Univ. Grenoble Alpes, CEA, CNRS, IRIG, SyMMES, F-38000, Grenoble, France.
Service Recherche Pharmaco-Clinique, Département Recherche Appliquée, Centre R&D Pierre Fabre, 31000, Toulouse, France.
Anal Bioanal Chem. 2022 Nov;414(26):7705-7720. doi: 10.1007/s00216-022-04302-1. Epub 2022 Aug 31.
UV-induced formation of photoproducts in DNA is a major initiating event of skin cancer. Consequently, many analytical tools have been developed for their quantification in DNA. In the present work, we extended our previous liquid chromatography-mass spectrometry method to the quantification of the short DNA fragments containing photoproducts that are released from cells by the repair machinery. We designed a robust protocol including a solid-phase extraction step (SPE), an enzymatic treatment aimed at releasing individual photoproducts, and a liquid chromatography method combining on-line SPE and ultra-high-performance liquid chromatography for optimal specificity and sensitivity. We also added relevant internal standards for a better accuracy. The method was validated for linearity, repeatability, and reproducibility. The limits of detection and quantification were found to be in the fmol range. The proof of concept of the use of excreted DNA repair products as biomarkers of the genotoxicity of UV was obtained first in in vitro studies using cultured HaCat cells and ex vivo on human skin explants. Further evidence was obtained from the detection of pyrimidine dimers in the urine of human volunteers collected after recreational exposure in summer. An assay was designed to quantify the DNA photoproducts released from cells within short fragments by the DNA repair machinery. These oligonucleotides were isolated by solid-phase extraction and enzymatically hydrolyzed. The photoproducts were then quantified by on-line SPE combined with UHPLC-MS/MS with isotopic dilution.
紫外线诱导的 DNA 光产物的形成是皮肤癌的主要起始事件。因此，已经开发出许多分析工具来定量检测 DNA 中的光产物。在本工作中，我们扩展了以前的液相色谱-质谱法，用于定量分析从细胞中由修复机制释放出来的含有光产物的短 DNA 片段。我们设计了一个稳健的方案，包括固相萃取（SPE）步骤、旨在释放单个光产物的酶处理以及液相色谱方法，该方法结合在线 SPE 和超高效液相色谱法以获得最佳的特异性和灵敏度。我们还添加了相关的内标以提高准确性。该方法经过线性、重复性和重现性验证。检测限和定量限被发现处于飞摩尔范围内。首先通过体外培养的 HaCat 细胞和人体皮肤外植体的离体研究获得了将排泄的 DNA 修复产物用作紫外线遗传毒性生物标志物的概念验证。从夏季休闲暴露后收集的人类志愿者尿液中检测到嘧啶二聚体进一步证实了这一观点。设计了一种测定由 DNA 修复机制从细胞内短片段中释放的 DNA 光产物的方法。这些寡核苷酸通过固相萃取分离，并通过酶解进行水解。然后通过在线 SPE 与 UHPLC-MS/MS 结合同位素稀释法定量分析光产物。
