Oxidative damage to biological macromolecules in Prague bus drivers and garagemen: Impact of air pollution and genetic polymorphisms

DNA integrity was investigated in the lymphocytes of 50 bus drivers, 20 garagemen and 50 controls using the comet assay with excision repair enzymes. In parallel, 8-oxo-7,8-dihydro-2′-deoxyguanosine and 15-F_2t-isoprostane levels in the urine and protein carbonyl levels in the plasma were assessed as markers of oxidative damage to DNA, lipids and proteins. Exposure to carcinogenic polycyclic aromatic hydrocarbons (cPAHs) and volatile compounds was measured by personal samplers for 48 and 24 h, respectively, before the collection of biological specimens. Both exposed groups exhibited a higher levels of DNA instability and oxidative damage to biological macromolecules than the controls. The incidence of oxidized lesions in lymphocyte DNA, but not the urinary levels of 8-oxodG, correlated with exposure to benzene and triglycerides increased this damage. Oxidative damage to lipids and proteins was associated with exposure to cPAHs and the lipid peroxidation levels positively correlated with age and LDL cholesterol, and negatively with vitamin C. The carriers of at least one variant hOGG1 (Cys) allele tended to higher oxidative damage to lymphocyte DNA than those with the wild genotype, while XPD23 (Gln/Gln) homozygotes were more susceptible to the induction of DNA strand breaks. In contrast, GSTM1 null variant seemed to protect DNA integrity.     

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.     

Microcystin-LR induces oxidative DNA damage in human hepatoma cell line HepG2.

Microcystins are naturally occurring hepatotoxins produced by strains of Microcystis aeruginosa. They are involved in promoting primary liver tumours and a previous study showed that they might also be tumour initiators. In this study we demonstrate that microcystin-LR (MCLR) at doses that were not cytotoxic (0.01-1 microg/ml), induced dose and time dependent DNA strand breaks in human hepatoma cell line HepG2. These DNA strand breaks were transient, reaching a maximum level after 4h of exposure and declining with further exposure. In the presence of the DNA repair inhibitors cytosine arabinoside (AraC) and hydroxyurea (HU), together with MCLR, DNA strand breaks accumulated after prolonged exposure. These results suggest that DNA strand breaks are intermediates, produced during the cellular repair of MCLR induced DNA damage. Digestion of DNA with purified, oxidative DNA damage specific enyzmes, endonuclease III (Endo III) and formamidopyrimidine-DNA glycosylase (Fpg) markedly increased DNA strand breaks in MCLR treated cells, providing evidence that a substantial portion of the MCLR induced DNA strand breaks originate from excision of oxidative DNA adducts. A hydroxyl radical scavenger (DMSO) significantly reduced MCLR induced DNA damage. From these results we conclude that MCLR induces formation of reactive oxygen species that cause DNA damage, and that MCLR may act as an initiator of liver cancer.     

Effect of vitamin C and iron chelation on diesel exhaust particle and carbon black induced oxidative damage and cell adhesion molecule expression in human endothelial cells

Exposure to particulate matter is associated with oxidative stress and risk of cardiovascular diseases. We investigated if vitamin C and desferrioxamine (iron chelator) altered the levels of oxidative stress and expression of cell adhesion molecules upon exposure to diesel exhaust particles (DEP) and carbon black in cultured human umbilical vein endothelial cells (HUVECs). We found that the particles were only slightly cytotoxic in the high concentration ranges. Particle-induced intracellular reactive oxygen species (ROS) production was attenuated by vitamin C administration or iron chelation and particularly when combined (p < 0.001). Only desferrioxamine protected the DNA from oxidative damage in terms of strand breaks and formamidopyrimidine DNA glycosylase sensitive sites induced by carbon black (p < 0.01). Carbon black and small sized DEP generated from an Euro4 engine increased the surface expression of VCAM-1 and ICAM-1, whereas DEP from an engine representing an old combustion type engine (SRM2975) with larger particles did not affect the expression of cell adhesion molecules. These effects were also attenuated by desferrioxamine but not vitamin C. The study shows that exposure to carbon black and DEP in HUVECs can generate both oxidative stress and expression of cell surface adhesion molecules and that these effects can in part be attenuated by vitamin C and desferrioxamine.     

Dietary exposure to diesel exhaust particles and oxidatively damaged DNA in young oxoguanine DNA glycosylase 1 deficient mice

Pulmonary exposure to diesel exhaust particles (DEP) has been associated with high levels of oxidized DNA in lung cells, whereas long-term oral DEP exposure appears to induce the DNA repair system with concomitant unaltered levels of oxidized DNA in the colon and liver of rats. Here we studied the generation of oxidatively damaged DNA in young wild type (WT) and oxoguanine DNA glycosylase 1 (OGG1) deficient mice after dietary exposure to 0mg/kg, 0.8 mg/kg, or 8 mg/kg Standard Reference Material 1650 in the feed for 21 days. The ingestion of DEP did not increase the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine and comet assay endpoints in terms of strand break, endonuclease III, and formamidopyrimidine glycosylase (FPG) in the colon, liver, and lung tissue of WT or Ogg1(-/-) mice. The level of OGG1 mRNA could only be measured in WT mice and it was not increased by DEP feeding. On the contrary, the level of FPG sites was twofold higher in the liver and lung of Ogg1(-/-) mice compared to the levels in the WT mice tissues. In conclusion, although Ogg1(-/-) mice have high levels of oxidized guanine lesions, they do not appear to be markedly vulnerable to the genotoxicity by oral administration of DEP.     

DNA adducts and strand breaks in workers exposed to vapours and aerosols of bitumen: associations between exposure and effect

To study the associations between exposure to vapours and aerosols of bitumen and genotoxic effects, a cross-sectional and cross-shift study was conducted in 320 exposed workers and 118 non-exposed construction workers. Ambient air measurements were carried out to assess external exposure to vapours and aerosols of bitumen. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine, whereas (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide ((+)-anti-BPDE), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodGuo) and DNA strand breaks were determined in blood. Significantly higher levels of 8-oxodGuo adducts and DNA strand breaks were found in both pre- and post-shift blood samples of exposed workers compared to those of the referents. No differences between exposed workers and referents were observed for (+)-anti-BPDE. Moreover, no positive associations between DNA damage and magnitude of airborne exposure to vapours and aerosols of bitumen could be observed in our study. Additionally, no relevant association between the urinary metabolites of PAH and the DNA damage in blood was observed. Overall, our results indicate increased oxidative DNA damage in workers exposed to vapours and aerosols of bitumen compared to non-exposed referents at the group level. However, increased DNA strand breaks in bitumen workers were still within the range of those found in non-exposed and healthy persons as reported earlier. Due to the lack of an association between oxidative DNA damage and exposure levels at the workplaces under study, the observed increase in genotoxic effects in bitumen workers cannot be attributed to vapours and aerosols of bitumen.     

Oxidative DNA damage and repair in skeletal muscle of humans exposed to high-altitude hypoxia

Recent research suggests that high-altitude hypoxia may serve as a model for prolonged oxidative stress in healthy humans. In this study, we investigated the consequences of prolonged high-altitude hypoxia on the basal level of oxidative damage to nuclear DNA in muscle cells, a major oxygen-consuming tissue. Muscle biopsies from seven healthy humans were obtained at sea level and after 2 and 8 weeks of hypoxia at 4100 m.a.s.l. We found increased levels of strand breaks and endonuclease III-sensitive sites after 2 weeks of hypoxia, whereas oxidative DNA damage detected by formamidopyrimidine DNA glycosylase (FPG) protein was unaltered. The expression of 8-oxoguanine DNA glycosylase 1 (OGG1), determined by quantitative RT-PCR of mRNA levels did not significantly change during high-altitude hypoxia, although the data could not exclude a minor upregulation. The expression of heme oxygenase-1 (HO-1) was unaltered by prolonged hypoxia, in accordance with the notion that HO-1 is an acute stress response protein. In conclusion, our data indicate high-altitude hypoxia may serve as a good model for oxidative stress and that antioxidant genes are not upregulated in muscle tissue by prolonged hypoxia despite increased generation of oxidative DNA damage.     

Protective effects of pulmonary epithelial lining fluid on oxidative stress and DNA single-strand breaks caused by ultrafine carbon black,ferrous sulphate and organic extract of diesel exhaust particles

Pulmonary epithelial lining fluid (ELF) is the first substance to make contact with inhaled particulate matter (PM) and interacts chemically with PM components. The objective of this study was to determine the role of ELF in oxidative stress, DNA damage and the production of proinflammatory cytokines following physicochemical exposure to PM. Ultrafine carbon black (ufCB, 15 nm; a model carbonaceous core), ferrous sulphate (FeSO₄; a model transition metal) and a diesel exhaust particle (DEP) extract (a model organic compound) were used to examine the acellular oxidative potential of synthetic ELF and non-ELF systems. We compared the effects of exposure to ufCB, FeSO₄ and DEP extract on human alveolar epithelial Type II (A549) cells to determine the levels of oxidative stress, DNA single-strand breaks and interleukin-8 (IL-8) production in ELF and non-ELF systems. The effects of ufCB and FeSO₄ on the acellular oxidative potential, cellular oxidative stress and DNA single-strand breakage were mitigated significantly by the addition of ELF, whereas there was no decrease following treatment with the DEP extract. There was no significant effect on IL-8 production following exposure to samples that were suspended in ELF/non-ELF systems. The results of the present study indicate that ELF plays an important role in the initial defence against PM in the pulmonary environment. Experimental components, such as ufCB and FeSO₄, induced the production of oxidative stress and led to DNA single-strand breaks, which were moderately prevented by the addition of ELF. These findings suggest that ELF plays a protective role against PM-driven oxidative stress and DNA damage.     

Chloroform,carbon tetrachloride and glutathione depletion induce secondary genotoxicity in liver cells via oxidative stress

Chemical carcinogens are generally classified as genotoxic or non-genotoxic. However, weak genotoxicity at high concentrations is sometimes observed and interpretation is often problematic. In addition, certain rodent carcinogens exert their effects at doses associated with cytotoxicity and compensatory hyperplasia may be a contributing factor to tumourogenesis. We hypothesise that certain substances, at high concentrations, can induce an oxidative stress via the depletion of glutathione (GSH) and other antioxidant defences and that this may lead to indirect genotoxicity, that could contribute to carcinogenicity. In support of this, human HepG2 cells treated with buthionine sulphoximine (BSO) to deplete GSH, exhibited DNA strand breaks alongside elevated 8-oxodeoxyguanosine (8-oxodG) and malondialdehyde deoxyguanosine (M(1)dG) adducts under conditions associated with lipid peroxidation. Chloroform and carbon tetrachloride are rodent carcinogens with characteristics as described above. In female rat hepatocytes, chloroform treatment resulted in a small dose-dependent increase in M(1)dG adducts (4 mM and above), DNA strand breakage (8 mM and above) and lipid peroxidation, in the absence of any associated increase in DNA oxidation. GSH depletion only occurred in association with cytotoxicity (20 mM; lactate dehydrogenase release). Alongside lipid peroxidation, carbon tetrachloride (1 and 4 mM) produced a small elevation in M(1)dG adducts and DNA strand breaks and increases in 8-oxodG were observed at the threshold of, and concomitant with, cytotoxicity (4 mM). These effects may contribute to high dose genotoxicity and carcinogenicity. Non-linearity in the dose response is expected on the basis of depletion of antioxidants, and therefore, a pragmatic threshold for biologically relevant responses should exist.     

Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile

Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses. Received: 15 December 1997 / Accepted: 10 February 1998     

Oxidative damage to nuclear DNA in streptozotocin-diabetic rat liver

1. Accumulating evidence suggests that oxidative and glycative stress is enhanced in diabetes. Oxidative stress induces DNA damage. In the present study, we assessed the 8-oxo-2'-deoxyguanosine (8-oxodG) content of DNA, an indicator of oxidative DNA damage, in streptozotocin (STZ)-induced diabetic (n = 21) and control rats (n = 18). 2. Rats were rendered diabetic by intraperitoneal administration of STZ at a dose of 65 mg/kg. Glucose was determined by glucose oxidase and glycated haemoglobin (GHb), an indicator of glycative stress, was determined by agarose-boronate affinity chromatography. 8-Oxo-2'-deoxyguanosine within the DNA (ratio of 8-oxodG to deoxyguanosine (dG)) was assessed by HPLC in conjunction with both electrochemical (8-oxodG) and diode array (dG) detection. 3. Glucose, GHb and the extent of oxidative DNA damage in the liver of STZ-diabetic rats were much higher compared with control rats. There was a correlation between GHb and 8-oxodG/10(5) dG levels in control (r = 0.756, P < 0.001) and diabetic groups (r = 0.468, 0.02 < P < 0.05). 4. These results clearly show that oxidative damage to hepatic nuclear DNA increases in the diabetic state and that this increase is correlated with glycative stress.     

Pulmonary exposure to particles from diesel exhaust,urban dust or single-walled carbon nanotubes and oxidatively damaged DNA and vascular function in apoE-/- mice

This study compared the oxidative stress level and vasomotor dysfunction after exposure to urban dust, diesel exhaust particles (DEP) or single-walled carbon nanotubes (SWCNT). DEP and SWCNT increased the production of reactive oxygen species (ROS) in cultured endothelial cells and acellullarly, whereas the exposure to urban dust did not generate ROS. The apoE^-/- mice, which were exposed twice to 0.5 mg/kg of the particles by intratracheal (i.t.) instillation, had unaltered acetylcholine-elicited vasorelaxation in aorta segments. There was unaltered pulmonary expression level of Vcam-1, Icam-1, Hmox-1 and Ogg1. The levels of oxidatively damaged DNA were unchanged in lung tissue. The exposure to SWCNT significantly increased the expression of Ccl-2 in the lung tissue of the mice. The exposure to DEP and SWCNT was associated with elevated ROS production in cultured cells, whereas i.t. instillation of the same particles had no effect on biomarkers of pulmonary oxidative stress and dilatory dysfunction in the aorta.     

Systemic effects of acute cigarette smoke exposure in mice

Abstract

Context: Cigarette smoke (CS) causes both pulmonary and extrapulmonary disorders.

Objective: To determine the pulmonary and extrapulmonary effects of acute CS exposure in regard to inflammation, oxidative stress and DNA damage.

Materials and methods: Mice were exposed to CS for 10 days and then their lungs, heart, liver, pancreas, kidneys, gastrocnemius muscle and subcutaneous (inguinal and flank) and visceral (retroperitoneum and periuterus) adipose tissues were excised. Bronchoalveolar lavage fluid samples were obtained for differential cell analysis. Inflammatory cell infiltration of the tissues was assessed by immunohistochemistry for Mac-3⁺ cells, F4/80⁺ cells and CD45⁺ cells. Oxidative stress was determined by immunohistochemistry for thymidine glycol (a marker of DNA peroxidation) and 4-hydroxy hexenal (a marker of lipid peroxidation), by enzyme-linked immunosorbent assay for protein carbonyls (a marker of protein peroxidation) and by measurements of enzyme activities of glutathione peroxidase, superoxide dismutase and catalase. DNA double-strand breaks were assessed by immunohistochemistry for γH2AX.

Results: CS exposure-induced inflammatory cell infiltration, oxidative stress and DNA damage in the lung. Neither inflammatory cell infiltration nor DNA damage was observed in any extrapulmonary organs. However, oxidative stress was increased in the heart and inguinal adipose tissue.

Discussions: Induction of inflammatory cell infiltration and DNA damage by acute CS exposure was confined to the lung. However, an increased oxidative burden occurred in the heart and some adipose tissue, as well as in the lung.

Conclusions: Although extrapulmonary effects of CS are relatively modest compared with the pulmonary effects, some extrapulmonary organs are vulnerable to CS-induced oxidative stress.     

Aristolochic acid I induced oxidative DNA damage associated with glutathione depletion and ERK1/2 activation in human cells

Aristolochic acid I (AAI) has been widely found in herbal remedies and linked to the development of nephropathy and urothelial carcinoma in humans. This study elucidated the mechanism of oxidative stress and DNA damage mediated by AAI in human cells. Treatment of human promyelocytic leukemia cells (HL-60) and human renal proximal tubular cells (HK-2) with AAI led to a dose-dependent increase of reactive oxygen species (ROS). AAI also elevated the levels of DNA strand breaks and 8-hydroxy guanosine in HL-60 and HK-2 cells. Antioxidants, including Tiron, N-acetyl-l-cysteine (NAC) and glutathione (GSH), effectively suppressed the AAI-induced ROS and AAI-elicited genotoxicity, indicating that AAI induced the DNA damage through oxidative stress. GSH depletion was also found in AAI-treated cultures and proceeded prior to ROS formation. Exposure of HL-60 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only MEK1/2 inhibitor, U0126, significantly decreased AAI-mediated ROS. Preincubation of cells with thiol-containing compounds (NAC and GSH) inhibited the caspase 3 activity triggered by AAI, but non-thiol Tiron did not show a similar effect. This study demonstrated that AAI treatment results in oxidative stress-related DNA damage through GSH depletion and ERK1/2 activation; AAI-induced apoptosis is associated with GSH loss, but is independent of ROS generation.     

Evaluation of genotoxic risk and oxidative DNA damage in mammalian cells exposed to mycotoxins,patulin and citrinin

Mycotoxins are fungal secondary metabolites with very diversified toxic effects in humans and animals. In the present study, patulin (PAT) and citrinin (CTN), two prevalent mycotoxins, were evaluated for their genotoxic effects and oxidative damage to mammalian cells, including Chinese hamster ovary cells (CHO-K1), human peripheral blood lymphocytes, and human embryonic kidney cells (HEK293). PAT, but not CTN, caused a significant dose-dependent increase in sister chromatid exchange (SCE) frequency in both CHO-K1 and human lymphocytes. PAT also elevated the levels of DNA gap and break in treated CHO-K1. In the single cell gel electrophoresis (SCGE) assay, exposure of HEK293 to concentrations above 15 microM of PAT induced DNA strand breaks; the tail moment values also greatly increased after posttreatment with formamidopyrimidine-DNA glycosylase (Fpg). This suggests that in human cells PAT is a potent clastogen with the ability to cause oxidative damage to DNA. However, no significant change in the tail moment values in CTN-treated cultures was found, suggesting that CTN is not genotoxic to HEK293. Incubation of HEK293 with CTN increased the mRNA level of heat shock protein 70 (HSP70), but not that of human 8-hydroxyguanine DNA glycosylase 1 (hOGG1). PAT treatment did not modulate the expression of either HSP70 or hOGG1 mRNA.     

Cell-specific oxidative DNA damage induced by estrogen in rat testicular cells in vitro

17 alpha-Ethinylestradiol (EE) can induce oxidative DNA damage in terms of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in rat testicular cells by an apparent estrogen receptor-mediated mechanism. We investigated differential susceptibility to EE in cell sub-populations from rat testes and the role of rat 8-oxo-guanine DNA glycosylase (rOGG1). Isolated rat testicular cells were incubated with EE concentrations ranging from 0.1 to 1000 nM. Single strand DNA breaks and oxidised purines as fapyguanine glycosylase (FPG) sensitive sites were assessed by the comet assay. In the total cell population and in round haploid cells, oxidised purines showed a bell-shaped concentration-response relationship with a maximally increased levels at 10 nM EE, whereas, no significant effects were seen in diploid, S-phase or tetraploid cells. The mRNA level of rOGG1 in testes cells was unaffected by EE, whereas, baseline levels were higher than in liver tissue and similar to colon tissue.     

Oxidative damages in the DNA, lipids, and proteins of rats exposed to isofluranes and alcohols

DNA damage, lipid peroxidation and protein oxidation were evaluated in rats exposed to a 1% isoflurane atmosphere with or without alcohol administration (administrated by gastric intubation at 4 g/kg body weight as a 50% solution). Single cell gel electrophoresis assays were performed in order to evaluate DNA damage occurring in the lymphocytes, spleen, bone marrow, brain, livers and lung of rats exposed to 1% isoflurane for 30 or 60 min with/without ethanol. Levels of malondialdehydes (MDA), a metabolite of lipid peroxidation, were determined in plasma and tissues. Carbonyl contents were also analyzed to determine levels of protein oxidation in plasma and tissues.

Levels of DNA damage in lymphocytes, bone marrow, and the organ tissues of rats exposed to isoflurane were found to increase time dependently, and alcohol increased DNA damage. Lipid peroxidation and protein oxidation results showed patterns that differed from those of DNA damage. Levels of MDA in plasma, bone marrow, spleen, and the livers of rats exposed to isoflurane with/without ethanol were found to be time dependently increased, but this was not observed in the brain or lung.

However, protein oxidation levels were significantly increased in the plasma, brains, and lungs of rats exposed to isoflurane, and exposure to isoflurane and alcohol, significantly increased these levels in plasma and brain.

The present study demonstrates that isoflurane exposure results in significant DNA damage in rat lymphocytes, bone marrow, spleen, brain, livers, and lung. Moreover, alcohol was found to be as a strong inducer of DNA damage, lipid peroxidation and protein oxidation. However, no evidence in association between DNA damage, lipid peroxidation and protein oxidation was found. Regarding the effects of isoflurane and alcohol on oxidative damages, single strand DNA damages may be a useful biomarkers and blood cells and plasma appear to be more sensitive targets to oxidative damage than other tissues.     

Chemoprotective effect of lipoic acid against cyclophosphamide-induced changes in the rat sperm

Treatment with cyclophosphamide (CP), a commonly used anticancer and immunosuppressive agent, may result in oligospermia and azoospermia. CP administration induces oxidative stress and is cytotoxic to normal cells. In this context, we have studied the effect of an established antioxidant, lipoic acid on its influence on CP-induced oxidative injury in rat sperm. In this study, we have assessed the possible protective efficacy of lipoic acid on the sperm characteristics, peroxidative damages and abnormal antioxidant levels in the epididymal sperm of CP-administered rats. Male Wistar rats of 140+/-20 g were categorized into four groups. Two groups of rats were administered CP (15 mg/kg body weight once a week for 10 weeks by oral gavage) to induce testicular toxicity; one of these groups received lipoic acid treatment (35 mg/kg body weight intraperitoneally once a week for 10 weeks; 24 h prior to CP administration). A vehicle treated control group and a lipoic acid drug control group were also included. CP-treated rats showed a significant decrease in sperm count and motility with an increase in dead and abnormal sperms. The epididymal sperm of untreated CP-exposed rats showed 1.9-fold increase in lipid peroxidation, along with a significant increase in protein carbonyl level. These changes were associated with significant increase in DNA damage in the sperm as evidenced by increased single strand breaks in fluorimetric analysis of DNA unwinding (FADU). In rats treated with CP, abnormal changes in the activities/levels of enzymic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymic (reduced glutathione, ascorbate and alpha-tocopherol) antioxidants, were also observed. Pretreatment with lipoic acid improved the semen quality and reduced the oxidative stress and DNA damage induced by CP, thereby demonstrating the protection rendered by lipoic acid.     

Oxidative DNA damage induced by di-(2-ethylhexyl) phthalate in HEK-293 cell line

Di-(2-ethylhexyl) phthalate (DEHP) is commonly employed as a plasticizer. We have found that exposure of human embryonic kidney cell line 293 (HEK-293) to DEHP resulted in a crucial dose-dependent increase of DNA strand breaks in a comet assay. To elucidate the role of glutathione (GSH) in the DNA damage, the cells were pretreated with buthionine-(S,R)-sulfoximine (BSO) and pretreated with N-acetylcysteine (NAC), a GSH precursor. Here we show that depletion of GSH in HEK-293 cells with BSO dramatically increased the susceptibility of HEK-293 cells to DEHP-induced DNA damage. Furthermore, when the intracellular GSH content was elevated by NAC, the DNA damage induced by DEHP was almost completely abolished. In addition, DEHP had effect on lysosomal or mitochondrial damage at high dose level. These results indicate that DEHP exerts genotoxic effects in HEK-293 cells, probably through DNA damage induced by oxidative stress; GSH is responsible for cellular defense against DEHP-induced DNA damage; lysosome and mitochondria may be the vital targets in DEHP-induced DNA damage.