DNA In Comet Tail Research Articles

The Association of Sperm DNA Fragmentation With Serum Lipid Profile Among Males With Abnormal Semen Parameters: An Analytical Cross-Sectional Study.

Through the ages, infertility, affecting 8% to 12% of couples worldwide, has been a perturbing clinical problem. Approximately 40% to 50% of all infertility cases are due to 'male factor' infertility. Semen analysis is crucial in routinely evaluating idiopathic male infertility. Studies support the idea that semen parameters are associated with serum lipids and sperm DNA fragmentation (SDF). Therefore, it is possible to evaluate male infertility by serum lipid levels, especially before assisted reproduction technology, and modify it by bringing about lifestyle modifications. This study aimed to measure the correlation of SDF with levels of total cholesterol (TC), triglycerides (TG),very low-density lipoprotein (VLDL),low-density lipoprotein (LDL), and high-density lipoprotein (HDL) among males with abnormal semen parameters. A cross-sectional analytical study was conducted in the infertility clinic of a tertiary care hospital. A total of 106 infertile males with abnormal semen analysis as per the WHO criteria (2010) were enrolled in the study. After routine semen analysis, SDF was studied using the comet assay. The serum fasting lipid profile was analyzed using the spectrophotometric kit in the autoanalyzer. The relationship of SDF with serum lipid profile parameters was analyzed. Out of 106 infertile men, 52% (n = 55) had severe SDF. A modest positive correlation was observed between SDF (percentage of DNA in comet tail) and serum lipid values (serum TG, serum LDL, and serum VLDL). Our study is novel in its research on the correlation between SDF and serum lipid values. Based on the findings of our study, it can be concluded that a significant level of SDF was observed in men with high levels of serum TG, LDL, and VLDL. This provokes a potential relationship between sperm DNA integrity and serum lipid profile, which warrants further research.

Abstract 4749: Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment

Abstract ACR-368 (prexasertib) is a clinically advanced CHK1/2 inhibitor which has demonstrated durable activity across a proportion of patients with advanced solid tumors. Genomic biomarkers have proven unsuccessful in predicting response to ACR-368, limiting its clinical success. Using AP3, we previously developed a response-predictive proteomics-based test for ACR-368 (ACR-368 OncoSignature) for the identification of patients sensitive to ACR-368 monotherapy treatment as demonstrated in blinded preclinical studies. A Phase 2 clinical trial is ongoing where patients are treated with ACR-368 monotherapy based on OncoSignature-predicted sensitivity (NCT05548296). Here, we demonstrate the utility of AP3 for the identification of a key druggable resistance mechanism to ACR-368 and how to overcome that with low dose gemcitabine (gem), providing OncoSignature negative patients with a new potential therapeutic option. Five ovarian cancer cell lines were rendered durably resistant to ACR-368 by culturing in the presence of clinically relevant concentrations of ACR-368. Matched parental and ACR-368 resistant cell line pairs were profiled using AP3 mass spectrometry. Comprehensive pathway reconstitution and kinase activity analyses were performed to identify drug resistance mechanisms in an unbiased manner. Downregulation of DNA damage repair pathway activity was causally linked to the ACR-368-resistant phenotype, suggesting agents that restore replication stress around the CHK1/2 signaling axis may re-sensitize to ACR-368. To test this, a panel of ovarian cancer cell lines with intrinsic or drug-induced resistance to ACR-368 were screened for cell growth inhibition by ACR-368 combined with gem. Gem synergized with ACR-368 in 12/13 cell lines at low doses (1-40 nM). Moreover, Western blot analysis demonstrated protein markers of replication stress were induced by low dose gem (3-30 nM), suggesting a correlation between gem-induced replication stress and synergy with ACR-368. Comet assays showed that DMSO, gem (3 nM), or ACR-368 (100 nM) had minimal impact (4.4%, 4.5%, and 11.4%, respectively) on % comet tail DNA in ACR-368 resistant cells, while the gem combination led to 35% comet tail DNA (p<0.001). Finally, in a human tumor xenograft mouse model, low dose gem demonstrated a dose-dependent increase in replication stress markers (Cyclin E, pCHK1 S345) from 0.3-3 mg/kg, which allometrically scales to 1-10 mg/m2 in humans. These data supported a dose escalation Phase 1b/2 clinical study of low dose gem with ACR-368 to evaluate the efficacy and safety of the combination in ACR-368 OncoSignature negative patients (NCT05548296). This shows the potential of AP3 for unbiased elucidation of actionable drug resistance mechanisms and rapid clinical implementation in our trials, which have recently confirmed clinical activity. Citation Format: Helén Nilsson, Lei Shi, Magnus E. Jakobsson, Joelle Baddour-Sousounis, Shahrzad Rafiei, Uthira Muralitharan, Zachary Best, Valentina Siino, Francisco J. Santana, Ignacio Arribas Diez, Kailash Singh, Portia Lombardo, William Dahlberg, Subodh Kumar, Ahmed Youssef, Reina Improgo, Corey Xu, Joon Jung, Jung-Min Lee, Ayesha Murshid, Michail Shipitsin, Jesper V. Olsen, Kristina Masson, David A. Proia, Caroline Wigerup, Peter Blume-Jensen. Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment [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 4749.

Abstract 5941: Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer

Abstract PARP inhibitors (PARPi) targeting poly (ADP-ribose) polymerase are the first clinically approved drugs designed to apply synthetic lethality in BRCA1 mutant/deficient cancer. However, the treatment can cause reversion of BRCA mutation which results in drug resistance. To explore new strategies to improve antitumor efficacy of PARPi, we applied PARPi in combination with a small molecule NSC59984 for cancer therapy. NSC59984 is a small molecular targeting mutant p53 degradation and activating p73 via ROS-ERK2-MDM2 pathway. The treatments with radiation or hydrogen peroxide showed that NSC59984 enhanced DNA comet tails and gamma-H2AX, correlated to reduction of rad51 foci in cancer cells. These results suggest that NSC59984 impairs DNA damage repair via abrogation of homologous recombination (HR). We further applied the combinational treatment of NSC59984 and PARPi in BRCA1 wild-type ovarian cancer cells and found that NSC59984 synergized with PARPi to reduce cell viability, inhibit colony formation, and increase cell death. The combination treatment enhanced DNA damage and correlated with reduction of BRCA1 at the protein level. These results, taken together, suggest that the induction of BRCAness causes a synthetical lethality with PARPi in BRCA1 wild-type cancer cells. Cell cycle profiling showed that the cells were arrested at the G2/M phase in response to combinational treatment. At the G2 phase arrest, the high doses of the combinational drugs led to cell death via mitotic catastrophe, and the intermediate doses induced cellular senescence defined by senescence phenotypic hallmarks including senescence-associated beta-gal staining and a secretome consistent with senescence-associated secretory phenotype (SASP). Therapy-induced senescence (TIS) in cancer cells is considered as one of the mechanisms of tumor recurrence and drug resistance. To reduce the risk of TIS in treatment, we applied senolytic treatment to target senescent cells in the combinational treatment. The senolytic drug ABT263 treatment eliminated the senescent cancer cells from the combinational treatment. ABT263, NSC59984 + PARPi combinational treatment further reduced cell viability. Our study provides a rationale for small molecular compounds targeting HR deficiency in combination with PARPi to treat BRCA1wild-type ovarian cancer cells, and additional senolytic treatment may be required to limit resistance by removal of the TIS. Citation Format: Shengliang Zhang, Lanlan Zhou, Leiqing Zhang, Maximilian Pinho-Schwermann, Benedito Carneiro, John Sedivy, Wafik S. El-Deiry. Small molecule NSC59984 synergizes with PARP inhibitors in BRCA1 wild type ovarian cancer [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 5941.

The role of RLIP76 in oxidative stress and mitochondrial dysfunction: Evidence based on autopsy brains from Alzheimer's disease patients

Several converging lines of evidence from our group support a potential role of RLIP76 (AKA Rlip) in neurodegenerative disorders, including Alzheimer's Disease (AD). However, the role of Rlip in Alzheimer's and other neurodegenerative diseases is not well understood. The purpose of the present study is to determine the role of Rlip in the brains of AD patients and control subjects. To achieve our goals, we used frozen tissues and formalin-fixed paraffin-embedded postmortem brains from AD patients of different Braak stages and age-matched control subjects. Our immunohistology and immunoblotting blotting analysis revealed that expression of Rlip protein gradually and significantly decreased (p = 0.0001) with AD progression, being lowest in Braak stage IV-V. Rlip was colocalized with Amyloid beta (Aβ) and phosphorylated tau (p-Tau) as observed by IHC staining and co-immunoprecipitation studies. Lipid peroxidation (4-HNE generation) and H2O2 production were significantly higher (p = 0.004 and 0.0001 respectively) in AD patients compared to controls, and this was accompanied by lower ATP production in AD (p = 0.0009). Oxidative DNA damage was measured by 8-Hydroxyguanosine (8-OHdG) in tissue lysates by ELISA and COMET assay. AD 8-OHdG levels were significantly higher (p = 0.0001) compared to controls. COMET assay was performed in brain cells, isolated from frozen postmortem samples. The control samples showed minimal DNA in comets representing few DNA strand breaks (<20 %), (score-0-1). However, the AD group showed an average of 50 % to 65 % of DNA in comet tails (score-4-5) indicating numerous DNA strand breaks. The difference between the two groups was significant (p = 0.001), as analyzed by Open Comet by ImageJ. Elevated DNA damage was further examined by western blot analysis for phosphorylated histone variant H2AX (γH2AX). Induction of γH2AX was very significant (p < 0.0001) and confirmed the presence of double-strand breaks in DNA. Overall, our results indicate an important role for Rlip in maintaining neuronal health and homeostasis by suppressing cellular oxidative stress and DNA damage. Based on our findings, we cautiously conclude that Rlip is a promising therapeutic target for Alzheimer's disease.

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P &lt; 0.001) and 3 h (P &lt; 0.0001), the optimum viability index (P &lt; 0.0001), SPMI (P &lt; 0.05), acrosome integrity (P &lt; 0.0001), non-fragmented DNA (P &lt; 0.01), DNA in the comet head (P &lt; 0.05), minimum DNA in the comet tail (P &lt; 0.05), shortest comet tail length (P &lt; 0.0001), and lowest comet tail moment (P &lt; 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Evaluation of gamma radiation–induced genotoxicity in the workers of Italian honey bees, Apis mellifera ligustica, using single cell gel electrophoresis (comet) assay

The environment exposed to radionuclide faces detrimental effects of ionizing radiations and these radiations can damage DNA by inducing genotoxicity in living organisms. The gamma radiation induced genotoxicity was evaluated by employing Single cell gel electrophoresis (comet) assay in the foragers of Apis mellifera ligustica by exposing to different doses (1 to 60 Gy) of gamma radiation. DNA damage was measured as the percentage of comet tail DNA. The results showed a dose dependent increase and post irradiation time (1-24 h) dependent decrease in % tail DNA level in bee foragers. A significant increase in DNA damage was observed in all the samples and the extent of damage was proportional to the dose of gamma radiation received by the bees except in those treated with the lower doses (1, 5 and 10 Gy) of gamma radiation. The highest amount of DNA damage was recorded at 1 h post-irradiation which decreased gradually with time, that is, at 3 h and 6 h post-irradiation. However, there were no significant differences in the % tail DNA of bees of 12 h and 24 h post irradiation. The present study shows gamma radiation induced DNA damage in foragers of A. m. ligustica and also the comet assay is a suitable technique for detection of gamma radiation induced DNA damage.

Genome response of hippocampal cells to stress in male rats with different excitability of the nervous system

Changes of genome stability in hippocampal cells of male rats with hereditary high and low thresholds of nervus tibialis response to electric stimuli (HT and LT strains, respectively) were studied in unstressed and stressed animals. HT and LT originated from Wistar strain, males of which were also used as a control. The comet assay was used after prolonged emotional painful stressor action. There were no interstrain differences in the spontaneous percentage of DNA in comet tails (tDNA). However, the prolonged emotional pain stressor induced genome instability differently in animals of different strains. The highest level of DNA damage in hippocampal cells was shown in highly sensitive animals of LT strain. Males of Wistar strain had intermediate levels of tDNA, while HT animals had the lowest stress reactivity.

Ecogenotoxicological studies for an early toxicity screening and monitoring in Epinephalus chlorostigma and Scamberomorus commerson

The study was planned to investigate DNA fragmentation in fish to screen aquatic toxicity and in Epinephalus chlorostigma and Scamberomorus commerson collected from Red sea near Jizan, Saudi Arabia from three locations “(Corniche North park: “16.92161, 42.54631; Jizan Port: 16.874, 42.54952” N and Jizan Economic City: 17.26589, 42.34738“ ”)“ were used as a case study for the application of comet assay. The study area of the Red Sea is polluted due to anthropogenic activities and the disposal of wastes from multiple sources. Comet and micronucleus assays were used to detect genotoxicity in these fish species harvested from three sites. The concentration of Pb, Cr, Zn, Mn, Cu, Cd, Sn, and Hg was higher in the water samples collected from the polluted site compared to the non-polluted site of the Red sea. Comet assay for S. commerson showed significant (p < 0.05) genetic damage about 44.33 ± 3.03% DNA in comet tail at site S1. It was subsequently reduced to 31.71 ± 3.52% and 22.11 ± 2.52% at sites S2 and S3. E. chlorostigma also showed significant DNA in comet tail as 17.34 ± 2.19%, 11.87 ± 3.01%, and 36.41 ± 3.98% at site S1-S3, respectively. Significant (p < 0.05) DNA damage was observed in the fishes procured from non-polluted locations and upstream locations. The micronucleus induction in E. chlorostigma was recorded as 23.20 ± 4.19 and 2.20 ± 0.58%, respectively, non-polluted and polluted sites. S. commerson exhibited significant differences between polluted and non-polluted sites (44.80 ± 3.73 and 8.20 ± 2.20‰) polluted and upstream (44.80 ± 3.73 and 20.60 ± 4.02‰), respectively. A significant difference was obtained between E. chlorostigma and S. commerson for nuclear abnormalities S. commerson showed higher frequencies for nuclear deformities than E. chlorostigma. S. commerson showed substantial micronucleus induction frequencies collected from an area of low pollution intensity (upstream). This study showed that E. clorostigma and S. commerson could be successfully used as a bioindicator to determine the health of the Red Sea through the most specific assays such as comet and micronucleus tests as an early warning and to devise the monitoring strategies to ensure a safe supply of fish for human consumption.

Mutant p53 reactivator SLMP53-2 hinders ultraviolet B radiation-induced skin carcinogenesis

The growing incidence of skin cancer (SC) has prompted the search for additional preventive strategies to counteract this global health concern. Mutant p53 (mutp53), particularly with ultraviolet radiation (UVR) signature, has emerged as a promising target for SC prevention based on its key role in skin carcinogenesis. Herein, the preventive activity of our previously disclosed mutp53 reactivator SLMP53-2 against UVR-induced SC was investigated. The pre-treatment of keratinocyte HaCaT cells with SLMP53-2, before UVB exposure, depleted mutp53 protein levels with restoration of wild-type-like p53 DNA-binding ability and subsequent transcriptional activity. SLMP53-2 increased cell survival by promoting G1-phase cell cycle arrest, while reducing UVB-induced apoptosis through inhibition of c-Jun N-terminal kinase (JNK) activity. SLMP53-2 also protected cells from reactive oxygen species and oxidative damage induced by UVB. Moreover, it enhanced DNA repair through upregulation of nucleotide excision repair pathway and depletion of UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPD) levels. SLMP53-2 further suppressed UVB-induced inflammation by inhibiting the nuclear translocation and DNA-binding ability of NF-κB, and promoted the expression of key players involved in keratinocytes differentiation. Consistently, the topical application of SLMP53-2 in mice skin, prior to UVB irradiation, reduced cell death and DNA damage. It also decreased the expression of inflammatory-related proteins and promoted cell differentiation, in UVB-exposed mice skin. Notably, SLMP53-2 did not show signs of skin toxicity for cumulative topical use. Overall, these results support a promising protective activity of SLMP53-2 against UVB-induced SC.

Abstract P030: Chemoprevention by the mutant p53 reactivator SLMP53-2 on ultraviolet radiation-induced skin cancer

Abstract The growing incidence of skin cancer (SC) is a global public health concern. Preventive approaches mostly embrace the sunscreen use against ultraviolet radiation (UVR), the main SC etiologic factor. Yet, the increasing SC incident have suggested that additional preventive agents could complement the sunscreen, helping to reverse this trend. Mutant p53 (mutp53) represents a promising target in SC prevention, since its occurrence is a key early event in UVR-induced SC. Thus, the inhibition of mutp53 formation by mutp53 reactivators, which are able to restore the wild-type-like function to p53, would be a valuable strategy for SC prevention. Recently, our group identified the tryptophanol-derived oxazoloisoindolinone SLMP53-2 as a new mutp53 reactivator. Herein, we aimed to study the potential of SLMP53-2 as a chemopreventive drug against UVR-induced SC. For that, assays with normal human keratinocyte HaCaT cells were performed. Cells were pre-treated with SLMP53-2 for 24h, followed by exposure to distinct UVB doses and further analysis. For in vivo studies, FVB/N mice were topically pre-treated for 1h prior to UVB radiation (180 mJ/cm2) and the skin was collected 24h after for immunohistochemistry/immunofluorescence analysis. Pre-treatment of HaCaT cells with SLMP53-2 enhanced cell survival in response to UVB with promotion of cell cycle arrest in G0/G1 phase, upregulation of p21 protein levels, reduction of UVB-induced apoptosis, and inhibition of JNK activity. SLMP53-2 also protected cells from UVB-induced ROS generation and from its consequent lipid peroxidation and protein carbonylation. It enhanced DNA repair through upregulation of the protein and mRNA levels of XPC (xeroderma pigmentosum complementation group C) and DDB-2 (DNA damaged binding protein 2) from the NER (nucleotide excision repair) pathway. In fact, SLMP53-2 depleted UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPDs) levels. SLMP53-2 also showed promising activity in suppressing UVB-induced inflammation, decreasing COX-2, IL-6 and TNF-α protein levels and reducing NF-kB nuclear translocation and DNA binding ability. Moreover, SLMP53-2 enhanced the expression of key players in keratinocytes differentiation, namely NOTCH1 and keratin 1. Consistently, pre-treatment with SLMP53-2 reduced mutp53 protein levels by potentially restoring its wild-type p53 form, which may underlie the increased resistance of HaCaT cells to UVB harmful effects. Importantly, in vivo studies supported that SLMP53-2 pre-treatment of UVB-irradiated mice promoted the survival of epidermal cells, reducing the DNA damage by enhancement of DNA repair and downregulating mutp53 expression levels. Further, it reduced the expression of inflammatory-related proteins and promoted cell differentiation. Collectively, these results support a promising chemopreventive activity for SLMP53-2 against UVB-induced skin damage. We thank FCT/MCTES for the projects UIDB/50006/2020, UIDP/04423/2020 and fellowship SFRH/BD/128673/2017 (J.B. Loureiro). Citation Format: Joana B. Loureiro, Rita Ribeiro, Nair Nazareth, Tiago Ferreira, Adelina Quaresma, Valentina Barcherini, Laura Marabini, Paula A. Oliveira, Maria M. M. Santos, Lucília Saraiva. Chemoprevention by the mutant p53 reactivator SLMP53-2 on ultraviolet radiation-induced skin cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P030.

AMLODIPINE-INDUCED TESTICULAR DNA DAMAGE AND ITS MODIFICATION BY A HERBAL PREPARATION IN WISTAR ALBINO RATS

The present study originated from the reports of adverse effects on male reproductive potential caused by the dihydropyridine derivative type calcium channel blockers, which are widely used agents for different cardiovascular disorders. The study was conducted to verify whether the repeated administration of this type of calcium channel blockers can cause any testicular DNA damage and if so, whether the damage can be modified by the co-administration of a herbal protective agent. The study was conducted in male Wistar albino rats. The testicular DNA damage of different groups of rats was evaluated by comet assay taking percentage DNA in comet tail as the main parameter. It was found that oral administration of the widely used dihydropyridine type calcium channel blocker amlodipine produced a duration- dependent and significant (P&lt;0.001) DNA damage in the testes of Wistar albino rats. The co- administration of the alcoholic extract of seeds of Asteracantha longifolia produced a dose dependent and significant ((P&lt;0.001) protective effect against this amlodipine-induced testicular DNA damage.

Abstract 2350: xCT overexpression induces metabolic reprogramming, epigenetic alterations, and DNA damage in precancerous airway epithelial cells

Abstract Metabolic reprogramming and genomic instability are two hallmarks of cancer known to drive tumorigenesis. While each has been investigated individually, there is a growing interest in their interplay during early cancer development. High expression of the cystine/glutamate antiporter SLC7A11 (xCT) is frequently observed in lung cancer. Our laboratory previously demonstrated that overexpressing xCT in normal bronchial airway epithelial cells induced metabolic reprogramming and caused cells to display precancerous properties, such as increased proliferation and colony formation in soft agar. In this study, we further investigated the effects of xCT overexpression in the airway epithelium with the goal of elucidating the role of xCT in influencing cancer-driving hallmarks and the interplay of these hallmarks during early tumorigenesis. We hypothesized that xCT overexpression-induced metabolic reprogramming leads to epigenetic changes through the perturbation of epigenetic-related metabolite levels. Furthermore, we posited that this altered epigenome changes the expression levels of key genes involved in genome stability maintenance, ultimately inducing genomic instability. Utilizing a LC-MS/MS-based metabolomic analysis, we observed significantly altered levels of 224 different metabolites in our xCT overexpressing cells, including metabolites involved in epigenetic processes such as S-adenosylhomocysteine and acetyl-CoA. We found that xCT overexpression alters several global histone modifications, increasing H3K9me3 and H3K9ac by 3.2 (P = 0.02) and 3.9-fold (P = 0.02) respectively. We also showed that xCT overexpression increased global DNA methylation by 56% (P = 0.04). Additionally, RNA-seq revealed significant alterations (logFC&amp;gt;2 or &amp;lt;0.5, pAdj&amp;lt;0.05) in the expression of over 100 genes, including a significant decrease in the expression of certain tumor suppressor genes such as TRIM29, and a significant increase in the expression of certain proto-oncogenes, such as CCND1. Finally, we discovered that xCT overexpression induces DNA damage, increasing the percentage of damaged DNA in comet tails in a comet assay by 15.1% (P = 0.002). Taken together, xCT overexpression in normal bronchial airway epithelial cells induces metabolic reprogramming, alters the epigenetic landscape and gene expression, and causes higher levels of DNA damage. Future experiments elucidating causal links between these processes, including ChIP-seq and WGBS are underway. This study provides a promising foundation for the connections between xCT-induced metabolic reprogramming, epigenetic alterations, and genomic instability, an imperative step in elucidating an effective, potentially multifaceted approach to preventing lung cancer development. Citation Format: Dalton Hill, Jamshedur Rahman, Christien Kluwe, Jamey Young, Emily Hodges, Pierre P. Massion. xCT overexpression induces metabolic reprogramming, epigenetic alterations, and DNA damage in precancerous airway epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2350.

Assessment of DNA Damage and Apoptosis in the Brain of Freshwater Fish &lt;i&gt;Oreochromis mossambicus&lt;/i&gt; Exposed to Tannery Effluent Wastewater

The present study was designed to evaluate the effect of tannery effluent on the fingerlings of freshwater fish Oreochromis mossambicus. The fishes were maintained at 10% concentration of tannery effluent for a period of 30 days. Apoptosis was studied by activity of caspases. Caspase 3 and 9 activity showed significant alteration. DNA seperation by agarose gel electrophoresis shows difference in intensity of genomic DNA between 750-1000 bp in fishes exposed to tannery effluent. DNA damage was also confirmed by Comet assay which showed an increase in DNA damage and tail migration was indicated by increase in percentage of mean DNA comet tail length formation.

Sterigmatocystin-induced DNA damage triggers cell-cycle arrest via MAPK in human neuroblastoma cells

Sterigmatocystin (STE) is a common mycotoxin found in food and feed. Many studies showed that STE is genotoxic. However, up to now, the potential genotoxicity of STE on human neuronal system remains unknown. In this study, we explored the effect of STE on DNA damage and cell-cycle progression on human neuroblastoma SH-SY5Y cells exposed to various concentrations of STE (0.78, 1.56 and 3.12 µM) for 24 h. The results indicated that STE exposure induced DNA damage, as evidenced by DNA comet tails formation and increased γH2AX foci. Additionally, genotoxicity was confirmed by micronuclei (MN) analysis. Furthermore, we found that STE exposure led to cell-cycle arrest at the S and the G2/M phase. Considering the important role played by MAPK and p53 signaling pathways in cell-cycle arrest, we explored their potential involvement in STE-induced cell-cycle arrest by using specific inhibitors. The inhibition of JNK and ERK resulted to attenuate S and G2/M arrest, whereas the inhibition of p38 and p53 attenuated only STE-induced S phase arrest. In conclusion, the present study demonstrates that STE induced DNA damage and triggered MAPK and p53 pathways activation, resulting in cell-cycle arrest at the S and the G2/M phase.

Hypothyroidism induces oxidative stress and DNA damage in breast.

Breast cancer and thyroid dysfunctions have been associated for decades. Although many studies suggest a biological correlation, the mechanisms linking these two pathologies have not been elucidated. Reactive oxygen species (ROS) can oxidize lipids, proteins, and DNA molecules and may promote tumor initiation. Hence, we aimed at evaluating the mammary redox balance and genomic instability in a model of experimental hypothyroidism. Female Wistar rats were treated with 0.03% methimazole for 7 or 21 days to evaluate ROS generation, antioxidant enzyme activities, and oxidative stress biomarkers, as well as genomic instability. After 7 days, lower catalase, GPX, and DUOX activities were detected in the breast of hypothyroid group compared to the control while the levels of 4-hydroxynonenal (HNE) were higher. In addition, hypothyroid group showed an increase in γH2Ax/H2Ax ratio. Twenty-one days hypothyroid group had increased catalase and SOD activities, without significant differences between groups in the levels of oxidative stress biomarkers and DNA damage. TSH-treated MCF10A cells showed a higher extracellular, intracellular, and mitochondrial ROS production. Additionally, greater DNA damage was observed in these cells, demonstrated by a higher comet tail DNA percentage and increased 53BP1 foci. Finally, we found that TSH treatment was not able to alter cell viability. The Genome Cancer Atlas (TGCA) data showed that high TSHR expression is associated with more invasive breast cancer types. In conclusion, we demonstrate that oxidative stress and DNA damage in breast are early events of experimental hypothyroidism. Moreover, high TSH levels induce oxidative stress and genomic instability in mammary cells.

Evaluation of irradiation quarantine dose – induced DNA damage in Rhyzopertha dominica and its effect on wheat grains using comet assay

Irradiation is a quarantine treatment option for stored product pests. In the present studies, we try to investigate the effect of low gamma irradiation doses in the range of 20–300 Gy on the different developmental stages of Rhyzopertha dominica in wheat grains. It was found that no adults emerged after irradiation of eggs, larvae, and pupae at 140, 240, and 260 Gy, respectively. Treatment of adult weevils at the radiation dose of 180 Gy resulted in 71.33% mortality after 14 days post-treatment and there was no adult emergence in the F1 generation. When the adult emergence of the F1 generation was used for the measuring effective irradiation dose and phytosanitary efficacy, 280 Gy was required for quarantine security of wheat weevils. The comet assay serves to verify the DNA damage induced by the phytosanitary irradiation dose, 280 Gy on the R. dominica, and wheat grains. DNA damage was measured as the percentage of comet tail DNA. A significant linear increase in DNA damage was observed in the irradiated adult stage of R. dominica but there is no DNA damage in the wheat grains. Also, the wheat grains viability (in terms of wheat germination) was not affected by the dose level, 280 Gy. Thus, an irradiation dose of 280 Gy is recommended as an effective phytosanitary irradiation dose for quarantine security to control lesser grain borer.

Waterborne Exposure of Adult Sand Dollar, Scaphechinus Mirabilis (Agassiz, 1864), to Zinc Ions and Zinc Oxide Nanoparticles Affects Early Development of its Offspring

Early development of the sand dollar, Scaphechinus mirabilis, and damage to DNA molecules in spermatozoa and larval cells were studied after a waterborne exposure of adult individuals to zinc ions (Zn2+) and zinc oxide nanoparticles (ZnO NP). It was found that the proportion of retarded and abnormal larvae increases if the parental individuals have been kept in aqueous solutions containing ionic and nanoform of zinc at concentrations of 100 and 200 μg/L. The percentage of DNA in comet tail and GDI was higher after the exposure to ZnO NP, compared with the Zn2+ exposure, at a concentration of 200 μg/L. Accumulation of zinc in soft tissues of S. mirabilis was also studied. The zinc concentrations in the soft tissues of both experimental groups of sand dollars proved to be higher than that in control groups. The results of the study have shown that the ionic form of zinc exhibits a lower bioavailability compared with ZnO NP and supported the higher toxic effect of the high concentrations ZnO NP.

Sclerotinia Sclerotiorum (White Mold): Cytotoxic, Mutagenic, and Antimalarial Effects In Vivo and In Vitro.

This work aimed includes performing the sclerotia chemical profile and evaluates their biological effects on mutagenesis, oxidative stress, cancer, and malaria. A chemical profile was determined by ultraperformance liquid chromatography mass spectrometry (UHPLC-HRMS) analysis dereplicating norditerpenoid dilactone, sclerolide, and other compounds. The GI50 values to cancer cells (19.8 to 277.6µg/mL) were higher than normal (16.05µg/mL), meaning high cytotoxicity. Regarding the oxidative stress, the results showed that the all AcOET fraction concentrations tested on IMR90 noncancer cell increased reactive oxygen species (ROS) production in more intense way (by fivefold) than in tested cancer cells. The in vivo study showed an increase of the following biomarkers (by 296.00%): % DNA in comet tail in peripheral blood and liver cells; micronucleated erythrocytes and colon cells and lipid serum peroxidation. These results indicate the sclerotia as genotoxic and mutagenic agent and its contamination may lead to fungal toxic effects with a risk to human health.

Usnic acid induces apoptosis in human gastric cancer cells through ROS generation and DNA damage and causes up-regulation of DNA-PKcs and γ-H2A.X phosphorylation

Usnic acid, a dibenzofuran derivative found in many lichen species, is reported to have anticancer activity against human gastric cancer. We investigated the molecular alterations associated with anticancer effects of usnic acid against human gastric adenocarcinoma AGS and gastric carcinoma SNU-1 cells. Usnic acid (10–25 μM) treatment to these cells caused a significant increase in mitochondrial membrane depolarization and apoptotic cells. Apoptosis induction was accompanied by an increase in the ratio of Bax:Bcl-2 expression and cleaved-PARP. Usnic acid increased the comet tail length and tail DNA in alkaline comet assay indicating DNA double-strand breaks which was also evidenced by an increase in γH2A.X (Ser139) phosphorylation. The expression of DNA damage response proteins including DNA-PKcs, pATM (Ser1981), Chk-2 and p53 were increased. Further, N-acetyl cysteine, a known reactive oxygen species (ROS) scavenger, reversed the effects of usnic acid on expression of DNA damage response proteins and γH2A.X (Ser139) phosphorylation. This reversal was also observed in comet assay in a time and dose-dependent manner suggesting that usnic acid-induced DNA damage was caused by ROS. In addition, the non-toxic concentrations (1–10 μM) of usnic acid inhibited colony forming potential of AGS cells indicating its anti-proliferation activity. More importantly, the concentration of usnic acid that caused significant death in gastric cancer cells, did not show any considerable toxicity to normal human embryonic kidney HEK293 cells, human keratinocyte HaCaT cells and mouse primary gastric cells. Collectively, these results for the first time demonstrated the selective apoptotic effect of usnic acid (10–25 μM) through ROS generation and DNA damage on human gastric cancer cells accompanied with upregulation of γH2A.X (Ser139) phosphorylation, DNA-PKcs and p53.

Toxicity assessment of magnesium oxide nano and microparticles on cancer and non-cancer cell lines

Testing of magnesium oxide nanoparticles (MgO NPs) on established cell lines at cellular levels using toxicological endpoints provide valuable information about their adverse effects upon exposure. In vitro toxicity assessment of MgO NPs and their microparticles was carried out at 50, 100, 200 and 400 µg/mL concentrations by using cytotoxicity, genotoxicity, oxidative stress, cellular apoptosis and cellular uptake studies in cancer (HepG2) and non-cancer (NRK 49F) cell lines after 24 h of treatment. IC50 concentration for MgO NPs was found to be > 400 µg/mL in both cell lines after 24 h treatment. A concentration dependent toxicity was noted in genotoxic studies and oxidative stress parameters. A significant increase in the comet tail DNA was recorded at 200 and 400 µg/mL concentrations of MgO NPs when compared with controls in HepG2 and NRK 49F cells. Exposure to MgO NPs led to an increase in the generation of reactive oxygen species (ROS) in both the cell types. Genotoxicity results were further supported by apoptotic analysis. MgO particles were found adhered to the cell membrane when assayed by ICP-OES. The results of this study showed that the MgO NPs were toxic at high concentrations only. Furthermore, MgO NPs are more toxic to cancerous cells compared to non-cancerous cells. ROS mediated genotoxicity was observed when treated with MgO NPs. The current study adds to the information on MgO particles. The results of this investigation may help in advancement of understanding of toxicological nature of MgO NPs and aid in their use.