Comparative gene expression analysis after exposure to 123I-iododeoxyuridine, γ- and α-irradiation

Gene expression analysis was carried out in human T-lymphoma Jurkat cells in order to identify candidate genes showing significant gene expression alterations allowing robust discrimination of the Auger emitter 123I, incorporated into the DNA as 123I-iododeoxyuridine (123IUdR), compared to α- and γ-radiation, which may be useful for biodosimetry purposes. Comparative gene expression analysis was performed employing whole human genome DNA microarrays. The gene expression of all candidate genes was validated by quantitative real-time PCR. 155, 316 and 982 genes were exclusively regulated after exposure to 123IUdR, α-particles and γ-rays, at equi-effect doses/activity, respectively. Applying the stringent requirements for candidate genes, four , one and one gene(s) were identified allowing a reliable discrimination between γ- versus 123IUdR exposure, γ- versus α-radiation and α- versus 123IUdR exposure, respectively. Gene expression analysis might be an effective tool for the general discrimination of radiation qualities and might help to elucidate different biological effectiveness on the mechanistic level. The formation of γ-H2AX foci after exposure to different radiation qualities was analyzed to determine equi-effect doses/activities and consequently to compare gene expression on a similar DNA damage effect level. A similar DNA damage effect level was found after exposure to 10 Gy γ-rays, 1 Gy α-particles and after 20 h exposure to 123IUdR, which corresponded roughly to 2600 accumulated 123I decays per cell. Genes with a fold-change > 1.5 and a FDR < 0.15 were used for the functionally categorization and the venn diagram. A candidate gene allowing the discrimination of different radiation qualities had to show a significant expression fold change of > 1.5 fold after exposure to a specific radiation quality and display no altered (1-fold ± 0.1) or even opposing (>1.1-fold) regulation in response to the other radiation qualities. Concomitantly, we used P values, which were not adjusted using the FDR, for the analysis of the significance in order to consider more genes. However, this loss of statistical stringency was justified because the gene expression level of the candidate genes were verified and validated by quantitative real-time PCR .

本研究以人类T淋巴瘤Jurkat细胞为模型开展基因表达分析，旨在筛选出可显著区分俄歇电子发射体（Auger emitter）¹²³I（以¹²³I-碘脱氧尿苷（¹²³I-iododeoxyuridine，¹²³IUdR）形式掺入DNA）与α、γ辐射的差异表达候选基因，相关成果可用于生物剂量学相关研究。本研究采用全人类基因组DNA微阵列（whole human genome DNA microarray）开展对比基因表达分析，所有候选基因的表达水平均通过实时定量聚合酶链式反应（quantitative real-time PCR，qPCR）完成验证。在等效剂量/活度条件下经¹²³IUdR、α粒子和γ射线照射后，仅受对应辐射特异性调控的基因数量分别为155、316和982个。通过严格的候选基因筛选标准，我们分别筛选出可可靠区分γ射线与¹²³IUdR照射、γ射线与α辐射、α粒子与¹²³IUdR照射的基因各4个、1个和1个。基因表达分析可作为通用区分辐射品质的有效工具，有助于从分子机制层面阐明不同辐射的生物学效应差异。本研究通过分析不同辐射照射后γ-H2AX焦点（γ-H2AX foci）的形成情况，确定等效剂量/活度，从而在相似DNA损伤效应水平下开展基因表达对比分析。实验发现，经10 Gy γ射线、1 Gy α粒子照射以及¹²³IUdR暴露20小时后，细胞达到了相似的DNA损伤效应水平，对应每细胞累积¹²³I衰变次数约为2600次。本研究选取表达倍数变化>1.5且错误发现率（False Discovery Rate，FDR）<0.15的基因，用于功能分类分析及韦恩图（Venn diagram）绘制。可用于区分不同辐射品质的候选基因需满足以下条件：在特定辐射照射后呈现>1.5倍的显著表达变化，而在其他辐射照射下仅出现1倍±0.1范围内的表达波动，甚至呈现与目标辐射相反（>1.1倍）的调控趋势。同时，为纳入更多候选基因，本研究未采用FDR校正的P值进行显著性分析。尽管此举降低了统计严格性，但由于所有候选基因的表达水平均通过qPCR完成验证，因此该处理方式具备合理性。