Benzo[a]pyrene and its metabolites combined with ultraviolet A synergistically induce 8-hydroxy-2'-deoxyguanosine via reactive oxygen species

We previously reported that benzo[a]pyrene (BaP) and UVA radiation synergistically induced oxidative DNA damage via 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in vitro. The present study shows that microsomal BaP metabolites and UVA radiation potently enhance 8-OHdG formation in calf thymus DNA about 3-fold over the parent compound BaP. Utilization of various reactive oxygen species scavengers revealed that singlet oxygen and superoxide radical anion were involved in the 8-OHdG formation induced by microsomal BaP metabolites and UVA. Two specific BaP metabolites, benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (+/-) (anti) (BPDE) and BaP-7,8-dione, were further tested for synergism with UVA. BaP-7,8-dione showed an effect on 8-OHdG formation induced by UVA radiation that was similar to that of the parent BaP, whereas BPDE exhibited significantly higher induction of 8-OHdG than BaP. At as low as 0.5 microM, BPDE plus UVA radiation substantially increased 8-OHdG levels about 25-fold over the parent BaP. BPDE increased the formation of 8-OHdG levels in both BPDE concentration- and UVA dose-dependent manners. Additionally, singlet oxygen was found to play a major role in 8-OHdG induction by BPDE and UVA. These results suggest that BaP metabolites such as BPDE synergize with UVA radiation to produce ROS, which in turn induce DNA damage.     

α‐Lipoic acid inhibits testicular and epididymal oxidative damage and improves fertility efficacy in arsenic‐intoxicated rats

The present study evaluates the protective effect of α‐lipoic acid (LA) against arsenic‐induced testicular and epididymal oxidative damage in rats. Arsenic caused significant reduction in the reproductive organ weights, serum testosterone levels, testicular daily sperm count, epididymal sperm count, sperm motility, sperm viability, and sperm membrane integrity. Significant reduction in the activity levels of superoxide dismutase, catalase, and glutathione levels with a concomitant increase in the lipid peroxidation and protein carbonyl content in the testis and the cauda epididymis of arsenic‐exposed rats. Arsenic intoxication also enhanced the testicular caspase‐3 mRNA levels, disorganization of testicular and cauda epididymal architecture as well as increased arsenic content in the testis and the cauda epididymis of rats. Arsenic exposure also deteriorated fertility ability in male rats over controls. Conversely, α‐LA negated the testicular and cauda epididymal oxidative stress and restored the male reproductive health in arsenic‐exposed rats.     

The associations among semen quality,oxidative DNA damage in human spermatozoa and concentrations of cadmium,lead and selenium in seminal plasma

To explore the associations among semen quality, oxidative DNA damage in human spermatozoa and concentrations of cadmium, lead and selenium in seminal plasma, 56 non-smoking subjects were asked to collect semen by masturbation into a sterile wide-mouth metal-free plastic container after 3 days of abstinence. The conventional semen parameters were analysed. The concentrations of Cd, Pb and Se in seminal plasma were detected using atomic absorption spectrophotometer. 8-OHdG levels in sperm DNA were measured using HPLC-EC. The results showed that the geometric mean concentrations of Cd, Pb and Se were 0.78, 7.8 and 51.4 microg/l, respectively. The geometric mean of 8-OHdG/10(6) dG was 51.4 (95% CI: 21.5-123.0). A significant inverse correlation exists between Cd and sperm density (r=-0.28, P<0.05), and between Cd and sperm number per ejaculum (r=-0.27, P<0.05). In contrast, there was a significantly positive correlation between Se and sperm density (r=0.50, P<0.01), between Se and sperm number (r=0.49, P<0.01), between Se and sperm motility (r=0.40, P<0.01), and between Se and sperm viability (r=0.38, P<0.01). No statistically significant correlation was observed between Pb and semen quality. A significant inverse correlation was observed between 8-OHdG and sperm density (r=-0.34, P<0.01), between 8-OHdG and sperm number per ejaculum (r=-0.30, P<0.01), and 8-OHdG and sperm viability (r=-0.24, P<0.05). 8-OHdG was significantly correlated with Cd in seminal plasma (r=0.55, P<0.01). A significant but weak positive correlation was found between 8-OHdG and Pb concentration in seminal plasma (r=0.28, P<0.05). In contract, a significant inverse correlation was observed between 8-OHdG and Se concentration in seminal plasma (r=-0.40, P<0.01). The results indicate that Cd in seminal plasma could affect semen quality and oxidative DNA damage in human spermatozoa. Se could protect against oxidative DNA damage in human sperm cells. Pb did not appear to have any association with the semen quality when concentration of Pb in seminal plasma was below 10 microg/l.     

Characterization of the semen quality of postpuberal boars with spontaneous unilateral abdominal cryptorchidism on the right side.

In recent studies, we found that the ectopic testis from postpuberal boars with unilateral abdominal cryptorchidism does not produce sperm. Therefore, in these males, the seminal characteristics can be used as indicators of the activity of the scrotal testis and its epididymis and also the accessory glands. The semen quality (ejaculate volume, cell-rich fraction volume, sperm concentration, sperm vitality, sperm motility, sperm morphology and cephalic stability of spermatozoa) was evaluated in healthy postpuberal boars and in postpuberal boars with unilateral abdominal cryptorchidism on the right side. In comparison with the healthy boars, the unilateral abdominal cryptorchid boars showed a significant decrease of the ejaculate volume, sperm concentration and sperm motility. The low sperm concentration indicated that unilateral abdominal cryptorchidism severely impairs the sperm production of the scrotal testis. The decrease of ejaculate volume was attributed to an abnormal activity of the accessory glands. The alterations in sperm motility develop as a result of dysfunctions in the epididymal epithelium and/or the accessory glands. The sperm vitality, sperm morphology and cephalic stability of spermatozoa maintained normal values; therefore, at testicular level, despite the low sperm production, the germ cell differentiation is not disturbed. At epididymal level, the morphological maturation of spermatozoa is not altered.     

PUVA (8-methoxy-psoralen plus ultraviolet A) induces the formation of 8-hydroxy-2'-deoxyguanosine and DNA fragmentation in calf thymus DNA and human epidermoid carcinoma cells.

The objective of this study is to investigate if 8-methoxy-psoralen (8-MOP) plus ultraviolet A (UVA) radiation (PUVA) induces oxidative DNA damage. When calf thymus DNA was incubated with 8-MOP and irradiated with UVA (335-400 nm), the level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was substantially increased by approximately 6-fold. Formation of 8-OHdG proportionally correlated with both UVA fluence and 8-MOP concentrations. Human epidermoid carcinoma cells were incubated with 10 microg 8-MOP per milliliter, followed by irradiation of 25 kJ/m2 UVA. The level of 8-OHdG increased by nearly 3-fold in PUVA-treated cells compared to 8-MOP and UVA controls. The formation of 8-OHdG correlated with DNA fragmentation as determined by spectrofluorometry. To investigate the reactive oxygen species (ROS) involved in PUVA-induced oxidative DNA damage, less or more specific ROS quenchers were added to DNA solution prior to PUVA treatment. The results showed that only sodium azide and genistein significantly quenched PUVA-induced 8-OHdG, whereas catalase, superoxide dismutase, and mannitol exhibited no effect. The quencher study with cultured cells indicated that N-acetyl-cysteine and genistein protected oxidative DNA damage as well as DNA fragmentation by PUVA treatment. Our studies show that PUVA treatment is able to induce the formation of 8-OHdG in purified DNA and cultured cells and suggest that singlet oxygen is the principle reactive oxygen species involved in oxidative DNA damage by PUVA treatment.     

Chloroacetonitrile (CAN) induces glutathione depletion and 8-hydroxylation of guanine bases in rat gastric mucosa

Chloroacetonitrile (CAN) is detected in drinking-water supplies as a by-product of the chlorination process. Gastroesophageal tissues are potential target sites of acute and chronic toxicity by haloacetonitriles (HAN). To examine the mechanism of CAN toxicity, we studied its effect on glutathione (GSH) homeostasis and its impact on oxidative DNA damage in gastric mucosal cells of rats. Following a single oral dose (38 or 76 mg/Kg) of CAN, animals were sacrificed at various times (0-24 h), and mucosa from pyloric stomach were collected. The effects of CAN treatment on gastric GSH contents and the integrity of genomic gastric DNA were assessed. Oxidative damage to gastric DNA was evaluated by measuring the levels of 8-Hydroxydeoxyguanosine (8-OHdG) in hydrolyzed DNA by HPLC-EC. The results indicate that CAN induced a significant, dose- and time-dependent, decrease in GSH levels in pyloric stomach mucosa at 2 and 4 hours after treatment (56 and 39% of control, respectively). DNA damage was observed electrophoretically at 6 and 12 hours following CAN administration. CAN (38 mg/Kg) induced significant elevation in levels of 8-OHdG in gastric DNA. Maximum levels of 8-OHdG in gastric DNA were observed at 6 hours after CAN treatment [9.59+/-0.60 (8-OHdG/10(5)dG) 146% of control]. When a high dose of CAN (76 mg/Kg) was used, a peak level of 8-OHdG [11.59+/-1.30 (8-OHdG/10(5)dG) 177% of control] was observed at earlier times (2 h) following treatment. When CAN was incubated with gastric mucosal cells, a concentration-dependent cyanide liberation and significant decrease in cellular ATP levels were detected. These data indicate that a mechanism for CAN-induced toxicity may be partially mediated by depletion of glutathione, release of cyanide, interruption of the energy metabolism, and induction of oxidative stress that leads to oxidative damage to gastric DNA.     

Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis,Immaturity and Oxidative Stress

Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2′-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.     

Detection of oxidative DNA damage in human sperm and its association with sperm function and male infertility

The expanding research interest in the last two decades on reactive oxygen species (ROS), oxidative stress, and male infertility has led to the development of various techniques for evaluating oxidative DNA damage in human spermatozoa. Measurement of 8-hydroxydeoxyguanosine (8-OHdG) offers a specific and quantitative biomarker on the extent of oxidative DNA damage caused by ROS in human sperm. The close correlations of 8-OHdG level with male fertility, sperm function and routine seminal parameters indicate the potential diagnostic value of this technique in clinical applications. On the other hand, single cell gel electrophoresis (SCGE or comet assay) and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay have also been demonstrated to be sensitive, and reliable methods for measuring DNA strand breaks in human spermatozoa. As certain technical limitations were inherent in each of these tests, it is believed that a combination of these assays will offer more comprehensive information for a better understanding of oxidative DNA damage and its biological significance in sperm function and male infertility.     

Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats

Acrylamide (ACR), used in many fields from industrial manufacturing to laboratory personnel work is also formed during the heating process through interactions of amino acids. Therefore ACR poses a significant risk to human health. This study aimed to elucidate whether resveratrol (RVT) treatment could modulate ACR-induced oxidative DNA damage and oxidative changes in rat brain, lung, liver, kidney and testes tissues. Rats were divided into four groups as control (C); RVT (30 mg/kg i.p. dissolved in 0.9% NaCl), ACR (40 mg/kg i.p.) and RVT + ACR groups. After 10 days rats were decapitated and tissues were excised. 8-hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative DNA damage. 8-OHdG content in the extracted DNA solution was determined by enzyme-linked immunosorbent assay method. Malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in tissues, while oxidant-induced tissue fibrosis was determined by collagen contents. Serum enzyme activities, cytokine levels, leukocyte apoptosis were assayed in plasma. As an indicator of oxidative DNA damage, 8-OHdG levels significantly increased in ACR group and this was reversed significantly by RVT treatment. In ACR group, GSH levels decreased significantly while the MDA levels, MPO activity and collagen content increased in the tissues suggesting oxidative organ damage. In RVT-treated ACR group, oxidant responses reversed significantly. Serum enzyme activities, cytokine levels and leukocyte late apoptosis which increased following ACR administration, decreased with RVT treatment. Therefore supplementing with RVT can be useful in individuals at risk of ACR toxicity.     

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde–protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.     

Impact of cryopreservation and reactive oxygen species on DNA integrity, lipid peroxidation, and functional parameters in ram sperm

Assisted reproduction using frozen-thawed semen has practical advantages, although cryopreservation is detrimental to sperm fertility in most mammals. We examined the influence of cryopreservation and reactive oxygen species (ROS) on ram sperm DNA stability (using SCSA), lipid peroxidation (LPO), chlortetracycline fluorescence (CTC) patterns, motility and viability. In Experiment 1, DNA integrity, LPO, CTC, motility and viability tests were performed on fresh and cryopreserved sperm after 0, 6, and 24 hr in synthetic oviductal fluid (SOF). In Experiment 2, fresh sperm were incubated in serum-free SOF (SOF-S; 1, 4, and 24 hr) with 0, 50, 150, or 300 microM H2O2 then assayed. Cryopreservation increased the percentage of sperm with a high DNA fragmentation index (%DFI), decreased the percentages of motile and viable sperm at thawing (0 hr), but did not affect LPO. H2O2 (150 or 300 microM) increased %DFI after 24 hr. LPO or sperm viability were not affected by H2O2, although most motility parameters decreased. H2O2 decreased the percentage of chlortetracycline pattern F sperm at 4 hr and increased the percentage of acrosome-reacted sperm (pattern AR) after 1 hr. Pooled data of Experiment 2 showed LPO was positively correlated with SCSA (r = 0.29 to r = 0.59; P < 0.05 to P < 0.01), while most motility parameters and the percentage of viable sperm were negatively correlated with LPO (r = -0.30 to r = -0.38; P < 0.05 to P < 0.01). LPO was positively correlated with the percentage of pattern AR sperm (r = 0.33; P < 0.01). Cryopreservation and H2O2 promote DNA instability in ram sperm, though motility is a more sensitive indicator of oxidative stress than the other parameters investigated.     

Lycopene protects against cyclosporine A-induced testicular toxicity in rats

Cyclosporine A (CsA)-induced direct failures in hypothalamic-pituitary-gonadal axis and Sertoli cell phagocytic function have been considered for testicular toxicity so far. It has clearly been reported that oxidative stress leads to damage in sperm functions and structure of the testis. Therefore, this study was conducted to demonstrate whether CsA causes testicular and spermatozoal toxicity associated with the oxidative stress, and to investigate the possible protective effect of lycopene against CsA-induced damages in all reproductive organs and sperm characteristics in male rats. While the daily administration of CsA at the dose 15 mg/kg for 21 days significantly decreased the seminal vesicles weight, epididymal sperm concentration, motility, testicular tissue glutathione (GSH), glutathione peroxidase (GSH-Px) and catalase (CAT), diameter of seminiferous tubules and germinal cell thickness, it increased malondialdehyde (MDA) level and abnormal sperm rates along with degeneration, necrosis, desquamative germ cells in testicular tissue. However, the CsA along with simultaneous administration of lycopene at the dose of 10mg/kg markedly ameliorated the CsA-induced all the negative changes observed in the testicular tissue, sperm parameters and oxidant/antioxidant balance. In conclusion, CsA-induced oxidative stress leads to the structural and functional damages in the testicular tissue and sperm quality of rats and, lycopene has a potential protective effect on these damages.     

2,4,6-trinitrotoluene-induced reproductive toxicity via oxidative DNA damage by its metabolite

Several epidemiological studies and animal experiments showed that 2,4,6-trinitrotoluene (TNT), a commonly used explosive, induced reproductive toxicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of TNT on the male reproductive system in Fischer 344 rats. TNT administration induced germ cell degeneration, the disappearance of spermatozoa in seminiferous tubules, and a dramatic decrease in the sperm number in both the testis and epididymis. TNT increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in sperm whereas plasma testosterone levels did not decrease. These results suggest that TNT-induced toxicity is derived from direct damage to spermatozoa rather than testosterone-dependent mechanisms. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by TNT and its metabolic products in vitro . 4-Hydroxylamino-2,6-dinitrotoluene, a TNT metabolite, induced Cu(II)-mediated damage to 32 P-labeled DNA fragments and increased 8-oxodG formation in calf thymus DNA, although TNT itself did not. DNA damage was enhanced by NADH, suggesting that NADH-mediated redox reactions involving TNT metabolites enhanced toxicity. Catalase and bathocuproine inhibited DNA damage, indicating the involvement of H 2 O 2 and Cu(I). These findings suggest that TNT induces reproductive toxicity through oxidative DNA damage mediated by its metabolite. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by TNT and other nitroaromatic compounds.     

Glycochenodeoxycholate plays a carcinogenic role in immortalized mouse cholangiocytes via oxidative DNA damage

Bile acids have been suggested to be involved in biliary carcinogenesis, although the underlying mechanisms are yet to be established. The aim of this study was to investigate the carcinogenic effect of bile acids in the biliary tract in relation to oxidative stress. Immortalized mouse cholangiocytes were incubated with various bile acids, followed by measurement of reactive oxygen species (ROS) and the glutathione (GSH) level. As a marker of oxidative DNA damage, 8-hydroxydeoxyguanosine (8-OHdG) expression in cholangiocytes was analyzed by flow cytometry. Then the expression of oxidative DNA repair enzymes in cholangiocytes was examined by real-time PCR. In addition, the long-term effect of bile acid-induced oxidative DNA damage on cholangiocytes was investigated using a mouse oligo DNA microarray. It was found that glycochenodeoxycholate (GCDC) induced the generation of ROS and the depletion of GSH. In contrast, no marked changes were induced by the other bile acids. The percentage of 8-OHdG-positive cells was also increased by GCDC, but the expression of oxidative DNA repair enzymes was not up-regulated. DNA microarray analysis showed marked changes of various genes associated with carcinogenesis (genes related to cell proliferation, angiogenesis, invasion, and metastasis). In conclusion, the long-term effect of oxidative DNA damage due to GCDC may promote carcinogenesis in the biliary tract. Furthermore, accumulation of 8-OHdG due to GCDC might contribute to the dysfunction of oxidative DNA repair enzymes.     

Developmental expression and function of Bmp4 in spermatogenesis and in maintaining epididymal integrity

Bone morphogenetic proteins (BMPs) play essential roles in many aspects of developmental biology. We have previously shown that Bmp7, Bmp8a, and Bmp8b of the 60A class of Bmp genes have additive effects in spermatogenesis and in maintaining the epididymal integrity of the caput and caudal regions. Here we report that Bmp4 of the Dpp class has a unique expression pattern in the developing testis and epididymis. Bmp4 heterozygous males on a largely C57BL/6 background show compromised fertility due to degeneration of germ cells, reduced sperm counts, and decreased sperm motility. More interestingly, some of these males show extensive degeneration of the epididymal epithelium in the corpus region, rather than in the caput and cauda regions as for Bmp7 and Bmp8 mutants. Thus, these genetic data reveal a region-specific requirement of different classes of BMPs for epididymal epithelium to survive and have significant implications on male reproductive health and perhaps birth control.     

Protective Role of Cactus Cladodes Extract on Sodium Dichromate-Induced Testicular Injury and Oxidative Stress in Rats

Cactus (Opuntia ficus-indica) is a xerophyte plant that belongs to the Cactaceae family. The present study was designed to investigate the possible protective effects of cactus cladodes extract (CCE) on sodium dichromate-induced testis damage in adult male Wistar rats. For this purpose, CCE at a dose of 100 mg/kg was orally administrated, followed by 10 mg/kg sodium dichromate (intraperitoneal injection). After 40 days of treatment, the rats were sacrificed, and the testes were excised for histological, lipid peroxidation (LPO), and antioxidant enzyme analyses. Sodium dichromate treatment significantly (P?<?0.01) decreased the body, testis, and accessory sex organ weights, sperm count and motility, and serum testosterone level. In addition, histological analysis revealed pronounced morphological alterations with tubular necrosis and reduction in the number of gametes in the lumen of the seminiferous tubules of sodium dichromate-intoxicated rats. Furthermore, exposure to sodium dichromate significantly (P?<?0.01) increased LPO level and decreased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in testis. Interestingly, pretreatment with CCE significantly (P?<?0.01) restored the serum testosterone level, sperm count, and motility to the levels of the control group. Moreover, CCE administration was capable of reducing the elevated level of LPO and significantly (P?<?0.01) increased SOD, CAT, and GPx activities in testis. Cactus cladodes supplementation minimized oxidative damage and reversed the impairment of spermatogenesis and testosterone production induced by sodium dichromate in the rat testis.     

Methacryloxylethyl Cetyl Ammonium Chloride Induces DNA Damage and Apoptosis in Human Dental Pulp Cells via Generation of Oxidative Stress

The polymerizable antibacterial monomer methacryloxylethyl cetyl ammonium chloride (DMAE-CB) has provided an effective strategy to combat dental caries. However, the application of such material raises the question about the biological safety and the question remains open. The mechanism of this toxic action, however, is not yet clearly understood. The present study aims at providing novel insight into the possible causal link between cellular oxidative stress and DNA damage, as well as apoptosis in human dental pulp cells exposed to DMAE-CB. The enhanced formation of reactive oxygen species and depletion of glutathione, as well as differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase in DMAE-CB-treated cells indicated oxidative stress. By using substances that can alter GSH synthesis, we found that GSH was the key component in the regulation of cell response towards oxidative stress induced by DMAE-CB. The increase in oxidative stress-sensitive 8-Oxo-2''-deoxyguanosine (8-OHdG) content, formation of γ-H₂AX and cell cycle G1 phase arrest indicated that DNA damage occurred as a result of the interaction between DNA base and ROS beyond the capacities of antioxidant mechanisms in cells exposed to DMAE-CB. Such oxidative DNA damage thus triggers the activation of ataxia telangiectasia-mutated (ATM) signaling, the intrinsic apoptotic pathway, and destruction of mitochondrial morphology and function.     

Deterioration of semen quality and sperm-DNA integrity as influenced by cigarette smoking in fertile and infertile human male smokers—A prospective study

In modernized lifestyle smoking is one of the trendy, psychological, and socioeconomic scenarios of young adolescents mainly in the age of the reproductive stage. Based on a number of cigarettes smoked, age, and duration of the smoke, the study aims to search for the profound effects of smoking and its impact on semen parameters, sperm-DNA integrity, and fragmentation of sperm DNA with cotinine and apoptotic caspase-3 marker in the seminal plasma of fertile and infertile smokers. To determine oxidative damage by 8-hydroxy deoxyguanosine (8-OHdG) from isolated sperm DNA (steps: reactive oxygen species washing by nitro blue tetrazolium (NBT), sperm lysis, salt digestion, ethanol washing, and finally with high-performance liquid chromatography analysis). Level of DNA fragmentation (percentage) in native and intact DNA, the activity of caspase-3 in infertile smokers will be compared with the control group of nonsmokers. Also, the sperm viability was visualized by eosin-nigrosin and aniline blue staining. Cotinine is one of the best markers of smoking. The cotinine level (2224.24 ± 1.19 *** ng/mL), when abundant it negative correlates with morphology and rapid motility in infertile smokers than nonsmokers. Gel preprogram measured the sperm integrity and was found to be less in smokers than nonsmokers. The spermatic oxidative marker 8-OHdG was high and gave an R ² value of 0.9104 with morphology and 0.9007 for rapid motility of infertile sperm, respectively. Infertile smoking subjects (<10 cigarettes/day) had significant changes increase in sperm fragmentation, caspase-3, and cotinine while negative impact with motility, morphology, and pH of semen compared with fertile, infertile nonsmoking subjects.     

Oxidative DNA damage induced by toluene is involved in its male reproductive toxicity

Toluene is widely used as an organic solvent in various industries and commercial products. Recent investigations have shown that toluene may induce male reproductive dysfunctions and carcinogenicity. To clarify whether the toxicity results from the interference of endocrine systems or direct damage to reproductive organs, we examined the effects of toluene on the male reproductive system in rats, comparing to those of diethylstilbestrol (DES), a potent synthetic estrogen. Toluene (50, 500 mg/kg) or DES (2 mg/kg) injected subcutaneously to male Sprague-Dawley rats once a day for 10 days decreased the epididymal sperm counts and the serum concentrations of testosterone. The mRNA level for gonadotropin-releasing hormone receptor in the pituitary was decreased by DES, but not by toluene. On the contrary, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation in testes, the biological marker for oxidative DNA damage, was increased by toluene but not by DES. These results suggest that toluene induces reproductive toxicity via direct oxidative damage of spermatozoa, whereas DES affects endocrine systems via the hypothalamo-pituitary-gonadal axis. Morphological findings supported the idea. To determine the mechanism of 8-oxodG formation in vivo , we examined DNA damage induced by toluene metabolic products in vitro . Minor toluene metabolites, methylhydroquinone and methylcatechols, induced oxidative DNA damage, and the methylcatechols induced NADH-mediated 8-oxodG formation more efficiently than methylhydroquinone did. We propose that oxidative DNA damage in the testis plays a role in reproductive toxicity induced by toluene.     

Decomposition of N-nitrosamines,and concomitant release of nitric oxide by Fenton reagent under physiological conditions

Our previous work has shown that prooxidant treatment has the propensity to induce male-mediated dominant lethal (DL) type mutations in mice. The present investigation is aimed to understand the effect of oxidative stress (OS) on DNA damage in testis, epididymal sperms and its propensity to induce sperm head abnormalities as well as its implications on male fertility in mice. Initially, employing two organic hydroperoxides, (t-butyl hydroperoxide, t-bHP and cumene hydroperoxide, cHP) as model prooxidants, induction of oxidative stress was ascertained following single/multiple sublethal doses. Further, the multiple exposure model was utilized to characterize effects on testicular weights, histoarchitecture, caudal sperm counts, lipid peroxidation, DNA damage and frequency of abnormal sperms. Single sublethal doses (1/20, 1/10 and 1/5 LD(50)) of t-bHP and cHP administered (i.p.) to adult mice resulted in only a marginal increase (20% at the highest dosage) in testicular MDA levels. However, multiple doses (1/10 and 1/5 LD(50) per day for 5 days) induced marked OS in testis and epididymal sperms as evidenced by a marked increase in lipid peroxidation at 24h after the last dose. This was associated with significant increase in the DNA damage (FADU assay) in the testicular tissue. While caudal sperm counts determined at all sampling weeks showed no treatment related alterations, analysis for head abnormalities revealed nearly 2-3-fold increase in the percent abnormal sperms among the hydroperoxide treated mice during the first 3 weeks. Furthermore, mating of prooxidant treated males sequentially for a period of 5 weeks with untreated females resulted in a significant reduction in average pup number per litter during the first 3 weeks. These results suggest that oxidative stress in testicular milieu is associated with DNA damage and produces higher frequency of abnormal sperms with significant effect on male fertility.