Flórez María R, Costas-Rodríguez Marta, Grootaert Charlotte, Van Camp John, Vanhaecke Frank
Department of Chemistry, Atomic & Mass Spectrometry - A&MS Research Unit, Ghent University, Krijgslaan 281-S12, 9000, Ghent, Belgium.
Department of Food Safety and Food Quality, Food Chemistry and Human Nutrition - nutriFOODchem Research Unit, Ghent University, Coupure Links 653-A, 9000, Ghent, Belgium.
Anal Bioanal Chem. 2018 Mar;410(9):2385-2394. doi: 10.1007/s00216-018-0909-x. Epub 2018 Feb 5.
Reactive oxygen species (ROS) are generated in biological processes involving electron transfer reactions and can act in a beneficial or deleterious way. When intracellular ROS levels exceed the cell's anti-oxidant capacity, oxidative stress occurs. In this work, Cu isotope fractionation was evaluated in HepG2 cells under oxidative stress conditions attained in various ways. HepG2 is a well-characterised human hepatoblastoma cell line adapted to grow under high oxidative stress conditions. During a pre-incubation stage, cells were exposed to a non-toxic concentration of Cu for 24 h. Subsequently, the medium was replaced and cells were exposed to one of three different external stressors: HO, tumour necrosis factor α (TNFα) or UV radiation. The isotopic composition of the intracellular Cu was determined by multi-collector ICP-mass spectrometry to evaluate the isotope fractionation accompanying Cu fluxes between cells and culture medium. For half of these setups, the pre-incubation solution also contained N-acetyl-cysteine (NAC) as an anti-oxidant to evaluate its protective effect against oxidative stress via its influence on the extent of Cu isotope fractionation. Oxidative stress caused the intracellular Cu isotopic composition to be heavier compared to that in untreated control cells. The HO and TNFα exposures rendered similar results, comparable to those obtained after mild UV exposure. The heaviest Cu isotopic composition was observed under the strongest oxidative conditions tested, i.e., when the cell surfaces were directly exposed to UV radiation without apical medium and in absence of NAC. NAC mitigated the extent of isotope fractionation in all cases.
活性氧（ROS）在涉及电子转移反应的生物过程中产生，其作用可能有益也可能有害。当细胞内ROS水平超过细胞的抗氧化能力时，就会发生氧化应激。在这项工作中，我们评估了在通过各种方式达到的氧化应激条件下，HepG2细胞中的铜同位素分馏情况。HepG2是一种特征明确的人肝癌细胞系，适合在高氧化应激条件下生长。在预孵育阶段，细胞暴露于无毒浓度的铜中24小时。随后，更换培养基，细胞暴露于三种不同的外部应激源之一：过氧化氢（HO）、肿瘤坏死因子α（TNFα）或紫外线辐射。通过多接收电感耦合等离子体质谱法测定细胞内铜的同位素组成，以评估细胞与培养基之间铜通量伴随的同位素分馏情况。在这些实验设置中，一半的预孵育溶液还含有N-乙酰半胱氨酸（NAC）作为抗氧化剂，以评估其通过影响铜同位素分馏程度对氧化应激的保护作用。氧化应激导致细胞内铜的同位素组成比未处理的对照细胞中的更重。过氧化氢和肿瘤坏死因子α暴露产生了相似的结果，与轻度紫外线照射后获得的结果相当。在测试的最强氧化条件下，即当细胞表面直接暴露于紫外线辐射且无顶培养基且无NAC时，观察到最重的铜同位素组成。在所有情况下，NAC都减轻了同位素分馏的程度。
