Nuclear DNA Fragmentation Research Articles

Antineoplastic with DNA fragmentation assay and anti-oxidant, anti-inflammatory with gene expression activity of Lactobacillus plantarum isolated from local Egyptian milk products

Many lactic acid bacteria (LAB), known for their human health benefits, are derived from milk and utilized in biotherapeutic applications or for producing valuable nutraceuticals. However, the specific role of milk-associated LAB in biotherapeutics remains underexplored. To address this, eight milk product samples were randomly selected from the Egyptian market, diluted, and then cultured anaerobically on MRS agar. Subsequently, 16 suspected LAB isolates were recovered and underwent rapid preliminary identification. Among these isolates, the Lactobacillus plantarum strain with accession number (OQ547261.1) was identified due to its strong antioxidant activity depending on the DPPH assay, L. plantarum displayed notable antioxidant activities of 71.8% and 93.8% at concentrations of 125–1000 µg/mL, respectively. While ascorbic acid showed lower concentrations of 7.81, 3.9, and 1.95 µg/mL which showed activities of 45.1%, 34.2%, and 27.2%, respectively. The anti-inflammatory efficacy of L. plantarum was evaluated based on its capability to prevent hemolysis induced by hypotonic conditions. At a concentration of 1000 µg/mL, L. plantarum could reduce hemolysis by 97.7%, nearly matching the 99.5% inhibition rate achieved by the standard drug, indomethacin, at an identical concentration. Moreover, L. plantarum exhibited high hemolytic activity at 100 µg/mL (14.3%), which decreased to 1.4% at 1000 µg/mL. The abundance of phenolic acids and flavonoids was determined by high-performance liquid chromatography (HPLC) in L. plantarum. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) demonstrated that L. plantarum increased gene expression of the inflammatory marker TLR2 by 133%, and cellular oxidation markers SOD1 and SOD2 by 65% and 74.2%, respectively, while suppressing CRP expression by 33.3%. These results underscore L. plantarum’s exceptional anti-inflammatory and antioxidant activities. Furthermore, L. plantarum induces cancer cell death through necrotic nuclear DNA fragmentation. These findings suggest that L. plantarum is not only suitable for nutraceutical production but also holds potential as a probiotic strain. Future research should focus on enhancing the capacity of this strain across various industries and fostering innovation in multiple fields.

Apoptosis, Mitochondrial Autophagy, Fission, and Fusion Maintain Mitochondrial Homeostasis in Mouse Liver Under Tail Suspension Conditions

Microgravity can induce alterations in liver morphology, structure, and function, with mitochondria playing an important role in these changes. Tail suspension (TS) is a well-established model for simulating the effects of microgravity on muscles and bones, but its impact on liver function remains unclear. In the current study, we explored the regulatory mechanisms of apoptosis, autophagy, fission, and fusion in maintaining liver mitochondrial homeostasis in mice subjected to TS for 2 or 4 weeks (TS2 and TS4). The results showed the following: (1) No significant differences were observed in nuclear ultrastructure or DNA fragmentation between the control and TS-treated groups. (2) No significant differences were detected in the mitochondrial area ratio among the three groups. (3) Cysteine aspartic acid-specific protease 3 (Caspase3) activity and the Bcl-2-associated X protein (bax)/B-cell lymphoma-2 (bcl2) ratio were not higher in the TS2 and TS4 groups compared to the control group. (4) dynamin-related protein 1 (DRP1) protein expression was increased, while mitochondrial fission factor (MFF) protein levels were decreased in the TS2 and TS4 groups compared to the control, suggesting stable mitochondrial fission. (5) No significant differences were observed in the optic atrophy 1 (OPA1), mitofusin 1 and 2 (MFN1 and MFN2) protein expression levels across the three groups. (6) Mitochondrial autophagy vesicles were present in the TS2 and TS4 groups, with a significant increase in Parkin phosphorylation corresponding to the duration of the TS treatment. (7) ATP synthase and citrate synthase activities were significantly elevated in the TS2 group compared to the control group but were significantly reduced in the TS4 group compared to the TS2 group. In summary, the coordinated regulation of apoptosis, mitochondrial fission and fusion, and particularly mitochondrial autophagy preserved mitochondrial morphology and contributed to the restoration of the activities of these two key mitochondrial enzymes, thereby maintaining liver mitochondrial homeostasis in mice under TS conditions.

Biosynthesized silver nanoparticles of Cissus woodrowii inhibit proliferation of cancer cells through induction of apoptosis pathway

The purpose of this study was biogenic synthesis of silver nanoparticles using aqueous leaf extract of Cissus woodrowii (CW-AgNPs) and exploration of their role in inhibition cell proliferation of breast cancer cells. Inhibitory effect of biogenic CW-AgNPs on cell proliferation of MCF-7 cells was determined by MTT assay. Apoptotic cell death was demonstrated by caspase-3 assay, DNA fragmentation assay, Annexin-V-FITC/PI staining assay and cell cycle arrest assay by flow cytometry. Expression of apoptosis-related genes including caspase-3, p53 and Bcl2 was studied by western blot analysis and semi-quantitative RT-PCR. Results of UV–Vis spectrum of colloidal solution of CW-AgNPs showed surface plasmon resonance peak at 430 nm. TEM and XRD results confirmed spherical shaped, 20–30 nm sized nanoparticles. Cell proliferation results showed inhibitory effects of CW-AgNPs on MCF-7 with IC50 (24 h) 8.48 µg/mL and (48 h) 7.11 µg/mL. The CW-AgNPs altered cell morphology of MCF-7 cells and induced apoptosis significantly which was evidenced through caspase-3 activation and nuclear DNA fragmentation. The results were supported by the observation of Annexin-V-FITC/PI staining and cell cycle arrest assays. The up-regulated expression of both p53 and caspase-3 genes and down-regulation of Bcl2 at both protein and mRNA level supported their role in induction of apoptosis in MCF-7 cells in response to CW-AgNPs. The findings obtained in current study supported the role of biogenic CW-AgNPs in inhibition of cell proliferation in breast cancer cells through induction of apoptosis.Graphical

Unveiling the mechanism of hesperidin-induced LdTopI-mediated cell death pathway in protozoan parasite Leishmania donovani

Unicellular protozoan parasite Leishmania donovani is the causative agent for visceral leishmaniasis (VL) or Kala-azar, a neglected fatal parasitic disease. The conventional treatment of VL consists of therapeutic agents having several shortcomings such as toxicity, high cost, efficacy variance and increased drug resistance. Therefore, there is a desperate need to develop an effective treatment against the parasite. Previous reports suggested that flavonoids can inhibit the enzyme Leishmania donovani DNA topoisomerase I (LdTopILS). Therefore, for the first time in this present study, we divulge HSP (one of the natural sources of flavonoids), as a potent natural antileishmanial compound with efficacy in BALB/c mice at 20 mg/kg of body weight, inhibits LdTopILS at 97 % of its activity at 160 μM in preincubation condition (competitively). It binds with free enzyme and does not allow it to bind with the substrate DNA. Moreover, HSP does not stabilize DNA-topoisomerase I cleavable complex. Thus, HSP acts a catalytic topoisomerase I inhibitor, which inhibits complete activity by binding with Lys269 and Thr411 of large subunit of the enzyme. On the other hand, HSP induces the topo I-mediated programmed cell death process by the formation of cellular reactive oxygen species, resulting in depolarization of mitochondrial membrane potential, followed by fragmentation of nuclear DNA. Therefore, the present study illuminates a natural flavonoid that in future might be a promising lead for the treatment of VL.

GC/MS Analysis and Compounds Isolation ofLycium shawii Petroleum Ether Seeds Extract for Regulating Nrf2/OH-1 Pathway, Oxidative Stress and Inflammation in Acrylamide- Induced Infertility in Female Rats.

Acrylamide is formed during heating of starchy foods at high temperature and induces reproductive toxicity. Our study is designed to evaluate the chemical constitution and anti-infertility effect of Lycium shawii seeds extract on female rats. Nutritional profile was estimated, and major active compounds were isolated and identified. Biological evaluation of Lycium shawii extract on female rats was performed and measured by prolactin, follicular stimulating hormone, luteinizing hormone, estradiol, progesterone, tumor necrosis factor-α, interleukin-6, heme oxygenase-1, nuclear respiratory factor-2, malondialdehyde, glutathione, DNA fragmentation and ovarian architecture parameters. Data revealed that presence of ɤ-tocopherol, vitamin C, magnesium and thirty-eight bioactive compounds in the fractions of Lycium shawii. Major constituents from GC/MS, were 9, 12-octadecadienoic acid (Z, Z), methyl ester, 2,7-Octadiene-1,6-diol and 2,6-dimethyl hydroxy linalool but further five compounds (i.e. lupenone, betulin, lupeol acetate, stigmasterol and β-sitosterol-D-glucoside) were isolated and identified. Treatment of rats with the seeds extract post acrylamide administration ameliorated female sex hormones, oxidative stress, inflammation, DNA damage, and ovarian structure. In conclusion, Lycium shawii petroleum ether seeds fraction may be considered as a nutraceutical agent for improving infertility disorders, oxidative stress and inflammation due to its richness with biologically active phenolic and flavonoids compounds.

An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA–MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields.

The growth factors amphiregulin and epiregulin are novel stimulators of feline ovarian granulosa cell functions.

This study aimed to investigate the impact of the epidermal growth factor receptor ligands amphiregulin (AREG) and epiregulin (EREG) on the fundamental functions of feline ovarian granulosa cells. Granulosa cells isolated from feline ovaries were incubated with AREG and EREG (0, 0.1, 1 or 10 ng/mL). The effects of these growth factors on cell viability, proliferation (assessed through BrdU incorporation), nuclear apoptosis (evaluated through nuclear DNA fragmentation) and the release of progesterone and estradiol were determined using Cell Counting Kit-8 assays, BrdU analysis, TUNEL assays and ELISAs, respectively. Both AREG and EREG increased cell viability, proliferation and steroid hormone release and reduced apoptosis. The present findings suggest that these epidermal growth factor receptor ligands may serve as physiological stimulators of feline ovarian cell functions.

Electron-beam generated X-ray irradiation could retard the senescence of postharvest Hericium erinaceus via regulating reactive oxygen metabolism

Fresh Hericium erinaceus, a type of white mushroom, is prone to browning and microbial invasion after harvest due to high rate of metabolism. This study aimed to clarify how electron-beam generated X-ray (EBGX) irradiation (0, 1.0, 1.5, and 2.0 kGy) affects the senescence of postharvest fresh H. erinaceus during storage at 15 °C for 9 d. The effects of EBGX irradiation on biological properties of H. erinaceus were evaluated. Results indicated that EBGX irradiation reduced the incidence of browning in H. erinaceus, increased firmness, inhibited the respiration rate, lowered malondialdehyde (MDA) content and electrolyte leakage relative to untreated samples. Moreover, the natural microflora presents on H. erinaceus were prominently inhibited (P < 0.05) in 1.0 kGy compare to control group after storage, such as total bacterial count (35.6%), molds and yeasts (31.4%), and Pseudomonas (33.2%). Meanwhile, exposure to 1.0 kGy of irradiation treatment exhibited a minimum hydrogen peroxide content (51.2 nmol kg−1) and superoxide anion production rate (1.88 μmol kg−1 min−1) at 9 d, enhanced the activities of antioxidative enzymes (superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase), as well as declined oxidative stress by regulating the expression of related genes (HeSOD1, HeSOD2, HeCAT1, HeCAT2, HePOX1, and HePOX2).TUNEL analysis also observed that H. erinaceus treated with 1.0 kGy remarkably retained a lower nuclear DNA fragmentation. This finding suggests that the dose of 1.0 kGy EBGX irradiation could postpone the senescence process of fresh H. erinaceus and has the potential to be a promising physical preservation technology for fresh H. erinaceus.

Hortensins, Type 1 Ribosome-Inactivating Proteins from Seeds of Red Mountain Spinach: Isolation, Characterization, and Their Effect on Glioblastoma Cells.

Ribosome inactivating proteins (RIPs) are specific N-β-glycosylases that are well-characterized in plants. Their enzymatic action is to damage ribosomes, thereby blocking protein translation. Recently, several research groups have been working on the screening for these toxins in edible plants to facilitate the use of RIPs as biotechnological tools and biopesticides and to overcome public prejudice. Here, four novel monomeric (type 1) RIPs have been isolated from the seeds of Atriplex hortensis L. var. rubra, which is commonly known as edible red mountain spinach. These enzymes, named hortensins 1, 2, 4, and 5, are able to release the β-fragment and, like many other RIPs, adenines from salmon sperm DNA, thus, acting as polynucleotide:adenosine glycosidases. Structurally, hortensins have a different molecular weight and are purified with different yields (hortensin 1, ~29.5 kDa, 0.28 mg per 100 g; hortensin 2, ~29 kDa, 0.29 mg per 100 g; hortensin 4, ~28.5 kDa, 0.71 mg per 100 g; and hortensin 5, ~30 kDa, 0.65 mg per 100 g); only hortensins 2 and 4 are glycosylated. Furthermore, the major isoforms (hortensins 4 and 5) are cytotoxic toward human continuous glioblastoma U87MG cell line. In addition, the morphological change in U87MG cells in the presence of these toxins is indicative of cell death triggered by the apoptotic pathway, as revealed by nuclear DNA fragmentation (TUNEL assay).

Cytotoxicity and Apoptosis Studies of Brucein D against T24 Bladder Cancer Cells.

Brucein D (BrD), a quassinoid isolated from Brucea javanica fruit, reportedly demonstrates anti-cancer activity. This study's objective is to evaluate the cytotoxicity of Brucein D and its ability to induce apoptosis in T24 bladder cancer cells. We investigated the cytotoxic activity of BrD against the T24 cell through the induction of apoptosis in vitro. This cytotoxic activity was evaluated with ΜΤΤ assay and followed by Calcein-AM/PI viability staining. Apoptotic activity was determined with Hoechst 33342 nuclear staining and DNA fragmentation. Doxorubicin and docetaxel were used as a positive control. Evaluation of apoptotic-related gene expression, Bax, Bak, Bcl2, and p53 was also performed using semi-quantitative PCR analysis. Statistical analysis was conducted using One-way ANOVA followed by post hoc test Turkey's HSD (Honestly Significance Difference). Results show that BrD had high toxicity against T24 bladder cancer cells with an IC50 value of 7.65 ± 1.2 µg/mL but relatively less toxic to 1BR3 normal skin fibroblast cells compared to the doxorubicin and docetaxel treated cells. The viability assay shows that BrD significantly increases the percentage of dead cells relative to control in a dose-dependent manner. Furthermore, the percentage of cells with apoptotic appearance was significantly higher in group treated with BrD IC50 (56.04±3.09%) compared to control (9.42±2.88). The result was similar to doxorubicin IC50 (58.97±12.31) but lower than docetaxel IC50 (74.42±9.79). DNA fragmentation in gel electrophoresis was also observed in T24 cells treated with BrD. Apoptosis was also verified by an alteration in the expression of apoptosis-related genes, upregulation of Bax, Bak, and p53, and downregulation of Bcl-2. BrD has shown a cytotoxic effect against T24 bladder cancer cells. Hence, it is a promising natural compound for the management of bladder cancer by induction of apoptosis through activation of the intrinsic pathway, with low toxicity to normal cells.

Investigation of Potential Effects of Ibuprofen on the Storage Cells and Anhydrobiosis Capacity of the Tardigrade Paramacrobiotus experimentalis

The surge in pharmaceutical consumption, particularly non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, has raised concerns about their presence in aquatic ecosystems. This study investigated the potential ecological impact of ibuprofen, focusing on the ultrastructure of storage cells in the tardigrade Paramacrobiotus experimentalis, renowned for its resilience to environmental stressors. Individuals were exposed to three ibuprofen concentrations (0.1 μg/L, 16.8 μg/L, and 1 mg/L) over 7 and 28 days. Storage cells were examined using light microscopy, transmission electron microscopy, and confocal microscopy. This study also explored ibuprofen’s impact on the process of anhydrobiosis. In the short-term experiment, no ultrastructural changes in tardigrade storage cells were observed across ibuprofen concentrations. However, in the long-term incubation, autophagic structures in storage cell cytoplasm were identified, indicating potential adaptive responses. Individual mitochondria exhibited degeneration, and the rough endoplasmic reticulum displayed slight swelling. No evidence of increased oxidative stress or nuclear DNA fragmentation was observed in any research group. This study elucidates the complex responses of tardigrade storage cells to ibuprofen exposure. The findings emphasize the importance of understanding pharmaceutical impacts on aquatic organisms, highlighting the resilience of tardigrades to specific environmental stressors.

Acetaminophen Hepatotoxicity: Paradigm for Understanding Mechanisms of Drug-Induced Liver Injury.

Acetaminophen (APAP) overdose is the clinically most relevant drug hepatotoxicity in western countries, and, because of translational relevance of animal models, APAP is mechanistically the most studied drug. This review covers intracellular signaling events starting with drug metabolism and the central role of mitochondrial dysfunction involving oxidant stress and peroxynitrite. Mitochondria-derived endonucleases trigger nuclear DNA fragmentation, the point of no return for cell death. In addition, adaptive mechanisms that limit cell death are discussed including autophagy, mitochondrial morphology changes, and biogenesis. Extensive evidence supports oncotic necrosis as the mode of cell death; however, a partial overlap with signaling events of apoptosis, ferroptosis, and pyroptosis is the basis for controversial discussions. Furthermore, an update on sterile inflammation in injury and repair with activation of Kupffer cells, monocyte-derived macrophages, and neutrophils is provided. Understanding these mechanisms of cell death led to discovery of N-acetylcysteine and recently fomepizole as effective antidotes against APAP toxicity.

Addendum to: The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation and copy number in normal and abnormal human ejaculates.

While the kinetics of human sperm nuclear DNA fragmentation (SDF-nDNA) following ejaculation have been described, the dynamics and relationships of mitochondrial DNA copy number per spermatozoon (mtDNAcn) and fragmentation (SDF-mtDNA) remain unexplored. To compare post-ejaculatory kinetics of mtDNAcn, SDF-mtDNA and SDF-nDNA, global, single-strand DNA breaks (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs) in normozoospermic and non-normozoospermic samples. 28 normozoospermic and 43 non-normozoospermic ejaculates were evaluated at 0, 6, 24 and 48h of incubation in vitro. SDF-nDNA was determined by sperm chromatin dispersion (SCD) assays. mtDNAcn and SDF-mtDNA were analysed by dPCR. SDF-nDNA-global values increased as a consequence of quadratic SDF-SSBs and linear SDF-DSBs kinetics. Non-normozoospermic samples showed a slower SDF-global rate between 6-24h, due to lesser SSBs production. Regarding SDF-DSBs, non-normozoospermic samples exhibited a faster initial increase rate, followed by a slower final increment. The mtDNAcn median value decreased linearly, being 3.2× higher in non-normozoospermics at all time points; mtDNAcn in both cohorts reduced to half of the baseline by 48h. mtDNAcn was identified as a risk factor for discriminating non-normozoospermia, a finding that was further strengthen when combined with SDF-Global or SDF-DSBs values. SDF-mtDNA frequencies were identical, increasing over time correspondingly in both cohorts. The mtDNA fragmentation rate was initially fast, decreasing progressively with time for both cohorts; half of the initially unfragmented copies were fragmented after 48h. Rates of mtDNAcn loss and SDF-mtDNA increase were only marginally correlated with the rates of nuclear fragmentation. mtDNA fragmentation and loss occur post ejaculation. Their dynamics are likely to be associated with different and/or uncoupled mechanisms to that which cause nuclear DNA fragmentation. Our results indicate that while mtDNA fragmentation is not influenced by the sperm quality, the number of copies of sperm mtDNAcn can potentially serve as a risk factor for predicting non-normozoospermia.

Exploration of the aptitude to alleviate oxidative impairment and curb colorectal cancer manifestation by Nostoc calcicola in HT-29 adenocarcinoma cells

BackgroundMarine cyanobacteria have been known to contain several unique bioactive compounds which have different therapeutic potentials. The current research focuses to identify the efficacy of Nostoc calcicola to counteract the harmful effects of free radicals and testing its anticancer activity against colorectal adenocarcinoma cells (HT-29).ResultsMethanol is used as a solvent for the extraction of bioactive metabolites from Nostoc calcicola followed by phytochemical screening representing the presence of flavonoids, phenols, tannins, saponins, and steroids to find out bioactive metabolites. Furthermore, evaluation of the extract efficacy revealed the profound ability of Nostoc calcicola to scavenge free radicals by neutralizing different reactive oxygen species. At 100 µg/mL concentration, it inhibited DPPH radicals (73.4%), enhanced phosphomolybdenum reduction (53.5%), displayed ferric-reducing power (55.1%), and finally the extract revealed remarkable hydroxyl radicals scavenging capacity (94.8%), compared to the standards. These compelling results emphasize the robust antioxidant potential of the Nostoc calcicola extract. In vitro, studies demonstrated the selective cytotoxic effects of methanol extracts of Nostoc calcicola on the HT-29 human colorectal cancer cell line, as indicated by IC50 values of 25 µg/mL for the extracts. Treatment with me Nostoc calcicola decreased the cell viability of HT-29 cells followed by consistent morphological changes leading to cytotoxicity. Nuclear condensation and DNA fragmentation were observed using AO/EtBr and DAPI staining. Flow cytometry analysis further confirmed the incidence of apoptosis during the S phase of the cell cycle. Furthermore, western blotting analysis confirmed the activation of caspase 9, a pivotal enzyme in the intrinsic apoptosis pathway, suggesting the ability of Nostoc calcicola to induce apoptosis in HT-29 colorectal cancer cells.ConclusionThese findings underscore the potential of Nostoc calcicola as a valuable source of bioactive compounds with antioxidant and anticancer properties, warranting further investigation for their potential therapeutic applications in colorectal cancer.Graphical

The Role of Mechanistic Biomarkers in Understanding Acetaminophen Hepatotoxicity in Humans.

Our understanding of the fundamental molecular mechanisms of acetaminophen (APAP) hepatotoxicity began in 1973 to 1974, when investigators at the US National Institutes of Health published seminal studies demonstrating conversion of APAP to a reactive metabolite that depletes glutathione and binds to proteins in the liver in mice after overdose. Since then, additional groundbreaking experiments have demonstrated critical roles for mitochondrial damage, oxidative stress, nuclear DNA fragmentation, and necrotic cell death as well. Over the years, some investigators have also attempted to translate these mechanisms to humans using human specimens from APAP overdose patients. This review presents those studies and summarizes what we have learned about APAP hepatotoxicity in humans so far. Overall, the mechanisms of APAP hepatotoxicity in humans strongly resemble those discovered in experimental mouse and cultured hepatocyte models, and emerging biomarkers also suggest similarities in liver repair. The data not only validate the first mechanistic studies of APAP-induced liver injury performed 50 years ago but also demonstrate the human relevance of numerous studies conducted since then. SIGNIFICANCE STATEMENT: Human studies using novel translational, mechanistic biomarkers have confirmed that the fundamental mechanisms of acetaminophen (APAP) hepatotoxicity discovered in rodent models since 1973 are the same in humans. Importantly, these findings have guided the development and understanding of treatments such as N-acetyl-l-cysteine and 4-methylpyrazole over the years. Additional research may improve not only our understanding of APAP overdose pathophysiology in humans but also our ability to predict and treat serious liver injury in patients.

Mortality caused by extracellular freezing is associated with fragmentation of nuclear DNA in larval haemocytes of two drosophilid flies.

The great complexity of extracellular freezing stress, involving mechanical, osmotic, dehydration and chemical perturbations of the cellular milieu, hampers progress in understanding the nature of freezing injury and the mechanisms to cope with it in naturally freeze-tolerant insects. Here we show that nuclear DNA fragmentation begins to occur in larval haemocytes of two fly species, Chymomyza costata and Drosophila melanogaster, before or at the same time as the sub-zero temperature is reached that cause irreparable freezing injury and mortality in freeze-sensitive larval phenotypes. However, when larvae of the freeze-tolerant phenotype (diapausing-cold acclimated-hyperprolinemic) of C. costata were subjected to severe freezing stress in liquid nitrogen, no DNA damage was observed. Artificially increasing the proline concentration in freeze-sensitive larvae of both species by feeding them a proline-enriched diet resulted in a decrease in the proportion of nuclei with fragmented DNA during freezing stress. Our results suggest that proline accumulated in diapausing C. costata larvae during cold acclimation may contribute to the protection of nuclear DNA against fragmentation associated with freezing stress.

The relationship between sperm nuclear DNA fragmentation, mitochondrial DNA fragmentation, and copy number in normal and abnormal human ejaculates.

While it is common to clinically evaluate sperm nuclear DNA fragmentation, less attention has been given to sperm mitochondrial DNA. Recently, a digital PCR assay has allowed accurate estimation of the proportion of fragmented mtDNA molecules and relative copy number. To determine the correlation of classical sperm parameters, average mtDNA copies per spermatozoon and the level of mtDNA fragmentation (SDF-mtDNA) to that of nuclear DNA fragmentation (SDF-nDNA), measured as the proportion of global, single-strand DNA (SDF-SSBs) and double-strand DNA breaks (SDF-DSBs). To determine whether the level of nuclear and mitochondrial DNA fragmentation and/or copy number can differentiate normozoospermic from non-normozoospermic samples. Ejaculates from 29 normozoospermic and 43 non-normozoospermic were evaluated. SDF was determined using the sperm chromatin dispersion assay. mtDNA copy number and SDF-mtDNA were analyzed using digital PCR assays. Relative mtDNA copy increased as sperm concentration or motility decreased, or abnormal morphology increased. Unlike SDF-mtDNA, mtDNA copy number was not correlated with SDF-nDNA. SDF-mtDNA increased as the concentration or proportion of non-vital sperm increased; the higher the mtDNA copy number, the lower the level of fragmentation. Non-normozoospermic samples showed double the level of SDF-nDNA compared to normozoospermic (median 25.00vs. 13.67). mtDNA copy number per spermatozoon was 3× higher in non-normozoospermic ejaculates (median 16.06vs. 4.99). Although logistic regression revealed SDF-Global and mtDNA copy number as independent risk factors for non-normozoospermia, when SDF-Global and mtDNA copy number were combined, ROC curve analysis resulted in an even stronger discriminatory ability for predicting the probability of non-normozoospermia (AUC=0.85, 95% CI 0.76-0.94, p<0.001). High-quality ejaculates show lower nuclear SDF and retain less mtDNA copies, with approximately half of them fragmented, so that the absolute number of non-fragmented mtDNA molecules per spermatozoon is extremely low.

Investigation of nuclear DNA content and cell cycle phases in rat liver cells under chlorpromazine administration

Hepatotoxicity of antipsychotic drugs remains an urgent problem of modern medicine. Therefore, the purpose of the study was to investigate the nuclear DNA content and cell cycle phases of rat liver cells under Сhlorpromazine administration at doses ranging from 3.5 mg/kg to 28 mg/kg for 30 and 60 days. The study was conducted on 60 sexually mature female rats. Chlorpromazine was administered once daily for 30 and 60 days at doses of 3.5 mg/kg, 7 mg/kg, 14 mg/kg, 21 mg/kg and 28 mg/kg. The DNA content in the nuclei of rat liver cells was determined by flow cytometry. Cytological analysis of cells was performed using FloMax software (Partec, Germany), where the percentage of nuclei in the G0G1 interval of the cell cycle, in the S phase, G2M interval, and the apoptosis index – SUB-G0G1 area on DNA histograms were determined. Statistical processing of the results was performed using the Mann-Whitney U test. The results of the study showed that Сhlorpromazine has a dose-dependent hepatotoxic effect: with an increase in the dose of this drug in rats from 7 to 28 mg/kg, the percentage of fragmented nuclei in liver tissue significantly increased, which is a sign of hepatocyte death by apoptosis. It was found that Сhlorpromazine at a dose of 3.5 mg/kg did not increase hepatocyte apoptosis, while at a dose of 21 and 28 mg/kg the drug showed the highest hepatotoxicity, increasing the level of apoptosis by 1.9 and 2.1 (p˂0.05) times, respectively. The hepatotoxic effect is enhanced by the use of Сhlorpromazine for 60 days, which is manifested in a significant increase in hepatocyte nuclear DNA fragmentation, which, in our opinion, should be taken into account when conducting long-term therapy in patients.

Effects of Ginkgo biloba Extract on Cognitive Function Recovery and Inflammatory Factors in Rats After Anesthesia

This research was aimed to investigated the impacts of Ginkgo biloba extract (GbE) on cognitive function and inflammatory factors in rats after anesthesia. Firstly, the primary cultured cortical neurons of rats were prepared in culture medium, to which different concentrations of GbE were added, and cell viability was observed. Twenty SD rats were selected, among which 10 rats were made into a model of neurological dysfunction, and the other 10 were used as controls. The correlation between neurological dysfunction and inflammatory factors was analyzed using Spearman rank correlation (PRC) analysis. Another 40 SD rats were screened and grouped into 4 (a model (M) group, a treatment (T) group, a prevention (P) group, and a blank (B) group). Rats in the P and T treatments were injected intraperitoneally with 10 mg/kg GbE injection before or after anesthesia induction, while an equal amount of 0.9% NaCl solution was administrated in M and B groups once a day for 3 days. Morris water maze (MWM) and open filed test (OFT) were employed to assess the cognitive function of rats in different groups. Furthermore, the levels of inflammatory factors of rats in various groups were compared on day 1 and day 7. Results revealed that GbE can enhance neural cell viability, reduce lactate dehydrogenase (LDH) release, and alleviate changes in cell nuclear morphology and DNA fragmentation. In terms of correlation, the NIHSS score was positively associated with TNF-α and IL-1β, and negatively correlated with IL-10 (all P &lt;0.05). Besides, the escape latency was greatly shortened, memory time was prolonged, and the TNF-α and IL-1β were downshifted in P and T groups to the conditions in the M group, showing great differences with P &lt;0.05. This study demonstrated that GbE could inhibit neural cell apoptosis (NCA), remarkably improve the cognitive dysfunction in rats after anesthesia, and regulate the inflammatory factors.

Central Mechanisms of Acetaminophen Hepatotoxicity: Mitochondrial Dysfunction by Protein Adducts and Oxidant Stress.

Acetaminophen (APAP) is an analgesic and antipyretic drug used worldwide, which is safe at therapeutic doses. However, an overdose can induce liver injury and even liver failure. Mechanistic studies in mice beginning with the seminal papers published by B.B. Brodie's group in the 1970s have resulted in important insight into the pathophysiology. Although the metabolic activation of APAP with generation of a reactive metabolite, glutathione depletion, and protein adduct formation are critical initiating events, more recently, mitochondria have come into focus as an important target and decision point of cell death. This review provides a comprehensive overview of the induction of mitochondrial superoxide and peroxynitrite formation and its propagation through a mitogen-activated protein kinase cascade, the mitochondrial permeability transition pore opening caused by iron-catalyzed protein nitration, and the mitochondria-dependent nuclear DNA fragmentation. In addition, the role of adaptive mechanisms that can modulate the pathophysiology, including autophagy, mitophagy, nuclear erythroid 2 p45-related factor 2 activation, and mitochondrial biogenesis, are discussed. Importantly, it is outlined how the mechanisms elucidated in mice translate to human hepatocytes and APAP overdose patients, and how this mechanistic insight explains the mechanism of action of the clinically approved antidote N-acetylcysteine and led to the recent discovery of a novel compound, fomepizole, which is currently under clinical development. SIGNIFICANCE STATEMENT: Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in western countries. Extensive mechanistic research over the last several decades has revealed a central role of mitochondria in the pathophysiology of APAP hepatotoxicity. This review article provides a comprehensive discussion of a) mitochondrial protein adducts and oxidative/nitrosative stress, b) mitochondria-regulated nuclear DNA fragmentation, c) adaptive mechanisms to APAP-induced cellular stress, d) translation of cell death mechanisms to overdose patients, and e) mechanism-based antidotes against APAP-induced liver injury.