DNA Single-strand Breaks Research Articles

Pharmacologic inhibition of ataxia telangiectasia and Rad3-related (ATR) in the treatment of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) poses significant treatment challenges, with high recurrence rates for locally advanced disease despite aggressive therapy typically involving a combination of surgery, radiation therapy, and/or chemotherapy. HNSCCs commonly exhibit reduced or absent TP53 function due to genomic alterations or human papillomavirus (HPV) infection, leading to dependence on the S- and G2/M checkpoints for cell cycle regulation. Both of these checkpoints are activated by Ataxia Telangiectasia and Rad3-related (ATR), which tends to be overexpressed in HNSCC relative to adjacent normal tissues and represents a potentially promising therapeutic target, particularly in combination with other treatments. ATR is a DNA damage signaling kinase that is activated in response to replication stress and single-stranded DNA breaks, such as those induced by radiation therapy and certain chemotherapies. ATR kinase inhibitors are currently being investigated in several clinical trials as part of the management of locally advanced, recurrent, or metastatic HNSCC, along with other malignancies. In this review article, we summarize the rationale and preclinical data supporting incorporation of ATR inhibition into therapeutic regimens for HNSCC.

DNA strand break induction of aqueous plasmid DNA exposed to 30 MeV protons at ultra-high dose rate.

Radiation cancer therapy with ultra-high dose rate exposure, so called FLASH radiotherapy, appears to reduce normal tissue damage without compromising tumor response. The aim of this study was to clarify whether FLASH exposure of proton beam would be effective in reducing the DNA strand break induction. We applied a simple model system, pBR322 plasmid DNA in aqueous 1 × TE solution, where DNA single strand breaks (SSBs) and double strand breaks (DSBs) can be precisely quantified by gel electrophoresis. Plasmid DNA were exposed to 27.5 MeV protons in the conventional dose rate of 0.05 Gy/s (CONV) and ultra-high dose rate of 40 Gy/s (FLASH). With both dose rate, the kinetics of the SSB and DSB induction were proportional to absorbed dose. The SSB induction of FLASH was significantly less than CONV, which were 8.79 ± 0.14 (10−3 SSB per Gy per molecule) and 10.8 ± 0.68 (10−3 SSB per Gy per molecule), respectively. The DSB induction of FLASH was also slightly less than CONV, but difference was not significant. Altogether, 27.5 MeV proton beam at 40 Gy/s reduced SSB and not DSB, thus its effect may not be significant in reducing lethal DNA damage that become apparent in acute radiation effect.

Antibiotic toxicity on human spermatozoa assessed using the sperm DNA fragmentation dynamic assay.

Sperm DNA fragmentation (SDF) dynamic assays were piloted on 4 fresh ejaculates to examine the possible sperm toxicity of three common antibiotics, ciprofloxacin, doxycycline and ampicillin, incubated at a concentration estimated to be reached in semen in vivo, and 100×, for 24h. SDF was assessed in terms of single-strand DNA breaks (SSBs) and double-strand DNA breaks (DSBs). Low and high concentrations of ciprofloxacin and high concentration of doxycycline significantly increased the SDF rate, due to sperm containing SSBs. Ampicillin did not affect SDF dynamics at any dose. Based on these results, the effect of antibiotics on the global-SDF dynamics was further examined in 21 ejaculates assessed at 0, 4 and 6h. Ciprofloxacin increased the rate of SDF at the low concentration in 17 from 21subjects; the high concentration resulted in a stronger effect in all individuals. A significant increase in the rate of SDF in 17 ejaculates was also noted when spermatozoa were incubated with the high concentration of doxycycline. The dynamic SDF assay is a rapid and sensitive tool to evidence sperm toxicity. Ciprofloxacin should be avoided when it is necessary to preserve sperm quality for reproductive purposes and as additive in semen diluents.

Cryo-EM structures and biochemical insights into heterotrimeric PCNA regulation of DNA ligase

DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.

XRCC1 protects transcription from toxic PARP1 activity during DNA base excision repair

Genetic defects in the repair of DNA single-strand breaks (SSBs) can result in neurological disease triggered by toxic activity of the single-strand-break sensor protein PARP1. However, the mechanism(s) by which this toxic PARP1 activity triggers cellular dysfunction are unclear. Here we show that human cells lacking XRCC1 fail to rapidly recover transcription following DNA base damage, a phenotype also observed in patient-derived fibroblasts with XRCC1 mutations and Xrcc1−/− mouse neurons. This defect is caused by excessive/aberrant PARP1 activity during DNA base excision repair, resulting from the loss of PARP1 regulation by XRCC1. We show that aberrant PARP1 activity suppresses transcriptional recovery during base excision repair by promoting excessive recruitment and activity of the ubiquitin protease USP3, which as a result reduces the level of monoubiquitinated histones important for normal transcriptional regulation. Importantly, inhibition and/or deletion of PARP1 or USP3 restores transcriptional recovery in XRCC1−/− cells, highlighting PARP1 and USP3 as possible therapeutic targets in neurological disease.

Cytogenetic follow-up studies on humans with internal and external exposure to ionizing radiation

Cells exposed to ionizing radiation have a wide spectrum of DNA lesions that include DNA single-strand breaks, DNA double-strand breaks (DSBs), oxidative base damage and DNA-protein crosslinks. Among them, DSB is the most critical lesion, which when mis-repaired leads to unstable and stable chromosome aberrations. Currently, chromosome aberration analysis is the preferred method for biological monitoring of radiation-exposed humans. Stable chromosome aberrations, such as inversions and balanced translocations, persist in the peripheral blood lymphocytes of radiation-exposed humans for several years and, therefore, are potentially useful tools to prognosticate the health risks of radiation exposure, particularly in the hematopoietic system. In this review, we summarize the cytogenetic follow-up studies performed by REAC/TS (Radiation Emergency Assistance Center/Training site, Oak Ridge, USA) on humans exposed to internal and external radiation. In the light of our observations as well as the data existing in the literature, this review attempts to highlight the importance of follow-up studies for predicting the extent of genomic instability and its impact on delayed health risks in radiation-exposed victims.

PARP Inhibition with Talazoparib Enhances DNA Damage and Anti-Leukemic Activity of Venetoclax in Preclinical Human Acute Myeloid Leukemia (AML) Models

Background: BCL-2 is an anti-apoptotic protein associated with chemotherapy resistance and survival of acute myeloid leukemia (AML) cells following cell stress or DNA damage. Venetoclax (Ven) is a BH3-mimetic that competitively binds to BCL-2, allowing BH3 proteins to bind to pro-apoptotic proteins (BAX, BAK) and induce cell death. The combination of Ven with Azacitidine (Aza), is now standard of care therapy for older patients with AML. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent the repair of single stranded DNA breaks by blocking the nicotinamide adenine dinucleotide (NAD) catalytic domain of the PARP protein and preventing dissociation of PARP from the DNA (termed PARP trapping). The PARP inhibitor, talazoparib (Tala) displays 10,000-fold increased PARP trapping as compared to other clinical PARP inhibitor agents in tumor models. We hypothesized that Tala could enhance the anti-leukemic activity of Ven on AML cells specifically by increasing DNA damage.Methods: Human AML (Molm13) cells were continuously exposed to increasing doses of Ven (1nM - 10mM), Tala (1nM - 10mM), and Aza (0.1-5mM) alone and in combination. Synergy reports were generated using CompuSyn software. Cell viability was measured using a WST colorimetric assay. Apoptosis (annexin /PI), DNA damage (pH2AX), and cell cycle arrest (propidium iodide) were quantified by flow cytometric analysis. DNA damage was also evaluated using the Fast-Halo assay. In vivo efficacy was assessed in NSG mice systematically engrafted with stably luciferase transfected Molm13 BLIV cells via tail vein injection. Cohorts of mice (5-10) were treated with vehicle, Ven (100 mg/kg given 5 days/week for 3 weeks), Tala (0.33 mg/kg for 5 days/week over 3 weeks), Ven + Tala (same doses), and Ven+ Aza (1.25 or 2.5 mg/kg daily for 7 days). Treatment effects on leukemia burden, toxicity, and overall survival were determined using weekly whole animal bioluminescent imaging, total animal weights, and time to morbidity, respectively.Results: Human AML cells treated with Ven displayed dose and time-dependent induction of DNA damage, apoptosis, and cell death. Similarly, treatment with single agent Tala resulted in dose-dependent anti-proliferative effects and PARP trapping. Combination in vitro therapy with Ven (0.1 - 1 μM) + Tala (10- 100 nM) or Ven (same doses) + Aza (1 μM) resulted in significant and synergistic anti-leukemic effects improving upon monotherapy with Ven, Tala, or Aza alone. However, in contrast to Ven+Aza, increased levels of DNA damage (as determined by phosphorylated H2AX and Fast Halo assays) were detected as well as induction of apoptosis, cell cycle arrest, decreased viability, and increased cell death vs vehicle and single agent therapy.We then evaluated the effects of in vivo treatment with vehicle, Ven, Tala, Ven + Tala, and Ven +Aza in NSG mice systemically engrafted with human luciferase-tagged AML cells (Molm13 BLIV). Single agent Ven or Tala therapy decreased systemic AML burden but did not prolong overall survival over vehicle treated mice. Combination Ven + Tala significantly prolonged overall survival of leukemia xenografted mice over vehicle, Ven, or Tala monotherapy. Of note, Ven + Tala treatment appeared to result in similar disease burden reduction and prolonged overall survival as Ven + Aza treated animals (Figure 1). Preliminary experiments of iv vivo triple therapy with Ven + Aza + Tala in AML xenografted mice resulted in further reduction in AML disease burden but was associated with marked weight loss leading to early morbidity and shortened overall survival (data not shown). Additional doses and schedules of this triplet approach are under investigation.Conclusions: Our results demonstrate that addition of the potent PARP inhibitor, talazoparib, to the BCL-2 inhibitor, venetoclax, enhances DNA damage and results in potent in vitro and in vivo activity in preclinical human AML models. Further evaluation of this combination in primary AML samples with varying genetic backgrounds as well as in confirmatory primary xenograft models is underway. These data strongly support future clinical investigation of PARP inhibitors as a novel class of agents with the potential to significantly enhance the efficacy of novel targeted therapies for acute leukemia. [Display omitted] DisclosuresWang: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; DAVA Oncology: Consultancy, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Advisory Board; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.

A Phase I/II Study of Sapacitabine and Venetoclax in Relapsed/Refractory Acute Myeloid Leukemia

A Phase I/II Study of Sapacitabine and Venetoclax in Relapsed/Refractory Acute Myeloid Leukemia

Phase 1b Trial of Talazoparib and Gemtuzumab Ozogamicin in Adult Patients with CD33+ Relapsed or Refractory Acute Myeloid Leukemia

Phase 1b Trial of Talazoparib and Gemtuzumab Ozogamicin in Adult Patients with CD33+ Relapsed or Refractory Acute Myeloid Leukemia

APE2 Is a General Regulator of the ATR-Chk1 DNA Damage Response Pathway to Maintain Genome Integrity in Pancreatic Cancer Cells.

The maintenance of genome integrity and fidelity is vital for the proper function and survival of all organisms. Recent studies have revealed that APE2 is required to activate an ATR-Chk1 DNA damage response (DDR) pathway in response to oxidative stress and a defined DNA single-strand break (SSB) in Xenopus laevis egg extracts. However, it remains unclear whether APE2 is a general regulator of the DDR pathway in mammalian cells. Here, we provide evidence using human pancreatic cancer cells that APE2 is essential for ATR DDR pathway activation in response to different stressful conditions including oxidative stress, DNA replication stress, and DNA double-strand breaks. Fluorescence microscopy analysis shows that APE2-knockdown (KD) leads to enhanced γH2AX foci and increased micronuclei formation. In addition, we identified a small molecule compound Celastrol as an APE2 inhibitor that specifically compromises the binding of APE2 but not RPA to ssDNA and 3′-5′ exonuclease activity of APE2 but not APE1. The impairment of ATR-Chk1 DDR pathway by Celastrol in Xenopus egg extracts and human pancreatic cancer cells highlights the physiological significance of Celastrol in the regulation of APE2 functionalities in genome integrity. Notably, cell viability assays demonstrate that APE2-KD or Celastrol sensitizes pancreatic cancer cells to chemotherapy drugs. Overall, we propose APE2 as a general regulator for the DDR pathway in genome integrity maintenance.

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways.

Endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs compromise genome integrity and are eliminated by multiple repair pathways. Aberrant Top1-DNA crosslinks, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between diverse mechanisms of DPC repair. Here, we show that several SUMO biogenesis factors (Ulp1, Siz2, Slx5, and Slx8) control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1Δ wss1Δ mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation inhibits alternative DPC repair mechanisms, including Ddi1. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

Causes and Consequences of Nuclear Envelope Rupture and DNA Damage in Micronuclei

Micronuclei are small, aberrant nuclear compartments containing mis-segregated chromosomes or chromosomal fragments. During telophase, dysfunctional micronuclear envelope reassembly leaves the micronuclear envelope highly unstable and rupture-prone. Following rupture, micronuclei attempt to repair membrane gaps, but the process is typically unsuccessful and may promote the invasion of ER tubules into the interior of micronuclei. These abnormalities cause ruptured micronuclei to accumulate significant DNA damage in the form of both single-stranded DNA and double-stranded breaks. Because micronuclei are capable of promoting genome instability, it is essential to understand the sources of DNA damage and the mechanism through which it arises in these structures. In this review, I will explore the causes and consequences of micronuclear envelope rupture, beginning with the processes surrounding improper micronuclear envelope reassembly. I will then discuss micronuclear envelope rupture, attempted micronuclear envelope repair and its consequences, and the proposed causes of micronuclear DNA damage.

Methylmercury chronic exposure affects the expression of DNA single-strand break repair genes, induces oxidative stress, and chromosomal abnormalities in young dyslipidemic APOE knockout mice

Mercury (Hg) is one of the most toxic environmental pollutants, especially when methylated, forming methylmercury (MeHg). MeHg affects DNA repair, increases oxidative stress, and predisposes to cancer. MeHg neurotoxicity is well-known, but recently MeHg-associated cardiovascular effects were recognized. This study evaluated circulating lipids, oxidative stress, and genotoxicity after MeHg-chronic exposure (20 mg/L in drinking water) in C57BL/6J wild-type and APOE knockout (ko) mice, the latter, being spontaneously dyslipidemic. Experimental mice were assigned to four groups: non-intoxicated and MeHg-intoxicated wild-type mice and non-intoxicated and MeHg-intoxicated APOE ko mice. Plasma levels of triglycerides, total cholesterol (TC), HDL, and LDL were analyzed. Liver lipid peroxidation and splenic gene expression of xeroderma pigmentosum complementation groups A, C, D, and G (XPA, XPC, XPD, and XPG), X-ray repair cross-complementing protein 1 (XRCC1), and telomerase reverse transcriptase (TERT) were measured. Fur Hg levels confirmed chronic MeHg intoxication. MeHg exposure raises TC levels both in wild-type and APOE ko mice. HDL and LDL-cholesterol levels were increased only in the MeHg-challenged APOE ko mice. MeHg increased liver lipid peroxidation, regardless of the genetic background. Unintoxicated APOE ko mice showed higher expression of TERT than all other groups. APOE deficiency increases XPA expression, regardless of MeHg intoxication. Furthermore, MeHg-intoxicated mice had more cytogenetic abnormalities, effect which was independent of APOE deficiency. More studies are needed to dissect the interactions between circulating lipids, MeHg intoxication, and DNA-repair pathways even at young age, interactions that likely play critical roles in cell senescence and the risk for chronic disorders later in life.

Anti-HBs titers are not decreased after treatment with oral Cladribine in patients with Multiple Sclerosis vaccinated against Hepatitis B virus

BackgroundOral cladribine is a novel treatment for Multiple Sclerosis (MS). It is a purine nucleoside antimetabolite analogue that is incorporated into the DNA, resulting in single-strand breaks in DNA and apoptosis of replicating lymphocytes. Specifically, Cladribine induces limited depletion of CD4 and CD8 T cell subsets and more marked depletion of memory B cell subsets. Therefore, natural and acquired humoral responses against pathogens may be potentially reduced. The aim of this study was to assess longitudinal variation of antiHBs titers in patients with MS treated with Cladribine. MethodsPatients with MS treated with 1 cycle of Cladribine (3,5 mg/kg) and previously vaccinated against Hepatitis B virus (HBV) were enrolled. Anti-HBs titers were compared before and after 12 months from Cladribine treatment. Total lymphocyte count was also analysed. ResultsAmong the 13 RMS patients (10 F, 3 M, mean age 33,8, SD 5,9) enrolled, all had anti-HBs titers >10 mg/dl at baseline. Anti-HBs titer dropped below the reference value at 12 months after Cladribine only in 1 case. Pre-post Cladribine mean anti-HBs values were not significantly different considering the whole cohort (Wilcoxon-Mann-Whitney Test p = 0,762). Four patients had grade 1 and 1 patient grade 2 lymphocytopenia at 12 months. Conclusions: Cladribine does not seem to reduce humoral immune responses in subjects previously vaccinated against HBV, even in case of lymphocytopenia. These results, if confirmed in larger populations, appear reassuring also for other vaccinations (i.e. COVID19). The low impact of Cladribine on plasma cells may explain such findings.

BRCA2 associates with MCM10 to suppress PRIMPOL-mediated repriming and single-stranded gap formation after DNA damage

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.

Anti-HBS titers are not decreased after treatment with oral cladribine in patients with multiple sclerosis vaccinated against hepatitis B virus

Oral cladribine is a novel treatment for Multiple Sclerosis (MS). It is a purine nucleoside antimetabolite analogue that incorporates into the DNA, resulting in single-strand breaks in DNA and apoptosis of replicating lymphocytes. Specifically, Cladribine induces limited depletion of CD4 and CD8 T subsets and a more marked depletion of memory B cell subsets. Therefore, natural and acquired humoral response against pathogens may be potentially impaired. The aim of this study was to assess longitudinal variation of antiHBs titers in patients with MS treated with Cladribine.

TOP1 modulation during melanoma progression and in adaptative resistance to BRAF and MEK inhibitors

In melanomas, therapy resistance can arise due to a combination of genetic, epigenetic and phenotypic mechanisms. Due to its crucial role in DNA supercoil relaxation, TOP1 is often considered an essential chemotherapeutic target in cancer. However, how TOP1 expression and activity might differ in therapy sensitive versus resistant cell types is unknown. Here we show that TOP1 expression is increased in metastatic melanoma and correlates with an invasive gene expression signature. More specifically, TOP1 expression is highest in cells with the lowest expression of MITF, a key regulator of melanoma biology. Notably, TOP1 and DNA Single-Strand Break Repair genes are downregulated in BRAFi- and BRAFi/MEKi-resistant cells and TOP1 inhibition decreases invasion markers only in BRAFi/MEKi-resistant cells. Thus, we show three different phenotypes related to TOP1 levels: i) non-malignant cells with low TOP1 levels; ii) metastatic cells with high TOP1 levels and high invasiveness; and iii) BRAFi- and BRAFi/MEKi-resistant cells with low TOP1 levels and high invasiveness. Together, these results highlight the potential role of TOP1 in melanoma progression and resistance.

Abstract P487: Impact Of Oxidative Dna Damage On Heart Health

The accumulation of DNA damage in human cardiomyocytes causes apoptosis which can lead to heart failure or other cardiovascular diseases. Although the effects of oxidative stress on heart health and on the DNA Damage Response network are well-known, the two fields have evolved as separate areas of research. The precise impact of oxidative DNA damage on cardiomyocyte contractile function still remains poorly understood. The human FANCJ helicase participates in multiple DNA repair pathways, including interstrand crosslink repair and double-stranded break repair. We have shown previously that FANCJ targets and unfolds 8-oxoguanine modified DNA secondary structures that arise from oxidative damage. We predict that human cardiomyocytes expressing mutations of FANCJ would be more susceptible to oxidative DNA damage and will negatively influence their contractile motion. To test this, hiPSC-CMs were treated with hydrogen peroxide, camptothecin, or bleomycin to induce different forms of DNA damage. The relative abundance of single-stranded DNA breaks and double-stranded DNA breaks were determined by modified comet assays, while contractile function was monitored using video-based detection methods. Cells that overexpress FANCJ protein were able to overcome the chemical stress from hydrogen peroxide. On the contrary, cells that produce a FANCJ K141/K142AA variant, which was previously characterized in the lab, resulted in a hypersensitivity to double-stranded DNA breaks. Based on this evidence, FANCJ plays a vital role in alleviating the effects of oxidative stress. Our long-term goal is to use the established methods to develop functional assays that characterize the cardiovascular risks of other FANCJ variants. These assays can be used to develop screening methods to identify patients who may be predisposed to FANCJ-associated cardiovascular diseases.

Reactive oxygen species, DNA damage and Solar-Earth communications

Reactive oxygen species play a significant role in regulating the main functions of the cell both in the normal conditions and when exposing the cell to exogenous and endogenous factors under electromagnetic and radiation emission at various levels of solar activity. The research studied the impact of low-intensity alternating magnetic field; the intensity level is comparable to the geomagnetic field, in the frequency range from 3 to 60 Hz and γ-radiation on the generation of the most stable ROS which are hydrogen peroxide, in vitro. A degree of oxidative DNA damage in the blood serum of health donors, after being exposed to the alternating magnetic field, was investigated by determining the content of 8- hydroxy-2-deoxyguanosine (8-OHdG) in the blood serum and DNA single breaks in the lymphocytes. An increase of the content of 8-OHdG in DNA by a factor of 1.5 – 2 in comparison with non-emitted samples, and non-linear change in the level of lipid peroxide in the blood serum with maxima at MF frequencies of 8 and 50 Hz. In lymphocytes are observed. When exposed to gamma radiation, the number of single-strand breaks (SSB) of DNA increased with increasing exposure time from 44.3 ± 0.4% (irradiation for 30 minutes) to 68.3 ± 0.8% (irradiation for 90 minutes) compared to the control samples. It is concluded that the obtained effects can be associated with the generation of reactive oxygen species in the aquatic environment with variations in solar activity and affect the adaptive capabilities of the human body as a whole.

The dynamic changes of Nrf2 mediated oxidative stress, DNA damage and base excision repair in testis of rats during aging.

Accumulation of oxidative stress, DNA damage and impaired DNA repair appear to play critical roles in the decline of testicular function with aging. However, when those factors begin to lose control in testis during aging has not yet been well understood. This study was designed to assess the changes of oxidative stress and DNA damage status, and DNA repair capacity in testis during aging. Thus, male Sprague-Dawley rats at 3, 9, 15 and 24months of age were used to delineate the dynamic changes in testicular weight and index, testosterone concentration, testicular histology, Nrf2-mediated oxidative stress, DNA damage, DNA repair and apoptosis. Results showed that testicular weight and index, testosterone concentration and spermatid number progressively declined from 9 to 24months of age. Similarly, seminiferous tubule diameters and seminiferous epithelium heights gradually diminished with aging. Nrf2-mediated antioxidant defense ability was significantly impaired in testis with increasing age including decreased the activity of SOD and the expression levels of Nrf2, HO-1 and NQO-1, and increased the contents of MDA. In addition, DNA damage including DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) also progressively increased accompanied by increased levels of 8-hydroxydeoxyguanosine (8-OHdG) and γ-H2AX, and activated ATM/Chk2 and ATR/Chk1 pathway. Consistent with the results of Nrf2 pathway, the expression levels of APE1, OGG1 and XRCC1 involved in base excision DNA repair (BER) pathway increased from 3 to 9months of age, and then gradually decreased after 9months of age. Finally, TUNEL and Western blot results further confirmed germ cell apoptosis progressively increased from 3 to 24months of age as evidenced by decreased ratio of Bcl-2/Bax and levels of Bcl-2 expression, and increased Bax expression levels. Taken together, our results suggest that downregulation of antioxidant ability mediated by Nrf2 pathway and impairment of BER capacity might correlate with increased DNA damage, and then induce declining testicular function during aging after adult.