Comet Assay Research Articles

The potential effect of temulawak (Curcuma xanthorrhiza Roxb.) and garlic (Allium sativum L.) as a radioprotective agent against 6 Gy total body irradiation in rats

The main impact of gamma radiation on a biological system is the formation of Reactive Oxygen Species (ROS) and free radicals. The study aimed to explore the potential effect of temulawak (Curcuma xanthorrhiza Roxb.) and garlic (Allium sativum L.) against 6 Gy total body irradiation (TBI) in rats by observing malondialdehyde (MDA), glutathione (GSH) and comet assay. Twenty male rats were divided into five groups: control, 6 Gy, temulawak extract + 6 Gy, garlic extract + 6 Gy, and n-acetyl cysteine (NAC) + 6 Gy. MDA and GSH were measured on liver and spleen tissue homogenates, while comet assay was on lymphocyte cells. Gamma irradiation at 6 Gy significantly increased the MDA level and comet assay compared to the control group, while the GSH level decreased (p < 0.05). Temulawak extract significantly reduced MDA levels and comet assay compared to the 6 Gy group while increasing GSH levels in the liver. Garlic extract significantly drops comet assay while increasing GSH levels in the liver. NAC decreases MDA levels in the liver and comet assay while increasing GSH levels in the spleen. It could be concluded that temulawak extract has a better radioprotective agent than garlic extract and is almost identical to NAC as a standard antioxidant.

Increased Sperm DNA Damage in Sprague-Dawley Rats Exposed to Dextromethorphan as an Antitussive

Introduction: Male reproductive functions have been observed to be negatively impacted by the antitussive dextromethorphan (DM). It was also discovered that DM has an impact on micronutrients (such as zinc, calcium, and selenium) which are crucial for both capacitation and the acrosome reaction, which can result in infertility. Using Sprague-Dawley rats as models, we investigated the effects of quercetin and rutin on DM-induced toxicity in males in relation to apoptotic protein markers. Methodology: For a period of 16 weeks, 80 male rats, weighing 150 ± 30 g, were utilized and split into four groups. Twenty animals were employed in each group. For a period of 16 weeks, Group B received 20 mg/kg, Group C received 40 mg/kg, and Group D received 80 mg/kg of DM. Group A acted as the control group and received 1 ml of distilled water (DW). Five randomly chosen animals from each group were put to sleep at the end of the DM treatment period, and their testes were taken out to measure DNA fragmentation using comet assay. To ascertain the pace of recovery, the animals were then split into three groups, E–G, and kept for a total of 16 weeks. Group G received 1 ml of DW, Group F was given rutin (25 mg/kg), and Group E was given quercetin (50 mg/kg). Results: Comparing DM-treated groups to control revealed a significant dose-dependent decrease in DNA fragmentation. When DM-treated and recovery-alone groups were contrasted with rutin and quercetin groups, an improvement in DNA was seen. Conclusion: Significant improvements in the parameters were observed when rutin and quercetin supplements were used, which may lessen the harmful effects of DM and enhance male fertility.

Investigation of Genotoxic and Development Effects of Tetramethrin on Drosophila melanogaster

The adverse effects of tetramethrin residues, a synthetic pyrethroid used in many insecticide formulations, on environmental health and living organisms are a matter of concern. The aim of this study was to evaluate the genotoxic and developmental effects of tetramethrin in a non-target organism, Drosophila melanogaster. Thus, its effect on DNA damage was evaluated using the Comet assay in hemocytes, and its mutagenic and recombinogenic effects were evaluated using Drosophila wing SMART. Also, the effects of tetramethrin on Drosophila development were evaluated by measuring larval weight, larval length, and fecundity. Results showed that tetramethrin induced a decrease in the larval weight and length only at a high concentration. Moreover, a decrease in fecundity in a dose-dependent manner was observed. According to the Comet assay results, DNA damage was not induced because there was no significant increase in % DNA. However, tetramethrin caused genotoxicity by inducing mitotic recombination in the SMART assay.

Anticancer Effects of Ethanolic Extracts of Macaranga Peltata Leaves on Human Oral Squamous Carcinoma Cell Lines: An In Vitro Study

ABSTRACT Cancer is a disease resulting from the disruption of cell cycle regulation, leading to the abnormal and unchecked proliferation of cells. Medicinal plants are rich in various bioactive phytochemicals or nutritional compounds. The aim is to determine the cytotoxic and genotoxic effects of ethanolic extracts of Macaranga peltata leaves on human oral cancer cell lines. The study setting was centre for Research on Molecular and Applied Sciences, Azeezia College of Dental Sciences and Research. The study design is a Comparative In Vitro study. Shade dried leaves of Macaranga peltata were subjected to Soxhlet extraction, and ethanolic extract was prepared. In vitro cytotoxic effects on human oral cancer cell lines were evaluated by (3-(4,5-dimethyl thiazole-2yl)-2,5–diphenyl tetrazolium bromide) MTT assay, and genotoxic effect was evaluated by comet assay. Untreated cell lines were used as control, and 5-fluorouracil was used as positive control. All experiments were performed in triplicates, and results were represented as Mean+/- SE. One-way ANOVA and Dunnet test were performed to analyze data. ***P < 0.001 compared with the control group. The ethanolic extract of Macaranga peltata exhibited cytotoxicity against oral cancer cells (LC50: 40.193089 μg/ml). There was a concentration-dependent increase in cell death, and at 100 μg/ml, the extract was most effective, causing 50% inhibition of viability. The comet assay showed significant genotoxic effects compared with 5-fluorouracil and untreated oral cancer cell lines. The ethanolic extract of Macaranga peltata leaves was subjected to MTT assay and comet using KB cell lines. The study concludes that the extract gave promising result for the anticancer activity on the KB cell lines producing cytotoxicity and genotoxicity.

Enhanced Biocompatibility by Evaluating the Cytotoxic and Genotoxic Effects of Magnetic Iron Oxide Nanoparticles and Chitosan on Hepatocellular Carcinoma Cells (HCC)

Hepatocellular carcinoma (HCC), the fifth most prevalent cancer worldwide, is influenced by a myriad of clinic-pathological factors, including viral infections and genetic abnormalities. This study delineates the synthesis, characterization, and the biological efficacy of iron oxide nanoparticles (Fe3O4) and chitosan-coated iron oxide nanoparticles (Fe3O4-CS) against HCC. Analytical methods confirmed the successful synthesis of both nanoparticles, with Fe3O4-CS demonstrating a smaller, uniform spherical morphology and distinct surface and magnetic properties attributable to its chitosan coating. The prepared materials were analyzed using various techniques, and their potential cytotoxic effects on HepG2 cancer cells line for HCC were investigated. In biological evaluations against HepG2 cells, a notable distinction in cytotoxicity was observed. Fe3O4 showed modest anticancer activity with an IC50 of 383.71 ± 23.9 µg/mL, whereas Fe3O4 exhibited a significantly enhanced cytotoxic effect, with a much lower IC50 of 39.15 ± 39.2 µg/mL. The Comet assay further evidenced Fe3O4-CS potent DNA damaging effect, showcasing its superior ability to induce apoptosis through extensive DNA fragmentation. Biochemical analyses integrated into our results reveal that Fe3O4-CS not only induces significant DNA damage but also markedly alters oxidative stress markers. Compared to control and Fe3O4-treated cells, Fe3O4-CS exposure significantly elevated levels of oxidative stress markers: superoxide dismutase (SOD) increased to 192.07 U/ml, catalase (CAT) decreased to 0.03 U/L, glutathione peroxidase (GPx) rose dramatically to 18.76 U/gT, and malondialdehyde (MDA) levels heightened to 30.33 nmol/gT. These results underscore the potential of Fe3O4-CS nanoparticles not only in inducing significant DNA damage conducive to cancer cell apoptosis but also in altering enzymatic activities and oxidative stress markers, suggesting a dual mechanism of action that may underpin their therapeutic advantage in cancer treatment. Our findings advocate for the further exploration of Fe3O4-CS nanoparticles in the development of anticancer drugs, emphasizing their capability to trigger oxidative stress and enhance antioxidant defense mechanisms.

In Vitro Antiviral and Anticancer Effects of Tanacetum sinaicum Essential Oil on Human Cervical and Breast Cancer.

Cervical cancer has been linked to human papillomavirus (HPV) types 16 and 18. Essential oils (EOs) are vital natural products of plants with various therapeutic and biological properties. The purpose of this study is to investigate and assess Tanacetum sinaicum essential oil's possible antiviral and anticancer properties, with a focus on its in vitro effects on human cervical cancer and human breast adenocarcinoma cell lines. Tanacetum sinaicum EO was extracted via hydrodistillation (HD) and characterized using gas chromatography-mass spectrometry (GC-MS). MTT assay was used to determine the cell viability of Hela (a human epithelial cervical cancer) and MCF-7 (human breast adenocarcinoma) cell lines. Quantitative real-time polymerase chain reaction (PCR) was utilized to assess the antiviral efficacy of EO against HPV-16 and 18, and anti-metastatic characteristics. The biological activity of EO was assessed using Autophage and Cell genotoxicity via the comet assay. EO is mostly composed of chrysanthenyl acetate, thujone, and verbenol. The cell viability was reduced after 24 hours of incubation at doses from 100 to 400 µg/ml. Concentrations of 800 to 3,200 µg/ml significantly inhibit cell growth. After a 24-hour incubation period, doses ranging from 100 to 400 µg/ml reduced cell viability from 62 to 72%. Concentrations of 800 to 3,200 µg/ml significantly suppress cell growth by over 95%. In MCF7 and HeLa cell lines, EO lowered virus copy numbers in a dose-dependent manner, with higher concentrations of the oil inhibiting virus replication more effectively. EO treatment increased the number of autophagosomes/autolysosomes and acidic vesicular organelles in both cell lines. On the HeLa and MCF7 cell lines, EO demonstrated antiproliferative and antimetastatic effects. The results demonstrated that EO had dose-dependent genotoxic effects on both cancer cell lines, as evidenced by DNA damage. Tanacetum sinaicum EO is a prospective source of natural bioactive compounds that can be employed in pharmaceutical and medicinal applications due to its antiviral, antiproliferative, anti-metastatic and genotoxic properties.

Does the Symbiotic Relationship Between Hydra Viridissima and Photoautotrophic Alga Provide an Evolutionary Advantage in Protecting DNA against Damage by the Cytotoxic or Genotoxic Mode of Action of Environmental Stressors?

The aim of this paper was to evaluate whether symbiotic cooperation between green hydra (Hydra viridissima) and photoautotrophic alga gives higher resistance of the preservation of DNA integrity compared to brown hydra (Hydra oligactis). Norflurazon concentrations were 0.061 or 0.61mg/L and UV-B light 254nm, 0.023mWcm- 2 applied separately or simultaneously. By alkaline comet assay primary DNA damage was assessed and cytotoxicity by fluorescent staining. Norflurazon at 0.61 mg L- 1 significantly increased DNA damage in brown hydras compared to the control (6.17 ± 0.6μm, 5.2 ± 1.7% vs. 2.9 ± 0.2μm, 1.2 ± 0.2%). Cytotoxicity was significantly elevated, being higher in brown hydras (25.7 ± 3.5% vs. 8.2 ± 0.2%). UV-B irradiation induced significant DNA damage in brown hydras (13.5 ± 1.0μm, 4.1 ± 1.0%). Simultaneous exposure to UV-B and norflurazon led to a synergistic DNA damaging. The frequency of cytotoxicity and hedgehog nucleoids was more pronounced in brown (78.3 ± 9.4%; 56.4 ± 6.0%) than in green hydras (34.7 ± 2.5%; 24.2 ± 0.6%). Evolutionary established symbiotic cooperation proved to provide resistance against cyto/genotoxicity.

DNA Damage in Bat Blood Leukocytes Using a Chromatin Dispersion Test (CDT): Biomarker of Environmental Genotoxicity.

Environmental pollutants produce adverse effects on organisms and ecosystems. Biomonitoring and biomarkers offer a reasonable approach to make these assessments. Induced genetic changes can be using as a biomarker in organisms that react to a given compound in the ecosystem. Monitoring environmental genotoxicity necessitates the choice of model animals known as "sentinels or biological monitors" and the suitability of validated tests for DNA damage evaluation. We aimed to estimate the DNA damage produced by thermal stress in the leukocytes of the Mexican free-tailed bat (Tadarida brasiliensis). The DNA damage in bat leukocytes exposed to different temperatures (35°C, 45°C, and 55°C) was evaluated by the adapted chromatin dispersion test (CDT) and the results were confirmed by the alkaline comet test. The CDT permitted a clear representation of leukocytes with fragmented DNA and of nonfragmented DNA. In addition, we detected nuclear anomalies in relation to cell death cellular swelling, nuclear fragmentation, and chromatin lysis. The alkaline comet assay revealed that the halos of diffuse chromatin include fragmented DNA. The assay of the method employing the CDT is well established, precise, and cost-effective for the routine quantitative analysis of DNA damage on the effect of the leukocytes of bats exposed to thermal stress. This could also apply as a sensitive screening tool for the evaluation of genotoxicity in environmental protection programs.

Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Genotoxicand biomarker response of mussel gill cells, haemocyte and A549 human lung cells to radiation from X-rays

A comparative study on the genotoxic effect of radiation from X-rays of different levels on Mussel gill cells, haemocyte and human lung cell A549 using the comet assay was carried out. The results revealed an increasing DNA damage with increasing doses of X-ray, single strand breaks and alkali labile sites in the haemocyte of the control had a mean value of 5.92 ± 1.27 while the highest treatment group 15Gray (Gy) had a mean value of 78.65 ± 4.36,withsignificant differences (P <0.05), A mean value of 21.10 ± 13.10 was recorded for Mussel gill cells in the control group while the highest treatment group 15 Gray (Gy) had 85.55 ± 1.43. Statistical analysis revealed significant differences (P <0.05) for univariate tests of the control group and the treatment groups. The A549 human lung cells had a minimal damage in the control group 2.30 ± 0.30 while the highest exposed group had a mean value from the median % tail intensity of 43.10 ± 2.81. Pair wise comparison of gills cells, haemocytes from mussels and A549 human lung cells exposed to 15 Gy of X-rays shows that the highest damaging effect was observed on the gill cells followed by the haemocytes with the least damaging effect on the A549 human lung cells exposed to the same dose rate of X-rays with significant differences (P <0.05) between them.This study revealed that A549 human lung cells were more resistant to DNA damage induced by the X-rays when compared to mussel gill and haemocyte cells and also indicates that mussel gill cells and haemocytes can serve as biomarkers of genotoxicity of radiation exposure using the comet assay.

First biochemical and behavioural analysis of the response of the scorpion Urophonius brachycentrus (Thorell: 1876) upon exposure to an organophosphate.

Scorpionism is an increasing public health problem in the world. Although no specific methodology or product is currently available for the control of those arachnids, the use of insecticides could be an effective tool. Chlorpyrifos is one of the insecticides used, but to date, whether scorpions recognise surfaces with that insecticide and how it affects their physiology and/or biochemistry is unknown. In the present study, we observed that scorpions recognise surfaces with 0.51 and 8.59 μg/cm2 of chlorpyrifos and avoid those areas. The 0.51 μg/cm2 concentration produced a decrease in acetylcholinesterase and an increase in catalase, superoxide dismutase and glutathione S-transferase, whereas the 8.59 μg/cm2 concentration evoked a decrease in acetylcholinesterase and an increase in catalase and glutathione S-transferase. Using the comet assay, we observed that the insecticide at 0.17, 0.51 and 8.59 μg/cm2 caused DNA damage. Finally, we found that the insecticide does not generate significant variations in glutathione peroxidase, glutathione reductase, the amount of protein or lipid peroxidation. The present results offer a comprehensive understanding of how scorpions respond, both at the biochemical and behavioural levels, when exposed to insecticides.

Indigo dyes: Toxicity, teratogenicity, and genotoxicity studies in zebrafish embryos

Wastewater released by textile dyeing industries is a major source of pollution. Untreated wastewater released from indigo dyeing operations affects aquatic ecosystems and threatens their biodiversity. We have assessed the toxicity of natural and synthetic indigo dye in zebrafish embryos, using the endpoints of teratogenicity, genotoxicity, and histopathology. The zebrafish embryo toxicity test (ZFET) was conducted, exposing embryos to ten concentrations of natural and synthetic indigo dyes; the 96-hour LC50 values were approximately 350 and 300 mg/L, respectively. Both dyes were teratogenic, causing egg coagulation, tail detachment, yolk sac edema, pericardial edema, and tail bend, with no significant difference in effects between the natural and synthetic dyes. Both dyes were genotoxic (using comet assay for DNA damage). Real-time RT-PCR studies showed upregulation of the DNA-repair genes FEN1 and ERCC1. Severe histological changes were seen in zebrafish larvae following exposure to the dyes. Our results show that indigo dyes may be teratogenic and genotoxic to aquatic organisms, underscoring the need for development of sustainable practices and policies for mitigating the environmental impacts of textile dyeing.

Exosome miR-101–3p derived from bone marrow mesenchymal stem cells promotes radiotherapy sensitivity in non-small cell lung cancer by regulating DNA damage repair and autophagy levels through EZH2

Background and objectiveThe morbidity rate of non-small cell lung cancer (NSCLC) increases with age, highlighting that NSCLC is a serious threat to human health. The aim of this study was mainly to describe the role of exosomal miR-101–3p derived from bone marrow mesenchymal stem cells (BMSCs) in NSCLC. MethodsA549 or NCI-H1703 cells (1×105/mouse) were injected into nude mice to establish an NSCLC animal model. RTqPCR, Western blotting and comet assays were used to assess the changes in gene expression, proteins and DNA damage repair. ResultsmiR-101–3p and RAI2 were found to be expressed at low levels in NSCLC, while EZH2 was highly expressed. In terms of function, miR-101–3p downregulated EZH2. In addition, exosomal miR-101–3p derived from BMSCs promoted the expression of RAI2, inhibited DNA damage repair, and inhibited the activation of the PI3K/AKT/mTOR signaling pathway by inhibiting EZH2, thereby promoting autophagy and decreasing cell viability and finally enhancing the sensitivity of NSCLC to radiotherapy and inhibiting the malignant biological behavior of NSCLC. ConclusionExosomal miR-101–3p derived from BMSCs can inhibit DNA damage repair, promote autophagy, enhance the radiosensitivity of NSCLC, and inhibit the progression of NSCLC by inhibiting EZH2.

DNA damage and ALAD polymorphism in high blood lead (Pb) levels of pregnant women attending a tertiary care teaching hospital

Pregnant women are particularly vulnerable to lead toxicity due to increased absorption and decreased elimination of lead from their bodies. The δ-aminolevulinic acid dehydratase (ALAD) gene plays a crucial role in lead metabolism, and its polymorphisms have been implicated in modifying the susceptibility to lead toxicity. A cross-sectional study was conducted involving 90 pregnant women and blood samples were collected to measure blood lead levels (BLL) and assessed DNA damage using the comet assay. ALAD polymorphisms were genotyped using PCR-RFLP analysis with MspI restriction enzyme. Statistical analysis, including chi-square tests, logistic regression, and correlation analysis, was performed to determine associations between ALAD polymorphisms, BLL, and DNA damage. From 90 pregnant women the participants, 16 had high BLL (≥5μg/dL), while the remaining 74 had normal levels (<5μg/dL). The ALAD 1-2 genotype was found to be significantly associated with high BLL (p<0.001). Pregnant women with the ALAD 1-2 genotype exhibited higher levels of DNA damage compared to those with other genotypes (p<0.001). Furthermore, a positive correlation was observed between the transfer of lead concentration from mother to infant and DNA damage severity (r=0.511, p<0.001). The combination of comet assay and polymorphism analysis offers a comprehensive approach to understanding the impact of lead exposure during pregnancy. These findings underscore the urgent need for effective regulatory measures to reduce lead exposure in the environment and mitigate its adverse effects of lead on maternal and child health.

Celtis australis Exhibits Cytotoxic Effects by Inducing DNA Damage in Human Ovarian Cancer Cells

Native to the Mediterranean and southwest Asia, Celtis australis is used in folk medicine to treatment of many diseases such as peptic ulcer, diarrhea, dysentery, pain, and colic. However, there are very few studies on the effects of this species on cancer. We report for the first time the cytotoxic and genotoxic properties of C. australis on the human ovarian cancer cell line A2780.&#x0D; In the study, C. australis extract prepared in ethanol/phosphate buffer was applied to A2780 cells. The change in viability level in A2780 cells after treatment was determined by MTT assay and DNA damage was determined by single-cell gel electrophoresis (Comet) analysis.&#x0D; Our results showed that plant extract application at doses above 200 µg/ml significantly decreased A2780 cell viability (p

Ginsenoside Rb1 Deters Cell Proliferation, Induces Apoptosis, Alleviates Oxidative Stress, and Antimetastasis in Oral Squamous Carcinoma Cells.

There are numerous therapeutic applications for ginsenoside Rb1 (GRb1), the primary saponin derived from ginseng root. According to earlier research, ginsenoside Rb1 causes apoptosis and reduces the cell cycle. Its adverse effects, especially those on the development of the embryo, still need to be thoroughly studied. A host's lifestyle choices, including smoking, drinking too much alcohol, using tobacco products, and having an HPV infection, can increase the risk of oral squamous cell carcinoma (OSCC), one of the most prevalent malignancies of the oral cavity. To address this challenge, this investigation focuses on the design of GRb1 for treating OSCC. In vitro cytotoxicity studies confirmed that GRb1 was more effective in PCI-9A and PCI-13 cells, with reduced toxicity in non-cancerous cells. Further verification of cellular morphology was achieved through various biochemical staining methods. The mechanism of cell death was investigated by Annexin V-FITC and PI methods. Additionally, the antimetastatic attributes of GRb1 have been evaluated using both migration scratch and Transwell migration assays, which have collectively revealed excellent antimetastatic potential. The DNA fragmentation of the PCI-9A and PCI-13 cells was assessed using a comet assay. Ginsenoside Rb1 improved ROS levels and caused mitochondrial membrane potential alterations and DNA damage, which resulted in apoptosis. OSCC administration significantly reduced the levels of SOD, GSH, GPx, and CAT, increasing the levels of PCI-9A and PCI-13 cells, while GRb1 improved this situation. Therefore, we propose that Ginsenoside Rb1 could be an alternative therapeutic strategy for OSCC therapy.

Comprehensive toxicity assessment of nanodiamond on Blaps polychresta: implications and novel findings.

With the increasing development of nanomaterials, the use of nanodiamonds (NDs) has been broadly manifested in many applications. However, their high penetration into the ecosystem indubitably poses remarkable toxicological risks. This paper investigates the toxic effects of NDs on the darkling beetle, Blaps polychresta Forskal, 1775 (Coleoptera: Tenebrionidae). Survival analysis was carried out by monitoring the beetles for 30 d after the injection of four different doses of NDs. A dose of 10.0 mg NDs/g body weight, causing less than 50% mortality effect, was assigned in the analysis of the different organs of studied beetles, including testis, ovary, and midgut. Structural and ultrastructural analyses were followed using light, TEM, and SEM microscopes. In addition, a variety of stress markers and enzyme activities were assessed using spectrophotometric methods. Furthermore, cell viability and DNA damage were evaluated using cytometry and comet assay, respectively. Compared to the control group, the NDs-treated group was exposed to various abnormalities within all the studied organs as follows. Significant disturbances in enzyme activities were accompanied by an apparent dysregulation in the antioxidant system. The flow cytometry results indicated a substantial decrease of viable cells along with a rise of apoptotic and necrotic cells. The comet assay demonstrated a highly increased level of DNA damage. Likewise, histological analyses accentuated the same findings showing remarkable deformities in the studied organs. Prominently, the research findings substantially contribute for the first time to evaluating the critical effects of NDs on B. polychresta, adopted as the bioindicator in this paper.

Redox-activity and in vitro effects of regional atmospheric aerosol pollution: Seasonal differences and correlation between oxidative potential and in vitro toxicity of PM1

Particulate Matter (PM) is a complex and heterogeneous mixture of atmospheric particles recognized as a threat to human health. Oxidative Potential (OP) measurement is a promising and integrative method for estimating PM-induced health impacts since it is recognized as more closely associated with adverse health effects than ordinarily used PM mass concentrations. OP measurements could be introduced in the air quality monitoring, along with the parameters currently evaluated. PM deposition in the lungs induces oxidative stress, inflammation, and DNA damage.The study aimed to compare the OP measurements with toxicological effects on BEAS-2B and THP-1 cells of winter and summer PM1 collected in the Po Valley (Italy) during 2021. PM1 was extracted in deionized water by mechanical agitation and tested for OP and, in parallel, used to treat cells. Cytotoxicity, genotoxicity, oxidative stress, and inflammatory responses were assessed by MTT test, DCFH-DA assay, micronucleus, γ-H2AX, comet assay modified with endonucleases, ELISA, and Real-Time PCR. The evaluation of OP was performed by applying three different assays: dithiothreitol (OPDTT), ascorbic acid (OPAA), and 2′,7′-dichlorofluorescein (OPDCFH), in addition, the reducing potential was also analysed (RPDPPH).Seasonal differences were detected in all the parameters investigated. The amount of DNA damage detected with the Comet assay and ROS formation highlights the presence of oxidative damage both in winter and in summer samples, while DNA damage (micronucleus) and genes regulation were mainly detected in winter samples. A positive correlation with OPDCFH (Spearman's analysis, p < 0.05) was detected for IL-8 secretion and γ-H2AX.These results provide a biological support to the implementation in air quality monitoring of OP measurements as a useful proxy to estimate PM-induced cellular toxicological responses. In addition, these results provide new insights for the assessment of the ability of secondary aerosol in the background atmosphere to induce oxidative stress and health effects.

The ameliorative effect of rosmarinic acid and epigallocatechin gallate against doxorubicin-induced genotoxicity

Doxorubicin (Dox), an effective anticancer agent, is known for its genotoxic effects on normal cells. Phenolic compounds, renowned for their antitumor, antioxidant, and antigenotoxic properties, have gained prominence in recent years. This study investigates the individual and combined protective effects of rosmarinic acid (RA) and epigallocatechin gallate (EGCG) against Dox-induced genotoxicity using various in vitro test systems. The synergistic/antagonistic interaction of these combinations on Dox’s chemotherapeutic effect is explored in breast cancer cell lines. Both RA and EGCG significantly mitigate Dox-induced genotoxicity in comet, micronucleus, and Ames assays. While Dox exhibits higher selectivity against MCF-7 cells, EGCG and RA show greater selectivity against MDA-MB-231 cells. The coefficient of drug interaction reveals a synergistic effect when RA or EGCG is combined with Dox in breast cancer cells. In conclusion, both EGCG and RA effectively reduce Dox-induced genetic damage and enhance Dox’s cell viability-reducing effect in breast cancer cells.

Abstract 2099: PELP1 inhibition enhances the therapeutic efficacy of topoisomerase inhibitors in triple-negative breast cancer

Abstract BACKGROUND: Triple negative breast cancers (TNBCs) account for 15-24% of breast cancer deaths and are characterized by aggressive clinical course, a worse prognosis, and a higher propensity to metastasize. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) is an oncogene, its expression is deregulated in TNBC, and its status is a prognostic indicator of poor TNBC survival. Recently, a small molecule inhibitor of PELP1 (SMIP34) which binds to and degrades PELP1 was developed. The Objective of this proposal is to define the mechanism of action of PELP1 inhibitor SMIP34 in TNBC progression and to pave the path for a novel combination therapy. METHODS: We tested the effectiveness of PELP1 inhibitor, SMIP-34, in combination with 140 FDA-approved medications on cell survival of multiple TNBC cell lines. The effect of SMIP34 combination therapy on cell survival was measured using MTT, colony formation, and Annexin V apoptosis assays. Mechanistic studies were done using RT-qPCR, immunoprecipitation, proximity ligation, mass spectrometry, gene reporter, immunofluorescence, and comet assays. Furthermore, the preclinical utility of combination therapy was evaluated using MDA-MB-231 xenografts, patient-derived organoids (PDOs) and explants (PDEs). RESULTS: We performed an in vitro screening of 140 FDA-approved drugs in combination with SMIP34. Results revealed that SMIP34 sensitized TNBC cells to five FDA approved drugs. Of the five drugs, the three drugs Gemcitabine, Valrubicin, and Mitoxantrone, induced DNA damage by inhibiting topoisomerase, a protein that cleaves and reconnects DNA during replication. We validated the ability of SMIP34 to enhance the efficacy of TIs using two additional TNBC cell lines and confirmed their synergistic activity using several in vitro assays including cell viability, colony formation, and apoptosis. In agreement with the role of PELP1 in DNA damage response, Western blotting analyses showed higher levels of γ-H2AX in SMIP34+TI combination therapy when compared to monotherapy. Results from the comet assays further demonstrate increased DNA damage caused by the SMIP-34 + TIs combination therapy. Mechanistic studies using Proximity labelling followed by immunoprecipitation identified topoisomerase 2A and 2B (TOP2A and TOP2B) as potential interactors of PELP1. Furthermore, gene correlation analysis using TCGA datasets revealed that PELP1 expression is positively correlated with TOP2A, and TOP2B in TNBC. SMIP34 plus Mitoxantrone (TI) combination treatment significantly reduced the growth of TNBC PDEXs, and PDOs in vitro, and MDA-MB-231 xenograft tumor growth in vivo, compared to vehicle or monotherapy treatment. CONCLUSION: Together, these results suggest a novel targeted therapy for the treatment of TNBC, involving the combination of topoisomerase inhibitors and the PELP1 inhibitor SMIP34. Citation Format: Khaled Mohamed Nassar, John R. Sanchez, Durga Meenakshi Panneerdoss, Behnam Ebrahimi, Yang Xue, Uday P. Pratap, Salvador C. Alejo, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. PELP1 inhibition enhances the therapeutic efficacy of topoisomerase inhibitors in triple-negative breast 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 2099.