Saleh Amer F, Priestley Catherine C, Gooderham Nigel J, Fellows Mick D
*Genetic Toxicology, Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG, UK and Biomolecular Medicine, Imperial College, London SW7 2AZ, UK.
*Genetic Toxicology, Drug Safety and Metabolism, AstraZeneca, Macclesfield, Cheshire SK10 4TG, UK and Biomolecular Medicine, Imperial College, London SW7 2AZ, UK
Toxicol Sci. 2015 May;145(1):169-76. doi: 10.1093/toxsci/kfv043. Epub 2015 Feb 20.
The degradation of phosphorothioate oligonucleotides (PS-ONDs) and the release of potentially genotoxic modified mononucleotides raise a safety concern for OND-based therapeutics. Deoxyadenosine monophosphorothioate (dAMPαS), a PS nucleotide analog, has been reported to be a potent in vitro mutagen at the thymidine kinase (TK) locus in human TK6 lymphoblastoid cells. This led us to explore the mechanism behind the apparent positive response induced by dAMPαS in the TK gene-mutation assay in TK6 cells. In this work, treatment of TK6 cells with dAMPαS produced a dose-dependent increase in cytotoxicity and mutant frequency at the TK locus. Surprisingly, when the colonies from dAMPαS were re-challenged with the selective agent trifluorothymidine (TFT), the TFT-resistant phenotype was lost. Moreover, dAMPαS-induced colonies displayed distinct growth kinetics and required longer incubation time than 4-nitroquinoline-1-oxide-induced colonies to start growing. Treatment of TK6 cells with dAMPαS induced cell cycle arrest at the G1 phase, enabling cells to grow, and form a colony after the efficacy of TFT in the culture medium was lost. Our findings suggest that a fraction of parental "nonmutant" TK6 cells escaped the toxicity of TFT, possibly via G1 arrest, and resumed growth after the degradation of TFT. We conclude that dAMPαS did not induce real TFT-resistant mutants and caution should be taken with interpretation of mutation data from TK gene-mutation assay in TK6 cells when assessing modified nucleotides.
硫代磷酸酯寡核苷酸（PS-ONDs）的降解以及潜在基因毒性修饰单核苷酸的释放引发了基于OND的治疗方法的安全性担忧。脱氧腺苷单硫代磷酸酯（dAMPαS），一种PS核苷酸类似物，据报道在人TK6淋巴母细胞系的胸苷激酶（TK）位点是一种强效的体外诱变剂。这促使我们探究dAMPαS在TK6细胞的TK基因突变试验中诱导的明显阳性反应背后的机制。在这项工作中，用dAMPαS处理TK6细胞会导致细胞毒性呈剂量依赖性增加以及TK位点的突变频率增加。令人惊讶的是，当用选择剂三氟胸苷（TFT）再次挑战来自dAMPαS的菌落时，TFT抗性表型消失了。此外，dAMPαS诱导的菌落表现出不同的生长动力学，并且比4-硝基喹啉-1-氧化物诱导的菌落需要更长的孵育时间才能开始生长。用dAMPαS处理TK6细胞会诱导细胞周期停滞在G1期，使细胞能够生长，并在培养基中TFT的效力丧失后形成菌落。我们的研究结果表明，一部分亲本“非突变”TK6细胞可能通过G1期停滞逃避了TFT的毒性，并在TFT降解后恢复生长。我们得出结论，dAMPαS并未诱导真正的TFT抗性突变体，在评估修饰核苷酸时，对TK6细胞中TK基因突变试验的突变数据进行解释时应谨慎。
