Genes and Environment最新文献

Symposium 3 of the 53rd Annual Meeting of the Japanese Environmental Mutagen and Genome Society (JEMS), entitled "Potential for Computational Genotoxicity," was held at Shujitsu University, Okayama, Japan, on December 8, 2024. The symposium discussed the application of advanced informatics technologies, such as (quantitative) structure-activity relationship ((Q)SAR) and error-corrected next-generation sequencing (ecNGS), to the field of genotoxicity within the framework of computational genotoxicity. In this symposium, we invited three scientists who are global leaders in the field of computational genotoxicity. This report summarizes the key discussions and presentations from the symposium. The organizers hope this summary will increase awareness of computational genotoxicity.

Background: Genetic effects due to long term exposure to low doses of ionizing radiation (LDIR) in humans are not well understood. Human population living in high level natural radiation areas (HLNRAs) of Kerala coast in India are continuously exposed to chronic LDIR emanating from monazite containing beach sand for many generations. The background radiation level in this area varies from < 1.0 to 45mGy/year. The people residing in HLNRAs sometimes receives background radiation dose which is approximately 10-40 times higher than the people living in adjacent normal level natural radiation areas (NLNRAs). This population provides a unique opportunity to identify, if present, a mutational signature due to chronic low-dose radiation exposure in humans. We have employed whole exome sequencing approach to determine germline mutational changes in the lymphocytes of healthy individuals from HLNRAs (mean background dose: 31.8 ± 5.4 mGy/year, mean age: 43.0 ± 5.9 years) and compared them with healthy individuals from NLNRAs (mean background dose: 0.9 ± 0.2 mGy/year, mean age: 43.0 ± 11.3 years).

Results: Our results revealed that the overall number of single nucleotide variants (SNVs) and insertions/deletions (indels) were not significantly different in HLNRA (7744 SNVs, 880 indels) and NLNRA (7951 SNVs, 856 indels) groups. A similar number of protein affecting mutations (PAMs) were observed in HLNRA (1925) and NLNRA (2082) individuals. Interestingly, several unique SNVs were identified in both the groups. In HLNRA, unique SNVs were overrepresented in genes involved in important biological pathways such as DNA repair (EXO1, PARP2, DDB1, POLQ, LIG1), epigenetic modification (KDM5D, SETDB2, KMT2B, BRD8, SIRT1), cell cycle progression (CDK14, CCND1) etc. Furthermore, significant predominance of C > T transitions which were unique to HLNRA group was observed preferentially at CpG dinucleotide regions. Analysis with REVEL and AloFT tools did not show any increase in potentially pathogenic mutations including those involved in carcinogenesis in HLNRA individuals exposed to chronic radiation.

Conclusion: This study did not show any significant changes in genetic variants due to long term exposure to LDIR in human population living in HLNRAs of Kerala coast. However, presence of unique SNVs and C > T transitions in CpG islands of HLNRA individuals indicate the possible role of epigenetic mechanisms i.e. DNA methylation in response to chronic LDIR in this population. This study significantly enhances the current understanding of radiation induced genetic changes and associated cancer risk in human population.

Background: Overcoming species differences in metabolism between humans and animals remains a critical challenge in toxicological studies. Rat liver S9 fraction has long been the gold standard for exogenous metabolic activation in in vitro genotoxicity tests. Experiences with human S9 or human primary hepatocytes have suggested that the human materials are unsuitable for standardized testing due to high variability. Nevertheless, there is growing interest in genotoxicity evaluation using metabolic systems that more closely mimic human physiology.

Results: We developed an in-cell ELISA system to measure γH2AX as a DNA damage marker in stable human hepatocytes (γH2AX-SHE). HepaSH cells are consistently available human hepatocytes that stably express a range of metabolic enzymes and drug transporters in vitro. Due to their highly differentiated and non-proliferative nature, conventional genotoxicity endpoints such as micronuclei formation, chromosomal aberrations, or mutant colony assays are not applicable. We used γH2AX, a sensitive DNA damage marker, in this assay system. Indirect mutagens including benzo(a)pyrene, aristolochic acid, and 2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine induced dose-dependent increases in γH2AX across all three HepaSH strains. Time-course analysis following benzo(a)pyrene exposure indicated that a treatment duration of 16 hours or longer was necessary to detect genotoxic responses. Prolonged exposure for 48 hours resulted in extensive cell death, which may interfere with γH2AX quantification.

Conclusions: We demonstrated that γH2AX-SHE can serve as a valuable tool for detecting DNA damage under conditions that mimic human metabolic activity. Based on the findings in this study, we recommend the following assay conditions for γH2AX-SHE: a 24-hour treatment period, a DMSO concentration not exceeding 1%, and careful interpretation of positive responses observed at highly cytotoxic doses - defined as approximately less than 60% cell survival - as these may lack biological relevance.