Synergistic Effects of Polymorphisms in DNA Repair Genes and Endogenous Estrogen Exposure on Female Breast Cancer Risk

Background  

Endogenous estrogen is suggested to initiate cell proliferation and cause oxidative DNA damage during breast tumorigenesis. Cells eliminate DNA damage by means of repair enzymes. Genotypic variants of DNA damage repair genes, participating in base excision repair (BER) and nucleotide excision repair (NER) pathways, may act as modifiers that affect the association between estrogen exposure and breast cancer.     

Single-Nucleotide Polymorphisms of DNA Repair Genes OGG1 and XRCC1: Association with Gallbladder Cancer in North Indian Population

Background  DNA damage by endogenous or exogenous source of reactive oxygen species (ROS) plays an important role in induction and progression of various cancers. Physiologically, gallbladder is likely to be exposed to various ROS which leads to extensive DNA damage. Cells overcome the DNA damage by repair mechanisms. Genetic variants of OGG1 and XRCC1, important enzymes participating in base excision repair pathway, may confer interindividual variations in susceptibility to gallbladder cancer (GBC). This study was aimed to examine the role of OGG1 Ser326Cys (rs1052133) and XRCC1 Arg194Trp (C > T) (rs25487) and Arg399Gln (G > A) (rs1799782) polymorphisms in GBC susceptibility. Methods  The study included 173 GBC patients and 204 controls. Genotyping was done by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Differences in the frequencies were estimated by chi-square test and risk was estimated by using unconditional logistic regression after adjusting for age and gender. Results   OGG1 Cys/Cys genotype frequency was significantly higher in GBC patients [odds ratio (OR) = 2.93; 95% confidence interval (CI) = 1.14–7.51]. The increased risk was more pronounced in female GBC patients (OR = 5.92; 95%CI = 1.20–29.13), patients with gallstone (OR = 5.50; 95%CI = 1.99–15.16), female gender, and late onset of disease (OR = 4.72, 95%CI = 1.43–15.53). In XRCC1 Arg399Gln polymorphism, significant differences in frequencies of Gln/Gln and Arg/Gln genotypes conferred significantly low risk for GBC (OR = 0.62; 95%CI = 0.39–0.97 and OR = 0.37; 95%CI = 0.19–0.71 respectively). However, XRCC1 Arg194Trp polymorphism was not associated with GBC. The carriers of Arg-Gln haplotype consisting of 194Arg and 399Gln alleles of XRCC1 were also at significant low risk for GBC (OR = 0.59, 95%CI = 0.42–0.82). Interaction of genotypes and tobacco usage did not modulate the risk. Conclusion  Results suggest that Cys/Cys genotype of OGG1 Ser326Cys polymorphism is associated with increased risk of GBC. However, Arg399Gln polymorphism and Arg-Gln haplotype comprising XRCC1 Arg194Trp and Arg399Gln polymorphisms conferred low risk for GBC susceptibility.     

The effect of sleep deprivation and disruption on DNA damage and health of doctors

Observational studies have highlighted the detrimental health effects of shift work. The mechanisms through which acute sleep deprivation may lead to chronic disease have not been elucidated, but it is thought that increased DNA damage or decreased repair can lead to disease. The objective of this study was to examine the effects of acute sleep deprivation on DNA damage. This was a cross-sectional observational study on 49 healthy, full-time doctors. Baseline blood was sampled from each participant after three consecutive days of adequate sleep. Participants (n = 24) who were required to work overnight on-site had additional blood sampled on a morning after acute sleep deprivation. DNA damage and expression of DNA repair genes were quantified. Information on health, working patterns and sleep diaries were collected. Independent t-tests were used to compare differences between groups and standardised mean differences expressed as Cohen's d. Overnight on-site call participants had lower baseline DNA repair gene expression and more DNA breaks than participants who did not work overnight (d = 1.47, p = 0.0001; and 1.48, p = 0.0001, respectively). In overnight on-site call participants, after acute sleep deprivation, DNA repair gene expression was decreased (d = 0.90, p = 0.0001) and DNA breaks were increased (d = 0.87, p = 0.0018). Sleep deprivation in shift workers is associated with adverse health consequences. Increased DNA damage has been linked to the development of chronic disease. This study demonstrates that disrupted sleep is associated with DNA damage. Furthermore, larger prospective studies looking at relationships between DNA damage and chronic disease development are warranted, and methods to relieve, or repair, DNA damage linked to sleep deprivation should be investigated.     

Effects of experimental left varicocele repair on hypoxia‐inducible factor‐1α and vascular endothelial growth factor expressions and angiogenesis in rat testis

This study was aimed to investigate the effects of experimental left varicocele (ELV) repair on hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) expressions and angiogenesis in rat testis. ELVs were surgically created in 26 adult male Sprague‐Dawley rats. Thirty days after surgery, ELV repair was performed in 13 of the rats. All rats subsequently underwent orchiectomy 30 days after the last laparotomy. Histology of ELV‐repaired testicles was compared to that of the unrepaired (ELV) group. The frequency of positive HIF‐1α findings was significantly lower in the ELV‐repaired than in the ELV group. The frequency of positive VEGF findings was also lower in the ELV‐repaired than in the ELV group, although the difference was not statistically significant (P = 0.238). The mean microvessel density in ELV‐repair group was significantly lower than that in the ELV group (P = 0.002). Our study demonstrated that ELV repair may protect tissues from hypoxia and hypoxia‐related pathophysiologic events, such as angiogenesis, in rat testis.     

Hypoxia pathway has more impact than inflammation pathway on etiology of infertile men with varicocele

This study aimed to compare main molecular markers of hypoxia (HIF1‐α and P53) and inflammation (TLR‐2, TLR‐4 and TNF‐α) pathways between infertile men with varicocele and fertile individuals. Sperm parameters such as sperm concentration, motility and morphology were assessed according to World Health Organization (Laboratory manual for the examination and processing of human semen. Geneva, Switzerland, 2010) guideline in 20 infertile men with grade II or III varicocele, and 20 fertile men candidate of family balancing. In addition, sperm DNA fragmentation and molecular markers involved in hypoxia and inflammation pathways were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay and real‐time PCR respectively. Mean of sperm parameters (concentration, motility and morphology) and DNA integrity were significantly lower in infertile men with varicocele compared to fertile individuals. Unlike markers involved in inflammation pathway, mean expression of markers of hypoxia pathway (HIF1‐α and P53) was significantly higher in infertile men with varicocele compared to fertile individuals (p < 0.05), and also a significant correlation was observed between expression of HIF1‐α and P53 (r = 0.461; p = 0.003). Overall, the result of this study suggests higher likelihood of involvement of hypoxia pathway, in comparison with inflammation pathway, in pathogenesis varicocele associated with male infertility.     

Effects of remote ischaemic preconditioning on intraportal islet transplantation in a rat model

Remote ischaemic preconditioning (RIPC), which is the intermittent interruption of blood flow to a site distant from the target organ, is known to improve solid organ resistance to ischaemia‐reperfusion injury. This procedure could be of interest in islet transplantation to mitigate hypoxia‐related loss of islet mass after isolation and transplantation. Islets isolated from control or RIPC donors were analyzed for yield, metabolic activity, gene expression and high mobility group box‐1 (HMGB1) content. Syngeneic marginal mass transplantation was performed in four streptozotocin‐induced diabetic groups: control, RIPC in donor only, RIPC in recipient only, and RIPC in donor and recipient. Islets isolated from RIPC donors had an increased yield of 20% after 24 h of culture compared to control donors (P = 0.007), linked to less cell death (P = 0.08), decreased expression of hypoxia‐related genes (Hif1a P = 0.04; IRP94 P = 0.008), and increased intra‐cellular (P = 0.04) and nuclear HMGB1. The use of RIPC in recipients only did not allow for reversal of diabetes, with increased serum HMGB1 at day 1; the three other groups demonstrated significantly better outcomes. Performing RIPC in the donors increases islet yield and resistance to hypoxia. Validation is needed, but this strategy could help to decrease the number of donors per islet recipient.     

Mechanism of oxidative DNA damage in diabetes: tuberin inactivation and downregulation of DNA repair enzyme 8-oxo-7,8-dihydro-2'-deoxyguanosine-DNA glycosylase

OBJECTIVE—To investigate potential mechanisms of oxidative DNA damage in a rat model of type 1 diabetes and in murine proximal tubular epithelial cells and primary culture of rat proximal tubular epithelial cells.RESEARCH DESIGN AND METHODS—Phosphorylation of Akt and tuberin, 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) levels, and 8-oxoG-DNA glycosylase (OGG1) expression were measured in kidney cortical tissue of control and type 1 diabetic animals and in proximal tubular cells incubated with normal or high glucose.RESULTS—In the renal cortex of diabetic rats, the increase in Akt phosphorylation is associated with enhanced phosphorylation of tuberin, decreased OGG1 protein expression, and 8-oxodG accumulation. Exposure of proximal tubular epithelial cells to high glucose causes a rapid increase in reactive oxygen species (ROS) generation that correlates with the increase in Akt and tuberin phosphorylation. High glucose also resulted in downregulation of OGG1 protein expression, paralleling its effect on Akt and tuberin. Inhibition of phosphatidylinositol 3-kinase/Akt significantly reduced high glucose–induced tuberin phosphorylation and restored OGG1 expression. Hydrogen peroxide stimulates Akt and tuberin phosphorylation and decreases OGG1 protein expression. The antioxidant N-acetylcysteine significantly inhibited ROS generation, Akt/protein kinase B, and tuberin phosphorylation and resulted in deceased 8-oxodG accumulation and upregulation of OGG1 protein expression.CONCLUSIONS—Hyperglycemia in type 1 diabetes and treatment of proximal tubular epithelial cells with high glucose leads to phosphorylation/inactivation of tuberin and downregulation of OGG1 via a redox-dependent activation of Akt in renal tubular epithelial cells. This signaling cascade provides a mechanism of oxidative stress–mediated DNA damage in diabetes.Systemic complications are the major causes of morbidity and mortality in patients with diabetes (1). Oxidative stress leads to protein, lipid, and DNA modifications that cause cellular dysfunction and contribute to the pathogenesis of macro- and microvascular complications of diabetes, including diabetic nephropathy (2–6). Mitochondrion and nucleus, two major targets of oxidative stress, contain a variety of DNA repair enzymes to repair oxidant-induced DNA modifications (7,8). Damage most likely occurs when the endogenous antioxidant network and DNA repair systems are overwhelmed (9–11). However, it is essential for the cell to repair DNA damage induced by oxidants.8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is a sensitive marker of reactive oxygen species (ROS)–induced DNA damage (12,13). There is an increase in 8-oxodG levels in tissue of diabetic rats and in the urine of patients with type 1 and type 2 diabetes (13–15), with the levels being significantly higher in patients with albuminuria or with other diabetic complications (16). The steady-state level of 8-oxodG in DNA reflects its rate of generation and of repair. 8-OxodG in DNA is repaired primarily via the DNA base excision repair pathway (17). The DNA repair enzyme that recognizes and excises 8-oxodG is 8-oxoG-DNA glycosylase (OGG1) (18). Deficiency in DNA repair enzyme OGG1 has important functional consequences, compromising the ability of cells to repair DNA. The steady-state levels of 8-oxodG are significantly higher in tissues of OGG1 knockout mice compared with wild-type animals (19). OGG1 expression in the kidney of rats heterozygotes for tuberin is lower than that in wild-type rats (20). Moreover, treatment of tuberin-deficient rats with an oxidative DNA damaging agent greatly decreases renal OGG1 expression, with concomitant increase in 8-oxodG levels compared with wild-type rats (20). We have recently shown that OGG1 is regulated by tuberin, the product of the tumor suppressor gene, TSC-2 (21). Tuberin normally exists in an active state physically bound to hamartin, the product of TSC-1 gene, to form a stable complex (22). These two proteins function within the same pathway(s) regulating cell cycle, cell growth, adhesion, and vesicular trafficking (23,24). Activation of phosphatidylinositol 3-kinase (PI 3-kinase) and phosphorylation of serine/threonine kinase Akt/protein kinase B (PKB) by certain agonists lead to inactivation of tuberin (25–28). The PI 3-kinase/Akt pathway is activated in diabetes (29), and there is evidence that this activation is redox dependent in different cell types (30–32), including renal cells.Little is known about DNA repair disturbances potentially contributing to DNA damage in diabetes. In the present study, we determined a potential mechanism by which ROS result in 8-oxodG accumulation and explored the role of tuberin phosphorylation and OGG1 in the kidney cortex of rats with type 1 diabetes. We also investigated the effect of high glucose on tuberin phosphorylation, OGG1 expression, and 8-oxodG accumulations in proximal tubular epithelial cells.     

Oxidative stress and male reproductive health

One of the major causes of defective sperm function is oxidative stress, which not only disrupts the integrity of sperm DNA but also limits the fertilizing potential of these cells as a result of collateral damage to proteins and lipids in the sperm plasma membrane. The origins of such oxidative stress appear to involve the sperm mitochondria, which have a tendency to generate high leve｜s of superoxide anion as a prelude to entering the intrinsic apoptotic cascade. Unfortunately, these cells have very little capacity to respond to such an attack because they only possess the first enzyme in the base excision repair （BER） pathway, 8-oxoguanine glycosylase 1 （OGG1）. The latter successfully creates an abasic site, but the spermatozoa cannot process the oxidative lesion further because they lack the downstream proteins （APE1, XRCC1） needed to complete the repair process. It is the responsibility of the oocyte to continue the BER pathway prior to initiation of S-phase of the first mitotic division. If a mistake is made by the oocyte at this stage of development, a mutation will be created that will be represented in every cell in the body. Such mechanisms may explain the increase in childhood cancers and other diseases observed in the offspring of males who have suffered oxidative stress in their germ line as a consequence of age, environmental or lifestyle factors. The high prevalence of oxidative DNA damage in the spermatozoa of male infertility patients may have implications for the health of children conceived in vitro and serves as a driver for current research into the origins of free radical generation in the germ line.     

Association between body mass index and sperm quality and sperm DNA integrity. A large population study

This study aimed to analyse whether the functional quality of spermatozoa is associated with body mass index (BMI). Semen samples were obtained from 1824 men undergoing fertility evaluation/treatment. Semen analysis was performed using World Health Organization (WHO) criteria, and morphology was evaluated with the motile sperm organelle morphology examination (MSOME). The percentages of sperm DNA fragmentation (using TdT (terminal deoxynucleotidyl transferase)‐mediated dUTP nick‐end labelling (TUNEL) assays), sperm chromatin packaging/underprotamination (using chromomycin A3/CMA₃), mitochondrial damage (using MitoTracker Green) and apoptosis (using annexin V) were also assessed. At least 200 spermatozoa were examined in each evaluation. The following BMI values were used as cut‐off points: ≤24.9 kg/m², 25–29.9 kg/m² (overweight) and ≥30 kg/m² (obese). High BMI negatively affects sperm concentration, vitality, motility and morphology (p < .05). Conversely, high BMI does not seem to be associated with impaired sperm DNA integrity, as assessed by DNA fragmentation, sperm protamination and sperm apoptosis (p > .05). However, increased BMI is associated with increased mitochondrial damage in spermatozoa (p < .05). In conclusion, given the adverse consequences of obesity and the possible effect of male BMI on assisted reproduction technology (ART) outcomes, the benefits of weight reduction should be discussed when counselling couples interested in fertility treatment.     

DNA repair gene expression in biological tissues exposed to low-intensity infrared laser

Special properties of laser light have led to its usefulness in many applications in therapy. Excitation of endogenous chromophores in biotissues and generation of free radicals could be involved in its biological effects. DNA lesions induced by free radicals are repaired by base excision repair pathway. In this work, we evaluated the expression of APE1 and OGG1 genes related to repair of DNA lesions induced by free radicals. Skin and muscle tissues of Wistar rats were exposed to low-intensity infrared laser at different fluences and frequencies. After laser exposition of 1 and 24 h, tissue samples were withdrawn for total RNA extraction, cDNA synthesis, and evaluation of APE1 and OGG1 gene expression by quantitative polymerase chain reaction. Data obtained show that laser radiation alters the expression of APE1 and OGG1 mRNA differently in skin and muscle tissues of Wistar rats depending of the fluence, frequency, and time after exposure. Our study suggests that low-intensity infrared laser affects expression of genes involved in repair of DNA lesions by base excision repair pathway.     

Clinical outcome and toxicity from taxanes in breast cancer patients with BRCA1 and BRCA2 pathogenic germline mutations

Germline variations in genes coding for proteins involved in the oxidative stress and DNA repair greatly influence drug response and toxicity. Because BRCA1 and BRCA2 proteins play a role in DNA damage repair, we postulated that taxane‐related toxicity is potentially higher and clinical outcome in different in patients with BRCA pathogenic variants (PV). Seven hundred nineteen women who underwent BRCA genetic testing and were treated with taxane‐containing chemotherapy for early‐stage breast cancer between 1997 and 2018 were included in the study. Patients with BRCA variants of uncertain significance were excluded. The Kaplan‐Meier product‐limit method was used to estimate recurrence‐free survival (RFS) and overall survival (OS) rates. Logistic regression models were used to assess the association between chemotherapy toxicity and factors of interest. Cox regression models were used to assess the association between RFS and OS and factors of interest. Ninety‐four (13%) and 54 (7%) patients had BRCA1 and BRCA2‐PVs, respectively. While anemia (P = .0025) and leukopenia (P = .001) were more frequently seen in BRCA noncarriers, there was no difference in regards to peripheral neuropathy or other toxicities between the groups. Increasing doses of taxane were associated with increased risk of neutropenia, stomatitis, nausea, vomiting, acne/rash, and peripheral neuropathy across all groups. In a multivariate logistic regression model, BRCA2 status remained as an independent significant predictor for decreased hematologic toxicity (HR: 0.36; 95% CI: 0.20‐0.67; P = .001) and increased gastrointestinal toxicity (HR: 1.93; 95% CI: 1.02‐3.67; P = .04). Being overweight, obese and African‐American race were significant predictors for peripheral neuropathy (P = .04; P = .03; P = .06, respectively). Total taxane dose received did not have any impact on survival outcomes. Our study demonstrates that taxane‐containing chemotherapy regimens do not increase risk of peripheral neuropathy or hematologic toxicity in patients with BRCA PVs. The mechanisms for this finding need to be further investigated as it may provide an opportunity to combine taxanes with other agents, such as platinum salts or PARP inhibitors, with less anticipated toxicity.     

The effect of cysteine and glutamine on human sperm functional parameters during vitrification

Assuming the adverse effects of reactive oxygen species (ROS) on sperm function, this study was conducted to assess the effects of cysteine and glutamine as effective antioxidants on human sperm parameters under vitrification. Twenty normozoospermic samples were used. The samples were subjected to a vitrification process and cysteine (5 and 10 mM) and glutamine (10 and 15 mM). The sperm motility parameters, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI), DNA damage and intracellular ROS damage were assessed for each sample. Statistical analyses showed that motility, mitochondrial membrane potential and DNA damage decreased in the vitrified groups with cysteine 5, 10 mM and glutamine 10, 15 mM separately. Also intracellular ROS increased significantly compared to the fresh group (p < .05). No significant differences were observed for PMI compared with the fresh group (p > .05). Supplementation of cysteine and glutamine in both concentrations separately decreased intracellular ROS and DNA damage of spermatozoa with significant increase in PMI, MMP and progressive motility compared to vitrified control group (p < .05). The results showed no significant effect of a specific concentration in cysteine and glutamine on sperm parameters compared to other concentrations. Both amino acids have the potential to improve the harmful effects of freezing on sperm parameters.     

Total flavonoids of Epimedium reduce ageing‐related oxidative DNA damage in testis of rats via p53‐dependent pathway

Increased DNA damage in testis is considered as a major factor for ageing‐related dysfunction. Total flavonoids of Epimedium (TFE), the main active compositions of Epimedium, have been used to treat sexual dysfunction and delay ageing. However, whether TFE could improve ageing‐related testicular dysfunction remains unknown. Therefore, we investigated the protection effects of TFE and its mechanisms of action in a naturally ageing rat model. Eighteen‐month‐old SD rats were randomised to receive either vehicle or TFE (10 and 20 mg/kg). Nine‐month‐old SD rats were used as adult controls. Morphology, protein expression and immunohistochemistry were determined. Compared with adult control group, intragastric administration of TFE for 6 months significantly improved testicular morphology, increased the activities of SOD and decreased the levels of MDA of testis. In addition, TFE decreased γH2AX expression levels and γH2AX focal formation in spermatogonia and primary spermatocyte with concomitant downregulation of 8‐OHdG levels. Furthermore, TFE inhibited p‐P53/p21 and chk1/chk2 expression levels. Collectively, TFE effectively reduce oxidative DNA damage in testis of ageing rats via a p53‐dependent pathway. Thus, inhibition of oxidative DNA damage is likely to represent a promising strategy for restoration of ageing‐related testicular dysfunction.     

Effect of cysteine and glutamine added to extender on post‐thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris‐egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post‐thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen–thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen–thawed spermatozoa.     

Can leukocytospermia predict prostate cancer via its effects on mitochondrial DNA?

Leukocytospermia was previously reported to affect sperm quality by the production of reactive oxygen species (ROS) leading to oxidative stress (OS). In turn, OS decreases sperm functional integrity, increases sperm DNA damage and ultimately alters fertility status. To elucidate the impact of leukocytospermia on sperm nuclear DNA integrity and mitochondrial DNA (mtDNA) structure, we conducted a study including 67 samples from infertile patients with low level of leucocytes (Group 1: n = 20) and with leukocytospermia (Group 2: n = 47). In addition to standard sperm parameters’ assessment, we measured the levels of inflammation biomarkers [interleukin-6 (IL-6) and interleukin-8 (IL-8)] and evaluated the oxidative status [malondialdehyde (MDA) and enzymatic and non-enzymatic antioxidants]. In addition, we evaluated the level of sperm nuclear DNA fragmentation and analysed mitochondrial DNA (mtDNA) of sperm cells by sequencing of 5 genes [cytochrome oxidase I (COXI), cytochrome oxidase II (COXII), cytochrome oxidase III (COXIII), adenosine triphosphate synthase 6 (ATPase 6) and adenosine triphosphate synthase 8 (ATPase 8)]. As expected, patients with leukocytospermia had significantly higher MDA levels (32.56 ± 24.30 nmole/ml) than patients without leukocytospermia (17.59 ± 9.60 nmole/ml) (p < .018). Also, sperm DNA fragmentation index (DFI) was significantly higher in Group 2 (33.05 ± 18.14%) as compared to Group 1 (14.19 ± 9.50%) (p < .001). The sequencing of mtDNA revealed a high number of substitutions in Group 2 (n = 102) compared to Group 1 (n = 5). These substitutions were observed mainly in COXI. Among COXI substitutions found in Group 2, twelve changes were previously described in patients with prostate cancer and six of them were shown associated with this pathology. These findings suggest that leukocytospermia may predispose to the manifestation of prostate cancer through modification of mitochondrial DNA and this may be promoted by OS.     

Tracing of zinc and iron in experimentally induced varicocele: correlation with oxidative,nitrosative and carbonyl stress

This study was designed to evaluate how varicocele (VCL) can time dependently induce zinc (Zn) and iron (Fe) toxicity in testicular tissue and to analyse the relation between heavy metals toxicity and lipid peroxidation, sperm DNA damage, nitrosative and carbonyl stresses. Twenty‐four mature male Wistar rats were divided into control‐sham and test groups, which were then submitted to experimentally induced VCL. Non‐VCL‐induced rats were considered as control‐sham. The test groups were subdivided into three groups based on the sample collecting date (2, 6 and 8 months after VCL induction). Zn and Fe distribution in testicles, DNA ladder for sperm DNA fragmentation, testicular total antioxidant capacity (TAC), malondialdehyde (MDA), nitrite oxide (NO) and carbonyl groups (CG) were analysed. A significant (p < .05) enhancement in the percentage of tubules with negative tubular repopulation, differentiation and spermiogenesis indices was revealed. The VCL increased Zn and Fe distribution in testicles. The VCL, time dependently, reduced sperm count, motility and enhanced sperm DNA damage (p < .05). The VCL downregulated the testicular TAC and enhanced the MDA, NO and CG contents. Our data showed that the VCL results in intensive Fe and Zn toxicities. Produced Zn‐ and Fe‐mediated‐oxidative stress increases sperm DNA damage associated with NO and CG‐induced stresses.     

Sperm DNA damage output parameters measured by the alkaline Comet assay and their importance

The alkaline Comet assay has shown high diagnostic value to determine male reproductive health and prognostic ability to predict ART success. Here, spermatozoon was analysed in 47 fertile donors and 238 patients, including 132 couples undergoing ART [semen was collected: Group I – within 3 months of their treatment (n = 79); and Group II – 3 months prior to their treatment (n = 53)]. We introduce four Comet distribution plots (A, B1, B2 and C) by plotting the level of DNA damage (x‐axis) and percentage of comets (y‐axis). Fertile donors had low mean DNA damage, olive tail moment and per cent of spermatozoa with damage and increased type A plots. Comet parameters were associated with clinical pregnancies in Group I. About 66% of couples with type A distribution plot were successful after ART, whereas couples with type B1, B2 and C distribution plots achieved 56%, 44% and 33% pregnancies respectively. The efficiency of the Comet assay was due to complete decondensation process, where the compact sperm nuclear DNA (28.2 ± 0.2 μm³) is decondensed to ~63 μm³ (before lysis) and ~1018 μm³ (after lysis). A combinational analysis of all the Comet output parameters may provide a comprehensive evaluation of patient's reproductive health as these parameters measure different aspects of DNA damage within the spermatozoa.     

DNA integrity and methylation changes of mouse spermatozoa following prolonged incubation

Sperm quality can be affected by different factors including the length of incubation time between sperm preparation and intracytoplasmic sperm injection. Here, we have evaluated the level of DNA methylation and expressions of related genes in mice spermatozoa. The spermatozoa were divided into three groups: fresh, spermatozoa incubated at room temperature (RT) and 37°C for 24 hr. The sperm chromatin structure assay was used to determine the DNA fragmentation index (DFI), and DNA methylation was analysed by flow cytometry. The expression levels of DNA methylation‐related genes were determined by quantitative real‐time PCR (qRT‐PCR). According to the results, we observed significantly higher sperm progressive motility and viability in the group incubated at RT compared to the spermatozoa incubated at 37°C (p < 0.05). Spermatozoa incubated at 37°C had a higher DFI compared to the other groups (p < 0.05), but the DNA methylation level significantly decreased (p < 0.05). qRT‐PCR analysis showed increased Dnmt‐1 expression in spermatozoa after 24‐hr incubation at 37°C. However, there were significantly higher expression levels of Dnmt‐3l, Dnmt‐3a and Dnmt‐3b after incubation at both RT and 37°C compared to the fresh group (p < 0.05). The 24‐hr incubation period affected both sperm DNA methylation and integrity. This study indicated that incubation at RT resulted in better sperm quality.