Human sperm DNA integrity assessed by the Comet and ELISA assays.

DNA integrity in sperm is essential for the accurate transmission of genetic information and therefore the maintenance of good health in future generations. The ELISA and Comet assays, two techniques that detect DNA damage in cells, are compared in this study of DNA integrity in human sperm. Both techniques rely on alkaline unwinding for the release of single strands of DNA from the nucleus. The ELISA detects single strands immunochemically whereas the Comet assay measures single strands drawn out by electrophoresis, stained with ethidium bromide and quantified by image analysis. The two techniques, both modified for use with sperm, detect similar levels of baseline DNA damage along with similar dose-dependent patterns of induced damage by X-ray irradiation at 10 and 30 Gy (P < 0.05). The assays are also comparable in the detection of a significant protective effect by ascorbic acid (300 and 600 microM) and alpha-tocopherol (30 and 60 microM) on DNA integrity, both at baseline levels and following X-ray irradiation (p < 0.01). The advantages and disadvantages of each technique are discussed.     

In vitro isoflavone supplementation reduces hydrogen peroxide-induced DNA damage in sperm

Isoflavones are plant compounds, proposed to have health benefits in a variety of human diseases, including coronary heart disease and endocrine-responsive cancers. Their physiological effects include possible antioxidant activity, therefore suggesting a role for isoflavones in the prevention of male infertility. The aim of this study was to test the antioxidant effects of the isoflavones genistein and equol on sperm DNA integrity, assessed in vitro after hydrogen peroxide-mediated damage, using the comet assay. Pre-treatment with genistein or equol at doses of 0.01-100 micromol/l significantly protected sperm DNA against oxidative damage. Both ascorbic acid (10-600 micromol/l) and alpha-tocopherol (1-100 micromol/l) also protected. Compared with ascorbic acid and alpha-tocopherol, added at physiological concentrations, genistein was the most potent antioxidant, followed by equol, ascorbic acid, and alpha-tocopherol. Genistein and equol added in combination were more protective than when added singly. Based on these preliminary data, which are similar to those observed previously in lymphocytes, these compounds may have a role to play in antioxidant protection against male infertility.     

Glutathione and hypotaurine in vitro: effects on human sperm motility, DNA integrity and production of reactive oxygen species

Sperm DNA integrity is of paramount importance for the accurate conveyance of genetic material. DNA damage may be a major contributory factor in male infertility as DNA from sperm of infertile men has been found to be more susceptible to induced DNA damage in vitro than DNA from fertile men. Reactive oxygen species (ROS) are a significant source of DNA damage and human sperm are extremely sensitive to ROS attack due to their high content of polyunsaturated fatty acids and lack of capacity for DNA repair. Seminal plasma, which contains a wealth of antioxidants, provides sperm with crucial protection against oxidative insult. However, during preparation for use in assisted conception techniques, sperm are separated from seminal plasma and deprived of that essential protection. The aim of this study was to determine the effects of supplementation with glutathione and hypotaurine during sperm preparation on subsequent sperm motility, DNA integrity, induced DNA damage and ROS generation. Semen samples (n = 45) were divided into aliquots and prepared by Percoll density centrifugation (95.0-47.5%) using medium which had been supplemented with these antioxidants to a number of different concentrations all within physiological levels. Control aliquots were included which had no glutathione or hypotaurine added. Sperm motility was determined using computer-assisted semen analysis. DNA damage was induced using H(2)O(2) and DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay, while ROS generation was measured using chemiluminescence. Addition of glutathione and hypotaurine, either singly or in combination, to sperm preparation medium had no significant effect on sperm progressive motility or baseline DNA integrity. Despite this, sperm were still afforded significant protection against H(2)O(2)-induced damage and ROS generation.     

Inhibitory effect of ascorbic acid post-treatment on radiation-induced chromosomal damage in human lymphocytes in vitro

In the present study, the effect of exposure to ascorbic acid (vitamin C) after gamma-ray-induced chromosomal damage in cultured human lymphocytes was examined to explore the mechanism by which this antioxidant vitamin protects irradiated cells Non-irradiated lymphocytes were exposed to increasing concentrations of ascorbic acid (1-100 micro g/ml) and DNA damage was estimated using chromosomal aberration analysis and the comet assay. The results showed that ascorbic acid did not influence the frequency of chromosomal aberrations in non-irradiated cells, except at the highest concentration (20 micro g/ml), which induced breakage-type chromosomal aberrations. Vitamin C at the concentration of 50 micro g/ml caused DNA damage detected by the comet assay. A significant (34%) decrease in the frequency of chromosomal aberrations was observed in lymphocytes exposed to gamma-radiation and then cultured in the presence of ascorbic acid (1 micro g/ml). The removal of DNA breaks in cells exposed to 2 Gy of gamma-radiation was accelerated in the presence of ascorbic acid as determined by the comet assay, suggesting that it may stimulate DNA repair processes.     

Antioxidant and anti-mutagenic effects of ebselen in yeast and in cultured mammalian V79 cells

Ebselen has a wide spectrum of interesting therapeutic actions including antioxidant, cytoprotective, neuroprotective and anti-inflammatory activities. Since its antioxidant effect is very well known, this paper links the effects of ebselen in redox cellular status to its possible involvement in the maintenance of the integrity of genomic information by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defences and the mammalian V79 cell line. Using the alkaline comet assay, we showed that 5-10 microM ebselen does not induce DNA damage in V79 cells. Similarly, these same concentrations diminished the extent of the DNA damage induced by hydrogen peroxide (H(2)O(2)). The modified comet assay using DNA glycosylases (formamidopyrimidine-DNA glycosylase and endonuclease II) showed that after pre-treatment with ebselen followed by exposure to H(2)O(2), oxidative damage as recognized by these enzymes was significantly lower. In the same way, ebselen showed strong activity against H(2)O(2)-induced oxidative damage in the anti-mutagenic assay using S. cerevisiae N123 strain and in the antioxidative assay by using S. cerevisiae strains lacking antioxidant defences. This antioxidant effect was more pronounced for the gpx3 delta mutant, which indicated that ebselen acts by mimicking the GPx3 catalytic activity. The results confirm that ebselen is involved in antioxidant defence and that its antioxidant ability contributes to its anti-mutagenic and anti-genotoxic action.     

Modulation by flavonoids of DNA damage induced by estrogen-like compounds

Reactive oxygen species (ROS) are produced by a wide variety of exogenous chemicals and metabolic processes and cause a broad spectrum of damage to biological systems. As a consequence, ROS react with DNA, among many other biological targets, disrupting its structure and functionality. Estrogen-like compounds mediate DNA damage by ROS generation, implying that their effects can be modulated by antioxidants such as catalase, superoxide dismutase, and vitamin C. We examined DNA damage in human lymphocytes and sperm after treatment with four estrogen-like compounds (beta-estradiol, diethylstilbestrol, daidzein, and genistein) and its modulation by flavonoids (quercetin and kaempferol) using the Comet assay. The results indicated that quercetin and kaempferol reduced the DNA damage produced in sperm and lymphocytes by the four estrogenic compounds. The flavonoids also reduced the DNA damage induced by hydrogen peroxide, which was used as a positive control. Our results demonstrate that the antioxidant properties of flavonoids can protect the integrity of human sperm and lymphocyte DNA from ROS induced by estrogenic compounds.     

Alterations of GSH and MDA levels and their association with bee venom‐induced DNA damage in human peripheral blood leukocytes

Bee venom (BV) has toxic effects in a variety of cell systems and oxidative stress has been proposed as a possible mechanism of its toxicity. This study investigated the in vitro effect of BV on glutathione (GSH) and malondialdehyde (MDA) levels, and their association with BV‐induced DNA strand breaks and oxidative DNA damage in human peripheral blood leukocytes (HPBLs). Blood samples were treated with BV at concentrations ranging from 0.1 to 10 μg/ml over different lengths of time, and DNA damage in HPBLs was monitored with the alkaline and formamidopyrimidine glycoslyase (FPG)‐modified comet assays, while GSH and MDA levels were determined in whole blood. Results showed a significant increase in overall DNA damage and FPG‐sensitive sites in DNA of HPBLs exposed to BV compared with HPBLs from controls. An increase in DNA damage (assessed with both comet assays) was significantly associated with changes in MDA and GSH levels. When pretreated with N‐acetyl‐L ‐cysteine, a source of cysteine for the synthesis of the endogenous antioxidant GSH, a significant reduction of the DNA damaging effects of BV in HPBLs was noted. This suggests that oxidative stress is at least partly responsible for the DNA damaging effects of BV. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.     

Lack of an association between environmental exposure to polychlorinated biphenyls and p,p'-DDE and DNA damage in human sperm measured using the neutral comet assay

BACKGROUND: Chlorinated organic chemicals, such as polychlorinated biphenyls (PCB), hexachlorobenzene (HCB), dichlorodiphenyl trichloroethane (DDT), and dichlorodiphenyl dichloroethene (DDE, the most stable daughter compound of DDT) are persistent lipophilic compounds found in a large portion of the general population. To explore the hypothesis that environmental exposure to these compounds is associated with altered DNA integrity in human sperm, a study of 212 male partners of a sub-fertile couple who presented to the Massachusetts General Hospital Andrology Laboratory was conducted. METHODS: The neutral single cell microgel electrophoresis assay (comet assay) was used to assess DNA integrity in sperm. VisComet image analysis software was used to measure total comet length, the proportion of DNA present in the comet tail, and tail distributed moment, an integrated measure of length and intensity. RESULTS: In the regression analyses, there were no statistically significant consistent associations between the comet assay parameters and any of the individual PCB congeners, sum of PCB, or p,p'-DDE. CONCLUSION: These results suggest that there are not strong relationships between adult levels of these chlorinated organic compounds and sperm DNA damage as measured by the comet assay.     

Characterization of sperm chromatin quality in testicular cancer and Hodgkin's lymphoma patients prior to chemotherapy

BACKGROUND: Although the incidences of testicular cancer and Hodgkin's lymphomahave increased in young men over the past decade, combinationchemotherapy has improved survival. As fertility is of importanceto these patients, characterization of sperm chromatin structureis needed. We assessed sperm chromatin in testicular cancerand Hodgkin's lymphoma patients prior to chemotherapy, in comparisonwith control community and idiopathic infertile volunteers. METHODS: DNA damage was assessed with the sperm chromatin structure assay(SCSA), terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling (TUNEL) and comet assays; reactive thiols(SH) and DNA compaction were determined with the monobromobimane(mBBr) and chromomycin A3 (CMA3) assays, respectively. RESULTS: Both testicular cancer (37%) and Hodgkin's lymphoma (81%) patientshad normospermic samples with increased DNA damage, comparedwith controls. Cancer patients also had higher reactive thiolsand CMA3 staining, indicating low DNA compaction. CONCLUSIONS: Sperm DNA integrity and compaction were affected in testicularcancer and Hodgkin's lymphoma patients prior to chemotherapy.Although SCSA, TUNEL and comet assays all detected DNA damage,the latter was optimal for use in cancer patients. A combinationof the comet assay with tests that evaluate sperm DNA compaction,such as flow cytometry-based CMA3 and mBBr assays, is a reliablestrategy to characterize sperm chromatin quality in cancer patientsat the time of sperm banking.     

Plasma ascorbate deficiency is associated with impaired reduction of sulfamethoxazole-nitroso in HIV infection

OBJECTIVE: The objective of these studies was to determine the role of ascorbate deficiency in HIV infection in the defective detoxification of sulfamethoxazole-nitroso, the metabolite thought to mediate sulfonamide hypersensitivity reactions. METHODS: Fifty-one HIV-infected patients and 26 healthy volunteers were evaluated. Vitamin supplementation histories were obtained, and blood samples were collected for determination of plasma ascorbate, dehydroascorbate, and cysteine concentrations, erythrocyte glutathione concentrations, and plasma reduction of sulfamethoxazole-nitroso in vitro. RESULTS: Plasma ascorbate concentrations were significantly lower in HIV-positive patients not taking vitamin supplements (29.5 +/- 22.3 microM) than in healthy subjects (54.8 +/- 22.3 microM; P = 0.0005) and patients taking 500-1000 mg of ascorbate daily (82.5 +/- 26.3 microM; P < 0.0001). Plasma ascorbate deficiency was strongly correlated with impaired reduction of sulfamethoxazole-nitroso to its hydroxylamine (r = 0.60, P < 0.0001), and during in vitro reduction, the loss of plasma ascorbate was strongly associated with the amount of nitroso reduced (r = 0.70, P < 0.0001). Ascorbate added ex vivo normalized this reduction pathway. Erythrocyte glutathione concentrations were significantly lower in HIV-positive patients (0.98+/-0.32 mM) than in healthy subjects (1.45+/-0.49 mM; P = 0.001), but this finding was unrelated to ascorbate supplementation. There was trend toward lower plasma cysteine concentrations in patients (8.4+/-3.9 microM) than in controls (10.3+/-4.3 microM), but this trend was similarly unrelated to ascorbate supplementation. Dehydroascorbate concentrations were not significantly higher in HIV-positive patients (7.4+/-10.5%) than in healthy controls (4.0+/-6.2%), even in the subset of patients taking ascorbate (8.4+/-9.4%). CONCLUSIONS: Ascorbate deficiency is common in HIV-positive patients and is associated with impaired detoxification of sulfamethoxazole-nitroso, the suspected proximate toxin in sulfonamide hypersensitivity. Patients taking daily ascorbate supplements (500-1000 mg) achieved high plasma ascorbate concentrations and did not show this detoxification defect. Ascorbate deficiency (or supplementation) was not associated with changes in glutathione or cysteine concentrations. These data suggest that ascorbate deficiency, independent of thiol status, may be an important determinant of impaired drug detoxification in HIV infection.     

Inhibition of telomerase activity in endometrial cancer cells by selenium-cisplatin conjugate despite suppression of its DNA-damaging activity by sodium ascorbate.

Telomerase activation can be considered as a critical step in cell immortalization. The enzyme elongates or maintains telomere length by adding to its end tandem TTAGGG repeats by using its endogenous RNA template. Telomerase is not detectable in most somatic cells but is upregulated in germ line cells and in 85-90% of human cancers, which suggests important role of telomerase in neoplastic transformation. Consequently, telomerase has been proposed as a potentially highly selective target for the development of antiproliferative agents. Platinum complexes are widely administrated in cancer therapy. A conjugate of selenite with diammineplatinum [(NH(3))(2)Pt(SeO(3))(2)] is a novel potential anticancer drug. Using alkaline single cell gel electrophoresis (comet assay), we showed that the drug at 5-30 microM induced concentration-dependent damage to DNA of endometrial cancer cells derived from tumor samples. Sodium ascorbate at 10 and 50 microM reduced the extent of the DNA damage evoked by the drug. (NH(3))(2)Pt(SeO(3)) reduced telomerase activity in the cells in a concentration-dependent manner as measured by using the telomere repeat amplification protocol (TRAP) assay. This effect was independent of sodium ascorbate. Therefore, mutagenic effects of the conjugate can be reduced by well-recognized antimutagen, sodium ascorbate, but it can still retain ability to affect neoplastic transformation. The results obtained indicate that (NH(3))(2)Pt(SeO(3)) may specifically inhibit telomerase activity in endometrial cancer cells.     

Urinary levels of insecticide metabolites and DNA damage in human sperm

BACKGROUND: Members of the general population are exposed to non-persistent insecticides at low levels. The present study explored whether environmental exposures to carbaryl and chlorpyrifos are associated with DNA damage in human sperm. METHODS: Subjects (n=260) were recruited through a Massachusetts infertility clinic. Individual exposures were measured as spot urinary metabolite concentrations of chlorpyrifos [3,5,6-trichloro-2-pyridinol (TCPY)] and carbaryl [1-naphthol (1N)], adjusted using specific gravity. Sperm DNA integrity was assessed by neutral comet assay and reported as comet extent, percentage DNA in comet tail (Tail%) and tail distributed moment (TDM). RESULTS: A statistically significant increase in Tail% was found for an interquartile range (IQR) increase in both 1N [coefficient=4.1; 95% confidence interval (CI) 1.9-6.3] and TCPY (2.8; 0.9-4.6), while a decrease in TDM was associated with IQR changes in 1N (-2.2; -4.9 to 0.5) and TCPY (-2.5; -4.7 to -0.2). A negative correlation between Tail% and TDM was present only when stratified by comet extent, suggesting that Tail% and TDM may measure different types of DNA damage within comet extent strata. CONCLUSIONS: Environmental exposure to carbaryl and chlorpyrifos may be associated with increased DNA damage in human sperm, as indicated by a change in comet assay parameters.     

Influence of vitamin C diet supplementation on endogenous antioxidant defences during exhaustive exercise

We determined the effects of dietary vitamin C supplementation on erythrocyte antioxidant enzymes and on plasma antioxidants during athletic competition and short-term recovery. Blood samples were taken from 16 volunteer endurance athletes, participating in a duathlon competition, under basal conditions and both immediately and 1 h after the competition. The results were analysed taking into account the individual vitamin C intake and the plasma levels. Athletes were assigned to either the vitamin C-supplemented or control groups (n=8 each). The control group had normal plasma ascorbate levels, the supplemented group high levels as a result of the higher vitamin C intake. Uric acid and lactate dehydrogenase increased after the competition only in the control group. Plasma ascorbate decreased after short-term recovery in the supplemented group. Erythrocyte catalase activity increased after the competition in the supplemented group. Glutathione peroxidase activity (determined with cumene hydroperoxide as substrate) increased only in the control group after short-term recovery. This pattern may suggest an important role for plasma ascorbate, and dietary vitamin C supplementation, in the defence against oxidative stress induced by exercise and in avoiding negative effects on erythrocyte integrity.     

Using the alkaline comet assay in prognostic tests for male infertility and assisted reproductive technology outcomes

Infertility affects one in six couples in Europe during their reproductive years with dysfunctional sperm being one of the most common causes. Conventional semen analysis has proven variable and lacking in prognostic value so, over the past decade, more useful molecular fertility biomarkers have been explored. Among the tests showing most promise are those measuring sperm DNA quality. Sperm DNA damage has been closely associated with numerous indicators of reproductive health, including, fertilization, embryo quality, implantation, spontaneous abortion and childhood diseases. It therefore has great potential as a prognostic test for assisted reproductive treatment (ART), when couples are presenting with male infertility. Unlike somatic cells, sperm have a unique tightly compacted chromatin structure. Our group has modified the alkaline comet assay for use with sperm. Sperm DNA also differs from somatic cells in its high susceptibility to oxidative damage; this is largely due to the presence of abundant polyunsaturated fatty acids acting as substrates for reactive oxygen species (ROS) and its lack of repair mechanisms. Consequently, the effects of ROS and antioxidant protection on sperm DNA fragmentation have been widely investigated. In this review, the relationship between actual sperm DNA damage as determined by the alkaline comet assay and potential DNA damage as measured by DNA adduct testing will also be examined and the potential of routine clinical practices such as cryopreservation and prolonged incubation to induce further DNA damage was investigated. Finally, the usefulness of sperm DNA tests as prognostic markers and in particular, the opportunities and challenges provided by DNA testing in male fertility determination will be discussed.     

DNA damage induced by a quinoxaline 1,4-di-N-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay

The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 microM in hypoxia; 20, 40 microM in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24-168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 +/- 13, 343 +/- 30 and 399 +/- 1; control comet score: 42 +/- 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 +/- 14 and 312 +/- 9; control comet score: 27 +/- 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time- and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.     

Enhanced testicular antioxidant system by ascorbic acid in alloxan diabetic rats

The diabetic subject is at significantly increased risk of developing testicular changes. Its etiology may involve oxidative damage by free radicals and protection against such damage can be offered by antioxidant supplementation. Alloxan elicited significant inhibition of antioxidants including superoxide dismutase, catalase and glutathione reductase activities and decreased glutathione content in testis. These effects were accompanied by significant elevation of testicular lipid peroxidation, decreased plasma testosterone level and a drop in copper and zinc concentrations in testis. The administration of ascorbic acid after alloxan treatment interfered and prevented alloxan action. Ascorbic acid blunted the increased testicular lipid peroxidation and the decreased plasma testosterone level probably by protecting antioxidants and the loss of copper and zinc from testes. The data suggested that ascorbic acid has a protective effect on alloxan-induced damage by maintaining the activity of cellular antioxidants.     

Caryocar brasiliense camb protects against genomic and oxidative damage in urethane-induced lung carcinogenesis

The antioxidant effects of Caryocar brasiliense Camb, commonly known as the pequi fruit, have not been evaluated to determine their protective effects against oxidative damage in lung carcinogenesis. In the present study, we evaluated the role of pequi fruit against urethane-induced DNA damage and oxidative stress in forty 8-12 week old male BALB/C mice. An in vivo comet assay was performed to assess DNA damage in lung tissues and changes in lipid peroxidation and redox cycle antioxidants were monitored for oxidative stress. Prior supplementation with pequi oil or its extract (15 µL, 60 days) significantly reduced urethane-induced oxidative stress. A protective effect against DNA damage was associated with the modulation of lipid peroxidation and low protein and gene expression of nitric oxide synthase. These findings suggest that the intake of pequi fruit might protect against in vivo genotoxicity and oxidative stress.     

Reactive oxygen species: potential cause for DNA fragmentation in human spermatozoa

The objective of this study was to evaluate the effect of the generation of reactive oxygen species (ROS) on the integrity of the DNA of human spermatozoa, and to determine if pretreatment with antioxidants can reduce DNA damage. Samples were obtained from 47 men undergoing infertility investigation. ROS were generated in the samples by the addition of xanthine/xanthine oxidase (X/XO) with or without antioxidants. After incubation at timed intervals (0-2 h) with X/XO, the percentage of spermatozoa with DNA fragmentation was determined using the method of TdT-mediated DNA end-labelling (TUNEL). Time intervals were selected to mimic the clinical situation in which spermatozoa are held for a period of time after swim-up while the oocytes are prepared for ICSI. A significant increase in sperm DNA damage was evident when samples were incubated in the presence of ROS for intervals of 1 and 2 h, but not when incubated with ROS for <1 h (P = 0.0001). The addition of antioxidants significantly decreased the amount of DNA damage induced by ROS generation (P < 0.04). ROS can cause an increase in DNA fragmentation and pretreatment with antioxidants can reduce DNA damage.      

Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants

Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.      

DNA damage and repair in human lymphocytes and gastric mucosa cells exposed to chromium and curcumin.

Human population can be considered as a subject of combined exposure to chemicals. Hexavalent chromium is a well-known mutagen and carcinogen. Curcumin, a popular spice and pigment, is reported to have antineoplastic properties. The single cell gel electrophoresis (Comet assay) is a sensitive technique that allows detecting double- and single-strand DNA breaks caused by a broad spectrum of mutagens. In the present work the ability of curcumin to reduce DNA damage induced by chromium in human lymphocytes and gastric mucosa (GM) cells was investigated by using the comet assay. Chromium at 500 microM evoked DNA damage measured as significant (P < 0.001), about a two-fold increase in comet tail moment of both lymphocytes and GM cells. Curcumin at 10, 25, and 50 microM also damaged DNA of both types of cells in a dose-dependent manner: the increase in the tail moment reached about twenty times of the control value (P < 0.001). The combined action of chromium at 500 microM and curcumin at 50 microM resulted in the significant (P < 0.001) increase in the comet tail moment of both types of cells. In each case, treated cells were able to recover within 60 min. Our study clearly demonstrates that curcumin does not inhibit DNA damaging action of hexavalent chromium in human lymphocytes and GM cells. Moreover, curcumin itself can damage DNA of these cells and the total effect of chromium and curcumin is additive. Further studies are needed to establish the role of interaction of curcumin with DNA in carcinogenesis.