Department of Chemistry, University of Utah, Salt Lake City, UT 84112-0850.
Proc Natl Acad Sci U S A. 2024 Dec 3;121(49):e2419175121. doi: 10.1073/pnas.2419175121. Epub 2024 Nov 27.
While hydroxyl radical is commonly named as the Fenton product responsible for DNA and RNA damage in cells, here we demonstrate that the cellular reaction generates carbonate radical anion due to physiological bicarbonate levels. In human and models, their transcriptomes were analyzed by RNA direct nanopore sequencing of ribosomal RNA and chromatography coupled to electrochemical detection to quantify oxidation products in order to follow the bicarbonate dependency in HO-induced oxidation. These transcriptomic studies identified physiologically relevant levels of bicarbonate focused oxidation on the guanine base favorably yielding 8-oxo-7,8-dihydroguanine (OG). In human cells, the bicarbonate-dependent oxidation was further analyzed in the metabolome by mass spectrometry, and a glycosylase-dependent qPCR assay to quantify oxidation sites in telomeres. These analyses further identify guanine as the site of oxidation when bicarbonate is present upon HO exposure. Labile iron as the catalyst for forming carbonate radical anion was demonstrated by repeating the bicarbonate-dependent oxidations in cells experiencing ferroptosis, which had a >fivefold increase in redox-active iron, to find enhanced overall guanine-specific oxidation when bicarbonate was present. The complete profiling of nucleic acid oxidation in the genome, transcriptome, and metabolome supports the conclusion that a cellular Fe(II)-carbonate complex redirects the Fenton reaction to yield carbonate radical anion. Focusing HO-induced oxidative modification on one pathway is consistent with the highly evolved base excision repair suite of enzymes to locate G-oxidation sites for repair and gene regulation in response to oxidative stress.
虽然羟基自由基通常被称为负责细胞内 DNA 和 RNA 损伤的 Fenton 产物，但在这里我们证明，由于生理碳酸氢盐水平，细胞反应会产生碳酸盐自由基阴离子。在人和 模型中，通过核糖体 RNA 的 RNA 直接纳米孔测序和色谱法与电化学检测相结合来分析它们的转录组，以定量氧化产物，从而跟踪 HO 诱导氧化中碳酸氢盐的依赖性。这些转录组研究确定了生理相关水平的碳酸氢盐聚焦氧化在鸟嘌呤碱基上，有利于生成 8-氧代-7,8-二氢鸟嘌呤（OG）。在人细胞中，通过质谱法在代谢组学中进一步分析了碳酸氢盐依赖性氧化，并通过依赖糖苷酶的 qPCR 测定来量化端粒中的氧化位点。这些分析进一步确定了当 HO 暴露时存在碳酸氢盐时，鸟嘌呤是氧化的位点。通过在经历铁死亡的细胞中重复碳酸氢盐依赖性氧化来证明作为形成碳酸盐自由基阴离子催化剂的不稳定铁，当存在碳酸氢盐时，发现整体鸟嘌呤特异性氧化增强。在基因组、转录组和代谢组中对核酸氧化的全面分析支持以下结论：细胞内的 Fe(II)-碳酸盐复合物将 Fenton 反应重定向以产生碳酸盐自由基阴离子。将 HO 诱导的氧化修饰集中在一条途径上与高度进化的碱基切除修复酶套件一致，以定位 G 氧化位点，以响应氧化应激进行修复和基因调控。
