Yauk Carole L, Polyzos Aris, Rowan-Carroll Andrea, Kortubash Igor, Williams Andrew, Kovalchuk Olga
Environmental Health Sciences and Research Bureau, Safe Environments Programme, Health Canada, Ottawa, Ontario, Canada.
Environ Mol Mutagen. 2008 Jan;49(1):26-35. doi: 10.1002/em.20359.
Mutations at expanded simple tandem repeat (ESTR) DNA sequences provide a useful tool for screening germline mutation. However, the mechanisms resulting in induced mutations are unknown and provide an impediment to the utility of the method. Induced ESTR mutations arise through a nontargeted mechanism resulting in destabilization of the repeat locus. We hypothesized that alterations in DNA methylation, or in DNA methyltransferase expression, may be associated with this indirect mechanism of mutation. DNA mutation frequency was measured in C3H/10T1/2 mouse embryonic fibroblast cells following chronic exposure to six chemicals exhibiting different modes of genotoxic action: N-nitroso-N-ethylurea (ENU); benzo(a)pyrene (BaP); etoposide (ETOP); okadaic acid (OA); cisplatin (CisPt); and 5-azacytidine (5azadC). Induced mutation ranged from 2-fold (ENU, BaP, ETOP), to 1.3-1.4 fold (OA, 5azadC), to nonresponsive (CisPt). Global DNA methylation, measured using the cytosine extension assay, revealed hypomethylation following exposure to ENU and 5azadC, hypermethylation following BaP and OA exposure, and no change following treatment with ETOP or CisPt. DNA methyltransferase transcription (Dnmt1, Dnmt3a, Dnmt3b) was significantly affected by all treatments except ETOP, with the vast majority of changes being downregulation. There was no direct correlation between ESTR mutation, global methylation, or DNA methyltransferase transcription. However, 4/5 ESTR mutagens caused changes in global methylation, while the noninducer (CisPt) did not cause changes in methylation. We hypothesize that chemicals that modify chromatin conformation through changes in methylation may compromise the ability of mismatch repair enzymes (or other enzymes) to access and repair secondary structures that may form across ESTR loci resulting in mutation.
扩展简单串联重复序列（ESTR）DNA序列的突变提供了一种筛选种系突变的有用工具。然而，导致诱导突变的机制尚不清楚，这阻碍了该方法的实用性。诱导的ESTR突变通过非靶向机制产生，导致重复基因座不稳定。我们假设DNA甲基化或DNA甲基转移酶表达的改变可能与这种间接突变机制有关。在慢性暴露于六种具有不同遗传毒性作用模式的化学物质后，测量C3H/10T1/2小鼠胚胎成纤维细胞中的DNA突变频率：N-亚硝基-N-乙基脲（ENU）；苯并(a)芘（BaP）；依托泊苷（ETOP）；冈田酸（OA）；顺铂（CisPt）；以及5-氮杂胞苷（5azadC）。诱导突变范围从2倍（ENU、BaP、ETOP）到1.3 - 1.4倍（OA、5azadC），再到无反应（CisPt）。使用胞嘧啶延伸测定法测量的全局DNA甲基化显示，暴露于ENU和5azadC后发生低甲基化，暴露于BaP和OA后发生高甲基化，而用ETOP或CisPt处理后无变化。除ETOP外，所有处理均显著影响DNA甲基转移酶转录（Dnmt1、Dnmt3a、Dnmt3b），绝大多数变化为下调。ESTR突变、全局甲基化或DNA甲基转移酶转录之间没有直接相关性。然而，5种ESTR诱变剂中有4种导致全局甲基化发生变化，而非诱导剂（CisPt）未导致甲基化变化。我们假设通过甲基化变化修饰染色质构象的化学物质可能会损害错配修复酶（或其他酶）访问和修复可能在ESTR基因座形成的二级结构的能力，从而导致突变。
