Cu isotope fractionation response to oxidative stress in a hepatic cell line studied using multi-collector ICP-mass spectrometry
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: H2O2, 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 H2O2 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.
KeywordsMulti-collector ICP-mass spectrometry MC-ICP-MS Isotope fractionation Oxidative stress Hep G2 cell line Copper isotope ratio Liver
The Flemish Research Foundation FWO-Vlaanderen (research project “G023014N”) is acknowledged for financial support. María R. Flórez thanks the Special Research Fund of Ghent University (BOF-UGent) for her postdoctoral grant and Marta Costas-Rodriguez thanks FWO-Vlaanderen for her postdoctoral grant.
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Conflict of interest
The authors declare that they have no conflict of interest.
- 29.Van Meerloo J, Kaspers GJL, Cloos J. Cell sensitivity assays: the MTT assay. In: Cree IA, editor. Cancer cell culture. Methods in molecular biology (methods and protocols). New York: Humana Press; 2011. p. 237–45.Google Scholar