Oxidative damage of U937 human leukemic cells caused by hydroxyl radical results in singlet oxygen formation.

Marek Rác,Michaela Sedlářová,Pavel Pospíšil,Svatopluk Binder,Zuzana Matušková,Milan Raška,Michal Křupka,Dariush Hinderberger

doi:10.1371/journal.pone.0116958

Abstract

The exposure of human cells to oxidative stress leads to the oxidation of biomolecules such as lipids, proteins and nuclei acids. In this study, the oxidation of lipids, proteins and DNA was studied after the addition of hydrogen peroxide and Fenton reagent to cell suspension containing human leukemic monocyte lymphoma cell line U937. EPR spin-trapping data showed that the addition of hydrogen peroxide to the cell suspension formed hydroxyl radical via Fenton reaction mediated by endogenous metals. The malondialdehyde HPLC analysis showed no lipid peroxidation after the addition of hydrogen peroxide, whereas the Fenton reagent caused significant lipid peroxidation. The formation of protein carbonyls monitored by dot blot immunoassay and the DNA fragmentation measured by comet assay occurred after the addition of both hydrogen peroxide and Fenton reagent. Oxidative damage of biomolecules leads to the formation of singlet oxygen as conformed by EPR spin-trapping spectroscopy and the green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. It is proposed here that singlet oxygen is formed by the decomposition of high-energy intermediates such as dioxetane or tetroxide formed by oxidative damage of biomolecules.

Highlights

Reactive oxygen species (ROS) are continuously produced as byproducts of various metabolic pathways localized in the different cellular compartments
Formation of hydroxyl radical detected by EPR spin-trapping spectroscopy
To study the oxidative damage of biomolecules caused by ROS, H2O2 and Fenton reagent (H2O2 and FeSO4) were added to cell suspension

Summary

Introduction

Reactive oxygen species (ROS) are continuously produced as byproducts of various metabolic pathways localized in the different cellular compartments. Superoxide anion radical (O2−) is produced by electron leakage to molecular oxygen in mitochondria, endoplasmic reticulum, microbodies and cell walls [1,2]. The spontaneous and enzymatic dismutation of O2− results. Formation of Singlet Oxygen in U937 Cells under Oxidative Stress and by the IGA_LF_2014_020, OPVK 2.3 (CZ.1.07/ 2.3.00/30.0004), NPU I MSMT-7778/2014, and PrF2013-003. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

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Results

Conclusion