Gault Nathalie, Rigaud Odile, Poncy Jean-Luc, Lefaix Jean-Louis
CEA/DSV/Département de Radiobiologie-Radiopathologie, CEA-FAR, Fontenay aux Roses, France.
Int J Radiat Biol. 2005 Oct;81(10):767-79. doi: 10.1080/09553000500515368.
Fourier transform infrared microspectroscopy (FT-IRM), which allows simultaneous detection of biochemical changes in the various cellular compartments, was used as a new analytical tool to study early radiation- and oxidation-induced cellular damage at the molecular level in single human cells.
HaCaT keratinocytes were given a single dose of 6 Gy (137Cs) or 650 microM H2O2, neither of which is cytotoxic (neutral red assay) but both of which result in less than 10% clonogenic survival, and deposited on zinc sulphur (ZnS) windows for infra-red (IR) spectra acquisition, immediately and 2 h after treatment. DNA damage was assessed by comet assays in alkaline conditions.
Comet assays showed that the yield of DNA damage was higher after H2O2 treatment than after gamma-irradiation. The comparison between spectra of irradiated and H2O2-treated cells showed common changes, but H2O2 treatment presented a broader spectrum of cellular oxidation than ionizing radiation. The bands characteristic of deoxyribose/ribose in nucleic acids centered at 966 and 997 cm(-1), the bands characteristic of nucleic acid bases centered at 1572, 1599, and 1691 cm(-1), as well as the bands characteristic of ordered secondary structure of DNA centered at 1713-1716 cm(-1), were changed in absorbance, sometimes accompanied by a shift. The bands characteristic of proteins centered at 1515, 1530, 1544 and 1640 cm(-1) were changed in absorbance indicating a decrease in secondary structure of proteins. Moreover, the absorbance of the bands at 1515 and 1630 cm(-1) was correlated the yield of reactive oxygen species. Two hours after both treatments most changes were persistent, suggesting either irreversible or not easily repaired damage or persistent oxidative stress.
As we previously demonstrated in radiation-induced apoptosis studies, these results show that FT-IRM, in correlation with other cellular biology techniques, might be useful for assessing immediate radiation- and oxidative-induced damage to nucleic acids and proteins in single human cells.
傅里叶变换红外显微光谱法（FT-IRM）能够同时检测细胞各个区室中的生化变化，被用作一种新的分析工具，在分子水平上研究单个活体细胞中早期辐射和氧化诱导的细胞损伤。
给HaCaT角质形成细胞单次给予6 Gy（137Cs）或650 μM过氧化氢，两者均无细胞毒性（中性红试验），但两者均导致克隆形成存活率低于10%，并在处理后立即及2小时后沉积在硫化锌（ZnS）窗口上以获取红外（IR）光谱。在碱性条件下通过彗星试验评估DNA损伤。
彗星试验表明，过氧化氢处理后DNA损伤的发生率高于γ射线照射后。照射细胞和过氧化氢处理细胞的光谱比较显示出共同变化，但过氧化氢处理呈现出比电离辐射更广泛的细胞氧化谱。核酸中脱氧核糖/核糖的特征峰位于966和997 cm-1处，核酸碱基的特征峰位于1572、1599和1691 cm-1处，以及DNA有序二级结构的特征峰位于1713 - 1716 cm-1处，其吸光度发生变化，有时还伴有位移。蛋白质的特征峰位于1515、1530、1544和1640 cm-1处，其吸光度发生变化，表明蛋白质二级结构减少。此外，1515和1630 cm-1处峰的吸光度与活性氧的产生率相关。两种处理后2小时，大多数变化持续存在，表明损伤要么不可逆，要么不易修复，要么存在持续的氧化应激。
正如我们之前在辐射诱导的细胞凋亡研究中所证明的，这些结果表明，FT-IRM与其他细胞生物学技术相结合，可能有助于评估单个活体细胞中辐射和氧化对核酸和蛋白质的即时损伤。
