Comet Assay Research Articles

Abstract A016: Targeting metabolic vulnerability of non-small cell lung cancer with annonacin and 2-deoxy-d-glucose in individual and combination modality

Abstract Non-small cell lung cancer (NSCLC) is a primary type of lung cancer and a leading cause of cancer-related mortality worldwide. The Warburg effect, characterized by increased glycolysis and reduced oxidative phosphorylation, is a hallmark of NSCLC and presents a therapeutic target. Annonacin, a natural compound found in fruits from the Annonaceae family, interferes with oxidative phosphorylation by inhibiting mitochondrial complex I. 2-deoxy-d-glucose (2DG), a glucose analog, disrupts glycolysis. This study examines the effects of Annonacin and 2DG on NSCLC A549 cells, both individually and in combination, with the hypothesis that inhibiting both metabolic pathways simultaneously will lead to more effective cancer treatment. In addition, we explored how these treatments might alter the tumor microenvironment, oxidative stress, and effects on normal epithelial bronchial cells. A549 NSCLC cells were exposed to different concentrations of Annonacin (0, 1, 2, 4, 6 µM) and 2DG (0,1.25, 2.5, 5, 10 mM), separately and together. Cell viability was measured using the MTT assay. Genotoxicity was assessed through the Comet Assay, and oxidative stress was evaluated by measuring superoxide dismutase and glutathione peroxidase activities. The long-term ability of the cells to proliferate was tested using colony-forming assays. The NL20 bronchial epithelial cell line was tested as a parallel control. Both Annonacin and 2DG showed a dose-dependent ability to kill A549 cells. However, when used together, these substances had a stronger effect, significantly lowering the number of surviving cells compared to when each was used alone. The response of this combination treatment on genotoxicity using Comet Assay is being pursued. Changes in oxidative stress responses were found, as shown by the altered superoxide dismutase and glutathione peroxidase enzyme activities. The ability of A549 cells to form colonies was greatly reduced following combination treatment, indicating a reduction in their long-term proliferative capacity. The combination of Annonacin and 2DG effectively inhibits the growth of NSCLC A549 cells by targeting their metabolic weaknesses, which may lead to increased DNA damage and oxidative stress. These results suggest simultaneous targeting glycolysis and oxidative phosphorylation could be a promising new approach for treating NSCLC. Future in vivo studies will evaluate this combination in live models and can offer insights into efficacy and possible translation to clinical application. Citation Format: Bhoj Raj Bhattarai, Cora Teets, Avinash Tope. Targeting metabolic vulnerability of non-small cell lung cancer with annonacin and 2-deoxy-d-glucose in individual and combination modality [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr A016.

Genotoxic impact of agricultural insecticides as contaminants of river Teesta on the resident fish Pethia Conchonius.

Fish, being highly sensitive to changes in the physico-chemical parameters of water, are good indicators of contamination. Teesta, a prominent northern West Bengal River system, is increasingly contaminated due to anthropogenic activities. This study aims to determine agricultural pesticide contamination and its genotoxic impact on the resident fish, Pethia conchonius, as an experimental organism. Sample water analysis from three riverine sites I, II & III, showed the presence of the insecticides imidacloprid (IMI), chlorpyrifos (CPF), bifenethrin (BF), cypermethrin (CP), difenthiuron, acetamiprid (AC) in the sites II and III only with adjoining agricultural lands. Comet assay revealed a significantly lower % Head DNA (~ 1.2 times), higher %Tail DNA (~ 16 times), and %Tail length (~ 3.1 times) in the gills of Pethia conchonius from sites II and III. About 4 and 10 times increase of micronuclei and other nuclear abnormalities were also noted in the erythrocytes of the fish from sites II and III than I, which was not contaminated. The antioxidant enzymes SOD, CAT, and GST activity and MDA levels were significantly higher (p < 0.05) in the liver samples from sites II and III, while AChE activity was significantly decreased (p < 0.001) in the brain tissues. Moreover, the sod, cat, and gpx expression in the hepatic cells were significantly upregulated compared to the β actin mRNA indicating increased oxidative stress. Increased genomic damage, antioxidant enzyme activity, higher MDA levels, decreased AChE activity in the brain, and the upregulation of hepatic genes strongly suggested the genotoxic effects of the detected insecticides in combination with other contaminants.

Investigating DNA damage caused by COVID-19 and influenza in post COVID-19.

The SARS-CoV-2 virus (termed COVID-19) was responsible for over 34 million global deaths. Although the COVID-19 pandemic has subsided, infection by emerging mutant variants of SARS-CoV-2 poses a continuing threat to public health. COVID-19 infection has been associated with the development of cytokine storm syndrome, hypercoagulability, immunological dysregulation and direct viral invasion of organs, and the long-term consequences for the health of COVID-19 survivors are currently unknown. Our research focuses on the possible mutagenic aspects of infection by COVID-19 and measures their harmful effects on DNA composition. DNA damage was investigated, using the comet assay method, during two periods: in the epidemic peak of COVID-19 and during the post-COVID-19 period, both in patients infected with COVID-19 and in those with influenza. During the epidemic peak, the levels of DNA damage ranged from the highest to the lowest levels in the following groups, respectively: intubated-ICU, non-intubated-ICU, non-ICU, and influenza, with a discernible increase in DNA damage in ICU-treated patients. The levels of DNA damage in the post-COVID-19 period were significantly lower compared to those in the epidemic peak period but there was still a discernible increase in DNA damage in the ICU group. Our results indicate that levels of DNA damage may be an effective indicator in prognostic decision-making and may therefore help to reduce mortality. Given that DNA damage and impaired repair processes can contribute to chronic diseases like diabetes, cancer, and neurodegenerative conditions, it will be crucial to investigate potential similar effects in patients with COVID-19.

Toxicity assessment following conventional radiation therapy and pulsed low dose rate radiation therapy: an in vivo animal study.

Pulsed low dose rate radiotherapy (PLDR) is a new radiation delivery method, in which the fractional dose is divided into sub-fractional doses with periodical time breaks in between. The goal of our study is to assess the toxicity on healthy tissues resulting from PLDR as compared to conventional radiotherapy (CRT) using the same physical X-ray dose. We analyzed the weight and survival time for CRT and PLDR groups and studied the inflammatory cytokine transforming Growth Factor-β (TGF-β), usually released following irradiation. Histopathological and immunohistochemical analyses were conducted for intestinal and bone marrow tissues from rats subjected to 8Gy whole- body irradiation using CRT and PLDR techniques. We investigated genotoxicity by performing a comet assay (CA) in splenic tissues. Our findings showed an improvement in survival time with PLDR versus CRT by 82%.The mean survival time for CRT rats' group was 6.3days, while it was 35.9days for PLDR group.The weight of CRT group decreased gradually by 3.7%, while weight of PLDR group increased gradually by 2.4%.CRT resulted in more cellular atrophy in bone marrow and intestinal tissues than in PLDR treatments as shown by hematoxylin and eosin staining analysis. In addition, the transforming growth factor-β (TGF-β) expression in bone marrow and intestinal tissues of CRT was higher than those expressed in tissues from PLDR as demonstrated by the Immuno reactive score (IRS). It was10(0.53) and 9.8(0.55) for BM and intestinal tissues, respectively from CRT group and 5.8(0.63) for PLDR for both tissues. The measured CA parameters were larger with CRT compared to PLDR, where the Tail Length (TL), Tail DNA % (TD%) and Tail Moment (TM) measurements were 25.4(3.4), 56.5(7.6)% and 20.5(3.5) for CRT, 7.3(1.9), 30.0(7.2)% and 5.7(1.8) for PLDR, with P value 0.000064, 0.0004 and 0.00017, respectively. This study indicates that PLDR can reduce the toxicity on normal tissues compared to CRT.

Cumulus cell DNA damage linked to fertilization success in females with an ovulatory dysfunction phenotype

Intracytoplasmic sperm injection (ICSI) is a widely used technique in fertility centers. ICSI success depends on both nuclear and cytoplasmic oocyte maturation. Cumulus cells, which surround the oocytes, play a pivotal role in oocyte competence. However, the significance of DNA damage in cumulus cells as a marker of fertilization success remains largely unexplored. This study aims to investigate the relationship between DNA damage in cumulus cells of females undergoing ICSI, and oocyte competence, with a focus on in vitro fertilization (IVF) outcomes. We employed the alkaline comet assay to assess DNA damage levels (%TDNA) in cumulus cells and whole blood from 22 potentially fertile females and 35 infertile females, including 20 with an ovulatory disfunction phenotype. Our results revealed significant differences between the levels of %TDNA in cumulus cells and blood. Females with an ovulatory dysfunction phenotype exhibited higher levels of %TDNA in cumulus cells compared to potentially fertile females. Additionally, within the group of females with ovulatory dysfunction, a significant correlation was observed between %TDNA levels and the number of oocytes with two pronuclei. Our findings suggest that blood does not accurately reflect DNA damage in cumulus cells, which was correlated with the fertilization success in females with ovulatory dysfunction. High levels of %TDNA in cumulus cells were associated with a higher likelihood of successful fertilization. Moreover, our results imply that low levels of %TDNA may be linked to oocytes that fail to complete maturation and, consequently, do not fertilize (oocytes with zero pronuclei). Further research with larger cohorts is necessary to validate these findings and to explore potential applications in female fertility. However, our study provides evidence that DNA damage in cumulus cells may serve as a valuable biomarker for predicting fertilization success and oocyte competence.

Enhancing the anticancer effect of metformin through nanoencapsulation: Apoptotic induction, inflammatory reduction, and suppression of cell migration in colorectal cancer cells.

Colorectal cancer (CRC) continues to be a significant health challenge, necessitating the development of efficient therapeutic strategies. Drug repurposing, which involves the use of existing medications for new purposes, presents a promising opportunity. Metformin, a widely used antidiabetic drug, has demonstrated potential anticancer effects. To enhance its efficacy, we formulated nano-metformin, metformin encapsulated within pectin nanoparticles. Our study aimed to evaluate the superiority of nano-metformin over free metformin in treating CRC. The cytotoxicity of both metformin and nano-metformin on Caco-2 CRC cells was assessed using the MTT assay, revealing a significant dose-dependent inhibition of cell growth using nano-metformin. The anti-inflammatory potential was evaluated by measuring the levels of nitric oxide and the pro-inflammatory cytokines IL-2 and IL-6 following lipopolysaccharide (LPS) induction, and the results revealed that treating LPS-induced cells with nano-metformin significantly reduced the production of these inflammatory mediators. To elucidate the mechanism of cell death, we employed an acridine orange/ethidium bromide staining assay, which revealed the enhancement of apoptotic cell death following treatment with nano-metformin. Additionally, we examined the expression of key apoptotic regulators using real-time qPCR. Nano-metformin, in particular, significantly downregulated the expression of the antiapoptotic markers Bcl-2 and Survivin while upregulating the proapoptotic caspases 3, 7, and 9. The comet assay revealed significant DNA damage induced by treatment with the nano-metformin compared with that in the free form. Moreover, nano-metformin significantly reduced the migration ability of cells. In conclusion, our work revealed the superior efficacy of our formulated nanoform over free metformin, highlighting its potential as a promising therapeutic agent for CRC treatment.

Effect of Mexel®432 on DNA Damage in Haliotis Discus Hannai and Mesocentrotus Nudus

Mexel®432 is a surfactant-like substance that is a biosuppressant against marine fouling organisms. Therefore, to study the toxic effect of Mexel®432 on marine organisms, the wrinkled abalone and echinococcus was exposed to concentrations of 0.175 mg·L-1, 0.350 mg·L-1, 0.700 mg·L-1, 0.275 mg·L-1, 0.550 mg∙L−1, 1.100 mg·L-1 Mexel®432 solution, and single-cell gel electrophoresis (comet experiment) was used to detect different exposure times (0, 10, 20 d) DNA damage effect of digestive gland cells and echinococcus pallidae intestinal cells in wrinkled disc abalone, and tail DNA content, tail length, tail distance, and Olive tail distance were used as evaluation indexes. The results showed that different concentrations of Mexel®432 could cause DNA disintegration and tailing in digestive gland cells and intestinal cells in a short period of time (10 d), and with the increase of Mexel®432 concentration and the extension of exposure time, DNA deformation was obvious, the fragmentation increased, and the degree of damage was aggravated. When the concentration of Mexel®432 reaches 0.350 mg·L-1, it produces certain genotoxicity to the digestive gland cells of wrinkled abalone, and when the concentration of Mexel®432 reaches 1.1000 mg·L-1, it also has certain genotoxicity to the intestinal cells of Mesocentrotus nudus. The experimental results showed that Mexel®432 had a significant dose effect relationship on DNA damage in the cells of two biological tissues.

Carvacrol modulates antioxidant enzymes, DNA integrity, and apoptotic markers in zearalenone-exposed fetal rat liver

Maternal exposure to zearalenone (ZEA), a mycotoxin, can impact fetal liver development. This study investigated the protective effects of carvacrol (CRV) against ZEA-induced fetal liver damage. Thirty-two pregnant rats were allocated to four groups (eight rats/group); control, CRV (75 mg/kg), ZEA (5 mg/kg), and co-treated group (ZEA + CRV). The animals were given their doses during the gestation period. Maternal exposure to ZEA revealed a significant increase in the malondialdehyde (MDA) level in the fetal liver. In contrast, glutathione S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) activities, besides glutathione (GSH) levels, were decreased in ZEA-intoxicated rats. Additionally, ZEA increased the expression of pro-apoptotic genes (P53, Bax, and caspase-9), elevated the immunoreactivity of caspase-3, decreased anti-apoptotic Bcl-2, and induced severe fatty degeneration, congestion, and necrosis in the fetal liver. The comet assays revealed significant DNA damage, as evidenced by reduced head DNA content and increased tail DNA content and tail moment in the ZEA-exposed rats. Surprisingly, co-treatment with CRV significantly mitigated fetal hepatic lipid peroxidation, antioxidant disturbance, apoptosis, and DNA damage after maternal exposure to ZEA. These findings highlight the potential of CRV as a promising approach to mitigate ZEA-associated developmental hepatotoxicity.

DDDR-06. ENHANCING GLIOBLASTOMA TREATMENT WITH MOLYBDENUM DISULFIDE(MOS2) NANOMATERIALS AND ULTRASOUND-TRIGGERED TUMOR GROWTH INHIBITION

Abstract INTRODUCTION Glioblastoma, known for its high mortality rate and limited treatment options, poses a persistent challenge in oncology. Despite advances in surgery, radiation, and chemotherapy, patient outcomes have seen only marginal improvements, with ongoing issues of high recurrence and low survival rates. The advent of nanotechnology, particularly the emergence of Molybdenum disulfide (MoS2) nanoparticles, offers new perspective in semiconductors, energy storage as well as cancer therapies. With its unique physical and chemical properties, MoS2 nanoparticles might be promising in its potential roles in the battle against cancer. We aimed to see whether ultrasound-treated MoS2 nanoparticles can interfere with growth of glioblastoma cells. METHODS MoS2 nanoparticles were added to the medium of glioblastoma cells and ultrasound treatments were given. We monitor the cell count as well as cell migration after the treatments. RESULTS MoS2 nanoparticles was found to significantly interfere with tumor cell proliferation and boost the production of reactive oxygen species (ROS). These reactive oxygen species were crucial for the apoptosis of brain tumor cells when in the right concentration. Our analysis also demonstrated that MoS2 impedes cell migration and infiltration, effectively halting tumor expansion. Additionally, Comet assay techniques confirmed extensive DNA damage in the treated cells, highlighting MoS2’s potent DNA-altering capabilities which promote programmed cell death. CONCLUSION The implications of these findings are significant, establishing a solid scientific basis for employing MoS2 nanoparticles as a targeted anti-cancer agent for brain tumors. While this study underscores the transformative potential of MoS2 nanoparticles in clinical oncology, it also acknowledges limitations such as the need for further clinical trials to evaluate efficacy and safety in humans. Key Words: MoS2 nanoparticles, Ultrasound treatment, Reactive oxygen species, Comet assay, Programmed cell death

Carboplatin-loaded zeolitic imidazolate framework-8: Induction of antiproliferative activity and apoptosis in breast cancer cell.

The challenge with breast cancer is its ongoing high prevalence and difficulties in early detection and access to effective care. A solution lies in creating tailored metal-organic frameworks to encapsulate anticancer drugs, enabling precise and targeted treatment with less adverse effects and improved effectiveness. Zeolitic imidazolate framework-8 (ZIF-8) and carboplatin (CP)-loaded ZIF-8 were synthesized and characterized using various analytical techniques. High Resolution-transmission electron microscopy of ZIF-8 and CP@ZIF-8 indicates that the particles had a spherical shape and were nanosized. The drug release rate of CP is 98% under an acidic medium (pH 5.5) because of the dissolution of ZIF-8 into its coordinating ions, whereas 35% in a physiological medium (pH 7.4) with the addition of CP, the high porosity, and pore diameter of ZIF-8 decrease from 1243 to 1041m2/g. Breast cancer MCF-7 cells were shown greater IC50 in CP@ZIF-8 (15.01±3.03µg/mL) than free CP (34.98±4.25µg/mL) in an in vitro cytotoxicity assessment. The cytotoxicity of the CP@ZIF-8 against MCF-7 cells was studied using the methylthiazolyldiphenyl-tetrazolium bromide method. The morphological changes were examined using fluorescent staining (acridine orange-ethidium bromide and Hoechst 33258) methods. The comet assay assessed the DNA fragmentation (single-cell gel electrophoresis). The results from the study revealed that CP@ZIF-8 can be used in the treatment of breast cancer.

Exploring antitumor activity of novel imidazo[2,1-b]thiazole and imidazo[1,2-a]pyridine derivatives on MDA-MB-231 cell line: Targeting VEGFR-2 enzyme with computational insight

Our study explored the anticancer potential of novel heterocyclic compounds, specifically 6a-d, 8a-d, 12a-d, and 13a-d, which are derived from imidazo[2,1-b]thiazole and imidazo[1,2-a]pyridine and linked through a hydrazide moiety. These compounds were assessed for their anticancer activity toward the MDA-MB-231 breast cancer cell line to evaluate their effectiveness in inhibiting cancer cell proliferation. Among the compounds tested, 13c and 13d emerged as the most active, with IC50 values of 4.40 ± 2.87 µM and 4.69 ± 2.55 µM, respectively. These two compounds were further investigated for their effects on VEGFR-2 enzymatic activity, cell cycle progression, and apoptosis, then 13c and 13d were further evaluated for their impact on DNA fragmentation using the comet assay. Both compounds demonstrated a significant increase in DNA fragmentation, with levels of damage being twice as high as those observed in the untreated control cells. Molecular docking studies through VEGFR-2 demonstrated that compounds 13c and 13d bind to VEGFR-2 in a manner comparable to the co-crystallized ligand sunitinib I. Further analysis using density functional theory highlighted their favorable physical properties. Additionally, computational evaluations of ADME and drug-likeness suggest that these derivatives hold promise and merit further investigation. The ultimate goal is to develop effective, safe, and orally bioavailable VEGFR-2 inhibitors for potential use in anticancer therapy.

Shikonin Stimulates Mitochondria-Mediated Apoptosis by Enhancing Intracellular Reactive Oxygen Species Production and DNA Damage in Oral Cancer Cells.

Phytotherapy has rendered a new insight towards the treatment of various cancers, including oral cancer with fewer side effects, over the traditional chemotherapeutic drugs to overcome chemoresistance. Shikonin (Shk) is a natural biologically active alkaloid found in the Lithospermum erythrorhizon plant's root. It has potent cytotoxic activities against various cancers. Our study revealed the release time and anticancer potential of Shk on the SCC9 and H357 oral cancer cell lines. We investigated the antiproliferative, antimigratory, cell cycle arresting and apoptosis promoting activity of Shk in oral cancer cells by performing MTT and morphological assay, colony, and tumor sphere formation assay, AO/EtBr and DAPI staining, Annexin V-FITC/PI staining, assay for reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) measurement, comet assay, qRT-PCR, and western blot analysis. We also checked the interaction of DNA and Shk by docking and CD spectroscopy and EtBr displacement assay. As a result, we found that Shk reduced the viability, proliferation, and tumorigenicity of SCC9 and H357 cells in a time and concentration-dependent manner. We obtained half-maximal inhibitory concentration (IC50) at 0.5 µM for SCC9 and 1.25 µM for H357. It promotes apoptosis via overexpressing proapoptotic Bax and caspase 3 via enhancing ROS that leads to MMP depletion and DNA damage and arrests cells at the G2/M & G2/S phase. The antimigratory activity of Shk was performed by analyzing the expression of markers of epithelial-mesenchymal transition like E-cadherin, ZO-1, N-cadherin, and vimentin. These overall results recommended that Shk shows potent anticancer activity against oral cancer cell lines in both in vitro and ex vivo conditions. So, it could be an excellent agent for the treatment of oral cancer.

Disperse Red 1 azo dye: Consequences of low-dose/low-concentration exposures in mice and zebrafish

Color Index Disperse Red 1 (DR1), an azo dye widely used in the textile industry and released into aquatic environments, is genotoxic in somatic cells, but little is known concerning its effects on the reproductive system or the early stages of embryonic development. We have assessed the effects on the spermatozoa of male mice following oral exposure to the dye, at low doses, for 14 days. Measured endpoints were DNA damage (comet assay), miRNA-34c levels, and sperm number, morphology, and motility. Exposure caused decreased miRNA-34c levels. We have also examined dye effects on zebrafish embryos and larvae, which included developmental impairment, altered glutathione transferase activity, and effects on reactive oxygen species and lipid peroxidation levels.

In vitro hepatic 3D cell models and their application in genetic toxicology: a systematic review

The rapid development of new chemicals and consumer products has raised concerns about their potential genotoxic effects on human health, including DNA damage leading to serious diseases. For such new chemicals and pharmaceutical products, international regulations require genotoxicity data, initially obtained through in vitro tests, followed by in vivo experiments, if needed. Traditionally, laboratory animals have been used for this purpose, however, they are costly, ethically problematic, and often unreliable due to species differences. Therefore, innovative more accurate in vitro testing approaches are rapidly being developed to replace, refine and reduce (3R) the use of animals for experimental purposes and to improve the relevance for humans in toxicology studies. One of such innovative approaches are in vitro three-dimensional (3D) cell models, which are already being highlighted as superior alternatives to the two-dimensional (2D) cell cultures that are traditionally used as in vitro models for the safety testing of chemicals and pharmaceuticals. 3D cell models provide physiologically relevant information and more predictive data for in vivo conditions. In the review article, we provide a comprehensive overview of 3D hepatic cell models, including HepG2, HepG2/C3A, HepaRG, human primary hepatocytes, and iPSC-derived hepatocytes, and their application in the field of genotoxicology. Through a detailed literature analysis, we identified 31 studies conducted between 2007 and April 2024 that used a variety of standard methods, such as the comet assay, the micronucleus assay, and the γH2AX assay, as well as new methodological approaches, including toxicogenomics, to assess the cytotoxic and genotoxic activity of chemicals, nanoparticles and natural toxins. Based on our search, we can conclude that the use of in vitro 3D cell models for genotoxicity testing has been increasing over the years and that 3D cell models have an even greater potential for future implementation and further refinement in genetic toxicology and risk assessment.

MiR-195-5p inhibits cisplatin resistance in lung adenocarcinoma by regulating DNA damage via targeting E2F7.

Lung adenocarcinoma (LUAD) emerges as one of the most lethal malignant tumors worldwide. Platinum-based combination chemotherapy remains one of the main methods for patients with advanced LUAD. Due to the resistance, the effect of this chemotherapy was not satisfactory. Therefore, studying the mechanism of cisplatin (DDP) resistance is essential for promoting the effect of this therapeutic strategy. Therefore, this work sought to probe the impact of E2F Transcription Factor 7 (E2F7) on LUAD resistance and the molecular regulatory mechanism. The mRNA expression level of the target gene E2F7 in LUAD was predicted by bioinformatics analysis, and regulatory miRNA upstream of the target gene was identified. The mRNA and protein expression of E2F7 in LUAD cells was detected through quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot, respectively. The expression of miR-195-5p in LUAD cells was measured via qRT-PCR. E2F7 high and low expression groups underwent enrichment analysis by utilizing Gene Set Enrichment Analysis software. The targeting relationship of miR-195-5p and E2F7 was validated by conducting a dual-luciferase reporter assay. The cell viability was tested through cell counting kit-8. The cell cycle was examined by flow cytometry. DNA damage level was determined via Comet assay and Western blot assay. The findings indicated that the mRNA and protein levels of E2F7 were high in LUAD. MiR-195-5p was the regulatory miRNA upstream of E2F7, and lowly expressed in LUAD. The cell experiments suggested that E2F7 advanced the DDP resistance of LUAD cells by repressing DNA damage. Finally, the rescue assay manifested that miR-195-5p overexpression could abate inhibition of E2F7 overexpression on the DNA damage and the DDP sensitivity of LUAD cells. MiR-195-5p raised the DDP sensitivity of LUAD cells by advancing the DNA damage in LUAD cells via inhibition of E2F7.

Antioxidant response fail to rescue growth of Hermetia illucens L. larvae induced by copper accumulated during long-term exposure

Antioxidant indices and hemocytes apoptosis in the 6th instar larvae of Hermetia illucens., and their correlation with larval growth were evaluated by exposing larvae to different concentrations of Cu2+ for 1, 3 and 5 generations. Cu2+ accumulated in larval hemolymph showed significant dose-dependent relationship with Cu2+ concentrations in diets within a generation. Larval growth was only promoted after low concentrations of Cu2+ exposure for 1 generation, while seriously affected after high concentrations of Cu2+ exposure. Though total antioxidant capacity activity in larval hemolymph in treatment groups was all higher than that in control, it was increased at lower levels of Cu2+, while decreased with increasing Cu2+ concentrations at higher levels of Cu2+ exposure. The catalase (CAT) activity and metallothioneins (MTs) levels were also characterized as improved at lower levels of Cu2+, and inhibited at higher levels of Cu2+ exposure. However, CAT activity and MTs levels at higher Cu2+ treatments were significantly lower than that in control. Apoptosis rate of hemocytes was increased with increasing Cu2+ concentrations. Annexin V - fluorescein isothiocyanate (FITC)/ propidium iodide (PI) staining was in accordance with the results exhibited in flow cytometer. Results from transmission electron microscope and comet assay further confirmed that membrane blebbing, nuclear condensation, and DNA fragmentation were gradually apparent with increasing Cu2+ concentration. All parameters in different generation had similar dose-dependent trends, but the effects were strongest in the fifth generation. This study indicated that at some extent growth of H. illucens were associated with antioxidant responses and apoptosis induced by Cu2+.

Fipronil exposure alters oxidative stress responses of Nile tilapia (Oreochromis niloticus) to acute moderate hypoxia

Acute hypoxia is known to increase the generation of reactive oxygen species (ROS), leading to modulation in antioxidant defenses. Pollutant exposure can potentiate ROS generation during hypoxic events and impair antioxidant defenses, increasing the susceptibility of hypoxia-tolerant fishes, such as the Nile tilapia (Oreochromis niloticus), to oxidative stress. The purpose of this study was to evaluate oxidative stress responses of O. niloticus to acute (3 and 8 h) moderate hypoxia (dissolved oxygen ≤2 mg/L−1) and how these responses are affected by simultaneous exposure to the insecticide fipronil (0.1 and 0.5 µg L−1). Hypoxia exposure for 3 h caused an increase in glutathione peroxidase (GPx) activity in the gill and also increased catalase (CAT) and glutathione S-transferase (GST) activities in the liver. After 8 h of hypoxia, glutathione reductase (GR) activity increased. DNA damage (comet assay) in erythrocytes was reduced by hypoxia after 3 and 8 h. Fipronil exposure for 3 h decreased CAT activity in the gill, both under normoxia and hypoxia. After 8h, the combination of fipronil and hypoxia increased GR activity in the gill. In the liver, fipronil exposure under hypoxia for 3 h increased CAT and GR activities; after 8 h, CAT was decreased, and GST increased. GR was also increased by fipronil under normoxia for 8 h. All treatments reduced lipid peroxidation levels in the gills, but in the liver, lipid peroxidation was increased by fipronil after 3 h under normoxia. Moreover, fipronil exposure under hypoxia for 3 and 8h increased DNA damage in erythrocytes, while 8 h of fipronil exposure under normoxia decreased it, suggesting the activation of DNA repair mechanisms. Results show that both fipronil and hypoxia exposure significantly modulate the oxidative stress parameters of O. niloticus and that the combination of these factors produces more pronounced effects.

Electrochemical voltammetry assessment of cellular DNA damage caused by drugs with different mechanisms: A comparative analysis with the γH2AX focus and alkaline comet assays

As industrialization accelerates, there is an increasing need to find a quick, easy, and affordable method to assess the potential DNA damage caused by newly developed chemicals. Electrochemical voltammetry using intracellular purine bases as markers shows promise for detecting DNA damage. However, it is unclear whether drugs with different DNA damage mechanisms will impact purine metabolism in cells and consequently disturb the electrochemical indicators of DNA damage. In this paper, the electrochemical behaviors of HepG2 cells exposed to the direct DNA-damaging agent (MNNG), the indirect DNA-damaging agent (CPT11), and the non-DNA-damaging agent (KCl) were studied. The findings showed that the electrochemical signals of cells increased with the rising concentration of DNA-damaging drugs. A time-dependent effect was observed, with a lower threshold dose for 4 h compared to 1 h. However, the non-DNA-damaging agent did not induce an increase in the cellular electrochemical signal. Despite the higher cytotoxicity of CPT11, MNNG triggered DNA damage at a lower dose, as detected by electrochemical indicators. The results of the electrochemical indicators assessment of DNA damage correlated significantly with those of the γH2AX focus and alkaline comet assays, with r > 0.98 and P < 0.01. Particularly, there was a stronger correlation with the alkaline comet assay. Furthermore, electrochemical voltammetry detected DNA damage at a lower threshold compared to other assessment methods, demonstrating a higher sensitivity to DNA damage. These findings suggest that by using purine bases as markers, electrochemical voltammetry enables precise assessment of DNA damage caused by various mechanisms.

Study of acute and subacute toxicities and genotoxic and mutagenic potentials of the lyophilized extract of Campomanesia sessiliflora (O.Berg) mattos leaves in wistar rats

Campomanesia sessiliflora (O.Berg) Mattos is a Brazilian native plant species used in a popular medicinal tea for treating gastrointestinal, urinary, and dermatological pathologies. This study evaluated the toxicity of Campomanesia sessiliflora (O.Berg) Mattos via acute and subacute toxicity tests. It also analyzed mutagenic and genotoxic potentials by the micronucleus test, which detects genetic material damage indicating mutagenicity, and the comet assay, which assesses DNA damage levels as a genotoxicity indicator. The plant extract initially originated from the ultrasonic maceration of Campomanesia sessiliflora (O.Berg) Mattos leaves in a hydroethanolic solution. The involved animals were adult Wistar rats. Ten females were available to evaluate acute toxicity and estimate the LD50, receiving a dose of 2000 mg/kg. The subacute toxicity evaluation used 35 females and 35 males divided into seven groups: negative control (saline control – SC), positive control (cyclophosphamide control – CC), 125 mg/kg (125), 250 mg/kg (250), 500 mg/kg (500), 1000 mg/kg (1000), and the satellite group (ST). Genotoxicity and mutagenicity experiments applied bone marrow micronucleus and comet assays. Acute and subacute toxicity tests did not present behavioral, physical, and physiological changes (p≥0.05). Administering the Campomanesia sessiliflora (O.Berg) Mattos extract reduced spleen size in male and female animals, without histopathological changes. However, doses above 500 mg/kg showed significant genotoxic and mutagenic effects in the comet and micronucleus assays compared to the control group. The extract did not exhibit acute or subacute toxicity, but doses higher than 500 mg/kg indicated some level of genotoxicity and mutagenicity.

Evaluating the impact of the combined acute toxicity of iron (Fe) and microplastics on Namalycastis jaya

The rising concern over heavy metals (HMs) and microplastics (MPs) pollution in marine ecosystems, primarily driven by anthropogenic activities, poses significant threats to ecological health. Understanding the combined exposure of HMs and MPs aids in toxicity assessment. In this study, we examined the combined effects of polystyrene microplastics (MPs) and iron (Fe) on oxidative stress, bioaccumulation, histopathology, and genotoxicity in Namalycastis jaya. Oxidative stress was assessed by analyzing the levels of Superoxide dismutase (SOD), Catalase (CAT), Peroxidase (POD), Malondialdehyde (MDA), and Bicinchoninic acid (BCA), while genotoxicity was evaluated using the comet assay. Bioaccumulation analysis, conducted via Inductively coupled plasma-optical emission spectrometry (ICP-OES), indicated that the highest values (4.790 µg/ml) were observed in combined exposure, emphasizing the significant increase in iron (Fe) accumulation in polychaetes facilitated by MPs. Biochemical analysis revealed that oxidative damage in polychaetes became evident within 48 h of exposure to individual contaminants. However, in the case of combined exposures, elevated stress levels were observed within just 24 h. The genotoxic assay further demonstrated a higher degree of DNA damage in the combined exposure compared to individual exposures. Similarly, histopathology revealed mild alterations in the gut epithelium in combined exposures. It is evident that MPs intensify both oxidative and DNA damage induced by Fe in polychaetes. The insights gained from this study provide valuable information for the risk assessment of Fe and MPs in environmental safety, contributing to our understanding of the complex interactions between these pollutants in marine ecosystems.