Environmental and Molecular Mutagenesis最新文献

Previously, we introduced an alternative adherent A375 cell line for clastogenicity and aneugenicity testing using a high content imaging platform. To further characterize the performance of A375 cells, we investigated the sensitivity and specificity of A375 and TK6 cells by directly comparing micronucleus (MN) induction, cytotoxicity (relative cell counts, viability, and apoptosis), clastogenicity (γH2AX), and aneuploidy markers (pH 3, MPM-2, and polyploidy) using flow cytometric methods. We evaluated 14 compounds across different mechanisms (non-genotoxic apoptosis inducers, clastogens, and aneugens with either tubulin binding or aurora kinase inhibiting phenotypes) at 4-h and 24-h post treatment. Both aneugens and clastogens tested positive for micronucleus induction in both cell lines. Apoptosis continued to be a confounding factor for flow cytometry-based micronuclei assessment in TK6 cells as evidenced by positive responses by the three cytotoxicants. Conversely, A375 cells were not affected by apoptosis-related false positive signals and did not produce a positive response in the in vitro micronucleus assay. Benchmark dose response (BMD) analysis showed that the induction of micronuclei and biomarkers occurred at similar concentrations in both cell lines for clastogens and aneugens. By showing that A375 cells have similar sensitivity to TK6 cells but a greater specificity, these results provide additional support for A375 cells to be used as an alternative adherent cell line for in vitro genetic toxicology assessment.

One type of epigenetic modification is genomic DNA methylation, which is induced by smoking, and both are associated with male infertility. In this study, the relationship between smoking and CHD5 gene methylation and semen parameters in infertile men was determined. After the MS-PCR of blood in 224 samples, 103 infertile patients (62 smokers and 41 non-smokers) and 121 fertile men, methylation level changes between groups and the effect of methylation and smoking on infertility and semen parameters in infertile men were determined. The results showed that there is a significant difference in the methylation status (MM, MU, UU) of the CHD5 gene between the patient and the control group, and this correlation also exists for the semen parameters (p < .001). The average semen parameters in smokers decreased significantly compared to non-smokers and sperm concentration was (32.21 ± 5.27 vs. 55.27 ± 3.38), respectively. MM methylation status was higher in smokers (22.5%) compared to non-smokers (14.6%). Smoking components affect the methylation pattern of CHD5 gene, and smokers had higher methylation levels and lower semen parameters than non-smokers, which can be biomarkers for evaluating semen quality and infertility risk factors. Understanding the epigenetic effects of smoking on male infertility can be very useful for predicting negative consequences of smoking and providing therapeutic solutions.

The aim of this study was twofold: (1) evaluate the effect of benzo[a]pyrene (BaP) on expression levels of AQP3 and Notch1 genes in HaCaT cells exposed “in vitro” and (2) investigate the possible biological role of assessed genes by bioinformatics methods. Cells were exposed to increasing concentrations of BaP (0.0–4.0 μM) for 1–4 days. After treatments, cell viability and expression levels of AhR, CYP1A1, AQP3, and Notch1 genes were evaluated. The possible biological role of assessed genes was evaluated using bioinformatics tools. Low cytotoxicity in HaCaT cells dosed with BaP was detected. A significant overexpression (p < .05) of CYP1A1, AQP3, and Notch1 was found in exposed HaCaT cells. The gene expression upregulation was dependent on AhR activation. The bioinformatics analysis showed that these genes were enriched in related cancer signaling pathways. The findings suggest that AQP3 and Notch1 are upregulated by AhR activation in HaCaT cells exposed to BaP.

Mutations in T lymphocytes (T-cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T-cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical-induced mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene of T-cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T-cell development and changes in chemical-induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase-polymerase chain reaction based method was developed to determine the specific T-cell receptor beta (Tcrb) gene mRNA expressed in mouse T-cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N-ethyl-N-nitrosourea-exposed mice permitted elucidation of the localization and timing of mutational events in T-cells, establishing that mutagenesis occurs primarily in the pre-rearrangement replicative period in pre-thymic/thymic populations. These findings demonstrate that chemical-induced mutagenic burden is determined by the combination of mutagenesis and T-cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

The blood cell phosphatidylinositol glycan class A (PIG-A) gene mutation assay has been extensively researched in rodents for in vivo mutagenicity testing and is now being investigated in humans. The PIG-A gene is involved in glycosyl phosphatidylinositol (GPI)-anchor biosynthesis. A single mutation in this X-linked gene can lead to loss of membrane-bound GPI anchors, which can be enumerated via corresponding GPI-anchored proteins (e.g., CD55) using flow cytometry. The studies published to date by different research groups demonstrate a remarkable consistency in PIG-A mutant frequencies. Moreover, with the low background level of mutant erythrocytes in healthy subjects (2.9–5.56 × 10⁻⁶ mutants), induction of mutation post genotoxic exposure can be detected. Cigarette smoking, radiotherapy, and occupational exposures, including lead, have been shown to increase mutant levels. Future applications of this test include identifying new harmful agents and establishing new exposure limits. This mutational monitoring approach may also identify individuals at higher risk of cancer development. In addition, identifying protective agents that could mitigate these effects may reduce baseline somatic mutation levels and such behaviors can be encouraged. Further technological progress is required including establishing underlying mechanisms of GPI anchor loss, protocol standardization, and the development of cryopreservation methods to improve GPI-anchor stability over time. If successful, this assay has the potential be widely employed, for example, in rural and low-income countries. Here, we review the current literature on PIG-A mutation in humans and discuss the potential role of this assay in human biomonitoring and disease detection.

Smoke flavorings are mixtures generated from wood pyrolysis that are filtered to remove tar and are often considered healthier alternatives to conventional smoking processes. While the latter is mostly unregulated, smoke-flavoring primary products (SFPPs) are undergoing the 10-year required re-evaluation in the European Union (EU). To comply with recent smoke flavor guidance, in vivo micronucleus studies in rats and transgenic rodent (TGR) mutation assays in Muta™Mice were conducted on three SFPPs. For most studies, typical limit doses were exceeded to comply with regulatory requests. Exposure to SFPPs by oral gavage did not result in significant increases in bone marrow micronucleus formation. Except for one group, exposure to SFPPs via feed for 28 days did not result in significant increases in mutant frequency (MF) in the glandular stomach or liver. One group exposed to a maximal feasible dietary dose of 50,000 ppm (>10,000 mg/kg bodyweight per day) exhibited a statistically significant increase in liver MF; however, the MF in all mice in this group were within the historical vehicle control 95% quantile confidence intervals and therefore not considered biologically relevant. Based on estimates of human dietary exposure to each SFPP, the margin of exposure (MOE) values in the TGR assays exceed 10,000. The MOE for one unintentionally present constituent, 2,5(H)-furanone, also exceeds 10,000. Collectively, these data indicate that these SFPPs pose no genotoxic risk and are safe alternatives to conventional smoking.

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose–response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

In this study, our aim was to investigate the epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) gene polymorphisms in oral squamous cell carcinoma (OSCC) patients and non-OSCC healthy controls. This case–control study comprised 89 OSCC and 107 healthy controls by using polymerase chain reaction (PCR) and restriction fragment length polymorphism methods, the genotypes for EGF + 61 A > G (rs4444903) and EGFR R497K (rs2227983) were analyzed. According to the EGF + 61 A > G genotype distribution, individuals with the GG genotype were more prevalent in the OSCC group when compared to the healthy controls. But the AA genotype frequency was significantly higher in the healthy control group. The frequency of G allele carriers was 2.3 times higher than A allele carriers in OSCC patients (p < .001). For the EGFR R497K genotype, there was no significant difference between the OSCC and healthy control groups. Regarding the study results, the G allele of EGF + 61 A > G polymorphism was associated with OSCC. Larger populations and functional investigations should be used to explore the nature of the interaction between EGF and OSCC.

In response to oxidative damage, base excision repair (BER) enzymes perturb the structural equilibrium of the VEGF promoter between B-form and G4 DNA conformations, resulting in epigenetic-like modifications of gene expression. However, the mechanistic details remain enigmatic, including the activity and coordination of BER enzymes on the damaged G4 promoter. To address this, we investigated the ability of each BER factor to conduct its repair activity on VEGF promoter G4 DNA substrates by employing pre-steady-state kinetics assays and in vitro coupled BER assays. OGG1 was able to initiate BER on double-stranded VEGF promoter G4 DNA substrates. Moreover, pre-steady-state kinetics revealed that compared to B-form DNA, APE1 repair activity on the G4 was decreased ~two-fold and is the result of slower product release as opposed to inefficient strand cleavage. Interestingly, Pol β performs multiple insertions on G4 substrates via strand displacement DNA synthesis in contrast to a single insertion on B-form DNA. The multiple insertions inhibit ligation of the Pol β products, and hence BER is not completed on the VEGF G4 promoter substrates through canonical short-patch BER. Instead, repair requires the long-patch BER flap-endonuclease activity of FEN1 in response to the multiple insertions by Pol β prior to ligation. Because the BER proteins and their repair activities are a key part of the VEGF transcriptional enhancement in response to oxidative DNA damage of the G4 VEGF promoter, the new insights reported here on BER activity in the context of this promoter are relevant toward understanding the mechanism of transcriptional regulation.