Alkaline Comet Research Articles

In vitro cytotoxic and genotoxic effects of Phyllanthus niruri extract loaded chitosan nanoparticles in TM4 cells and their influence on spermatogenesis

Purpose: This paper introduces a complete in vitro investigation of cytotoxic and genotoxic effects of Phyllanthus niruri extract loaded chitosan nanoparticles (PNNP) on mouse Sertoli cell line (TM4), as well as their impact on spermatogenesis. Methods: Chitosan nanoparticles (ChNP) and PNNP were prepared using an ionic gelation process, while their cytotoxicity on TM4 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. Comet and fast halo assays were used to quantify single-strand DNA breaks in TM4 cells. To detect changes in cell morphology during apoptosis, nuclear staining with Hoechst 33342 was performed. An immunofluorescence assay was employed to examine the expression level of proteins connexin 43 and Claudin 11 in TM4 cells after exposure to PNNP concentration of 125 µg/mL. Results: The synthesized PNNP had a size of 170.6 nm, a polydispersity index of 0.269, a zeta potential of +37.8 mV, and a good entrapment efficiency of 71.0%. Encapsulation of Phyllanthus niruri into ChNP induced DNA damage in TM4 cells as determined by alkaline comet and fast halo assay (FHA). Additionally, it stimulated apoptosis, as determined by changes in cell morphology by Hoechst 33342 staining. There was significant down regulation of blood-testis barrier (BTB) proteins in TM4 cells after exposure to PNNP which could compromise the integrity of BTB and subsequently disrupt spermatogenesis process in male. Conclusion: Our investigation confirms the cytotoxic and genotoxic effects of PNNP in TM4 cells, which could lead to spermatogenesis disruption and male infertility.

Abstract 5611: Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications

Abstract Glioblastoma (GBM), a highly aggressive brain cancer, poses a formidable challenge marked by its resistance to conventional therapies. F-actin, a crucial cytoskeletal protein, influences DNA repair and impacts genomic stability. F-actin dynamics also shape the glioma stem cell (GSC) phenotype, affecting self-renewal and migration. Altered F-actin organization contributes to GBM resistance to conventional therapies. Understanding F-actin's multifaceted role is vital for developing targeted strategies to overcome treatment resistance and enhance outcomes for GBM patients. This study goes deeper into the intricate interplay between the F-actin cytoskeleton and the acquisition of resistance in GBM cells. Our primary goal was to explore the effects of pharmacological or genetic F-actin depolymerization on the reversal of acquired resistance to ionizing radiation (IR) and temozolomide (TMZ) in GBM cells, particularly focusing on the underlying mechanisms involving DNA repair, the glioma stem cell (GSC) phenotype, and therapeutic implications. To establish resistant sublines, GBM U87-MG cells underwent cycles of IR and TMZ exposure, revealing a significant increase in IC50 values for TMZ and ID50 values for IR through viability assays. Subsequent characterization of these sublines growth was in both adherent and spheroid cultures uncovering a distinct sphere-forming capacity. Notably, alterations in DNA repair and the Rho GTPase pathway were observed, with evident disorganized F-actin polymerization in resistant cells, especially at the periphery of spheroids. Elevated expression of stemness markers (Nestin, CD133, and CD44) highlighted a GSC-like profile in the resistant cells, a characteristic further accentuated in spheroid cultures. To assess the relevance of the actin cytoskeleton in maintaining the resistant phenotype, cells were treated with Rho-GTPase inhibitor (C3) or actin polymerization inhibitor (Cytochalasin D). Both treatments resulted in reduced TMZ IC50 and IR ID50 values, partially attributed to the negative modulation of DNA repair capacity - observed by alkaline comet and host-cell reactivation assays. Additionally, F-actin disassembly was found to impact the expression of stemness markers, further reinforcing the connection between cytoskeletal dynamics and the GSC phenotype. Overall, our data strongly suggest that F-actin cytoskeleton dynamics plays a pivotal role in the development and maintenance of resistance in GBM. Targeting these cytoskeletal components sees a promising pharmacological strategy for resensitizing recurrent and resistant GBM tumors, not only by diminishing DNA repair capabilities but also by influencing the GSC-like phenotype, thus opening new avenues for therapeutic interventions. Citation Format: Yuli Thamires Magalhães, Fábio Luís Forti. Unraveling the role of F-actin in resistant GBM: Insights into DNA repair, GSC phenotype, and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5611.

Genotoxic and Cytotoxic Activity of Fisetin on Glioblastoma Cells.

Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells. We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis. Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 μM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells. Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials.

Myricetin, a natural inhibitor of CD147, increases sensitivity of cisplatin in ovarian cancer

ABSTRACT Background Ovarian cancer (OC) is the most lethal gynecological tumor, but it currently lacks effective therapeutic targets. CD147, which is overexpressed in OC, plays a crucial role in promoting malignant progression and is associated with poor prognosis in patients. Therefore, CD147 has been identified as a potential therapeutic target. However, there is a limited amount of research on the development of CD147 inhibitors. Methods Surface plasmon resonance (SPR) assay and virtual molecular docking analysis were performed to identify potential natural compounds targeting CD147. The anti‑tumor effects of myricetin were evaluated using various assays, including CCK8, Alkaline comet, immunofluorescence and xenograft mouse models. The underlying mechanism was investigated through western blot analysis and lentivirus short hairpin RNA (LV-shRNA) transfection. Results Myricetin, a flavonoid commonly found in plants, was discovered to be a potent inhibitor of CD147. Our findings demonstrated that myricetin exhibited a strong affinity for CD147 and down-regulated the protein level of CD147 by facilitating its proteasome-dependent degradation. Additionally, we observed synergistic antitumor effects of myricetin and cisplatin both in vivo and in vitro. Mechanistically, myricetin suppressed the expression of FOXM1 and its downstream DNA damage response (DDR) genes E×O1and BRIP1, thereby enhancing the DDR induced by cisplatin. Conclusion Our data demonstrate that myricetin, a natural inhibitor of CD147, may have clinical utility in the treatment of OC due to its ability to increase genomic toxicity when combined with cisplatin.

K2Cr2O7-induced DNA damage in HT1080 cells: Electrochemical signal response mechanism

The existing DNA damage detection technology cannot meet the current detection requirements. It is critical to build new methods and discover novel biomarkers. In this study, alkaline comet and 8-OHDG ELISA assays were used to identify DNA damage in HT-1080 cells exposed to K2Cr2O7, and electrochemical behaviors of HT-1080 cells with DNA damage was studied. With an increase in K2Cr2O7 exposure time, two electrochemical signals from HT-1080 cells at 0.69 and 1.01 V steadily grew before decreasing after reaching their highest values. The electrochemical signal's initial response time and peak time decreased as the concentration of K2Cr2O7 increased. The duration of the high dose group was 0.5 and 1 h, while the low dose group was 1.5 and 6 h. Western blotting analysis revealed that DNA damage increased the expression of proteins involved in catabolism and de novo purine synthesis, particularly de novo purine synthesis. Expressions of PRPP amidotransferase, IMPDH, and ADA were all higher than those of ADSS, XOD, and GDA, which resulted in larger concentrations of hypoxanthine, guanine, and xanthine, and in turn improved electrochemical signaling. These findings suggest that intracellular purine identified by linear scan voltammetry is predicted to evolve as a marker of early DNA damage.

Investigations of the novel checkpoint kinase 1 inhibitor SRA737 in non-small cell lung cancer and colorectal cancer cells of differing tumour protein 53 gene status

Aim: In response to DNA damage the serine/threonine-specific protein kinase checkpoint kinase 1 (CHK1) is activated allowing cells to enter S phase (S) and G2 phase (G2) cell-cycle arrest. CHK1 inhibitors are expected to prevent cells from entering such arrest, thereby enhancing DNA damage-induced cytotoxicity. In contrast, normal cells with intact ataxia–telangiectasia mutated (ATM), CHK2 and tumour suppressor protein 53 (P53) signalling are still able to enter cell-cycle arrest using the functioning G1/S checkpoint, thereby being rescued from enhanced cytotoxicity. The main objective of this work is to investigate the in vitro effects of the novel CHK1 inhibitor SRA737 on pairs of non-small cell lung cancer (NSCLC) and colorectal cancer (CRC) cell lines, all with genetic aberrations rendering them susceptible to replication stress but of differing tumour protein 53 (TP53) gene status, focusing on DNA damage induction and the subsequent effects on cell proliferation and viability. Methods: NSCLC cell lines H23 [TP53 mutant (MUT)] and A549 [TP53 wild-type (WT)] and CRC cell lines HT29 (TP53 MUT) and HCT116 (TP53 WT) were incubated with differing micromolar concentrations of SRA737 for 24 h and then analysed using alkaline comet and phosphorylated H2A.X variant histone (γH2AX)-foci assays to assess mostly DNA single strand break and double strand break damage, respectively. Cell-counting/trypan blue staining was also performed to assess cell proliferation/viability. Results: Clear concentration-dependent increases in comet formation and γH2AX-foci/cell were noted for the TP53 MUT cells with no or lower increases being noted in the corresponding TP53 WT cells. Also, greater anti-proliferative and cell killing effects were noted in the TP53 MUT cells than in the TP53 WT cells. Conclusions: This study’s data suggests that P53 status/functioning is a key factor in determining the sensitivity of NSCLC and CRC cancer cells towards CHK1 inhibition, even in circumstances conducive to high replicative stress.

CEP55 as a promising biomarker and therapeutic target on gallbladder cancer.

Gallbladder cancer (GBC) is a highly malignant biliary tumor with a poor prognosis. As existing therapies for advanced metastatic GBC are rarely effective, there is an urgent need to identify more effective targets for treatment. Hub genes of GBC were identified by bioinformatics analysis and their expression in GBC was analyzed by tissue validation. The biological role of CEP55 in GBC cell and the underlying mechanism of the anticancer effect of CEP55 knockdown were evaluated via CCK8, colony formation assay, EDU staining, flow cytometry, western blot, immunofluorescence, and an alkaline comet assay. We screened out five hub genes of GBC, namely PLK1, CEP55, FANCI, NEK2 and PTTG1. CEP55 is not only overexpressed in the GBC but also correlated with advanced TNM stage, differentiation grade and poorer survival. After CEP55 knockdown, the proliferation of GBC cells was inhibited with cell cycle arrest in G2/M phase and DNA damage. There was a marked increase in the apoptosis of GBC cells in the siCEP55 group. Besides, in vivo, CEP55 inhibition attenuated the growth and promoted apoptosis of GBC cells. Mechanically, the tumor suppressor effect of CEP55 knockdown is associated with dysregulation of the AKT and ERK signaling networks. These data not only demonstrate that CEP55 is identified as a potential independent predictor crucial to the diagnosis and prognosis of gallbladder cancer but also reveal the possibility for CEP55 to be used as a promising target in the treatment of GBC.

Occupational exposure to pesticides: DNA damage in horticulturist from Nativitas, Tlaxcala in Mexico

Mexico is a country where agricultural activity is of great importance, but biomonitoring data are still scarce. With more intensive pesticides use per unit area/surface in horticultural productivity, there is a higher impact on environmental contamination and workers’ health. Considering that exposure to various pesticide and pesticide mixtures represents an additional genotoxic risk, the appropriate characterization of exposure, confounding factors and the risk itself are very much needed. We compared genetic damage in 42 horticulturists and 46 unexposed controls (Nativitas, Tlaxcala) using alkaline comet (whole blood) and micronucleus (MN) test with nuclear abnormalities (NA) (buccal epithelial cells). Workers demonstrated significantly higher levels of damage (TI%=14.02 ± 2.49 vs. 5.37 ± 0.46; MN=10.14 ± 5.15 vs. 2.40 ± 0.20), with more than 90% of them not using protective clothing nor gloves during application. Combined DNA damage techniques and periodic monitoring together with educational programs for safe pesticide application is the best strategy to assess and prevent workers’ health risks.

Genome stability‑related lncRNA ZFPM2‑AS1 promotes tumor progression via miR‑3065‑5p/XRCC4 in hepatocellular carcinoma.

Long noncoding RNAs (lncRNAs) have a certain link to genomic stability (GS). However, the regulatory relationship of lncRNAs and GS has not been thoroughly investigated in hepatocellular carcinoma (HCC). In the present study, samples were retrieved from The Cancer Genome Atlas with somatic mutations and lncRNA expression data. Cox regression analysis was used to identify independent prognostic factors. The RNA levels were determined by reverse transcription‑quantitative PCR and protein levels were detected by western blot analysis. Cell Counting Kit‑8 and colony‑formation assays were used to assess cell viability. Cell migration was measured by wound‑healing and Transwell assays. Cell apoptosis and cell‑cycle progression were evaluated by flow cytometry. GS was detected by alkaline comet and chromosomal aberration assays. A xenograft model and lung metastasis model were used to assess the role of zinc finger protein, FOG family member 2 antisense 1 (ZFPM2‑AS1) in tumor growth invivo. The molecular mechanisms underlying the biological functions of ZFPM2‑AS1 were investigated through bioinformatics prediction, RNA pull‑down and luciferase reporter assays. A total of 85 genomic instability‑related lncRNAs were identified and a prognostic model was developed. The prognostic model exhibited good predictive power (area under the receiver operating characteristic curve, 0.786). ZFPM2‑AS1 was significantly upregulated in tumor tissues (P<0.001) and it promoted DNA damage repair (P<0.01) and tumor progression invitro and invivo. Luciferase reporter assays demonstrated that miR‑3065‑5p was able to bind directly with ZFPM2‑AS1 and X‑ray repair cross complementing 4 (XRCC4). ZFPM2‑AS1 upregulated XRCC4 expression by acting as a sponge (P<0.001). In the present study, a prognostic model for HCC was developed and validated, and one lncRNA of its components was experimentally investigated. ZFPM2‑AS1 regulates XRCC4 by sponging miR‑3065‑5p to promote GS and HCC progression.

EMS-induced cellular DNA damage detection by electrochemical method: A new biomarker of early DNA damage

It is difficult to screen the increasing numerous chemicals in the environment for DNA damage by traditional methods. Therefore, it is urgent to find new markers and establish simple, rapid and low-cost new DNA damage detection methods. In this paper, the electrochemical behavior of Ethyl methanesulfonate (EMS)-induced DNA damage in HT-1080 cells was investigated, and the two electrochemical signals of HT-1080 cells at 0.69 and 1.03 V, respectively, coming from mixture of guanine (G) and xanthine (X) and hypoxanthine (HX) displayed parabolic changes, showing a time- and dose-dependent increase within 4 h and 5 mM, which was completely different from the trend of cell proliferation. The time- and dose-dependent DNA damage of cells exposed to EMS was detected by both alkaline comet and micronucleus assay. The HPLC found that the levels of HX, G and X in HT-1080 cells quickly rose to the peak values within 4 h, and felt back. The changes of intracellular purine levels were reliable biomarkers of DNA damage, and monitoring the content changes of intracellular purines by electrochemical method could simply and quickly evaluate the DNA damage toxicity of chemicals. This study provided a new perspective for the evaluation of DNA damage.

Determination of double- and single-stranded DNA breaks in bovine sperm is predictive of their fertilizing capacity

BackgroundThe analysis of chromatin integrity has become an important determinant of sperm quality. In frozen-thawed bovine sperm, neither the sequence of post-thaw injury events nor the dynamics of different types of sperm DNA breaks are well understood. The aim of the present work was to describe such sperm degradation aftermath focusing on DNA damage dynamics, and to assess if this parameter can predict pregnancy rates in cattle.ResultsA total of 75 cryopreserved ejaculates from 25 Holstein bulls were evaluated at two post-thawing periods (0-2 h and 2-4 h), analyzing global and double-stranded DNA damage through alkaline and neutral Comet assays, chromatin deprotamination and decondensation, sperm motility, viability, acrosomal status, and intracellular levels of total ROS, superoxides and calcium. Insemination of 59,605 females was conducted using sperm from the same bulls, thus obtaining the non-return to estrus rates after 90 d (NRR). Results showed an increased rate of double-stranded breaks in the first period (0-2 h: 1.29 ± 1.01%/h vs. 2-4 h: 0.13 ± 1.37%/h; P < 0.01), whereas the rate of sperm with moderate + high single-stranded breaks was higher in the second period (0-2 h: 3.52 ± 7.77 %/h vs. 2-4h: 21.06 ± 11.69 %/h; P < 0.0001). Regarding sperm physiology, viability decrease rate was different between the two periods (0-2 h: − 4.49 ± 1.79%/h vs. 2-4 h: − 2.50 ± 3.39%/h; P = 0.032), but the progressive motility decrease rate was constant throughout post-thawing incubation (0-2 h: − 4.70 ± 3.42%/h vs. 2-4 h: − 1.89 ± 2.97%/h; P > 0.05). Finally, whereas no correlations between bull fertility and any dynamic parameter were found, there were correlations between the NRR and the basal percentage of highly-damaged sperm assessed with the alkaline Comet (Rs = − 0.563, P = 0.003), between NRR and basal progressive motility (Rs = 0.511, P = 0.009), and between NRR and sperm with high ROS at 4 h post-thaw (Rs = 0.564, P = 0.003).ConclusionThe statistically significant correlations found between intracellular ROS, sperm viability, sperm motility, DNA damage and chromatin deprotamination suggested a sequence of events all driven by oxidative stress, where viability and motility would be affected first and sperm chromatin would be altered at a later stage, thus suggesting that bovine sperm should be used for fertilization within 2 h post-thaw. Fertility correlations supported that the assessment of global DNA damage through the Comet assay may help predict bull fertility.

Characterization, antioxidant, ACE inhibition and toxicity evaluations of palm kernel cake-derived Alcalase® hydrolysate

Palm (Elaeis guineensis Jacquin) kernel cake protein (PKCP) was extracted and hydrolyzed using Alcalase® 2.4L to obtain hydrolysate (PKCPH), then fractionated using size exclusion chromatography. PKCPH consisted of predominantly fraction one (PKCPH1), containing two peptides (4.9 kDa and 6.3 kDa) with functional amide (6.75–7.04 ppm) and amine (1.5–2.0 ppm) groups. PKCP and PKCPH shared similar amino acid profiles. PKCPH1 had moderate amounts of hydrophobic (23.60%) and antioxidant (26.10%) amino acids, with high hydrophobicity index (Ho 79.60), thus exhibiting the highest antioxidant activity in mostly all the antioxidant assays. On the other hand, fraction two, PKCPH2 (58.8 kDa), possessed strong angiotensin converting enzyme (ACE) inhibitory activity (77.29%), but undetectable antioxidant activity. Furthermore, high viability (87–92%) and negligible cytotoxic activity were revealed with the highest dosages of PKCPH (2 mg/mL) and PKCPH1 (1 mg/mL) in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Salmonella reverse mutation (AMES), alkaline comet and micronucleus (MNi) assays. Besides, in vivo acute oral toxicity test on PKCPH using Sprague Dawley albino rats showed negative outcome according to the consistent body and organ weights as well as normal morbidity. In short, PKCPH is safe for potential applications in the formulation of functional food and nutraceutical products with health benefits.

Increase of Dna Fragmentation Evaluated Through The Alkaline Comet Is Concomitant With A Decrease In The Quality of Frozen-Thawed Bovine Sperm

Increase of Dna Fragmentation Evaluated Through The Alkaline Comet Is Concomitant With A Decrease In The Quality of Frozen-Thawed Bovine Sperm

Genotoxic impact of aluminum-containing nanomaterials in human intestinal and hepatic cells

Exposure of consumers to aluminum-containing nanomaterials (Al NMs) is an area of concern for public health agencies. As the available data on the genotoxicity of Al2O3 and Al0 NMs are inconclusive or rare, the present study investigated their in vitro genotoxic potential in intestinal and liver cell models, and compared with the ionic form AlCl3. Intestinal Caco-2 and hepatic HepaRG cells were exposed to Al0 and Al2O3 NMs (0.03 to 80 μg/cm2). Cytotoxicity, oxidative stress and apoptosis were measured using High Content Analysis. Genotoxicity was investigated through γH2AX labelling, the alkaline comet and micronucleus assays. Moreover, oxidative DNA damage and carcinogenic properties were assessed using the Fpg-modified comet assay and the cell transforming assay in Bhas 42 cells respectively.The three forms of Al did not induce chromosomal damage. However, although no production of oxidative stress was detected, Al2O3 NMs induced oxidative DNA damage in Caco-2 cells but not likely related to ion release in the cell media. Considerable DNA damage was observed with Al0 NMs in both cell lines in the comet assay, likely due to interference with these NMs. No genotoxic effects were observed with AlCl3. None of the Al compounds induced cytotoxicity, apoptosis, γH2AX or cell transformation.

Direct but Not Indirect Methods Correlate the Percentages of Sperm With Altered Chromatin to the Intensity of Chromatin Damage.

Although sperm chromatin damage, understood as damage to DNA or affectations in sperm protamination, has been proposed as a biomarker for sperm quality in both humans and livestock, the low incidence found in some animals raises concerns about its potential value. In this context, as separate methods measure different facets of chromatin damage, their comparison is of vital importance. This work aims at analyzing eight techniques assessing chromatin damage in pig sperm. With this purpose, cryopreserved sperm samples from 16 boars were evaluated through the following assays: TUNEL, TUNEL with decondensation, SCSA, alkaline and neutral sperm chromatin dispersion (SCD) tests, alkaline and neutral Comet assays, and chromomycin A3 test (CMA3). In all cases, the extent of chromatin damage and the percentage of sperm with fragmented DNA were determined. The degree of chromatin damage and the percentage of sperm with fragmented DNA were significantly correlated (p < 0.05) in direct methods (TUNEL, TUNEL with decondensation, and alkaline and neutral Comet) and CMA3, but not in the indirect ones (SCD and SCSA). Percentages of sperm with fragmented DNA determined by alkaline Comet were significantly (p < 0.05) correlated with TUNEL following decondensation and CMA3; those determined by neutral Comet were correlated with the percentage of High DNA Stainability (SCSA); those determined by SCSA were correlated with neutral and alkaline SCD; and those determined by neutral SCD were correlated with alkaline SCD. While, in pigs, percentages of sperm with fragmented DNA are directly related to the extent of chromatin damage when direct methods are used, this is not the case for indirect techniques. Thus, the results obtained herein differ from those reported for humans in which TUNEL, SCSA, alkaline SCD, and alkaline Comet were found to be correlated. These findings may shed some light on the interpretation of these tests and provide some clues for the standardization of chromatin damage methods.

P–004 Effect of varicocelectomy on sperm DNA fragmentation rates in infertile men with clinical varicocele: a systematic review and meta-analysis

Abstract Study question Does varicocelectomy improve sperm DNA quality in men with infertility and clinically detected varicoceles? Summary answer Varicocelectomy reduces sperm DNA fragmentation (SDF) rates in infertile men with clinical varicocele. What is known already Varicocele has been linked to male infertility through various non-mutually exclusive mechanisms, including an increase in reactive oxygen species (ROS) production that may lead to sperm DNA damage. Damage to sperm DNA may result in longer time-to-pregnancy, unexplained infertility, recurrent pregnancy loss, and failed intrauterine insemination or in vitro fertilization/intracytoplasmic sperm injection. Therefore, interventions aimed at decreasing SDF rates, including varicocele repair, have been explored to improve fertility and pregnancy outcomes potentially, either by natural conception or using medically assisted reproduction. Study design, size, duration Systematic review and meta-analysis Participants/materials, setting, methods We followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Our systematic search included PubMed/Medline, EMBASE, Scielo, and Google Scholar to identify all relevant studies written in English and published from inception until October 2020. Inclusion criteria were studies comparing SDF rates before and after varicocelectomy in infertile men with clinical varicocele. Articles were included if the following SDF assays were utilized: SCSA, TUNEL, SCD test, or alkaline Comet. Main results and the role of chance Thirteen studies fulfilled the inclusion criteria and were selected for the analysis. The estimated weighted mean difference of SDF rates after varicocelectomy was –6.58% (13 studies, 95% CI –8.33%, –4.84%; I2=90% p &amp;lt; 0.0001). Subgroup analysis revealed a significant decrease in SDF rates using SCSA (eight studies, WMD –6.80%, 95% CI –9.31%, –4.28%; I2=89%, p &amp;lt; 0.0001), and TUNEL (three studies, WMD –4.86%, 95% CI –7.38%, –2.34%; I2=89%, p &amp;lt; 0.0001). The test for subgroup difference revealed that pooled results were conservative using the above SDF assays. Comet and SCD tests were used in only one study each; thus, a meta-analysis was not applicable. The studies were further categorized by the surgical technique (microsurgical versus non-microsurgical). This subgroup analysis showed a significant decrease in SDF rates using microsurgical technique (10 studies, WMD –6.70%, 95% CI –9.04%, –4.37%; I2=91%, p &amp;lt; 0.0001). After varicocelectomy, SDF rates were also decreased when non-microsurgical approaches were used, albeit the effect was not statistically significant (2 studies, WMD –6.84%, 95% CI –10.05%, 1.38%; I2=86%) (Figure 3). The heterogeneity was not materially affected by performing analyses by the above subgroups, suggesting that the SDF assay and surgical technique do not explain the inconsistency in the treatment effect across primary studies. Limitations, reasons for caution There were no randomized controlled trials comparing varicocelectomy to placebo for alleviating SDF levels. Heterogeneity was high, which may be explained by the low number of included studies. Pregnancy data are not available in most studies, thus the impact of reduced SDF after varicocelectomy on pregnancy rates unclear. Wider implications of the findings: Our study indicates a positive association between varicocelectomy and reduced postoperative SDF rates in men with clinical varicocele and infertility, independentetly of the assays used to measure SDF. These findings may help counsel and manage infertile men with varicocele and high SDF levels. Trial registration number Not applicable

Marine Cyclic Dipeptide Cyclo (L-Leu-L-Pro) Protects Normal Breast Epithelial Cells from tBHP-induced Oxidative Damage by Targeting CD151

Background: Oxidative stress plays a key role in breast carcinogenesis. Cyclo (L-Leu-L-Pro) (CLP) is a homodetic cyclic dipeptide with 2,5-diketopiperazine scaffold isolated from marine actinobacteria. This study aimed to evaluate the protective activity of CLP and linear - (L-Leu-L-Pro) (LP) from tert-butyl hydroperoxide (tBHP)-induced damage using normal breast epithelial cell line model (MCF-12A). Methods: The cytoprotective activity was evaluated by detecting the changes in intracellular ROS, mitochondrial superoxide, hydroxyl radical, hydrogen peroxide, and lipid peroxidation detection assays as well as cytotoxic assays of MTT, LDH assays and phase contrast microscopy. Genoprotective activity was evaluated by (Apurinic/Apyrimidinic) AP site, alkaline Comet, and 8-hydroxy-2-deoxyguanosine assays. Results: The marine cyclic peptide, CLP, significantly protected MCF-12A cells by scavenging tBHP induced intracellular ROS such as super oxide, hydroxyl radicals and hydrogen peroxide, and by reducing the cytotoxicity and genotoxicity effect compared to LP. Moreover, the results showed that CD151 gene silencing by shRNA significantly reduced the overexpression of CD151, tBHP-induced ROS generation, cytotoxicity and genotoxicity in MCF-12A cells. The overexpression of CD151 caused increased levels of cytochrome P450, but was reduced following the application of CD151shRNA and CLP which led to elevated levels of intracellular ROS. Conclusion: In the present study we noticed that CD151 gene silencing by shRNA and treatment with CLP have similar effects on reducing the intracellular ROS. This study uncovers the protective activity of CLP against a CD151-mediated oxidative stress-induced cellular damage. Our observations suggest that the anti-stress and anti-inflammation properties of CLP might have implications in cancer and are worth testing in cancer cell lines and tumor cells.

Complete Chromatin Decondensation of Pig Sperm Is Required to Analyze Sperm DNA Breaks With the Comet Assay.

Sperm quality is usually evaluated prior to artificial insemination in farm animals. In addition to conventional semen analysis, other biomarkers, such as mitochondrial activity, integrity and lipid disorder of plasma membrane, generation of reactive oxygen species (ROS) and sperm DNA integrity, have been found to be related to fertility rates in different species. While mounting evidence indicates that the Comet assay is a sensitive method for the detection of DNA breaks, complete sperm chromatin decondensation is required in order to properly analyze the presence of single- and double-strand DNA breaks. In this sense, a previous study showed that longer lysis treatment with proteinase K is needed to achieve complete chromatin decondensation. The current work sought to determine which specific lysis treatment leads to complete chromatin decondensation in pig sperm, as this is needed for the measurement of DNA damage in this species. With this purpose, incubation with a lysis solution containing proteinase K for 0, 30, and 180 min was added to the conventional protocol. The impact of the DNA damage induced by hydrogen peroxide (H2O2; 0.01 and 0.1%) and DNAse I (1U and 4U) was also evaluated. Complete chromatin decondensation was only achieved when a long additional lysis treatment (180 min) was included. Furthermore, olive tail moment (OTM) and percentage of tail DNA (TD) indicated that a higher amount of DNA breaks was detected when hydrogen peroxide and DNAse I treatments were applied (P < 0.05). The comparison of treated and control sperm allowed defining the thresholds for OTM; these thresholds revealed that the percentage of sperm with fragmented DNA determined by the alkaline Comet does not depend on chromatin decondensation (P > 0.05). In conclusion, complete chromatin decondensation prior to alkaline and neutral Comet assays is needed to analyze DNA breaks in pig sperm.

Evaluating the radioprotective effect of single dose and daily oral consumption of green tea, grape seed, and coffee bean extracts against gamma irradiation

IntroductionThe aim of this study was to investigate and compare the radio-protective effect of green tea, grape seed, and coffee bean extracts in different oral consumption methods in mice. Materials and methodsIn this experimental-quantitative study 150 mice in 15 equally sized groups were used. For each extract, two groups received 200 mg/kg of herbal extracts’ combination for 7 and 30 consecutive days before irradiation, and one group received 800 mg/kg of the extract 2 h before irradiation (3 Gy gamma-rays of Co-60). The similar groups were classified to receive a combination of the plant extracts (green tea, grape seed, and coffee bean). Irradiation without consuming plant extract (irradiated group), and a control group were also devised. Alkaline comet and micronucleus assays were used to investigate the radioprotective effect on mice blood and bone marrow cells, respectively. ResultsConsumption of all plant extracts significantly decreased the radiation damage to blood and bone marrow cells, compared to the irradiated group (p < 0.01), with grape seed extract showing higher protective effect. Continuous daily oral consumption (one week/month) showed a significant higher radioprotective effect compared to single consumption (p < 0.05). Continuous consumption of the combination of the extracts showed a higher radio-protection in comparison to each of the plant extracts (p < 0.03). ConclusionsThe radioprotective effect of continuous consumption (for one week/month) of the plant extracts was greater than single dose. In continuous consumption protocols, we found the synergetic property and higher radioprotective effect of the plant extract combination compared to each one.

Sperm selection during ICSI treatments reduces single- but not double-strand DNA break values compared to the semen sample.

To detect a possible bias in sperm DNA fragmentation (SDF) testing when performed on semen samples or on those few spermatozoa selected for Intracytoplasmic Sperm Injection (ICSI) treatments. A multimethodological analysis of Single- and Double-Strand DNA Breaks (SSB and DSB, respectively) was performed through the Neutral Comet, the Alkaline Comet, the Sperm Chromatin Dispersion (SCD) and the Terminal deoxynucleotidyl transferase dUTP Nick End Labelling (TUNEL) assays. SDF was evaluated in (i) semen samples from 23 infertile patients (not achieving pregnancy or suffering recurrent miscarriage); (ii) samples after a Swim-up and (iii) spermatozoa microselected for ICSI (ICSI-S). The analysis of 3217 ICSI-S revealed a significant reduction of SSB values compared to the Ejaculate and the Swim-up samples. On the contrary, DSB values were not reduced after any sperm selection method. The No-pregnancy group presented poorer semen parameters and higher SSB values. The Recurrent miscarriage group presented better semen parameters but also higher DSB values. The analysis of SDF on semen samples may not be fully representative of those few spermatozoa selected for ICSI. Since oxidative stress impairs sperm motility and causes SSB, selecting a motile sperm may intrinsically imply choosing a sperm not affected by this damage. DSB have an enzymatic origin which does not affect motility, making it difficult to select a sperm without this damage. Therefore, ICSI treatments could be effective in patients presenting high SSB values. Patients presenting high DSB values should expect bad ICSI results if this damage is not reduced through other specific methods.