Embryotoxicology Section and Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research CSIR-IITR, Lucknow 226001, Uttar Pradesh, India.
Mutat Res. 2013 Jul-Aug;747-748:28-39. doi: 10.1016/j.mrfmmm.2013.04.005. Epub 2013 Apr 27.
Hexavalent chromium [Cr(VI)] is a well known mutagen and carcinogen. Since genomic instability due to generation of double strand breaks (DSBs) is causally linked to carcinogenesis, we tested a hypothesis that Cr(VI) causes in vivo generation of DSBs and elicits DNA damage response. We fed repair proficient Drosophila melanogaster (Oregon R(+)) larvae Cr(VI) (20.0μg/ml) mixed food for 24 and 48h and observed a significant (p<0.05) induction of DSBs in their midgut cells after 48h using neutral Comet assay. Global gene expression profiling in Cr(VI)-exposed Oregon R(+) larvae unveiled mis-regulation of DSBs responsive repair genes both after 24 and 48h. In vivo generation of DSBs in exposed Drosophila was confirmed by an increased pH2Av immunostaining along with the activation of cell cycle regulation genes. Analysis of mis-regulated genes grouped under DSB response by GOEAST indicated the participation of non-homologous end joining (NHEJ) DSB repair pathway. We selected two strains, one mutant (ligIV) and another ku80-RNAi (knockdown of ku80), whose functions are essentially linked to NHEJ-DSB repair pathway. As a proof of principle, we compared the DSBs generation in larvae of these two strains with that of repair proficient Oregon R(+). Along with this, DSBs generation in spn-A and okr [essential genes in homologous recombination repair (HR) pathway] mutants was also tested for the possible involvement of HR-DSB repair. A significantly increased DSBs generation in the exposed ku80-RNAi and ligIV (mutant) larvae because of impaired repair, concomitant with an insignificant DSBs generation in okr and spn-A mutant larvae indicates an active participation of NHEJ repair pathway. The study, first of its kind to our knowledge, while providing evidences for in vivo generation of DSBs in Cr(VI) exposed Drosophila larvae, assumes significance for its relevance to higher organisms due to causal link between DSB generation and Cr(VI)-induced carcinogenesis.
六价铬[Cr(VI)]是一种众所周知的致突变剂和致癌物。由于双链断裂（DSB）引起的基因组不稳定性与致癌作用有因果关系，我们检验了一个假设，即 Cr(VI)导致体内产生 DSB 并引发 DNA 损伤反应。我们用 20.0μg/ml 的 Cr(VI)混合食物喂养修复能力强的黑腹果蝇（Oregon R(+)）幼虫 24 和 48 小时，并在 48 小时后使用中性彗星试验观察到其中肠细胞中 DSB 的显著（p<0.05）诱导。在 Cr(VI)暴露的 Oregon R(+)幼虫中进行的全基因组表达谱分析揭示了 24 和 48 小时后 DSB 反应性修复基因的失调。通过增加 pH2Av 免疫染色以及细胞周期调节基因的激活，在暴露的果蝇中体内产生 DSB 得到了证实。GOEAST 对失调基因的分析表明，非同源末端连接（NHEJ）DSB 修复途径的参与。我们选择了两个品系，一个是突变体（ligIV），另一个是 ku80-RNAi（ku80 的敲低），它们的功能主要与 NHEJ-DSB 修复途径有关。作为一个原理证明，我们比较了这两个品系幼虫与修复能力强的 Oregon R(+)幼虫的 DSB 产生情况。与此同时，还测试了 spn-A 和 okr[同源重组修复（HR）途径中的必需基因]突变体中 DSB 的产生，以确定 HR-DSB 修复的可能参与。由于修复受损，暴露的 ku80-RNAi 和 ligIV（突变体）幼虫中 DSB 的产生显著增加，而 okr 和 spn-A 突变体幼虫中 DSB 的产生则微不足道，这表明 NHEJ 修复途径的积极参与。这项研究是首次在黑腹果蝇幼虫中进行的体内 DSB 产生的研究，为六价铬致癌作用中 DSB 的产生提供了证据，由于 DSB 的产生与 Cr(VI)诱导的致癌作用之间存在因果关系，因此该研究对高等生物具有重要意义。
