Nuclear DNA Fragmentation Research Articles

The WRKY10-VQ8 module safely and effectively regulates rice thermotolerance.

WRKY transcription factors (TFs) play crucial roles in biotic and abiotic stress responses. However, their roles in thermal response are still largely elusive, especially in rice. In this study, we revealed the functions of WRKY10 TF and VQ8 protein containing VQ motif in rice thermotolerance. Overexpression of WRKY10 or loss of VQ8 function increases thermosensitivity, whereas conversely, overexpression of VQ8 or loss of WRKY10 function enhances thermotolerance. Overexpression of WRKY10 accelerates reactive oxygen species (ROS) accumulation in chloroplasts and apoplasts, and it also induces the expression of heat shock TF and protein genes. We also found that WRKY10 regulates nuclear DNA fragmentation and hypersensitive response by modulating NAC4 TF expression. The balance between destructive and protective responses in WRKY10-overexpression plant is more fragile and more easily broken by heat stress compared with wild type. In vitro and in vivo assays revealed that VQ8 interacts with WRKY10 and inhibits the transcription activity via repressing its DNA-binding activity. Our study demonstrates that WRKY10 negatively regulates thermotolerance by modulating the ROS balance and the hypersensitive response and that VQ8 functions antagonistically to positively regulate thermotolerance. The functional module of WRKY10-VQ8 provides safe and effective regulatory mechanisms in the heat stress response.

Alleviation of Endoplasmic Reticulum Stress Enhances Human Corneal Epithelial Cell Viability under Hyperosmotic Conditions.

Tear hyperosmolarity plays an essential role in the initiation and progression of dry-eye disease. Under a hyperosmotic environment, corneal epithelial cells experience perturbations in endoplasmic reticulum function that can lead to proinflammatory signaling and apoptosis. In this study, we investigated the effect of tauroursodeoxycholic acid (TUDCA), a chemical chaperone known to protect against endoplasmic reticulum stress, on corneal epithelial cells exposed to hyperosmotic conditions. We found that the expression of the genes involved in the activation of the unfolded protein response and the pro-apoptotic transcription factor DDIT3 were markedly upregulated in patients with Sjögren’s dry-eye disease and in a human model of corneal epithelial differentiation following treatment with hyperosmotic saline. Experiments in vitro demonstrated that TUDCA prevented hyperosmotically induced cell death by reducing nuclear DNA fragmentation and caspase-3 activation. TUDCA supplementation also led to the transcriptional repression of CXCL8 and IL5, two inflammatory mediators associated with dry-eye pathogenesis. These studies highlight the role of hyperosmotic conditions in promoting endoplasmic reticulum stress in the cornea and identify TUDCA as a potential therapeutic agent for the treatment of dry-eye disease.

Metabolic syndrome and losartan treatment effects in adult and pubertal rats

Introduction: Comparative estimation of losartan treatment effects in adult and pubertal rats with metabolic syndrome (MS). Material and methods: MS model was induced by full replacement of drinking water with 20% fructose solution at Wistar male rats of two age categories: young animals of 21–23 days age (50–70 g) and adults (160–180 g). Clinical hematology and biochemistry tests, blood pressure measurement, chromatin DNA fragmentation, and liver morphological investigations were carried out after 60 days of MS modeling and losartan treatment. Results: Effects of losartan on blood clotting time, lipid metabolism, and DNA fragmentation were more pronounced in pubertal rats, while more profound influence on high-density lipoprotein (HDL) contents, pancreas, and visceral fat relative weights was found in adults. Conclusions: In pubertal and adult rats with MS, losartan effects were age-dependent for lipid metabolism indices, blood clotting time, nuclear DNA fragmentation, relative organs weights, and liver morphologic structure. Losartan treatment normalized blood pressure independently of age, while its effects on other parameters in adults and young rats differed not only in their degree of manifestation but also in their very nature.

Apoptosis inducing potential of Cyclosporine C in human lung cancer cells (A549)

Cyclotides are a large group of plant based cyclic peptides that have attracted attention in the field of peptide drug design. Cyclic peptides exhibit a more potent biological activity when compared to linear peptides due to their stable cyclic structure. Cyclosporine C is a naturally occurring lipophilic cyclic peptide which is used as an immunosuppressant drug. The objective of this study was to establish Cyclosporine C as a viable novel anticancer drug in the treatment of lung cancer and to find the optimal concentration at which Cyclosporine C results in a significant decrease of viable cancer cells to determine the physiological changes caused by Cyclosporine C and also to find the pathway of apoptosis that is induced by Cyclosporine C. The cytotoxic effect of Cyclosporine C was assessed using techniques such as MTT, Hoechst staining, DNA fragmentation, Flowcytometry and Western blot which were carried out on Lung adenocarcinoma cell line A549. The results obtained showed a significant decrease in A549 viable cells at a concentration of 200μg/mL of Cyclosporine C as observed by the MTT assay. The nuclear condensation and DNA fragmentation of apoptotic cells were observed using Hoechst 33258 staining and DNA fragmentation assays respectively. Western blot confirms that Cyclosporine C treated cells expressed a significant level of caspase-9 enzyme. From the results obtained, we can conclude that Cyclosporine C induces apoptosis in Lung adenocarcinoma cell line A549. The apoptosis is induced by caspace-9 enzyme dependent pathway. The antitumor activity of Cyclosporine C suggests that it has potential as a novel anticancer drug for human lung cancer treatment.

Mapping the time dependent DNA fragmentation caused by doxorubicin loaded on PEGylated carbogenic nanodots using fluorescence lifetime imaging and superresolution microscopy.

Doxorubicin is an anthracycline drug most commonly used in cancer therapy. It intercalates with the nuclear DNA and induces toxicity by causing DNA breaks and histone eviction. However, the kinetics of its action on the nucleus has not been mapped effectively. This study shows successful PEGylation and DOX loading through π-π interaction onto carbogenic fluorescent nanodots (FNDs), which have an affinity for the nucleolus. Then the drug release from the nanoparticle and its action on the nuclear environment were aptly mapped using both fluorescence lifetime imaging and superresolution radial fluctuation (SRRF) techniques. Here for the first time, the nuclear degradation kinetics caused by the released DOX from the FNDs as a result of DNA double-strand breaks and histone eviction was visualized. This led to the observation of decreasing length, breadth, and complex structure of the nuclear clusters from 6 h to 24 h, resulting in isolated cluster visualization. However, the superresolution images for free DOX and untreated cells reveal no such drastic effects at the same concentration and time points, unlike DOX loaded particles.

Influence of storage at 5°C and vitrification on apoptotic changes in equine blastocysts

The aim of the study was to determine the extent of nuclear DNA fragmentation in equine embryos at the blastocyst stage stored at 5° C for 6 and 24 h or vitrified in straws or in the Rapid-i system. An additional aim was to determine the relationship between the age of fresh and stored embryos and the intensity of apoptosis. In this study, 42 embryos were analyzed by the TUNEL method and allocated to the following groups: fresh (control -14 embryos), stored at 5° C for 6 h (7 embryos), stored at 5° C for 24 h (7 embryos), vitrified in straws (7 embryos), and vitrified in the ultra-fast Rapid-i system (7 embryos). It was found that 36.7% of fresh equine embryos had apoptotic nuclei, and the mean dead cell index (DCI) was 0.3%. This was evident as an increase in DNA fragmentation in equine embryos after vitrification in straws (2.3%) compared to fresh embryos (0.3%) and those vitrified in the Rapid-i system (0.3%). Embryo storage at 5° C did not affect the apoptosis frequency in nuclei regardless of the time of storage. DCI for embryos stored for 6 h was 0.44%, and after 24 h of incubation it was 0.64%. The intensity of apoptotic changes observed in equine embryos depended on their age and size. The apoptosis frequency was negatively correlated with the age and diameter of fresh embryos, embryos stored at 5° C, and embryos vitrified in Rapid-I, whereas for embryos vitrified in straws the correlation was positive.

Abstract 10926: Importance of Mitochondrial DNA Damage in Cigarette Smoke Extract-Induced Activation of Innate Immune System

Objective: Smoking is a well-known risk factor for atherosclerosis, yet the mechanism by which smoking causes atherosclerosis remains unclear. It has been reported that inflammation and DNA damage are involved in the formation and development of atherosclerosis. We have previously reported that nuclear DNA damage was increased in peripheral mononuclear cells of smokers compared to those of non-smokers, and that DNA damage was accumulated in atherosclerotic lesions. In this study, to clarify the mechanism by which smoking induces inflammation in vascular endothelial cells, we investigated the effect of cigarette smoke extract (CSE) on both nuclear and mitochondrial DNA damage and the subsequent cellular response in human umbilical vein endothelial cells (HUVEC). Methods and Results: CSE increased double-strand breaks in HUVEC. Notably, this increase was detected relatively late (after 3 days of CSE exposure) compared to other genotoxic agents. CSE also increased oxidative DNA damage both in the nucleus and mitochondria and induced mitochondrial dysfunction. Mitochondrial dysfunction decreased cytosolic protein levels of ICAD, an inhibitor of caspase-activated DNase (CAD), which causes nuclear DNA fragmentation, and increased CAD in the nucleus, suggesting that this is one of the mechanisms of nuclear DNA double-strand breaks. In addition, continuous stimulation by CSE induced the accumulation of cytosolic DNA. Interestingly, real-time PCR analysis revealed that the accumulated DNA in the cytosol was derived not only from the nucleus but also from mitochondria. We further examined whether DNA accumulated in the cytosol activated cytosolic DNA-sensing pathways. The production of cGAMP, a second messenger in cGAS (a DNA-sensing receptor) signaling, was increased by CSE. We also found that the mRNA expression of IL-6 was increased by CSE, and that the increase was suppressed by si-cGAS. Conclusions: This study showed that continuous exposure to CSE induces not only nuclear but also mitochondrial DNA damage, which leads to cytosolic DNA accumulation, and evokes chronic inflammation via the cGAS-STING pathway.

Turnover of diacylglycerol kinase 4 by cytoplasmic acidification induces vacuole morphological change and nuclear DNA degradation in the early stage of pear self-incompatibility response.

Pear has an S-RNase-based gametophytic self-incompatibility (SI) system. Nuclear DNA degradation is a typical feature of incompatible pollen tube death, and is among the many physiological functions of vacuoles. However, the specific changes that occur in vacuoles, as well as the associated regulatory mechanism in pear SI, are currently unclear. Although research in tobacco has shown that decreased activity of diacylglycerol kinase (DGK) results in the morphological change of pollen tube vacuole, whether DGK regulates the pollen tube vacuole of tree plants and whether it occurs in SI response, is currently unclear. We found that DGK activity is essential for pear pollen tube growth, and DGK4 regulates pollen tube vacuole morphology following its high expression and deposition at the tip and shank edge of the pollen tube of pear. Specifically, incompatible S-RNase may induce cytoplasmic acidification of the pollen tube by inhibiting V-ATPase V0 domain a1 subunit gene expression as early as 30 min after treatment, when the pollen tube is still alive. Cytoplasmic acidification induced by incompatible S-RNase results in reduced DGK4 abundance and deposition, leading to morphological change of the vacuole and fragmentation of nuclear DNA, which indicates that DGK4 is a key factor in pear SI response.

Zerumbone mediates apoptosis and induces secretion of proinflammatory cytokines in breast carcinoma cell culture.

Objective(s):To investigate the potential anti-breast cancer activity of zerumbone in regulating apoptotic mediators and cytokines in comparison with paclitaxel (positive control). Materials and Methods: In this study, assays such as viability, apoptosis, reactive oxygen species, cell cycle, DNA fragmentation, and cytokines were carried out on MCF-7 cells after treatment with zerumbone and paclitaxel.Results:The results showed that zerumbone demonstrated a higher (18-fold) IC50 value (126.7 µg/ml) than paclitaxel (7.29 µg/ml) in order to suppress proliferation and induce cell death of MCF-7. The cell cycle arrest at the G0/G1 phase and excessive intracellular ROS production during the in vitro zerumbone treatment indicated occurrence of apoptotic cell death although nuclear DNA fragmentation was not observed. The flow cytometer analysis of treated cells revealed secretion of proinflammatory cytokines suggesting the potential immunomodulatory activity of zerumbone. Conclusion:Although, zerumbone exhibited a higher IC50 value compared with paclitaxel yet its anticancer activity against MCF-7 cells is still parallel to paclitaxel hence zerumbone has the potential to be an antineoplastic agent in the treatment of breast cancer especially the luminal type A.

Cytotoxic activity of Indigofera aspalathoides (Vahl.) extracts in cervical cancer (HeLa) cells: Ascorbic acid adjuvant treatment enhances the activity

The folkloric use of Indigofera aspalathoides (Vahl) in anti-inflammatory and anti-cancer treatment dates back to circa 300 BC. However, reports on scientific validation for folkloric usage are not available yet. The objective of the study is to assess the cytotoxic potential of I. aspalathoides and to provide a rationale for its ethnopharmacological use with lime juice (ascorbic acid) as an adjuvant. Using Soxhlet, the active principles of I. aspalathoides were extracted using methanol and ethanol as solvent and studied using gas chromatography coupled with mass spectrometry (GC–MS). We assessed the in vitro antioxidant efficacy by studying the DPPH radical scavenging activity, ferrous reducing antioxidant capacity (FRAC), and ABTS cation radical scavenging activity. Using MTT assay, the cytotoxic activity of SV-ME and EE against human cervical cancer (HeLa) and normal murine fibroblast (L929) cells were evaluated. The cytotoxicity and apoptosis induction was studied by dual acridine orange/ethidium bromide (AO/EB) fluorescent probe and Hoechst dye. Mitochondria membrane potential (Δψm) was assessed using JC1. Ascorbic acid (AA) additive/synergism was studied by co-treating HeLa cells with AA along with SV-ME and SV-EE at IC50 concentration. SV-ME and EE exert higher DPPH radical scavenging at 200 μg/mL. Both the extracts showed effective ABTS radical cation scavenging activity and ferrous reducing antioxidant capacity. Further, SV-ME and SV-EE treatment affected the cell viability and induced apoptosis, reduced cell viability with a concomitant increase in nuclear condensation and DNA fragmentation in a dose-dependent manner in cancer (HeLa) cells, but not in normal (L929) cells. Both the extracts increased the mitochondria membrane potential (Δψm) depolarization and altered the cellular redox state in HeLa cells. In addition, adjuvant ascorbic acid (AA) treatment increased the cytotoxic efficacy of the SV-ME and SV-EE in HeLa cells. I. aspalathoides used in the Siddha medicine for antitumor treatment have a very potent cytotoxic effect against cervical cancer cells (HeLa) in vitro. The cytotoxic and antitumor effect may have plausible involvement of Nrf2-ARE signaling pathway by restoration of cellular redox homeostasis.

APOPTOSIS INDUCTION OF ETHANOLIC EXTRACT OF AMOMUM SUBULATUM ROXB. IN HELA CELL LINES

Deficiency in apoptosis is one of the key factors that plays a pivotal role in cancer cell growth and proliferation. A procedure used in the treatment of cancer is the triggering of apoptosis in cancer cells. The current study aims to investigate the anticancer property of ethanolic extract of Amomum subulatum Roxb. against HeLa cell line. The MTT 3-(4, 5-di-methylthiazol-2-yl)-2,5-diphenyl-2H tetrazolium bromide assay is the quantitative cytotoxicity assay used, maximal inhibitory concentration (IC50) value was selected as the cytotoxicity parameter. The IC50 value of A. subulatum Roxb. extract is 62.5 μg/ mL and for 5-fluorouracil it is 7.8 μg/mL which indicates anticarcinogenic properties against HeLa cells. The apoptotic morphological changes in HeLa cells were observed using an inverted microscope and changes in HeLa cells were noticed after treatment with 62.5 mg/mL of ethanolic extracts, followed by acridine orange and ethidium bromide staining. The induction of apoptosis by extract of A. subulatum Roxb. was determined using the DNA fragmentation study. The results of the DNA fragmentation study, which exhibits prototypical intrinsic apoptotic characterization, also included fragmentation of nuclear DNA. We also found that the expression of Bcl2 and p53 mRNA were measured using RT-PCR. Overall, the current study results suggest that the

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

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

Simultaneous detection of sperm membrane integrity and DNA fragmentation by flow cytometry: A novel and rapid tool for sperm analysis.

Sperm DNA integrity has become one of the most discussed and promising biomarkers for the assessment of male fertility. However, an easy-to-apply method capable of estimating DNA fragmentation in the live fraction of spermatozoa has remained elusive, preventing this parameter from being fully applied in clinical settings. To validate a novel co-staining for the analysis of DNA fragmentation in membrane-intact spermatozoa. Normozoospermic semen samples were used to validate the co-staining consisting of acridine orange (AO) and LIVE/DEAD™ Fixable Blue Dead Cell Stain (LD), against established methods for the evaluation of cell viability, propidium iodide stain (PI), and DNA fragmentation, the sperm chromatin structure assay (SCSA), to rule out cross-interference. Furthermore, the accuracy of the method was tested by the evaluation of samples prepared with different amounts of membrane and DNA damage (20, 40, 60, 80, and 100%). No significant differences were observed between the co-staining and the established staining procedures (membrane integrity, p=0.755; DNA fragmentation p=0.976). Moreover, high R square values were obtained from the analysis of samples of known membrane (R2 =0.9959) and DNA damage (R2 =0.9843). The simultaneous assaying of sperm membrane integrity and nuclear DNA fragmentation allowed the analysis of four sperm categories and thereby to assess the proportion of membrane-intact spermatozoa with compromised DNA integrity. This new protocol has the potential to provide clinically relevant information about the DNA fragmentation in membrane-intact spermatozoa. Thus, it has the potential of improving the diagnostic of male infertility and enabling a better understanding of sperm dysfunction.

Bioallethrin enhances generation of ROS, damages DNA, impairs the redox system and causes mitochondrial dysfunction in human lymphocytes

Bioallethrin is a synthetic pesticide that is widely used to control insect pests. The wide use of bioallethrin has resulted in inevitable human exposure. In this study we report the effect of different concentrations of bioallethrin (10 to 200 µM, 2 h at 37 °C) on human lymphocytes under in vitro conditions. Bioallethrin treatment resulted in loss of cell viability (> 30% at 200 µM bioallethrin). Oxidative stress markers like lipid peroxidation and protein oxidation were significantly increased accompanied by lower ratio of reduced to oxidized glutathione. Enhanced ROS generation was observed through fluorescence spectroscopy and microscopy. Bioallethrin-induced oxidative stress also compromised the antioxidant defence as it reduced antioxidant capacity of cells and inhibited major antioxidant enzymes. Biomolecular modifications and systemic toxicity by bioallethrin resulted in plasma membrane damage with mitochondrial depolarization. Comet assay showed nuclear DNA fragmentation and strand scission with significant increase in tail length and olive tail moment. Apoptosis and necrosis of cells was confirmed through acridine orange/ethidium bromide dual staining and visualization under fluorescence microscope. Thus, bioallethrin causes oxidative damage and compromises the antioxidant system leading to DNA damage, cellular and organelle toxicity, resulting in apoptosis and necrosis of human lymphocytes.

Increased Levels of ER Stress and Apoptosis in a Sheep Model for Pulmonary Fibrosis Are Alleviated by In Vivo Blockade of the KCa3.1 Ion Channel.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease, characterized by progressive damage to the lung tissues. Apoptosis and endoplasmic reticulum stress (ER stress) in type II alveolar epithelial cells (AECs) and lung macrophages have been linked with the development of IPF. Therefore, apoptosis- and ER stress-targeted therapies have drawn attention as potential avenues for treatment of IPF. The calcium-activated potassium ion channel KCa3.1 has been proposed as a potential therapeutic target for fibrotic diseases including IPF. While KCa3.1 is expressed in AECs and macrophages, its influence on ER stress and apoptosis during the disease process is unclear. We utilized a novel sheep model of pulmonary fibrosis to demonstrate that apoptosis and ER stress occur in type II AECs and macrophages in sheep with bleomycin-induced lung fibrosis. Apoptosis in type II AEC and macrophages was identified using the TUNEL method of tagging fragmented nuclear DNA, while ER stress was characterized by increased expression of GRP-78 ER chaperone proteins. We demonstrated that apoptosis and ER stress in type II AECs and macrophages increased significantly 2 weeks after the final bleomycin infusion and remained high for up to 7 weeks post-bleomycin injury. Senicapoc treatment significantly reduced the rates of ER stress in type II AECs and macrophages that were resident in bleomycin-infused lung segments. There were also significant reductions in the rates of apoptosis of type II AECs and macrophages in the lung segments of senicapoc-treated sheep. In vivo blockade of the KCa3.1 ion channel alleviates the ER stress and apoptosis in type II AECs and macrophages, and this effect potentially contributes to the anti-fibrotic effects of senicapoc.

Chrysin inhibits propagation of HeLa cells by attenuating cell survival and inducing apoptotic pathways.

Chrysin, one of the main active constituents of flavonoids, is known for demonstrating protective effects against various types of cancer including cervical cancer. The aim of this study was to determine apoptosis induction and antiproliferative action of chrysin on human cervical cancer cells. In this study, attempts have been made to establish anticancer role of chrysin on HeLa cells. MTT, mitochondrial potential, DNA fragmentation, annexin V/propidium iodide assays, qPCR and protein profiling were performed. Chrysin treated HeLa cells showed time and dose dependent decrease in cell viability and demonstrated profound effects on nuclear morphology and DNA fragmentation. Chrysin treatment increased the expression of proapoptotic genes BAD, BAX, BID, BOK and APAF1, TNF, FASL, FAS, FADD and caspases (like caspase 3, caspase 7, caspase 8 and caspase 9), whereas it decreased the expression level of antiapoptotic genes MCL-1, NAIP, XIAP and Bcl-2 and cell cycle regulatory genes CCNB1, CCNB2, CCND1, CCND2, CCND3, CCNE2, CDK4 and CDK2 at transcript level. Furthermore, chrysin significantly upregulated pro-apoptotic proteins, like TRAILR2/DR5, TRAILR1/DR4, Fas/TNFRSF6/CD95, phosphoP53(S15), BAD, BAX, cleaved caspase 3, procaspase 3, HTRA2/Omi and SMAC/Diablo, while downregulated anti-apoptotic proteins like BCL-X, BCL2, XIAP and CIAPs that support chrysin mediated apoptosis in HeLa cells. Remarkably, chrysin downregulated the phosphorylated AKT pathway proteins, (p-473) AKT, (p-Ser 2448) mTOR, (p-Ser241) PDK1, (p-Ser112) BAD, and upregulated (p-Ser21) GSK3b, (p-Thr172) AMPKa, P27 (p-Thr198) and (p-Ser15) P53, which endorses chrysin mediated apoptosis. Chrysin significantly inhibited proliferation and induced apoptosis by modulation of various apoptotic genes and AKT/MAPK pathway genes.

Relationship between nuclear DNA fragmentation, mitochondrial DNA damage and standard sperm parameters in spermatozoa of infertile patients with leukocytospermia

The association of leukocytospermia with male fertility is still under debate. Our objective was to evaluate the association of leukocytospermia with sperm parameters, mitochondrial DNA (mtDNA) variations, and seminal concentration of several oxidative stress and inflammatory cytokines in Tunisian infertile men. The studied patients were divided into two groups: patients without leukocytospermia (Group 1) and patients with leukocytospermia (Group 2). DNA fragmentation significantly increased in group 2 (31.41 %) compared to group 1 (14.68 %) ; (p < 0.001). A total of 115 nucleotide substitutions in mitochondrial DNA were depicted, among which 113 were previously identified. The number of substitutions was more elevated in group 2. Leukocytospermic group had significantly higher MDA (nmole/mL) levels than patients without leukocytospermia (34±24.43 vs 18.94±15.96 ; p=0.001), GSH (μg/mL) levels were also higher compared to the control group (126.53±22.87 vs 79.4±19.38 ; p < 0.001), SOD (U/mg of protein) levels were higher but without reaching the statistical significance (89.74±74.85 vs 67.56±37.11 ; p = 0.25) ; whereas seminal CAT (μmole H2O2/min/mg of protein) levels were lower in this group (10.66±14.32 vs 27.35±25.28 ; p = 0.012). No statistically significant differences between the two groups of patients were found in the levels of inflammatory cytokines. However, IL-8 level was positively correlated with DNA fragmentation and negatively correlated with vitality. These findings confirm the association between leukocytospermia and sperm DNA damage.

Seasonal Changes of Nuclear DNA Fragmentation in Boar Spermatozoa in Spain.

Simple SummaryArtificial insemination is widely used in pig production and currently a boar performs several thousand matings per year. Traditionally sperm quality is focused on the number of spermatozoa, their motility and morphology. However, the quality of sperm DNA, which contains genetic information, is also related to fertility problems. The aim of this research was to study the effect of natural light hours and age of the boar on the status of the sperm DNA. After a powerful statistical analysis, it was found that the percentage of spermatozoa with fragmented DNA decreases within the observed age range as the boar gets older. On the other hand, the amount of spermatozoa with fragmented DNA was the lowest in autumn while it was the highest in summer. This study demonstrates the remaining seasonality of boars in Spain and highlights the importance of controlling the environmental conditions in the farms. Sperm DNA testing provides a basis for improving the selection of AI boars by excluding males with higher DNA fragmentation due to their very young reproductive age that may pose a potential subfertility.There are numerous cases when conventional spermiogram parameters are all within an acceptable range but boar subfertility persists. The total sperm nuclear DNA fragmentation index (tDFI) is a trait related to fertility and prolificacy problems that is not routinely evaluated in commercial AI boars. The aim of this research was to study the effect of the photoperiod, season and reproductive age of the boar on tDFI (measured by SCSA) of 1279 ejaculates from 372 different boars belonging to 6 different breeds located in 6 AI studs in Spain. tDFI data ranged from 0.018% to 20.1%. Although there was a significant single boar effect in the tDFI occurrence, a negative correlation between the tDFI and the age of the boar was found (p < 0.001). tDFI would decrease due to aging of the boar 0.66% each year old within the observed age range. After including age as a covariate in the ANCOVA, no differences were found in tDFI between photoperiods when the sperm collection date was evaluated. However, when the date of the production of semen in the testis was evaluated, the total percentage of spermatozoa with fragmented nuclear DNA was 1.46% higher in the increasing photoperiod in comparison to the decreasing photoperiod (p < 0.0001). On the other hand, for both dates, the lowest tDFI values corresponded to minimum day length for decreasing photoperiod phase (autumn), while the highest tDFI values were found in summer (maximum day length for decreasing photoperiod phase).

Biogenic synthesis of gold nanoparticles using Argemone mexicana L. and their cytotoxic and genotoxic effects on human colon cancer cell line (HCT-15)

BackgroundNanomedicine has evolved as precision medicine in novel therapeutic approach of cancer management. The present study investigated the efficacy of biogenic gold nanoparticles synthesized using Argemone mexicana L. aqueous extract (AM-AuNPs) against the human colon cancer cell line, HCT-15. ResultsBiosynthesis of AM-AuNPs was determined by ultraviolet-visible spectroscopy and further characterized by transmission electron microscopy, X-ray diffraction, and Fourier transition infrared spectroscopy analysis. The cytotoxic activity of AM-AuNPs was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, whereas genotoxicity was evaluated by the DNA fragmentation assay. The expression of apoptosis regulatory genes such as p53 and caspase-3 was explored through semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting to evidence apoptotic cell death in HCT-15 cells. Biogenic AM-AuNPs inhibited cell proliferation in HCT-15 cell line with a half maximal inhibitory concentration (IC50) of 20.53 μg/mL at 24 h and 12.03 μg/mL at 48 h of exposure. The altered cell morphology and increased apoptosis due to AM-AuNPs were also evidenced through nuclear DNA fragmentation and upregulated expression of p53 and caspase-3 in HCT-15 cells. ConclusionThe AM-AuNPs may exert antiproliferative and genotoxic effects on HCT-15 cells by cell growth suppression and induction of apoptosis mediated by activation of p53 and caspase-3 genes.

A novel synergistic anticancer effect of fungal cholestanol glucoside and paclitaxel: Apoptosis induced by an intrinsic pathway through ROS generation in cervical cancer cell line (HeLa)

In the search for efficient therapeutics with economically viable for cancer treatment, combination therapy has developed as a keystone in the pursuit of novel approaches for drug discovery. In this regard, we confirmed the presence of cholestanol glucoside (CG) in Lasiodiplodia theobromae culture filtrate and its production was estimated to be 20.01 mg/l. The purified fungal CG was obtained with a molecular mass of 550.18 m/z. The combination of CG and paclitaxel (PTX) was found to have potent cytotoxicity against HeLa cells. We revealed that the synergistic effect of CG and PTX induced apoptosis through the formation of nuclear fragments, DNA fragmentation and sub G1 cell cycle arrest. Further, it was proven that apoptosis took place by loss of the mitochondrial membrane potential (MMP) through reactive oxygen species (ROS) production and caspase 3/7 activity. Moreover, the data suggests that the synergistic effect of CG and PTX played a role in a mitochondrial intrinsic pathway through the apoptotic gene expression of Bax, caspase-9 and caspase-3. In addition, the down-regulation of Bcl-2 strongly described the induced apoptosis through an intrinsic pathway using the Western blot analysis. The conclusion of this study is that a combination of CG and PTX has synergistic apoptotic effects in HeLa cells, which provides a possible therapeutic strategy for cancer therapy in the future.