Desaulniers Daniel, Cummings-Lorbetskie Cathy, Leingartner Karen, Meier Matthew J, Pickles Jessica C, Yauk Carole L
Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9, Canada.
Brunel University London, Uxbridge, Middlesex UB8 3PH, UK.
Toxicology. 2023 Mar 15;487:153451. doi: 10.1016/j.tox.2023.153451. Epub 2023 Feb 6.
Current chemical testing strategies are limited in their ability to detect non-genotoxic carcinogens (NGTxC). Epigenetic anomalies develop during carcinogenesis regardless of whether the molecular initiating event is associated with genotoxic (GTxC) or NGTxC events; therefore, epigenetic markers may be harnessed to develop new approach methodologies that improve the detection of both types of carcinogens. This study used Syrian hamster fetal cells to establish the chronology of carcinogen-induced DNA methylation changes from primary cells until senescence-bypass as an essential carcinogenic step. Cells exposed to solvent control for 7 days were compared to naïve primary cultures, to cells exposed for 7 days to benzo[a]pyrene, and to cells at the subsequent transformation stages: normal colonies, morphologically transformed colonies, senescence, senescence-bypass, and sustained proliferation in vitro. DNA methylation changes identified by reduced representation bisulphite sequencing were minimal at day-7. Profound DNA methylation changes arose during cellular senescence and some of these early differentially methylated regions (DMRs) were preserved through the final sustained proliferation stage. A set of these DMRs (e.g., Pou4f1, Aifm3, B3galnt2, Bhlhe22, Gja8, Klf17, and L1l) were validated by pyrosequencing and their reproducibility was confirmed across multiple clones obtained from a different laboratory. These DNA methylation changes could serve as biomarkers to enhance objectivity and mechanistic understanding of cell transformation and could be used to predict senescence-bypass and chemical carcinogenicity.
当前的化学检测策略在检测非遗传毒性致癌物（NGTxC）方面能力有限。无论分子起始事件是与遗传毒性致癌物（GTxC）还是NGTxC事件相关，表观遗传异常都会在致癌过程中出现；因此，可以利用表观遗传标记来开发新的方法，以改进对这两类致癌物的检测。本研究使用叙利亚仓鼠胎儿细胞，确定了从原代细胞到衰老旁路（这是一个关键的致癌步骤）过程中致癌物诱导的DNA甲基化变化的时间顺序。将暴露于溶剂对照7天的细胞与未处理的原代培养细胞、暴露于苯并[a]芘7天的细胞以及处于后续转化阶段的细胞进行比较：正常集落、形态转化集落、衰老、衰老旁路以及体外持续增殖。通过简化代表性亚硫酸氢盐测序确定的DNA甲基化变化在第7天最小。在细胞衰老过程中出现了深刻的DNA甲基化变化，其中一些早期差异甲基化区域（DMR）在最终的持续增殖阶段得以保留。通过焦磷酸测序验证了一组这些DMR（例如，Pou4f1、Aifm3、B3galnt2、Bhlhe22、Gja8、Klf17和L1l），并在从不同实验室获得的多个克隆中证实了它们的可重复性。这些DNA甲基化变化可作为生物标志物，以增强对细胞转化的客观性和机制理解，并可用于预测衰老旁路和化学致癌性。
