This study aimed to establish a novel BRCA1 mutant cell line from Chinese hamster V79 cells and clarify the role of BRCA1 in the DNA damage response (DDR) by comparison with a BRCA2 mutant line, V-C8. Using CRISPR/Cas9 editing, we generated a hypomorphic BRCA1 mutant, designated B1-21, carrying a 27-bp in-frame deletion in exon 4. This mutation deletes nine amino acids within the RING domain. B1-21, V79, and V-C8 cells were analyzed for DDR phenotypes. Both mutants showed impaired RAD51 foci formation, defective homologous recombination repair, and increased sensitivity to DNA-damaging agents. B1-21 cells were particularly sensitive to camptothecin and the PARP inhibitor NU1025, whereas V-C8 cells showed higher sensitivity to etoposide, cisplatin, mitomycin C, and bleomycin. Although γH2AX and FANCD2 focus responses were similar between V79 and B1-21, RAD51 recruitment was only partially reduced in B1-21 and completely absent in V-C8. B1-21 also displayed chromosomal instability (19-20 chromosomes), while V79 and V-C8 maintained a stable karyotype. After gamma irradiation, V-C8 cells accumulated substantially more chromatid-type aberrations and retained unrepaired chromatin longer than B1-21. Neither mutant showed normal RAD51 foci formation, radiation-induced sister chromatid exchange or an effective G2/M checkpoint arrest, unlike wild-type cells. Mitotic index measurements further confirmed checkpoint failure: V79 cells suppressed mitotic entry after irradiation, while B1-21 and V-C8 continued to enter mitosis, with V-C8 showing the most complete checkpoint breakdown. These findings indicate that partial disruption of the BRCA1 RING domain results in a hypomorphic phenotype with impaired homologous recombination, defective checkpoint control, and enhanced genotoxic sensitivity. The isogenic BRCA1 and BRCA2 mutant V79 lines offer a valuable model for dissecting DDR pathway differences and developing mutation-specific therapeutic strategies targeting BRCA-mutant cancers.

Overweight and obesity have been increasing drastically in recent years due to the growing consumption of fast food. Obese individuals exhibit reduced antioxidant defenses, which can lead to DNA damage. Thus, studies have been conducted to mitigate obesity-related complications. Animal research has used the cafeteria diet (CAF) as an obesity induction model, as it mimics human consumption of ultra-processed foods. Currently, complementary dietary strategies are being explored to prevent and/or alleviate obesity-related complications, particularly through natural compounds with anti-obesity effects. Among these, melatonin (MEL) has gained attention due to its antioxidant and anti-inflammatory properties. This study aimed to evaluate the effects of MEL supplementation on biochemical, genotoxic, and inflammatory parameters in mice fed a CAF diet. A total of 60 male Swiss mice were divided into six experimental groups (n = 10): (i) Standard Diet-fed standard chow for 21weeks; (ii) SD + MEL-fed standard chow and supplemented with MEL for 24weeks; (iii) CAF-fed CAF for 21weeks; (iv) CAF + MEL-fed CAF and supplemented with MEL; (v) CAF/CAF + MEL-fed CAF for the first 17weeks, then continued CAF while starting MEL supplementation for the last 4 weeks, totaling 17weeks of CAF; (vi) CAF + MEL/CAF-fed CAF and supplemented with MEL for the first 17weeks, then stopped MEL supplementation for the last 4 weeks, totaling 21weeks. Blood samples were collected at 17 and 21weeks to assess DNA damage, lipid profile (triglycerides, total cholesterol, and HDL), liver function (ALT and AST), inflammatory markers (TNF-α and IL-10), fasting glucose, and insulin tolerance in all six groups. At the end of the experiment, animals were euthanized, and liver, kidney, adipose tissue, and bone marrow were collected for further analyses, including the Comet Assay, Micronucleus Test, oxidative stress evaluation, and Western blot. Results showed that CAF induced an inflammatory state, characterized by increased TNF-α and decreased IL-10, along with alterations in lipid and liver profiles. Additionally, CAF led to DNA damage in multiple tissues and insulin resistance. MEL supplementation for 17weeks reversed these changes. In the last 4 weeks of the experiment, CAF was associated with oxidative stress and damage in the liver, kidney, and bone marrow. MEL effectively attenuated these obesity-related alterations, primarily by modulating proteins involved in homologous and non-homologous DNA repair pathways. In conclusion, the findings demonstrate that MEL is a potent antioxidant and may be a promising candidate for reducing biochemical and genetic alterations associated with obesity.

This article summarizes the scientific contributions of Professor Sachi Prasad Ray-Chaudhuri and his unwavering commitment to advancing research and teaching in mutagenesis and genetics in post-independence India, following his postgraduate training in the United Kingdom under the mentorship of Professor H. J. Muller. His contributions in basic mutagenesis, radiation protection, animal cytogenetics and population genetics using a range of model and native species laid the foundation of scientific research in India in these contemporary field. This played a pivotal role in advancing genetics and biological sciences in India at a time when scientific infrastructure and resources were limited. He also took the initiative to modernize the curriculum of biology teaching in India in line with the developments in the subject globally. He expanded these efforts by teaching as a visiting professor in different, often remote parts of the country where he inspired the students. The seeds of his relentless effort to connect the advances in Indian science with those in the rest of the world have blossomed, and Indian science today contributes significantly to the global knowledge base. His pioneering scientific contributions and inspiring legacy continue unabated.

Nitrosamine drug substance-related impurities (NDSRIs) have raised significant concern among the pharmaceutical industry and regulatory agencies, prompting a need to establish methods to determine the mutagenic risks associated with these substances. As human, metabolically competent HepaRG cells previously showed promise in evaluating the mutagenicity of a small-molecule nitrosamine impurity, N-nitroso-dimethylamine, this study evaluated the suitability of HepaRG cells for mutagenesis assessment of NDSRIs. HepaRG cells, cultured in both two-dimensional (2D) and three-dimensional (3D) spheroid formats, were exposed to N-nitroso-fluoxetine and N-nitroso-varenicline for either 3days or 14days. DNA damage was evaluated using the comet assay, clastogenicity/aneugenicity was measured with the micronucleus assay, and mutagenesis was assessed using High-Fidelity sequencing (HiFi-seq). N-Nitroso-fluoxetine and N-nitroso-varenicline were cytotoxic, produced DNA damage, and increased mutation frequency in both 2D and 3D HepaRG models. Both compounds increased micronucleus formation only at the highest concentration in 3D spheroids after the 14-day exposure. Benchmark concentration (BMC) analysis indicated that, despite overlapping confidence intervals between 3D and 2D HepaRG cultures in most instances, BMC values tended to be lower in 3D than in 2D cultures after both 3-day and 14-day treatments. In addition, the 14-day treatments of both 2D and 3D HepaRG cultures with N-nitroso-fluoxetine resulted in higher levels of mutagenesis than the 3-day treatments, while 3-day and 14-day treatments with N-nitroso-varenicline produced similar mutagenesis responses. These findings demonstrate the feasibility of employing HepaRG cells with HiFi-seq as a mammalian cell assay for assessing NDSRI mutagenesis.

The comet assay is a widely used method for measuring DNA damage and DNA repair. When DNA strand breaks happen, the supercoiling of DNA is relaxed, and after alkaline or neutral electrophoresis, depending on the type of performed comet assay, DNA moves toward the anode, forming a comet tail. Thus, with increasing frequency of DNA strand breaks, an increase in the percentage of DNA in the tail is observed. The aim of this study was to compare systematically various steps like lysis, duration of electrophoresis, and pH of the electrophoresis solution and their effect on the comet tail with regard to sensitivity for detection and quantification of DNA damage. We treated human lymphoblastoid TK6 cells with known genotoxic substances with a different mode of action and then performed both standard and modified alkaline and neutral comet assays. The modifications included Fpg- and MspI-modified comet assays. Several aspects of this comparison are investigated for the first time here. The results obtained from these experiments showed a higher %DNA in tail in the alkaline comet assay compared to the neutral comet assay. Additionally, the lysis step was not critical in the alkaline comet assay, whereas it was essential for the neutral comet assay. Results from alkaline Fpg-modified comet assay showed higher sensitivity in detecting single strand breaks and the neutral MspI-modified comet assay was better in detecting DNA double-strand breaks. Overall, our findings provided valuable insight into the differences between alkaline and neutral electrophoresis conditions in the comet assay and indicated that the alkaline comet assay is more sensitive for measuring total DNA damage.

Aristolochic acid I (AAI) is a carcinogen associated with various human cancers. However, its causal relationship with hepatocellular carcinoma remains controversial, and inconsistent results from rodent studies have suggested species-specific differences. Here we evaluated AAI genotoxicity using functional human-induced hepatocyte-like cells (hiHep cells), a model that closely mimics primary human hepatocytes in gene expression and function, thereby shedding light on its potential hepatocarcinogenic risk in humans. First, we assessed AAI genotoxicity by evaluating AAI-DNA adducts and micronucleus frequency. In hiHep cells, AAI (0.7-2.5 µM) induced up to 105 adducts per 108 nucleotides, indicating high metabolic activation of AAI. A concentration-dependent increase in micronucleus frequency indicated a significant increase in chromosomal aberrations in hiHep cells. Considering the evidence of AAI inducing oxidative stress, we assessed 8-hydroxy-2'-deoxyguanosine and reactive oxygen species levels to evaluate DNA oxidative damage. For both indicators, significantly elevated levels were observed. A mechanism involving oxidative damage was further supported by observations of mitochondrial dysfunction, including changes in mitochondrial membrane potential and mitochondrial complex activity. Ascorbate treatment decreased AAI-induced oxidative DNA damage and DNA adduct formation, providing direct cellular evidence for free radical intermediates in AAI metabolic activation-a mechanism previously hypothesized but not experimentally validated in a human-relevant hepatocyte model. Our study findings revealed the genotoxic effects of AAI on hiHep cells and implicated oxidative stress as the key mechanism. These findings strengthen the association between AAI exposure and liver disease and highlight the potential role of antioxidant therapies in mitigating AAI-associated carcinogenesis.

In the comet assay, DNA damage is assessed by differences in DNA migration from gel-embedded nucleoids. Even a small difference in DNA migration between exposure groups can be statistically significant but may invite speculation about the biological significance of such slight increases in DNA migration. A small difference can be defined as a net difference of 1–2% Tail DNA, but background levels of DNA migration typically vary already more than 1–2% Tail DNA between studies. Here, we have used studies on ionizing radiation to assess the lowest detectable differences in DNA migration; variation in exposure-effect relationships; variation in central tendencies of DNA migration; unsystematic (residual) variation; and the actual number of lesions detectable with the comet assay. A total of 51 studies on ionizing radiation exposure in mammalian cells have been systematically reviewed, including results from ring-trial studies where the same batch of irradiated cells has been analysed in different laboratories. Ring-trial studies have shown that unsystematic variation is approximately 4% Tail DNA in studies on ionizing radiation. Studies on ionizing radiation in cell cultures have shown statistically significant effects when the net increase of DNA migration is 0.3–3.1% Tail DNA. Among those experiments, the ones with optimal assay conditions to detect low levels of DNA damage show statistically significant effects with doses of around 0.30 Gy, which corresponds to approximately 350 lesions per diploid cell. However, it has also been shown that the same dose of ionizing radiation can give rise to different levels of DNA migration (i.e. 0.7–7.8% Tail DNA per Gy) in different studies. In summary, the results show that even a small statistically significant difference in DNA migration has biological significance within the same experiment, but comparisons of DNA migration values between studies have limited biological implications.

Nucleotide excision repair (NER) is crucial for repairing bulky lesions and crosslinks in DNA caused by exogenous and endogenous genotoxins. The number of studies that have considered DNA repair as a biomarker is limited, and therefore one of the primary objectives of the European COST Action hCOMET (CA15132) was to assemble and analyse a pooled database of studies with data on NER activity. The database comprised 738 individuals, gathered from 5 laboratories that ran population studies using the comet-based in vitro DNA repair assay. NER activity data in peripheral blood mononuclear cells were normalized and correlated with various host-related factors, including sex, age, body mass index (BMI), and smoking habits. This multifaceted analysis uncovered significantly higher NER activity in female participants compared to males (1.08 ± 0.74 vs. 0.92 ± 0.71; P = .002). Higher NER activity was seen in older subjects (>30 years), and the effect of age was most pronounced in the oldest females, particularly those over 70 years (P = .001). Females with a normal BMI (<25 kg/m2) exhibited the highest levels of NER, whereas the lowest NER was observed in overweight males (BMI ≥ 25 kg/m2). No independent effect of smoking was found. After stratification by sex and BMI, higher NER was observed in smoking males (P = .017). The biological implication of higher or lower repair capacity remains unclear; the inclusion of DNA repair as a biomarker in molecular epidemiological trials should elucidate the link between health and disease status.

Porous silica nanoparticles (PSNs) hold immense promise as drug delivery carriers owing to their high surface area, accessible silanol groups, customisable pore and particle sizes, and facile surface modification capabilities.However, before their clinical translation, a thorough assessment of PSN toxicity is imperative to ensure their biocompatibility.This study undertook a comprehensive evaluation of various toxicological endpoints, encompassing cytotoxicity, genotoxicity, and immunogenicity, using three cell lines representing potential exposure routes of PSNs.Physicochemical characterisation of PSNs was conducted to identify the size, charge and surface functionalisation.Lymphoblastoid TK6, monocytic THP-1, and liver cancer HepG2 cells were exposed to five functionalised PSNs, polyethylenimine (PEI), amine, carboxyl, thiol or silanol at varying concentrations (0 to 200mg/ml) for 24, 48, or 72 hours.Cytotoxicity assessment via the MTT assay revealed varying toxicity among PSN types and cell lines.Notably, PSN-PEI showed significant toxicity towards THP-1 cells compared to TK6 cells.PSN-Carboxyl exhibited toxicity in HepG2 cells, whereas PSN-Thiol demonstrated minimal cytotoxicity across all cell types, suggesting differential cellular responses.Genotoxicity analysis using the cytokinesis block micronucleus assay identified an increased micronuclei frequency induced by PSN-PEI and PSN-Carboxyl in TK6 and THP-1 cells, underscoring the significance of DNA damage evaluation.This correlated with the cellular uptake observed in THP-1 cells.Immunogenicity studies using western blotting revealed interactions between PSNs and C3b protein.PSN-Silanol showed binding to multiple complement proteins C1q, C3 and MBL, thereby suggesting activation of all three complement pathways -classical, alternative and lectin, respectively.Complement activation could generate anaphylatoxins and PSN opsonisation, affecting the therapeutic effect of PSNs.This study provides crucial insights into PSN toxicity, ensuring the use of safe and effective nanocarriers for drug delivery.Future research will investigate intracellular signalling events and protein corona associated with toxicity to better understand the impact of PSNs, thus supporting safe nanocarriers.

DNA ligase (LIG) I and IIIα finalize base excision repair (BER) by sealing a nick product after nucleotide insertion by DNA polymerase (pol) β at the downstream steps. We previously demonstrated that a functional interplay between polβ and BER ligases is critical for efficient repair, and polβ mismatch or oxidized nucleotide insertions confound the final ligation step. Yet, how targeting downstream enzymes with small molecule inhibitors could affect this coordination remains unknown. Here, we report that DNA ligase inhibitors, L67 and L82-G17, slightly enhance hypersensitivity to oxidative stress-inducing agent, KBrO3, in polβ+/+ cells more than polβ-/- null cells. We showed less efficient ligation after polβ nucleotide insertions in the presence of the DNA ligase inhibitors. Furthermore, the mutations at the ligase inhibitor binding sites (G448, R451, A455) of LIG1 significantly affect nick DNA binding affinity and nick sealing efficiency. Finally, our results demonstrated that the BER ligases seal a gap repair intermediate by the effect of polβ inhibitor that diminishes gap filling activity. Overall, our results contribute to understand how the BER inhibitors against downstream enzymes, polβ, LIG1, and LIGIIIα, could impact the efficiency of gap filling and subsequent nick sealing at the final steps leading to the formation of deleterious repair intermediates.