Reynolds Mindy, Zhitkovich Anatoly
Department of Pathology and Laboratory Medicine, Brown University, 70 Ship Street, Room 507, Providence, RI 02912, USA.
Carcinogenesis. 2007 Jul;28(7):1613-20. doi: 10.1093/carcin/bgm031. Epub 2007 Feb 14.
Ascorbate (Asc) plays a key role in reductive activation of carcinogenic chromium(VI) in vivo. In addition to much higher rates (t(1/2) = 1 min for 1 mM Asc), its reactions at physiological conditions differ from other reducers by low yields of Cr(V) intermediates. Human cells in culture are severely Asc deficient, which results in distorted metabolism and potentially abnormal responses to Cr(VI). We found that restoration of physiological Asc levels in human lung cells (primary IMR90 fibroblasts and epithelial H460 cells) increased clonogenic lethality and apoptosis by Cr(VI). Enhanced cytotoxicity in mass cultures was more evident after normalization for lower Cr uptake caused by leakage of Asc into media. Asc did not change uptake-adjusted yields of Cr-DNA adducts and had no effect on cytotoxicity when delivered shortly after Cr(VI) exposure. Protein and Ser-15 phosphorylation levels of p53 did not show any association with the presence of Asc and there were no increases in p53-driven reporter activity in Cr-treated cells. Stable silencing of p53 expression by short hairpin RNA (shRNA) had no effect on toxicity of Cr(VI) in both -Asc and +Asc IMR90 and H460 cells. In contrast, shRNA-mediated depletion of essential components of MutS or MutL mismatch repair complexes greatly improved survival of all Cr-treated cells and eliminated Asc-potentiated effects on cell death. Thus, mismatch repair-mediated enhancement of Cr(VI) cytotoxicity by Asc should promote the selection of MSI+/wt-p53 phenotype found among chromate-induced human lung cancers. Our findings also indicate that Asc plays a dual role in Cr(VI) toxicity: protective outside and potentiating inside the cell.
抗坏血酸盐（Asc）在体内致癌性铬（VI）的还原活化过程中起着关键作用。除了反应速率高得多（1 mM抗坏血酸盐的半衰期t(1/2)=1分钟）外，其在生理条件下的反应与其他还原剂不同，Cr(V)中间体的产率较低。培养的人类细胞严重缺乏抗坏血酸盐，这导致代谢紊乱，并可能对铬（VI）产生异常反应。我们发现，恢复人类肺细胞（原代IMR90成纤维细胞和上皮H460细胞）中的生理抗坏血酸盐水平会增加铬（VI）诱导的克隆致死率和细胞凋亡。由于抗坏血酸盐泄漏到培养基中导致铬摄取降低，在对大量培养物进行标准化后，增强的细胞毒性更加明显。抗坏血酸盐不会改变经摄取调整后的Cr-DNA加合物产率，并且在铬（VI）暴露后不久给予时对细胞毒性没有影响。p53的蛋白质和Ser-15磷酸化水平与抗坏血酸盐的存在没有任何关联，并且在铬处理的细胞中p53驱动的报告基因活性没有增加。通过短发夹RNA（shRNA）稳定沉默p53表达对-Asc和+Asc的IMR90和H460细胞中铬（VI）的毒性没有影响。相比之下，shRNA介导的MutS或MutL错配修复复合物必需成分的缺失极大地提高了所有铬处理细胞的存活率，并消除了抗坏血酸盐对细胞死亡的增强作用。因此，错配修复介导的抗坏血酸盐增强铬（VI）细胞毒性应促进在铬酸盐诱导的人类肺癌中发现的MSI+/wt-p53表型的选择。我们的研究结果还表明，抗坏血酸盐在铬（VI）毒性中起双重作用：在细胞外具有保护作用，在细胞内具有增强作用。
