenols from grape seeds on oxidative damage to cellular DNA

Grape seed polyphenols have been reported to exhibit a broad spectrum of biological properties. In this study, eleven phenolic phytochemicals from grape seeds were purified by gel chromatography and high performance liquid chromatography (HPLC). The antioxidant activities of five representative compounds with different structure type were assessed by the free radical-scavenging tests and the effects of the more potent phytochemicals on oxidative damage to DNA in mice spleen cells were investigated. Procyanidin B4, catechin, epicatechin and gallic acid reduced ferricyanide ion and scavenged the stable free radical, alpha, alpha-diphenyl-beta-picrylhydrazyl (DPPH) much more effectively than the known antioxidant vitamin ascorbic acid, while epicatechin lactone A, an oxidative derivative of epicatechin, did not reduce ferricyanide ion appreciably at concentrations used and was only about half as effective on free radical-scavenging as epicatechin. Mice spleen cells, when pre-incubated with relatively low concentration of procyanidin B4, catechin or gallic acid, were less susceptible to DNA damage induced by hydrogen peroxide (H2O2), as evaluated by the comet assay. In contrast, noticeable DNA damage was induced in mice spleen cells by incubating with higher concentration (150 microM) of catechin. Collectively, these data suggest that procyanidin B4, catechin, gallic acid were good antioxidants, at low concentration they could prevent oxidative damage to cellular DNA. But at higher concentration, these compounds may induce cellular DNA damage, taking catechin for example, which explained the irregularity of dose-effect relationship.     

Effects of dietary antioxidants on DNA damage in lysed cells using a modified comet assay procedure

A modified version of the comet assay was employed to investigate the effect in vitro of dietary antioxidants in the subcellular environment. Human lymphocytes were isolated, embedded in agarose gel, lysed in high ionic strength solution with Triton X-100, and then incubated for 30 min with antioxidants at different concentrations. Gels were washed, and the comet assay performed on cells stressed by 5 min incubation with 45 microM hydrogen peroxide and on unstressed cells in parallel. Results showed that alpha-tocopherol was protective against oxidant stress, whereas caffeic acid did not protect, and at high concentration (100 microM) caused increased DNA damage. Results for quercetin suggested a direct damaging effect, but this did not reach statistical significance. However, at low concentration (3.1 microM), quercetin appeared protective. Thus some dietary antioxidants that have been shown previously to have a protective effect in the 'standard', whole-cell, comet assay cause DNA damage in this lysed-cell version. The cell membrane may have an important role in limiting cellular access of these 'double-edged' antioxidants. Furthermore, the absolute concentration and the presence of complementary or synergistic intracellular antioxidants may delineate the type of action of a putative antioxidant. We suggest that, used in conjunction with the standard comet assay, this lysed-cell version is useful for assessing the effect of the cell membrane and intracellular systems on susceptibility of DNA to oxidative damage, and will help determine the mechanism of protection or damage by phytochemicals.     

Genoprotection and genotoxicity of green tea (Camellia sinensis): Are they two sides of the same redox coin?

Abstract

Objectives

Regular intake of green tea associates with lower DNA damage and increased resistance of DNA to oxidant challenge. However, in vitro pro-oxidant effects of green tea have been reported. Both effects could be mediated by hydrogen peroxide (H₂O₂) which is generated by autoxidation of tea catechins. In large amounts, H₂O₂ is genotoxic, but low concentrations could activate the redox-sensitive antioxidant response element (ARE) via the Keap-1/Nrf2 redox switch, inducing genoprotective adaptations. Our objective was to test this hypothesis.

Methods

Peripheral lymphocytes from healthy volunteers were incubated for 30 minutes at 37°C in freshly prepared tea solutions (0.005, 0.01, 0.05%w/v (7, 14, 71 µmol/l total catechins) in phosphate buffered saline (PBS), with PBS as control) in the presence and absence of catalase (CAT). H₂O₂ in tea was measured colorimetrically. Oxidation-induced DNA lesions were measured by the Fpg-assisted comet assay.

Results

H₂O₂ concentrations in 0.005, 0.01, and 0.05% green tea after 30 minutes at 37°C were, respectively, ～3, ～7, and ～52 µmol/l. Cells incubated in 0.005 and 0.01% tea showed less (P < 0.001) DNA damage compared to control cells. Cells treated with 0.05% green tea showed ～50% (P < 0.001) more DNA damage. The presence of CAT prevented this damage, but did not remove the genoprotective effects of low-dose tea. No significant changes in expression of ARE-associated genes (HMOX1, NRF2, KEAP1, BACH1, and hOGG1) were seen in cells treated with tea or tea + CAT.

Conclusion

Genoprotection by low-dose green tea could be due to direct antioxidant protection by green tea polyphenols, or to H₂O₂-independent signalling pathways.     

Increased hydrogen peroxide concentration in human tumor cells due to a nitroxide free radical.

Evidence is presented that the nitroxide free radical, TEMPO, at concentrations commonly used to prevent oxidative damage, increases the intracellular hydrogen peroxide concentration. To investigate the origin of this increased hydrogen peroxide concentration, we have incubated various human tumor cell lines with compounds interfering with the generation of active oxygen metabolites. Sodium azide, inhibitor of the respiratory chain, the iron-chelating agent desferrioxamine, superoxide dismutase and catalase had no effect on the hydrogen peroxide concentration. Metyrapone, inhibitor of the cytochrome P450 system, was demonstrated to decrease, but not completely prevent, the hydrogen peroxide production. N-ethylmaleimide, a sulphydryl-bond alkylating agent, was able to completely prevent the increased hydrogen peroxide production. We conclude that, by increasing the cellular hydrogen peroxide concentration, TEMPO exerts a pro-oxidant effect. This increase in hydrogen peroxide production seems to be mediated by the induction of oxidase activity in the cytochrome P450 system, but other cellular systems involved in electron transport may also play a role.     

Mangiferin: A xanthone attenuates mercury chloride induced cytotoxicity and genotoxicity in HepG2 cells

Mangiferin (MGN), a dietary C-glucosylxanthone present in Mangifera indica, is known to possess a spectrum of beneficial pharmacological properties. This study demonstrates antigenotoxic potential of MGN against mercuric chloride (HgCl2)-induced genotoxicity in HepG2 cell line. Treatment of HepG2 cells with various concentrations of HgCl2 for 3 h caused a dose-dependent increase in micronuclei frequency and elevation in DNA strand breaks (olive tail moment and tail DNA). Pretreatment with MGN significantly (p < 0.01) inhibited HgCl2 -induced (20 μM for 30 h) DNA damage. An optimal antigenotoxic effect of MGN, both in micronuclei and comet assay, was observed at a concentration of 50 μM. Furthermore, HepG2 cells treated with various concentrations of HgCl2 resulted in a dose-dependent increase in the dichlorofluorescein fluorescence, indicating an increase in the generation of reactive oxygen species (ROS). However, MGN by itself failed to generate ROS at a concentration of 50 μM, whereas it could significantly decrease HgCl2 -induced ROS. Our study clearly demonstrates that MGN pretreatment reduced the HgCl2-induced DNA damage in HepG2 cells, thus demonstrating the genoprotective potential of MGN, which is mediated mainly by the inhibition of oxidative stress.     

Photo-irradiated titanium dioxide catalyzes site specific DNA damage via generation of hydrogen peroxide

Titanium dioxide (TiO₂) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO₂ was examined using [₃₂P]-5'-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO₂ (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2' -deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO₂ particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO₂.     

Bimodal pattern of killing of DNA-repair-defective or anoxically grown Escherichia coli by hydrogen peroxide.

Two modes of killing of Escherichia coli K-12 by hydrogen peroxide can be distinguished. Mode-one killing was maximal with hydrogen peroxide at a concentration of 1 to 2 mM. At higher concentrations the killing rate was approximately half maximal and was independent of H2O2 concentration but first order with respect to exposure time. Mode-one killing required active metabolism during the H2O2 challenge, and it resulted in sfiA-independent filamentation of both cells which survived and those which were killed by the challenge. This mode of killing was enhanced in xth, polA, recA, and recB strains and was accelerated in all strains by an unidentified, anoxia-induced cell function. A strain carrying both xth and recA mutations appeared to undergo spontaneous mode-one killing only under aerobic conditions. Mode-one killing appeared to result from DNA damage which normally occurs at a low, nonlethal level during aerobic growth. Mode-two killing occurred at higher doses of H2O2 and exhibited a multihit dependence on both H2O2 concentration and exposure time. Mode-two killing did not require active metabolism, and killed cells did not filament, although survivors demonstrated a dose-dependent growth lag. Strains with DNA-repair defects were not especially susceptible to mode-two killing.     

Antioxidant activity of diphenyl diselenide prevents the genotoxicity of several mutagens in Chinese hamster V79 cells

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organic selenium compounds. Studies have shown its antioxidant, hepatoprotective, neuroprotective, anti-inflammatory, and antinociceptive effects. We recently showed the antioxidant effect of DPDS in V79 cells, and established the beneficial and toxic doses of this compound in this cell line. Here, we report the antigenotoxic and antimutagenic properties of DPDS, investigated by using a permanent lung fibroblast cell line derived from Chinese hamsters. We determined the cytotoxicity by clonal survival assay, and evaluated DNA damage in response to several mutagens by comet assay and micronucleus test in binucleated cells. In the clonal survival assay, at concentrations ranging from 1.62 to 12.5microM, DPDS was not cytotoxic, while at concentrations up to 25microM, it significantly decreased survival. The treatment with this organoselenium compound at non-cytotoxic dose range increased cell survival after challenge with hydrogen peroxide, methyl-methanesulphonate, and UVC radiation, but did not protect against 8-methoxypsoralen plus UVA-induced cytotoxicity. In addition, the treatment prevented induced DNA damage, as verified in the comet assay. The mutagenic effect of these genotoxins, as measured by the micronucleus test, similarly attenuated or prevented cytotoxicity and DNA damage. Treatment with DPDS also decreased lipid peroxidation levels after exposure to hydrogen peroxide MMS, and UVC radiation, and increased glutathione peroxidase activity in the extracts. Our results clearly demonstrate that DPDS at low concentrations presents antimutagenic properties, which are most probably due to its antioxidant properties.     

The effect of some tannins on trout erythrocytes exposed to oxidative stress

In order to gain more knowledge on the role of tannins as antioxidants, their ability to protect (Salmo irideus) erythrocytes against oxidative stress was investigated. Antioxidant activity of different tannins (tannic, gallic and ellagic acid) was evaluated by chemiluminescence (CL) techniques using lucigenin and luminol as chemiluminogenic probes for the superoxide radical generated by the xanthine/xanthine oxidase system and hydrogen peroxide, respectively. The superoxide-scavenging activity of these tannins was shown for all the compounds; however, it is not clear if this is due to their ability of scavenging the superoxide radical or to their inhibitory activity on xanthine oxidase. Tannic and ellagic acid showed a marked effect on the reduction of H2O2-luminol chemiluminescence. The influence of these tannins on the rate of hemolysis in stressed trout erythrocytes was investigated and the results indicate that tannic acid accelerates the hemolytic event while gallic and ellagic acid have no significant effect. The possible protective action of these compounds against oxidative DNA damage was assessed using the comet assay, a rapid and sensitive single-cell gel electrophoresis technique, used to detect primary DNA damage in individual cells. The results here reported show that tannins under study are capable at low concentrations of protecting DNA breakage, while at high concentrations they can be genotoxic.