Alkaline Comet Assay Research Articles

Liver cancer is the third leading cause of cancer-related death. Plantderived therapeutics have played a significant role in preventing and treating many diseases, including cancers. The present study investigated the anticancer properties of protein fractions from the green leaf extract of Adenium obesum (A. obesum) in the laboratory. Protein fractions of leaf extract were separated using reversed-phase high-performance liquid chromatography (RP-HPLC). The cytotoxicity of protein fractions was studied by MTT and sulforhodamine B assays. The apoptotic cell death was examined using the alkaline comet assay, and redox-related indicators were assessed using the catalase enzyme activity assay, glutathione content, and nitric oxide release. The RBC hemagglutination test investigated the possible presence of ribosome-inactivating proteins (RIPs) in the most toxic protein fraction, and the LD50 of the protein fraction with the highest anticancer effects was determined. The amino acid sequence of fraction proteins was determined by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. The results showed that protein fraction 8 had the highest toxicity in the HepG2 cell line, with an IC50 of 0.16 μg/mL. This fraction induced hemagglutination in red blood cells at concentrations higher than 65 μg/mL. The apoptosis was induced in the HepG2 cells following treatment with the concentrations of 0.08, 0.16, 0.32, and 0.64 μg/mL. Moreover, the redox potential of the treated cells was changed after treatment. The in vivo cytotoxicity investigation of this fraction in mice showed that it is not toxic for animals in concentrations up to 800 μg/kg, indicating its safety potential for pharmaceutical applications. The protein extract in the aforementioned fraction contained two proteins (22 and 53 kD) as determined by electrophoresis and sequencing methods. The findings of this investigation demonstrated that the protein content of fraction 8 derived from A. obesum leaf extract possesses anticancer activity in the HepG2 cell line. The two isolated proteins from this fraction are novel and have been reported for the first time. Further investigations should be performed to evaluate the treatment potential in in vitro/vivo conditions.

Evaluation of potassium bromate as a positive control in the in vivo Fpg-modified comet assay for the detection of oxidised bases.

Heterozygous familial hypercholesterolemia (HeFH) is a common autosomal dominant genetic disease (1:250) characterized by elevated LDL-C. Patients with HeFH are at increased risk of premature atherosclerosis and have at least a 10-fold greater chance of cardiovascular disease (CVD). The present study examines the effect of PCSK9 inhibitor treatment (iPCSK9: arilocumab or evolocumab) on DNA damage in HeFH patients. Fifty-six patients were studied, with a normolipidemic group (control; n = 20) and patients with HeFH (study group; n = 36). DNA damage was determined by alkaline comet assay and PCSK9 protein level by ELISA. PCSK9i treatment was found to be associated with lower DNA damage, Lp(a), PCSK9, and lipid profile compared to before treatment. However, 16 of 36 patients still had Lp(a) values above 125 nmol/L, and reduced Lp(a) did not correlate with reduced DNA damage. Reduced PCSK9 demonstrated a moderately positive correlation (r = 0.48) with reduced DNA damage. PCSK9i therapy reduces the level of DNA damage in HeFH patients, regardless of the type of inhibitor. While our findings confirm that PCSK9 treatment can reduce DNA damage, the mechanism remains unclear.

Levothyroxine, the synthetic form of thyroxine, is widely prescribed for hypothyroidism, including in men of reproductive age. While generally considered safe, its potential direct effects on male reproductive somatic cells remain poorly defined. This study investigated the invitro reproductive toxicity of levothyroxine by evaluating its cytotoxic, oxidative, and genotoxic effects on murine TM3 Leydig and TM4 Sertoli cells. Cells were exposed to levothyroxine at 0.1-200 μM for 24 h. Cytotoxicity was assessed using MTT and neutral red uptake assays, intracellular ROS by DCFDA fluorescence, and DNA damage by alkaline comet assay. Levothyroxine significantly reduced cell viability in a concentration-dependent manner. Based on MTT results, the IC50 values were 121.22 μM for TM3 cells and 114.16 μM for TM4 cells, whereas neutral red uptake assays yielded IC50 values of 104.16 and 104.61 μM, respectively. ROS levels increased with dose but did not reach statistical significance. Comet assay results revealed mild, nonsignificant DNA damage in TM3 cells at 100 μM and a nonmonotonic pattern in TM4 cells, peaking at 1 μM. These findings reveal subtle, cell-type-specific responses to supraphysiological exposure to levothyroxine. Importantly, TM4 Sertoli cells exhibited slightly greater sensitivity than TM3 Leydig cells across assays, consistent with their critical role in supporting the seminiferous epithelium. Although direct clinical extrapolation is not possible, this represents the first comparative analysis of levothyroxine's effects on Leydig and Sertoli cells invitro. In conclusion, levothyroxine induces modest, dose-dependent cytotoxicity and subtle oxidative/genotoxic alterations in testicular somatic cells, with Sertoli cells being more susceptible.

Background Mutations in the cluster of differentiation (CD) 295 gene, which encodes a class I cytokine receptor, are associated with obesity and breast cancer (BC). Single-nucleotide polymorphisms (SNPs) in the adipocyte-inferred novel cytokine intelectin 1 (ITLN1) remain understudied in connection to CD295 polymorphisms and diabetes mellitus (DM) or a pre-diabetic state, as well as to DNA damage seen in BC. Aim To explore whether CD295 (rs6700986) and ITLN1 (rs952804) SNPs impact BC with or without DM, insulin resistance (IR), or obesity. Effects of ITLN1 or CD295 polymorphism(s) on DNA damage in BC were also examined. All of these are to be confirmed by bioinformatics/ in silico analysis. Subjects and methods Blood samples from 170 women with BC (including 33 and 48 with DM and pre-diabetes, respectively) and from 108 age-matched women in the control group were collected. Plasma insulin, leptin, CD295, and ITLN1 levels were measured by ELISA. rs6700986 and rs952804 were analyzed by RT-PCR. DNA damage was assessed using the alkaline comet assay. Results BC cases with clinical stage T II and positive LN, as well as tumor histologic grade III, presence of obesity, pre-diabetic events, DM, or IR, were associated with CD295 rs6700986 mutant homozygous (CC) and heterozygous (CT) genotypes and ITLN1 rs952804 mutant CT genotype ( p ≤ 0.05). Tail DNA (%) and tail moment units were significantly associated with the CD295 rs6700986 CT and the ITLN1 rs952804 TT genotypes. The C allele (CT + CC vs. TT) and T allele (TT + CT vs. CC) for CD295 rs6700986 and ITLN1 rs952804, respectively, were associated with BC risk ( p ≤ 0.05). Conclusion CD295 (rs6700986) and ITLN1 (rs952804) SNPs should be considered as BC-associated susceptibility risk factors in obese, insulin resistance, or pre-diabetic individuals.

Polluted water in the Chapala–Santiago basin (CSB) contains several genotoxic substances that present risks to human health, particularly among residents of communities surrounding Lake Chapala and the Santiago River, where a high prevalence of cancer has been documented. For this reason, it is necessary to study the genotoxic activity of these waters and the genetic damage in inhabitants of the surrounding populations. This study assessed the genotoxicity of water in various communities in the CSB and evaluated DNA damage to lymphocytes in residents of nearby locations. The alkaline comet assay was employed to evaluate water genotoxicity and DNA damage to lymphocytes in residents living near these waters. A standardized questionnaire was distributed to participants of this study to evaluate their exposure to polluted water. Significant genotoxic activity (p < 0.05) was observed in the lymphocytes of individuals exposed to contaminated water (tail length in Puente Grande 27.88 ± 5.4 compared to 3.77 ± 1.64 of negative control), along with notable DNA damage (p ≤ 0.05) to the lymphocytes of residents living in proximity to these waters (tail length in Juanacatlán 12.3 ± 3.4 compared to 1.4 ± 0.74 of negative control). The waters of the CSB possess the capacity to cause DNA damage; meanwhile, genotoxicity increases from Chapala to El Salto due to the additional input of genotoxic contaminants, thereby elevating the cancer risk for the exposed population. The comet test proved to be a useful tool that allowed data to be obtained quickly and reliably.

Fish is a primary protein source in Côte d'Ivoire, accounting for 39% of animal protein intake. However, the current local production from fisheries is insufficient to meet the annual national demand. Fish farming could address this gap, but the production of high-quality fry remains a key challenge. Water quality plays a crucial role in fry production, yet many tilapia farms in the country are susceptible to agrochemical contamination from nearby agricultural activities, which can affect both fish health and reproduction. This study aims to assess genotoxicity of a hatchery water in tilapia using the alkaline comet assay. Water samples from Modern Hatchery of Gonaté in the Haut-Sassandra Region of Côte d'Ivoire were collected in November 2021 and October 2022 for pesticides analysis. Simultaneously, fish broodstock samples were collected for genotoxicity assessment. Our results show that in 2021, when pesticides were used in the surrounding area, DNA damage in the erythrocytes of tilapia broodstock (41.6 ± 6.7% tail DNA) was significantly higher (ANOVA, F(3,31) = 222.9, P < 0.001) compared to individuals sampled in 2022 (15.5 ± 4.4%) after pesticide use in the adjacent food crops ceased. Pesticide residues, including glyphosate, acetamiprid, and mancozeb, were detected in water samples from 2021 but were absent in 2022, coinciding with the cessation of pesticide use near the hatchery. These findings underscore the impact of agrochemical exposure on DNA damage in tilapia, which may have detrimental effects on fry production.

Etofenprox is a synthetic pyrethroid pesticide widely used in agriculture, with significant ecotoxicological concern due to its potential for long-term persistence in the environment. This study aimed to evaluate the cytotoxic and genotoxic effects of etofenprox on Chinese hamster ovary (CHO) cells. Cytotoxicity was determined by measuring the cytokinesis-block proliferation index (CBPI) after exposure to various concentrations (1–800 µg/mL) of etofenprox. Genotoxicity was assessed using the micronucleus (MN) assay and alkaline comet assay. The results revealed significant reductions in CBPI, increases in MN frequency, and pronounced elevations in DNA damage parameters (tail length, % tail DNA, olive tail moment) at higher concentrations. These effects increased in a dose-dependent manner, particularly at concentrations ≥200 µg/mL, where the changes were statistically significant (p

Hydroxychloroquine sulfate (HCQ), widely prescribed for autoimmune disorders, carries unresolved concerns regarding potential genotoxic risks. This integrated invitro assessment comprehensively evaluated HCQ's cytogenotoxic profile using bacterial reverse mutation (Ames test in TA98/TA100 strains, 5-80 μg/plate), DNA strand break detection (alkaline Comet assay), chromosomal instability assessment (cytokinesis-block micronucleus test in human lymphocytes, 10-40 μg/mL), oxidative stress biomarkers (TOS/TAR/OSI), plasmid DNA protection (pBR322 under UV/H₂O₂), and molecular docking targeting DNA polymerase δ. Results demonstrated no mutagenicity in TA98. While a statistically significant (p ≤ 0.001) increase in revertants was observed in TA100 at a single concentration, this was transient, nondose-dependent, and biologically insignificant as it remained below the two-fold threshold defined by OECD criteria. No significant DNA damage occurred in mammalian systems, with Genetic Damage Index ≤ 0.14 and micronucleus frequency consistently below 9.75%. Molecular docking revealed weak binding affinity to DNA polymerase δ (ΔG = -5.6 kcal/mol). HCQ induced pronounced dose-dependent cytostasis, evidenced by a 20.5% reduction in Nuclear Division Index at 40 μg/mL, without accompanying genotoxicity. Redox modulation was confirmed through a 15.4% decrease in oxidative stress index. Crucially, HCQ exhibited a complex, biphasic effect on plasmid DNA, paradoxically exacerbating damage at a low concentration (10 μg/mL) while offering significant protection at higher concentrations under combined oxidative stress. These data establish HCQ as a cytostatic agent devoid of genotoxic risk, reinforcing its clinical safety profile while highlighting the necessity for environmental risk validation through invivo models.

Abstract BACKGROUND Glioblastoma is the primary brain tumor of the central nervous system, with a median survival of less than 15 months. Despite its approval by the FDA in 2005 as a first-line treatment for glioblastoma, TMZ has exhibited limited efficacy primarily due to the inherent heterogeneity and rapid emergence of resistance. Therefore, effective drugs with low toxicity are urgently to be developed or combined for glioblastoma treatment. MATERIAL AND METHODS The effects of AKT expression on the prognosis of TMZ were analyzed in the Chinese Glioma Genome Atlas (CGGA) database. CCK-8 and plate cloning experiments evaluated the effects of TMZ and perifosine on glioblastoma cell proliferation. SynergyFinder APP calculated the Synergy Score. RNA-seq was used to analyze the possible mechanism pathways and verified by Western blot and alkaline comet assay. Apoptosis was detected by Flow cytometry and Western blot after TMZ and perifosine treatment. Finally, the antitumor activity of the drug was detected in glioblastoma xenografted nude mice. RESULTS Bioinformatics analysis showed a poor prognosis of high-expression AKT in TMZ-treated glioblastoma patients. Therefore, we hypothesized that the combination of TMZ with AKT inhibitor perifosine can improve the sensitivity of TMZ and may be an effective treatment for glioblastoma. Perifosine stands as the pioneering allosteric AKT inhibitor in clinical development, showcasing commendable safety and notable permeability across the blood‒brain barrier. Consequently, we opted for the combination of TMZ and perifosine to investigate their collective impact on glioblastoma treatment at the preclinical stage. The ZIP Synergy scores for the combined treatment in U87MG and U251 were 20.349±1.92 and 14.766±1.33, respectively. We found that the combination of TMZ and perifosine synergistically inhibits glioblastoma. This co-treatment significantly decreases BRCA1 expression, leading to the inhibition of DNA repair. Additionally, TMZ and perifosine combination triggers caspase-dependent apoptosis and inhibits glioblastoma cell viability and proliferation. This synergy effect on glioblastoma treatment was further confirmed in vivo according to the sharp reduction in glioblastoma xenograft after the co-treatment with TMZ and perifosine. Lastly, higher BRCA1 expression is associated with a worse prognosis of recurrent glioma patients, especially the ones that received TMZ-treated. CONCLUSION Our preclinical study provides optimism for future clinical trials exploring the combination of perifosine with TMZ for glioma treatment, especially in patients with poor prognosis of glioblastoma and recurrence. In conclusion, our study suggests that the combination of TMZ and perifosine holds promise as a strategy for glioblastoma treatment.

Fabrication of silver nanoparticles by green approach is the most effective and eco-friendly technique in recent technologies. The current study aimed to generate a simple, valid, and justifiable method for biogenic synthesis of silver nanoparticles (HP-AgNPs) using aqueous extract of Hypecoum pendulum L.(HP) and to assess their invitro anticancer and genotoxic potentials on baby hamster kidney cell (BHK-21) and human blood lymphocytes using 3-(4,5-dimethylthiazol-2-yl-)-2,5-di-phenyltetrazolium bromide (MTT) and alkaline comet assay, respectively. HP-AgNP characterization was done using UV-vis spectrometry, EDX, SEM, XRD, and FTIR techniques. The crystalline nature of HP-AgNPs with a particle size of 36.3 nm was assessed using the XRD technique. The surface morphologies with a particle size of 80 nm were verified by SEM analysis. UV spectroscopy verified the existence of HP-AgNPs by yielding a sharp peak at 417 nm with an absorbance intensity of 1.54. FTIR assessment revealed the existence of different functional moieties that contribute to the HP-AgNPs stabilization and reduction. Similarly, EDX analysis revealed Ag as a principal element (49%). MTT assay showed significant cytotoxicity by Doxorubicin and HP-AgNPs with a smaller IC50 value of 104.21 ± 4.33 and 134.91 ± 6.33 μg/mL correlated to HP extract (229.84 ± 4.66 μg/mL). The outcomes of the comet assay revealed potential DNA damage in a positive trend with concentration (25-600 μg/mL). HP-AgNP-treated lymphocytes showed higher DNA damage as compared to HP extract-treated cells, but less damage as compared to a positive control, H2O2. These outcomes showed that HP-AgNPs have demonstrated promising anticancer and genotoxic action than HP extract due to their size and shape.

DNA damage and nuclear anomalies in peripheral blood lymphocytes from asthma patients and obese individuals.

The therapeutic benefits of Phyllanthus niruri (PN) are well-known, particularly in traditional medicine. Nanoemulsion technology has enhanced its bioavailability and efficacy, but potential nanotoxic effects, especially on male reproductive health, are not fully understood. This study examines the cytotoxic and genotoxic impacts of Phyllanthus niruri nanoemulsions (PNNE) of different sizes on TM4 Sertoli cells, focusing on their effects on the blood-testis barrier (BTB). PNNE of two distinct sizes, were prepared using spontaneous emulsification. The cytotoxicity of these formulations was assessed using a Cell Counting Kit-8 (CCK-8) assay, while genotoxicity was evaluated through an alkaline comet assay. Additionally, the expression of BTB proteins, claudin 11 and connexin 43, was examined via immunofluorescence analysis. PNNE were synthesized with droplet sizes of $16.9~\pm ~3.31$ nm and $163.7~\pm ~8.53$ nm. The smaller PNNE exhibited higher cytotoxicity (IC $50= 160.6~\pm ~8.3~\mu $ g/mL) compared to the larger PNNE (IC $50= 324.4~\pm ~12.5~\mu $ g/mL) and caused more significant DNA damage, as evidenced by the comet assay Both sizes led to a reduction in BTB protein expression, with the smaller nanoparticles causing more pronounced disruption. The study highlights the crucial role of nanoparticle size in determining the biological effects of PNNE on TM4 Sertoli cells. Smaller PNNE were found to be more detrimental to BTB integrity and cellular health, emphasizing the need for careful size optimization in the development of nanoemulsion-based therapies. These findings contribute to the understanding of nanotoxicity in the context of male reproductive health.

Protective effects of nicotinamide mononucleotide on DNA damage and cell death in A549 cells and aging in C. elegans caused by hydrogen peroxide.

Workers exposed to lead at a battery recycling plant in Mexico: Blood lead levels; DNA damage and repair in blood cells (comet assay).

E171-induced toxicity in human iPSC-derived colon organoids: Effects on cell viability, ROS generation, DNA damage, and gene expression changes.

Iohexol, a nonionic and low-osmolality iodinated contrast agent, is widely used in medical imaging applications. Although it is generally considered safe, its potential cytotoxic and genotoxic effects on neuronal cells have not been sufficiently elucidated. In this study, the cytotoxic, genotoxic, and oxidative stress-related effects of iohexol on the SH-SY5Y human neuroblastoma cell line were evaluated by both invitro and in silico approaches. SH-SY5Y neuroblastoma cells were exposed to increasing concentrations of iohexol in the range of 1.5-150 mg I/mL for 24 h. Cell viability was evaluated by the CCK-8 (Cell Counting Kit-8) method, and DNA damage was analyzed by alkaline comet assay. Morphological changes were examined by phase contrast microscopy. In addition, possible interactions of the iohexol molecule with double-stranded DNA (B-DNA) and the human thioredoxin reductase I (TrxR1) enzyme were investigated by molecular docking analyses. Iohexol significantly decreased cell viability with increasing concentrations; especially significant cytotoxicity was detected at concentrations ≥ 75 mg I/mL. Morphological analyses revealed cellular stress indicators such as cell rounding, shrinkage, and detachment from the surface. The comet assay revealed a mild but significant genotoxic potential at high concentrations (75 and 150 mg I/mL), indicated by an increased frequency of cells with DNA damage (Damaged Cell Index), but not in the overall severity of DNA damage (Genetic Damage Index). Molecular docking results revealed that iohexol showed weak binding affinity with B-DNA (-3.9 kcal/mol) but a moderate interaction potential with the TrxR1 enzyme (-5.99 kcal/mol). This suggests that the observed toxicity may be mediated through indirect mechanisms, such as oxidative stress, rather than direct DNA interaction. In conclusion, this study demonstrates that high concentrations of iohexol induce both cytotoxic and mild genotoxic effects in a neuronal cell model. The findings suggest that the safety profile of this contrast agent should be carefully evaluated, highlighting the importance of dose adjustment in clinical applications.

Carbofuran, a highly toxic N-methyl carbamate insecticide banned in many regions due to ecological and health risks, exerts genotoxic effects through oxidative stress and DNA damage. This study evaluated and compared its time-dependent genotoxicity on human and chicken lymphocytes using the Alkaline Comet Assay, exposing isolated peripheral blood lymphocytes from both species to 50 µM carbofuran for 1 or 2 hours and quantifying DNA strand breaks using CASP software. Results revealed moderate DNA damage in human lymphocytes after 1 hour (Tail DNA% = 12.65%, TM = 0.51, OTM = 2.32), increasing moderately after 2 hours (Tail DNA% = 18.92%, TM = 1.14, OTM = 2.40). In stark contrast, chicken lymphocytes exhibited high DNA damage after 1 hour (Tail DNA% = 40.17%, TM = 17.68, OTM = 13.11), which significantly increased after 2 hours (Tail DNA% = 60.08%, TM = 29.44, OTM = 18.94). These findings demonstrate carbofuraninduced time-dependent DNA damage in both species but crucially reveal significantly higher susceptibility in chicken lymphocytes compared to human lymphocytes at the same concentration. This heightened vulnerability underscores carbofuran's extreme toxicity to birds and highlights the value of chicken lymphocytes as a sensitive model for Eco toxicological genotoxicity assessment.

Fibrates, carboxylic acid derivatives used in hypercholesterolemia treatment, are classified as non-genotoxic carcinogens. However, induce oxidative stress and DNA damage. This study investigates the genotoxic effects of gemfibrozil (GMF), a hypolipidemic agent, on human peripheral blood lymphocytes through cytokinesis-block micronucleus (CBMN) and alkaline comet assays. Plasmid DNA (pBR322) assessed GMF's DNA protective effects, while total oxidant (TOS) and antioxidant (TAS) status quantified oxidative stress modulation. Molecular docking simulations evaluated non-covalent interactions of GMF against DNA and peroxisome proliferator-activated receptor alpha (PPAR-α). At 25-250µg/mL, GMF did not induce micronuclei (24-48h) but triggered significant DNA fragmentation at 250µg/mL (p < 0.01). GMF significantly reduced cytokinesis-block proliferation index (CBPI) across all concentrations and durations (p < 0.001), except 25µg/mL (48h). In a cell-free system, GMF exhibited a complex, bimodal protective effect against H₂O₂-induced plasmid damage, offering protection at 25 and 175µg/mL but not at 100µg/mL. However, TOS/TAS levels remained unaltered. Molecular docking demonstrated weak DNA binding (ΔG = -5.93kcal/mol) compared to mitomycin C (ΔG = -7.25kcal/mol), but strong PPAR-α affinity (ΔG = -7.40kcal/mol). These findings suggest GMF exerts cytotoxicity via disrupted cell division kinetics rather than direct DNA damage or oxidative stress. Despite a low genotoxic risk of GMF in vitro. In vivo studies are critical to confirm safety.

This study’s objective was to evaluate genotoxic effects on automotive paint workers who are exposed to a complex mixture of VOCs, heavy metals, and solvents. Biological samples, including blood, urine, and buccal epithelial cells, were collected from 80 exposed workers and 80 demographically matched control subjects. DNA damage was assessed using the alkaline COMET assay in lymphocytes and whole blood. The Buccal Micronucleus Cytome (BMCyt) assay was also employed to identify cytogenetic abnormalities. Additionally, trichloroacetic acid (TCA), hippuric acid (HA), phenol, and lead (Pb) levels were measured as biomarkers of exposure. A significant increase in DNA damage was observed in the lymphocytes and whole blood of exposed workers (p < 0.05) BMCyt analysis also revealed higher frequencies of micronuclei (MN), binucleated cells, condensed chromatin (CC), and karyorrhectic (KHC) and pyknotic cells (PYC) in buccal cells (p < 0.05). Elevated levels of urinary HA, phenol, TCA, and blood lead indicated systemic chemical exposure. DNA damage positively correlated with these biomarkers, supporting a strong link between chronic occupational exposure and genotoxicity. The findings from this study highlight the critical importance of implementing effective safety measures and consistent biomonitoring for paint workers to prevent adverse health effects.