Mutagenesis最新文献

Objective: To investigate the effects of polystyrene microplastics (PS-MPs) on human corneal epithelial cells (HCEP).

Methods: The cytotoxicity of PS-MPs on HCEP cells was evaluated using a CCK-8 assay to measure cell viability, flow cytometry to analyze cell cycle and status, immunofluorescence to detect reactive oxygen species (ROS) and γ-H2AX levels, and western blotting to assess protein expression.

Results: The effects of PS-MPs on HCEP cell morphology and viability were particle size- and concentration-dependent. Smaller particle sizes and higher concentrations of PS-MPs were associated with greater cytotoxicity. PS-MP exposure induced cell cycle arrest, necrosis, and apoptosis in HCEP cells, along with excessive ROS production and DNA damage. Furthermore, ROS scavengers significantly reduced PS-MP-induced ROS overproduction and DNA damage, thereby alleviating PS-MP-induced cell cycle arrest, necrosis, and apoptosis. At the molecular level, ROS scavengers reversed the PS-MP-induced changes in the expression of γ-H2AX, P53, cell cycle-related proteins (cyclin D1, CDK2, and CDK4), necrosis-related proteins (CypD, PARP-1, and SRX), and apoptosis-related proteins (Cyt C, AIF, and cleaved-caspase 3).

Conclusion: PS-MP exposure leads to cell cycle arrest, necrosis, and apoptosis in HCEP cells, which is associated with ROS overproduction and activation of the P53 pathway.

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.

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.

The proceedings of the 36th annual meeting of the Industrial Genotoxicology Group (IGG) are shared here. The meeting held at Lhasa Limited, Leeds, UK on 28 November 2023, focussed on two aspects; new approach methodologies (NAMs), including those for the assessment of non-standard modalities such as gas-vapour assessments and nanomaterials, and addressing the regulatory challenges associated with understanding the genotoxic and carcinogenic potential of N-nitrosamines and N-nitrosamine impurities. New approach methodologies, such as error-corrected sequencing and enhanced Ames tests that may help address these challenges were also discussed.

The in vitro bacterial reverse mutation (Ames) test is crucial for evaluating the mutagenicity of pharmaceutical impurities. For N-nitrosamines (NAs) historical data indicated that for certain members of this chemical class, the outcomes of the Ames test did not correlate with their associated rodent carcinogenicity outcomes. This has resulted in negative outcomes in an OECD (Organization for Economic Cooperation and Development)-aligned Ames test alone (standard or enhanced) no longer being considered sufficient by regulatory authorities to assess potential carcinogenic risk of NAs if present as impurities in drug products. Consequently, extensive follow-up in vivo testing can be required to characterize the potential mutagenicity and genotoxic carcinogenicity of NA impurities (i.e. beyond that defined in the ICH M7 guideline for non-NA impurities). We previously demonstrated that the mutagenicity of alkyl-nitrosamines can be detected by the appropriately designed, OECD-aligned Ames test and identified those conditions that contributed most to assay sensitivity. This OECD-aligned Ames test design was used to assess seven NAs, i.e. (methyl(neopentyl)nitrosamine, N-methyl-N-nitroso-2-propanamine, N-nitrosodiisopropylamine, bis(2-methoxyethyl)nitrosoamine, N-nitroso-N-methyl-4-fluoroaniline, dinitrosoethambutol, (R,R)- and mononitrosocaffeidine) that were reported to be negative in historical Ames tests but positive in rodent carcinogenicity studies. All seven of the NAs were demonstrated to be mutagenic in the OECD-aligned Ames test and therefore these compounds should no longer be considered as discordant (false negatives) with respect to the correlation of the Ames test and rodent carcinogenicity. These results confirm the sensitivity of the OECD-aligned Ames test for the detection of NA mutagenicity and provides further support of its pivotal placement within the ICH M7 framework for the assessment of mutagenic impurities in pharmaceuticals to limit potential carcinogenic risk. In addition, we present data for 1-cyclopentyl-4-nitrosopiperazine, that indicates it could serve as a suitable positive control to provide further confidence in the sensitivity of the Ames test for the NA chemical class.

The fetal brain is susceptible to programming effects during pregnancy, potentially leading to long-term consequences for offspring's cognitive health. Fructose (FRU) intake is thought to adversely affect fetal brain development, whereas physical exercise before and during pregnancy may be protective. Therefore, this study aimed to assess biochemical and genotoxic changes in maternal hippocampi and behavioral, genotoxic, and biochemical alterations in offspring hippocampi. Seventy female mice were exposed to FRU (20%/L) and/or voluntary physical exercise (VPE) pre-pregnancy for eight weeks, and then mated and exposure was continued until weaning. Offspring were evaluated at 60 days old using behavioral test, genotoxic, and biochemical markers. FRU-induced long-term memory impairment in male offspring, which was alleviated by VPE. VPE mitigated DNA damage from maternal FRU consumption in both maternal and offspring hippocampi in female offspring, VPE increased levels of apurine/apyrimidinic endonuclease 1, erythroid nuclear factor 2, and cAMP response element binding proteins, whereas in males, 8-oxoguanine DNA glycosylase-1 levels upregulate. FRU consumption led to oxidative stress and antioxidant defense alterations in offspring, while VPE mitigated these effects. Telomere shortening was observed in male offspring from mothers who consumed FRU during pregnancy. Our findings suggest that exposure to FRU during (pre)pregnancy and lactation has adverse effects on offspring's hippocampi later in life, and VPE has a protective effect. Overall, the study underscores the significance of maternal dietary and physical habits on long-term offspring health, with an emphasis on implications for adult cognitive function.

Doxorubicin, a well-known and widely used antineoplastic agent with direct ROS-accumulating activity, has proven effective in treating various cancer types. However, its non-specific cytotoxicity towards non-cancerous cells prompts concerns regarding potential adverse effects. Azithromycin is an antibiotic for treating bacterial infections and an anti-inflammatory agent, particularly beneficial in managing respiratory conditions like bronchitis and sinusitis. Despite azithromycin's well-documented antibacterial properties, its potential cellular/genomic protective effects remain unexplored. As an in vitro model, BEAS-2B cells (normal human bronchial epithelium cells) were employed in this study to assess whether azithromycin possesses any protective properties against doxorubicin-induced cellular toxicity. Cells in pretreatment culture were treated to various amounts of azithromycin (3.125, 6.25, 12.5, 25, and 50 μg/ml) in combination with doxorubicin at IC50 (0.08 μg/ml). Doxorubicin at 0.08 μg/ml highlighted cytotoxicity, oxidative stress, and genotoxicity. Azithromycin at 25 and 50 μg/ml markedly modulated oxidative stress and genomic damage by decreasing the ROS and LPO amounts and suppressing DNA fragmentation in the comet assay parameters. Consequently, azithromycin may be regarded as a cytomodulating, antigenotoxic, and antioxidant agent.

Micronuclei (MN) are cellular structures containing chromosome fragments or whole chromosomes that fail to be incorporated into the main nuclei during mitosis. MN measured in lymphocytes using the cytokinesis-block method and MN in buccal cells are among the most widely used methods for measuring DNA damage in humans. However, it remains unclear whether they correlate well with each other. This has important implications regarding whether existing evidence linking MN in lymphocytes to prospective cancer risk can also be extended to MN in buccal cells, a much less invasive approach. We therefore systematically reviewed results from published studies that reported MN frequencies simultaneously in buccal cells and lymphocytes. Data were extracted from a set of 81 study groups reported in 62 publications. The overall frequency of MN in groups exposed to increased risk of DNA damage was 2.54 times higher compared to controls (95% CI: 2.06-3.01) in buccal cells and 2.43 times higher (95% CI: 1.92-2.93) in lymphocytes. Frequencies of MN in populations investigated for occupational or environmental exposure to genotoxins, various diseases, and poor nutrition/lifestyle were also compared in each study and for each tissue (lymphocytes and buccal mucosa) with frequencies in control subjects using the Mean Ratio (MR). Concordance between the two MN assays was evaluated by comparing MRs for primary exposure in all studies using a correlation analysis. The overall Pearson correlation index was 0.768 (0.877 for case-control studies and 0.998 for intervention studies), showing that MR estimates from the two assays were highly and significantly correlated (p<0.001). The results from this investigation indicate that data obtained using the buccal MN assay reflect results obtained using the lymphocyte cytokinesis-block MN assay. This suggests that the buccal MN assay may also identify those at increased risk of tumorigenesis. Prospective studies will ultimately be required to completely verify this hypothesis.

Multidisciplinary molecular tumor boards (MTB) are already well established in many comprehensive cancer centers and play an important role in the individual treatment planning for cancer patients. Comprehensive genomic profiling of tumor tissue based on next-generation sequencing is currently performed for diagnostic and mainly predictive testing. If somatic genomic variants are identified, which are suspected to be pathogenic germline variants (PGVs), MTB propose genetic counseling and germline DNA testing. Commonly used comprehensive genomic profiling approaches of tumor tissue do not include a matched germline DNA control. Therefore, the detection of PGVs could be only predicted based on the content of tumor cells (CTC) in selected tumor area (%) and variant allele frequency score (%). For conclusion, the role of a medical geneticist is essential in these cases. The overall prevalence of PGVs in patients with pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) is approximately 10%. In this single-center study, we present 37 patients with PDAC and 48 patients with CRC who were presented at MTB and tested using the large combined DNA/RNA sequencing panel. Content of tumor cells and variant allele frequency scores were evaluated in all tested patients. In case of suspicion of PGV and no previous genetic testing based on the standard guidelines, genetic counseling was recommended regardless of age, sex, and family history. In the PDAC subgroup, five patients were recommended by MTB for genetic counseling based on suspicious genetic findings. Based on a medical geneticist's decision, germline DNA sequencing was performed in four of these cases, and all of them tested positive for PGV in the following genes: ATM, ATM, BRCA1, and BRCA2. In the CRC subgroup, no PGV was confirmed in the two patients genetically tested based on the MTB recommendations. Furthermore, we present data from our center's registry of patients with PDAC and CRC who underwent genetic counseling and germline DNA testing based on the standard screening criteria. Our data confirm that comprehensive genomic profiling of tumor tissue can identify patients with hereditary forms of PDAC, who could remain unidentified by standard screening for hereditary forms of cancer.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer, with a survival approaching only 11% at 5 years after diagnosis. In the last 15 years, telomere length (TL) measured in leukocyte (LTL) has been studied in relation to PDAC risk. The majority of the studies reported an association between short LTL and increased PDAC risk, but the results are heterogeneous. Genome-wide association studies have identified several single-nucleotide polymorphisms (SNPs) in the telomerase reverse transcriptase (TERT) gene as susceptibility loci for PDAC. Polygenic risk scores computed using SNPs associated with LTL have been tested in relation to PDAC susceptibility with various methods and giving contrasting results. The aim of this review is to analyze all publications carried out specifically on LTL, considering LTL measured with qPCR and with genetic proxies, and PDAC risk. Additionally, we will give an overview of the most relevant associations between SNPs in telomere-associated genes and PDAC, to answer the question shorter or longer? Which one of the two is associated with PDAC risk?