Characterization of Formaldehyde's Genotoxic Mode of Action by Gene Expression Analysis in TK6 Cells

Gene expression analysis has been established as a tool for the characterization of genotoxic mechanisms of chemical mutagens. This approach has been shown to differentiate between DNA reactive genotoxins and non-DNA reactive or indirectly-acting genotoxins. In this context, it has been suggested that expression analysis is capable of distinguishing compounds that cause DNA damage from those that interfere with mitotic spindle function. Formaldehyde (FA) is known to be a DNA-reactive substance which mainly induces chromosomal damage in cultured mammalian cells. However, there has been concern that FA might also act as an aneugen (i.e., induce aneuploidy) but recent cytogenetic studies did not support this assumption. To further characterize FA's genotoxic mode of action, we now used gene expression profiling as a molecular tool to differentiate between clastogenic and aneugenic activity. TK6 cells were exposed to FA for 4 and 24 h and changes in gene expression were analyzed using a whole-genome human microarray. Results were compared to the expression profiles of two DNA-damaging clastogens (methyl methanesulfonate [MMS] and ethyl methanesulfonate [EMS]) and two aneugens (colcemid [COL] and vincristine [VCR]). The gene expression profiles indicated that clastogens and aneugens induce discriminable gene expression patterns. The expression profile of FA showed more similarities to clastogens than to aneugens. Hierarchical clustering analysis as well as several class prediction algorithms revealed a much closer relationship of FA with clastogens than with aneugens. A pathway analysis of differentially regulated genes also demonstrated an overall better agreement of FA with clastogens than with aneugens. Altogether, the results of this study revealed great similarities in gene expression in response to FA and clastogens but did not support an aneugenic activity of FA. Gene expression analysis of untreated TK6 cells or TK6 cells treated with formaldehyde, the DNA-damaging clastogens methyl methanesulfonate or ethyl methanesulfonate, or the aneugens colcemid or vincristine.

基因表达分析已被确立为表征化学诱变剂遗传毒性机制的研究工具。该方法已被证实可区分DNA反应性遗传毒物与非DNA反应性或间接作用型遗传毒物。在此研究背景下，有观点认为基因表达分析能够区分引发DNA损伤的化合物与干扰有丝分裂纺锤体功能的化合物。甲醛（Formaldehyde, FA）是一种已知的DNA反应性物质，在体外培养的哺乳动物细胞中主要诱导染色体损伤。不过，此前有观点认为FA也可能作为致非整倍体剂（aneugen，即诱导非整倍体）发挥作用，但近期的细胞遗传学研究并未支持这一假设。为进一步阐明FA的遗传毒性作用模式，本研究采用基因表达谱分析作为分子工具，以区分致断裂剂（clastogen）与致非整倍体剂的活性特征。将TK6细胞分别暴露于FA中4小时与24小时，随后通过全基因组人类微阵列芯片分析其基因表达变化。将实验结果与两种DNA损伤型致断裂剂——甲基磺酸甲酯（methyl methanesulfonate, MMS）和乙基磺酸乙酯（ethyl methanesulfonate, EMS）——以及两种致非整倍体剂——秋水仙酰胺（colcemid, COL）和长春新碱（vincristine, VCR）——的表达谱进行对比。基因表达谱分析结果显示，致断裂剂与致非整倍体剂可诱导出可区分的基因表达模式。FA的基因表达谱与致断裂剂的相似性高于其与致非整倍体剂的相似性。层级聚类分析以及多种分类预测算法均显示，FA与致断裂剂的亲缘关系远较其与致非整倍体剂更为紧密。对差异表达基因进行的通路分析同样证实，FA整体上与致断裂剂的契合度优于其与致非整倍体剂的契合度。综上，本研究结果表明，FA诱导的基因表达与致断裂剂诱导的基因表达具有高度相似性，但并未证实FA具备致非整倍体活性。本数据集涵盖未经处理的TK6细胞，以及经甲醛、DNA损伤型致断裂剂（甲基磺酸甲酯或乙基磺酸乙酯）或致非整倍体剂（秋水仙酰胺或长春新碱）处理的TK6细胞的基因表达分析数据。