Effects Of Oxidative DNA Damage Research Articles

Assessing the antioxidant properties of Naringin and Rutin and investigating their oxidative DNA damage effects in breast cancer

This work examines the capacity of Naringin and Rutin to influence the DNA damage response (DDR) pathway by investigating their interactions with key DDR proteins, including PARP-1, ATM, ATR, CHK1, and WEE1. Through a combination of in silico molecular docking and in vitro evaluations, we investigated the cytotoxic and genotoxic effects of these compounds on MDA-MB-231 cells, comparing them to normal human fibroblast cells (2DD) and quiescent fibroblast cells (QFC). The research found that Naringin and Rutin had strong affinities for DDR pathway proteins, indicating their capacity to specifically regulate DDR pathways in cancer cells. Both compounds exhibited preferential cytotoxicity towards cancer cells while preserving the vitality of normal 2DD fibroblast cells, as demonstrated by cytotoxicity experiments conducted at a dose of 10 µM. The comet experiments performed particularly on QFC cells provide valuable information on the genotoxic impact of Naringin and Rutin, highlighting the targeted initiation of DNA damage in cancer cells. The need to use precise cell models to appropriately evaluate toxicity and genotoxicity is emphasized by this discrepancy. In addition, ADMET and drug-likeness investigations have emphasized the pharmacological potential of these compounds; however, they have also pointed out the necessity for optimization to improve their therapeutic profiles. The antioxidant capabilities of Naringin and Rutin were assessed using DPPH and free radical scavenging assays at a concentration of 10 µM. The results confirmed that both compounds have a role in reducing oxidative stress, hence enhancing their anticancer effects. Overall, Naringin and Rutin show potential as medicines for modulating the DDR in cancer treatment. They exhibit selective toxicity towards cancer cells while sparing normal cells and possess strong antioxidant properties. This analysis enhances our understanding of the therapeutic uses of natural chemicals in cancer treatment, supporting the need for more research on their mechanisms of action and clinical effectiveness.

Mediating Role of Glucose-Lipid Metabolism in the Association between the Increased Risk of Coronary Heart Disease and Exposure to Organophosphate Esters, Phthalates, and Polycyclic Aromatic Hydrocarbons.

There is a lack of human evidence concerning the cardiovascular effects of combined exposure to endocrine disruptors. This case-control study sought to investigate coronary heart disease (CHD) associations with exposure to organophosphate flame retardants (OFRs), phthalates (PAEs), and polycyclic aromatic hydrocarbons (PAHs) among 148 adults with coronary-angiography-diagnosed CHD and 320 healthy adults from southern China. The mediating role of glucose-lipid metabolism was also explored. Bayesian kernel machine regression suggested that when exposure status was fixed to the 75th percentile with the median value as the reference, exposure to OFRs, PAEs, and PAHs was associated with an 84% (95% CI: 36%-134%), 132% (12%-252%), and 214% (89%-331%) increased risk of developing CHD, respectively. Weighted quantile sum regression indicated urinary bis(2-butoxyethyl) phosphate (BBOEP), dibutyl phosphate (DBP), monoisononyl phthalate (miNP), and metabolites of phenanthrene may be major contributors to the overall effect of mixtures. In further analyses on identified chemical risk factors, mediation analyses suggested exposure to phenanthrene may increase the risk of CHD via elevating total cholesterol and blood glucose, while exposure to DiNP mainly associates with serum lipids. Besides, we observed a slight mediation effect of oxidative DNA damage between urinary BBOEP and risk of CHD. These results provide potential direction for further experimental studies. Longitudinal evidence is needed to clarify the causation of the results.

Role of Oxidative DNA Damage and Repair in Atrial Fibrillation and Ischemic Heart Disease.

Atrial fibrillation (AF) and ischemic heart disease (IHD) represent the two most common clinical cardiac diseases, characterized by angina, arrhythmia, myocardial damage, and cardiac dysfunction, significantly contributing to cardiovascular morbidity and mortality and posing a heavy socio-economic burden on society worldwide. Current treatments of these two diseases are mainly symptomatic and lack efficacy. There is thus an urgent need to develop novel therapies based on the underlying pathophysiological mechanisms. Emerging evidence indicates that oxidative DNA damage might be a major underlying mechanism that promotes a variety of cardiac diseases, including AF and IHD. Antioxidants, nicotinamide adenine dinucleotide (NAD+) boosters, and enzymes involved in oxidative DNA repair processes have been shown to attenuate oxidative damage to DNA, making them potential therapeutic targets for AF and IHD. In this review, we first summarize the main molecular mechanisms responsible for oxidative DNA damage and repair both in nuclei and mitochondria, then describe the effects of oxidative DNA damage on the development of AF and IHD, and finally discuss potential targets for oxidative DNA repair-based therapeutic approaches for these two cardiac diseases.

Oxidative DNA Damage Modulates DNA Methylation Pattern in Human Breast Cancer 1 (BRCA1) Gene via the Crosstalk between DNA Polymerase β and a de novo DNA Methyltransferase.

DNA damage and base excision repair (BER) are actively involved in the modulation of DNA methylation and demethylation. However, the underlying molecular mechanisms remain unclear. In this study, we seek to understand the mechanisms by exploring the effects of oxidative DNA damage on the DNA methylation pattern of the tumor suppressor breast cancer 1 (BRCA1) gene in the human embryonic kidney (HEK) HEK293H cells. We found that oxidative DNA damage simultaneously induced DNA demethylation and generation of new methylation sites at the CpGs located at the promoter and transcribed regions of the gene ranging from −189 to +27 in human cells. We demonstrated that DNA damage-induced demethylation was mediated by nucleotide misincorporation by DNA polymerase β (pol β). Surprisingly, we found that the generation of new DNA methylation sites was mediated by coordination between pol β and the de novo DNA methyltransferase, DNA methyltransferase 3b (DNMT3b), through the interaction between the two enzymes in the promoter and encoding regions of the BRCA1 gene. Our study provides the first evidence that oxidative DNA damage can cause dynamic changes in DNA methylation in the BRCA1 gene through the crosstalk between BER and de novo DNA methylation.

The Role of DNA Repair Glycosylase OGG1 in Intrahepatic Cholangiocarcinoma.

The effects of oxidative stress on various carcinomas were reported in previous studies, but those in intrahepatic cholangiocarcinoma (ICC) have not been fully elucidated. The purpose of this study was, thus, to reveal the effects of oxidative DNA damage and repair enzymes on ICC. The levels of 8-hydroxydeoxyguanosine (8-OHdG) and 8-OHdG DNA glycosylase (OGG1) were immunohistochemically evaluated in specimens resected from 63 patients with ICC. Low OGG1 expression was related to tumour depth T4 (p=0.04), venous invasion (p=0.0005), lymphatic vessel invasion (p=0.03), and perineural invasion (p=0.03). Compared to the high-OGG1-expression group, patients with low OGG1 expression had a significantly poorer prognosis (overall survival: p=0.04, recurrence-free survival: p=0.02). Unlike for OGG1, the expression levels of 8-OHdG showed no association with prognosis. Oxidative DNA damage and DNA repair enzymes may be closely related to ICC progression.

Effects of differently shaped TiO2NPs (nanospheres, nanorods and nanowires) on the in vitro model (Caco-2/HT29) of the intestinal barrier

BackgroundThe biological effects of nanoparticles depend on several characteristics such as size and shape that must be taken into account in any type of assessment. The increased use of titanium dioxide nanoparticles (TiO2NPs) for industrial applications, and specifically as a food additive, demands a deep assessment of their potential risk for humans, including their abilities to cross biological barriers.MethodsWe have investigated the interaction of three differently shaped TiO2NPs (nanospheres, nanorods and nanowires) in an in vitro model of the intestinal barrier, where the coculture of Caco-2/HT29 cells confers inherent intestinal epithelium characteristics to the model (i.e. mucus secretion, brush border, tight junctions, etc.).ResultsAdverse effects in the intestinal epithelium were detected by studying the barrier’s integrity (TEER), permeability (LY) and changes in the gene expression of selected specific markers. Using Laser Scanning Confocal Microscopy, we detected a different behaviour in the bio-adhesion and biodistribution of each of the TiO2NPs. Moreover, we were able to specifically localize each type of TiO2NPs inside the cells. Interestingly, general DNA damage, but not oxidative DNA damage effects, were detected by using the FPG version of the comet assay.ConclusionsResults indicate different interactions and cellular responses related to differently shaped TiO2NPs, nanowires showing the most harmful effects.

Levels of leukocyte oxidative DNA damage (8-OHdG), serum coenzyme Q10 and lipid peroxidation in the formation attacks of patients with multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease
 of the nervous system. Evidence about oxidative stress plays an important role
 in the pathogenesis of MS is increasing day by day. In our study, we aimed to
 investigate the effect of oxidative DNA damage and oxidative stress in the
 pathogenesis of MS disease. Blood samples were obtained from during an attack
 (Group 1), between attacks (Group 2) of MS patients (20 male and 10 female) and
 30 healthy volunteers (Group 3). Malondialdehyde (MDA) levels as indicator of
 oxidized lipids were detected using fluorescence dedector with high pressure
 liquid chromatograph (HPLC). DNA was extracted from leukocytes of control and
 patients with MS and then we measured 8-hydroxy- 2' -deoxyguanosine (8-OHdG) and deoxyguanosin (dG) by using HPLC method with electrochemical and
 UV detector, respectively. Measurement of oxidized coenzyme Q10 (CoQ10) and
 reduced CoQ (CoQ10H) was performed by using UV detector with HPLC method. Serum
 MDA level of group 1 was significantly higher than those in group 2 and group 3
 (p< 0.001). 8- OHdG/106 dG ratio
 of group 1 was significantly higher than those in group 2 and group 3 (p<
 0.001). CoQ10/CoQ10H rates of group 1 were significantly increased compared
 with group 2 and group 3 (p<0.001). In conclusion, we observed that
 oxidative DNA damage, lipid and mitochondria oxidative damage were high in
 blood of patients with MS. It seems that oxidative stress acts a play role the
 pathogenesis of MS patients as well as induces attacks.
 

Effects of 3-monochloropropane-1,2-diol (3-MCPD) and its metabolites on DNA damage and repair under in vitro conditions

3-monochloropropane-1,2-diol (3-MCPD) is a food contaminant that occurs during industrial production processes and can be found mainly in fat and salt containing products. 3-MCPD has exhibited mutagenic activity in vitro but not in vivo, however, a genotoxic mechanism for the occurrence of kidney tumors has not so far been excluded. The main pathway of mammalian 3-MCPD metabolism is via the formation of β – chlorolactatic acid and formation of glycidol has been demonstrated in bacterial metabolism. The aim of this study was to investigate genotoxic and oxidative DNA damaging effects of 3-MCPD and its metabolites, and to provide a better understanding of their roles in DNA repair processes. DNA damage was assessed by alkaline comet assay in target rat kidney epithelial cell lines (NRK-52E) and human embryonic kidney cells (HEK-293). Purine and pyrimidine base damage, H2O2 sensitivity and DNA repair capacity were assessed via modified comet assay. The results revealed in vitro evidence for increased genotoxicity and H2O2 sensitivity. No association was found between oxidative DNA damage and DNA repair capacity with the exception of glycidol treatment at 20 μg/mL. These findings provide further insights into the mechanisms underlying the in vitro genotoxic potential of 3-MCPD and metabolites.

Cytotoxicity and genotoxicity of orthodontic bands with or without silver soldered joints

Stainless steel bands, with or without silver soldered joints, are routinely used in orthodontics. However, little is known about the toxic biological effects of these appliances. The aims of this study were to evaluate the cytotoxic, cytostatic, genotoxic and DNA damage-inducing effects of non-soldered bands (NSB) and silver soldered bands (SSB) on the HepG2 and HOK cell lines and to quantify the amount of ions released by the bands. The 24-h metallic eluates of NSBs and SSBs were quantified by atomic absorption spectrophotometry. An MTT reduction assay was performed to evaluate the cytotoxicity, alkaline and modified comet assays were employed to measure genotoxicity and oxidative DNA damage effects, and cytokinesis-block micronucleus cytome (CBMN-Cyt) assays were used to verify DNA damage, cytostasis and cytotoxicity. Ag, Cd, Cr, Cu and Zn were detected in SSB medium samples, and Fe and Ni were detected in both the SSB and NSB medium samples. The SSB group induced stronger cytotoxic effects than the NSB group in both evaluated cell lines. NSB and SSB induced genotoxicity as evaluated by comet assays; stronger effects were observed in the SSB group. Both groups induced similar increases in the number of oxidative DNA lesions, as detected by the FPG and Endo III enzymes. Nucleoplasmic bridges, biomarkers of DNA misrepair and/or telomere end fusions, were significantly elevated in the SSB group. The SSB eluates showed higher amounts of Ni and Fe than NSB, and all the quantified ions were detected in SSB eluates, including Cd. The SSB eluates were more cytotoxic and genotoxic than the NSB samples. Based on these results, we propose that other brands, materials and techniques should be further investigated for the future manufacture of orthodontic appliances.

Oxidative DNA damage in human esophageal cancer: clinicopathological analysis of 8-hydroxydeoxyguanosine and its repair enzyme

Both internal and external oxidative stresses act on DNA and can induce carcinogenesis. 8-hydroxydeoxyguanosine (8-OHdG) is an indicator of oxidative stress and it leads to transversion mutations and carcinogenesis. 8-OHdG is excision-repaired by 8-OHdG DNA glycosylase (OGG1). The purpose of this study is to clarify the effect of oxidative DNA damage and repair enzymes on esophageal carcinogenesis. The levels of 8-OHdG and OGG1 were immunohistochemically evaluated in resected specimens, including squamous cell carcinoma (SCC) in 97 patients with esophageal cancer. Higher levels of 8-OHdG in normal esophageal epithelium were associated with a higher smoking index (P = 0.0464). The 8-OHdG level was higher in cancerous areas than in normal epithelia (P = 0.0061), whereas OGG1 expression was weaker in cancerous areas than in normal epithelia (P < 0.0001). An increase of OGG1 expression in normal epithelium was observed as 8-OHdG levels increased (P = 0.0011). However, this correlation was not observed in cancerous areas. High OGG1 expression in the cytoplasm was related to deeper tumors (P = 0.0023), node metastasis (P = 0.0065) and stage (P = 0.0019). Oxidative DNA damage, which is attributable to smoking as well as disturbances in DNA repair systems, appears to be closely related to esophageal carcinogenesis and its progression.

Oxidative DNA damage and β-catenin expression in colorectal cancer evolution

Oxidative DNA damage is one of the mechanisms associated to initial colorectal carcinogenesis, but how it interacts with β-catenin, an adherence protein related to cancer evolution, is not clear. This study investigates the relationship between oxidative DNA damage and β-catenin expression in normal mucosa and colon tumor tissue (adenoma and adenocarcinoma) in colorectal adenocarcinoma evolution. One hundred and 13 samples were studied. Hematoxylin-eosin determined histological grade. β-Catenin expression was analyzed by immunohistochemistry. The oxidative DNA damage was evaluated using comet assay technique. The coefficient for rejection of the nullity hypothesis was taken to 5 %. Kruskal-Wallis, Spearman test, and partial correlation were used to analyze the data. There was oxidative DNA damage increase in colorectal cancer evolution (p < 0.01). Histological grade was correlated with oxidative DNA damage (p < 0.01). There were differences in β-catenin expression among normal, adenoma, and adenocarcinoma tissue with progressive increase of β-catenin expression (p < 0.00). Histological grade was correlated to β-catenin expression (p < 0.00). There was a relationship (p < 0.00) between β-catenin and histological grade while controlling for the effect of oxidative DNA damage. The findings of this study make it possible to establish a relationship between oxidative DNA damage and β-catenin expression in normal mucosa and colorectal tumor tissue. Additionally, they show a causal relationship between variations of β-catenin in different tissues analyzed while controlling for the effect of oxidative DNA damage.

DNA Damage Due to Oxidative Stress in Chronic Obstructive Pulmonary Disease (COPD)

According to the American Thorasic Society (ATS)/European Respiratory Society (ERS) Statement, chronic obstructive pulmonary disease (COPD) is defined as a preventable and treatable disease with a strong genetic component, characterized by airflow limitation that is not fully reversible, but is usually progressive and associated with an enhanced inflammatory response of the lung to noxious particles or gases. The main features of COPD are chronic inflammation of the airways and progressive destruction of lung parenchyma and alveolar structure. The pathogenesis of COPD is complex due to the interactions of several mechanisms, such as inflammation, proteolytic/antiproteolytic imbalance, oxidative stress, DNA damage, apoptosis, enhanced senescence of the structural cells and defective repair processes. This review focuses on the effects of oxidative DNA damage and the consequent immune responses in COPD. In susceptible individuals, cigarette smoke injures the airway epithelium generating the release of endogenous intracellular molecules or danger-associated molecular patterns from stressed or dying cells. These signals are captured by antigen presenting cells and are transferred to the lymphoid tissue, generating an adaptive immune response and enhancing chronic inflammation.

Docosahexaenoic acid selectively induces human prostate cancer cell sensitivity to oxidative stress through modulation of NF‐κB

Oxidative burden is strongly implicated in the pathogenesis of age-related diseases, including prostate cancer tumor formation. As omega-3 fatty acids possess known antioxidant properties, we investigated the effects of docosahexaenoic acid (DHA-22:6n-3), one component of fish oil, in modulating the effects of oxidative DNA damage in LNCaP and PacMetUT1 human prostate adenocarcinoma cells and in a normal human prostate cell line, PrEC. Cell survival was determined by an inhibition of colony formation assay. DNA double-strand breaks, NF-κB subcellular localization and relative survivin expression levels were determined by immunofluorescence and survivin expression levels confirmed by immunoblot assay. Measurement of NF-κB transcriptional activity was investigated by dual luciferase assay. LNCaP and PacMetUT1 cells pretreated with DHA and pulsed with 32 µM H(2) O(2) exhibit decreased survival compared to PrEC. γ-H2AX foci, indicating DNA double-strand breaks, were associated with translocation of NF-κB into the nucleus, whereas exposure to DHA prior to H(2) O(2) treatment prevented NF-κB translocation. Further, DHA attenuated H(2) O(2) -induced NF-κB transcriptional activity and diminished expression of the downstream target, survivin. NF-κB is heavily implicated in promoting prosurvival signaling and may be critical for resistance to the chronic oxidative stress observed in the pathogenesis of prostate cancer. Our studies indicate that exposure of cells to physiologically achievable levels of DHA prior to treatment with H(2) O(2) results in decreased cancer cell survival which is associated with nuclear exclusion of NF-κB. We therefore propose that DHA selectively sensitizes prostate cancer cells to growth arrest through attenuation of the NF-κB survival pathway.

Role of nucleotide excision repair proteins in oxidative DNA damage repair: an updating

DNA repair is a crucial factor in maintaining a low steady-state level of oxidative DNA damage. Base excision repair (BER) has an important role in preventing the deleterious effects of oxidative DNA damage, but recent evidence points to the involvement of several repair pathways in this process. Oxidative damage may arise from endogenous and exogenous sources and may target nuclear and mitochondrial DNA as well as RNA and proteins. The importance of preventing mutations associated with oxidative damage is shown by a direct association between defects in BER (i.e. MYH DNA glycosylase) and colorectal cancer, but it is becoming increasingly evident that damage by highly reactive oxygen species plays also central roles in aging and neurodegeneration. Mutations in genes of the nucleotide excision repair (NER) pathway are associated with diseases, such as xeroderma pigmentosum and Cockayne syndrome, that involve increased skin cancer and/or developmental and neurological symptoms. In this review we will provide an updating of the current evidence on the involvement of NER factors in the control of oxidative DNA damage and will attempt to address the issue of whether this unexpected role may unlock the difficult puzzle of the pathogenesis of these syndromes.

Evaluation of cytotoxicity and oxidative DNA damaging effects of di(2-ethylhexyl)-phthalate (DEHP) and mono(2-ethylhexyl)-phthalate (MEHP) on MA-10 Leydig cells and protection by selenium

Di(2-ethylhexyl)-phthalate (DEHP) is the most abundantly used phthalate derivative, inevitable environmental exposure of which is suspected to contribute to the increasing incidence of testicular dysgenesis syndrome in humans. Oxidative stress and mitochondrial dysfunction in germ cells are suggested to contribute to phthalate-induced disruption of spermatogenesis in rodents, and Leydig cells are one of the main targets of phthalates’ testicular toxicity. Selenium is known to be involved in the modulation of intracellular redox equilibrium, and plays a critical role in testis, sperm, and reproduction. This study was aimed to investigate the oxidative stress potential of DEHP and its consequences in testicular cells, and examine the possible protective effects of selenium using the MA-10 mouse Leydig tumor cell line as a model. In the presence and absence of selenium compounds [30 nM sodium selenite (SS), and 10 μM selenomethionine (SM)], the effects of exposure to DEHP and its main metabolite mono(2-ethylhexyl)-phthalate (MEHP) on the cell viability, enzymatic and non-enzymatic antioxidant status, ROS production, p53 expression, and DNA damage by alkaline Comet assay were investigated. The overall results of this study demonstrated the cytotoxicity and genotoxicity potential of DEHP, where MEHP was found to be more potent than the parent compound. SS and SM produced almost the same level of protection against antioxidant status modifying effects, ROS and p53 inducing potentials, and DNA damaging effects of the two phthalate derivatives. It was thus shown that DEHP produced oxidative stress in MA-10 cells, and selenium supplementation appeared to be an effective redox regulator in the experimental conditions used in this study, emphasizing the critical importance of the appropriate selenium status.

The effects of oxidative DNA damage and mutations in the p53 protein on cells of the colonic mucosa with and without the fecal stream: an experimental study in rats

Objective. The aim of this study was to investigate the levels of oxidative DNA damage and p53 mutations in an experimental model of diversion colitis. Material and methods. Sixty rats were divided into three groups with 20 animals in accordance with the sacrifice was carried out 6, 12 and 18 weeks. For each group, 15 animals were subjected to diversion of the fecal stream through colostomy in the left proximal colon and distal mucous fistula (experimental group), and five to a laparotomy without deviation of the fecal stream (control group). The presence of colitis was evaluated by inflammatory grading scale. Mutations in the p53 protein were evaluated by immunohistochemistry with primary antibody with cross-reactivity for rats. The oxidative DNA damage was measured using the comet assay. To statistical analysis were used the Student's t, Mann-Whitney and Kruskal-Wallis test adopting a significance level of 5% (p < 0.05). Results. Colon segments without fecal stream showed greater degree of inflammation when compared to animals with preserved fecal stream (p = 0.01). The levels of oxidative stress were significantly higher in segments without fecal stream (p < 0.0001) and increased with the time of fecal diversion (p = 0.007). The levels of oxidative DNA damage are directly related to tissue degree of inflammation. There were no mutations in the p53 protein in the segments without fecal stream regardless of time of exclusion considered. Conclusion. Despite higher levels of oxidative damage to nuclear DNA on segments without fecal stream that developed colitis mutations in the p53 protein were not detected.

Myricetin, quercetin, (+)-catechin and (−)-epicatechin protect against N-nitrosamines-induced DNA damage in human hepatoma cells

The aim of this study was to investigate the protective effect of myricetin, quercetin, (+)-catechin and (−)-epicatechin, against N-nitrosodibutylamine (NDBA) and N-nitrosopiperidine (NPIP)-induced DNA damage in human hepatoma cells (HepG2). DNA damage (strand breaks and oxidized purines/pyrimidines) was evaluated by the alkaline single-cell gel electrophoresis or Comet assay. (+)-Catechin at the lowest concentration (10 μM) showed the maximum reduction of DNA strand breaks (23%), the formation of endonuclease III (Endo III, 19–21%) and formamidopyrimidine-DNA glycosylase (Fpg, 28–40%) sensitive sites induced by NDBA or NPIP. (−)-Epicatechin also decreased DNA strand breaks (10 μM, 20%) and the oxidized pyrimidines/purines (33–39%) induced by NDBA or NPIP, respectively. DNA strand breaks induced by NDBA or NPIP were weakly reduced by myricetin at the lowest concentration (0.1 μM, 10–19%, respectively). Myricetin also reduced the oxidized purines (0.1 μM, 17%) and pyrimidines (0.1 μM, 15%) induced by NDBA, but not the oxidized pyrimidines induced by NPIP. Quercetin did not protect against NDBA-induced DNA damage, but it reduced the formation of Endo III and Fpg sensitive sites induced by NPIP (0.1 μM, 17–20%, respectively). In conclusion, our results indicate that (+)-catechin and (−)-epicatechin at the concentrations tested protect human derived cells against oxidative DNA damage effects of NDBA and NPIP. However, myricetin at the concentrations tested only protects human cells against oxidative DNA damage induced by NDBA and quercetin against oxidative DNA damage induced by NPIP.

Continuous elimination of oxidized nucleotides is necessary to prevent rapid onset of cellular senescence

Reactive oxygen species (ROS) appear to play a role in limiting both cellular and organismic lifespan. However, because of their pleiotropic effects, it has been difficult to ascribe a specific role to ROS in initiating the process of cellular senescence. We have studied the effects of oxidative DNA damage on cell proliferation, believing that such damage is of central importance to triggering senescence. To do so, we devised a strategy to decouple levels of 8-oxoguanine, a major oxidative DNA lesion, from ROS levels. Suppression of MTH1 expression, which hydrolyzes 8-oxo-dGTP, was accompanied by increased total cellular 8-oxoguanine levels and caused early-passage primary and telomerase-immortalized human skin fibroblasts to rapidly undergo senescence, doing so without altering cellular ROS levels. This senescent phenotype recapitulated several salient features of replicative senescence, notably the presence of senescence-associated beta-galactosidase (SA beta-gal) activity, apparently irreparable genomic DNA breaks, and elevation of p21(Cip1), p53, and p16(INK4A) tumor suppressor protein levels. Culturing cells under low oxygen tension (3%) largely prevented the shMTH1-dependent senescent phenotype. These results indicate that the nucleotide pool is a critical target of intracellular ROS and that oxidized nucleotides, unless continuously eliminated, can rapidly induce cell senescence through signaling pathways very similar to those activated during replicative senescence.

Dietary polyphenols protect against N-nitrosamines and benzo(a)pyrene-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 human hepatoma cells

Dietary polyphenols have been reported to have a variety of biological actions, including anticarcinogenic and antioxidant activities. In the present study we investigated the protective effect of dietary polyphenols against N-nitrosodimethylamine (NDMA), N-nitrosopyrrolidine (NPYR) and benzo(a)pyrene (BaP)-induced DNA damage (strand breaks and oxidized purines/pyrimidines) in HepG2 cells. Human hepatocellular carcinoma (HepG2) cells, which retain many specialized liver functions and drug metabolizing enzyme activities, were used as in vitro model for human hepatocytes. NDMA, NPYR and BaP were employed to induce DNA damage. DNA damage (strand breaks, oxidized pyrimidines and oxidized purines) was evaluated by the alkaline single cell gel electrophoresis or comet assay. None of the polyphenols concentrations tested in presence or absence of Fpg (formamidopyrimidine-DNA glycosylase), or Endo III (Endonuclease III) caused DNA damage per se. Increasing concentrations of BaP (25-100 microM) induced a significant increase of DNA strand breaks, Fpg and Endo III sensitive sites in a dose dependent manner. Myricetin and quercetin decreased DNA strand breaks and oxidized pyrimidines induced by NDMA, but not oxidized purines. However, both flavonoids reduced oxidized pyrimidines and purines induced by NPYR. DNA strand breaks induced by NPYR were prevented by quercetin, but not by myricetin. BaP-induced DNA strand breaks and oxidized pyrimidines were strongly reduced by myricetin and quercetin, respectively. While oxidized purines induced by BaP were reduced by quercetin, myricetin had no protective effect. (+)-Catechin and (-)-epicatechin reduced DNA strand breaks, oxidized pyrimidines and oxidized purines induced by NDMA. DNA strand breaks, and oxidized purines induced by NPYR were also prevented by (+)-catechin and (-)-epicatechin, while the maximum reduction of oxidized pyrimidines was found by (+)-catechin and (-)-epicatechin at 10 microM. (+)-Catechin and (-)-epicatechin decreased also DNA strand breaks and oxidized pyrimidines but not oxidized purines induced by BaP. Our results clearly indicate that polyphenols protect human derived cells against DNA strand breaks and oxidative DNA damage effects of NDMA, NPYR or BaP, three carcinogenic compounds which occur in the environment.

Reducing oxidative DNA damage by adding antioxidants in human semen samples undergoing cryopreservation procedure

To evaluate the protective effects of oxidative DNA damage by adding antioxidants: ascorbate, catalase (CAT), and superoxide dismutase (SOD) in human semen samples undergoing cryopreservation procedure. Semen sample form 30 fertile men were mixed with modified cryoprotectant and divided into six groups according to the category and concentration of antioxidants: ascorbate 300 micromol/L, ascorbate 600 micromol/L, CAT 200 U/ml, CAT 400 U/ml, SOD 200 U/ml, and SOD 400 U/ml. Comet assay was conducted to measure the percentage of comet cells, and the nuclear DNA damaged parameters: tail DNA percentage (TD%) and Olive tail moment (OTM). Flow cytometry was used to detect the reactive oxidative species (ROS). The motility (a + b grade), viable recovery rate, nuclear DNA integrity and reactive oxidative species (ROS) of all groups were analyzed before and/or after freeze-thawing. (After cryopreservation, compared with the control group, the a + b grade sperm rates of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were all higher than that of the control group (all P < 0.05), however, the levels of reactive oxygen species (ROS) of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 30 +/- 13, 30 +/- 11, and 30 +/- 11 respectively, all significantly lower than that of the control group (37 +/- 17 , all P < 0.05). The viable recovery rates of the ascorbate 300 micromol/L , CAT 200 U, and CAT 400 U groups were 67% +/- 14%, 68% +/- 14%, and 69% -/+ 15% respectively, all significantly higher than that of the control group (59% +/- 10%, all P < 0.05). (2) The TD% levels of the ascorbate 300 micromol/L, CAT 200 U, and CAT 400 U groups were 41% +/- 4%, 40% +/- 7%, 40% +/- 6%, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (46% +/- 6%, all P < 0.01). The OTM levels of the ascorbate 300 micromol/ L, CAT 200 U, and CAT 400 U groups were 7.7 +/- 1.2, 7.5 +/- 1.6, and 7.8 +/- 1.9, all similar to that of the raw semen (all P > 0.05), but significantly lower than that of the control group (10.1 +/- 3.1, all P < 0.01) too. The TD% and OTM levels of the other groups were all significantly higher than that of the raw semen (all P < 0.01), but not significantly different from those of the control group (all P > 0.05). (3) ROS was significantly negatively correlated with the motility in all groups (P < 0.05 or P < 0.01). Apart from the ascorbate 600 micromol/L group, the TD% and OTM of the other groups were all significantly positively correlated with the ROS (P < 0.05 or P < 0.01). Supplementation of ascorbate or CAT reduces the level of ROS that induces sperm nuclear DNA damage, and improves the human sperm quality in the process of freeze-thawing.