Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Summary

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

Abstract

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H₂O₂ (hydrogen peroxide), NO (nitric oxide), O₂^- (oxide anion), peroxynitrite (ONOO^-), hydrochlorous acid (HOCl), and hydroxyl radical (OH^-).

Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases ¹, autoimmune diseases ², etc…) but also during physiological (non-pathological) situations such as cellular metabolism ^{3, 4}. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation ^{5, 6}, migration ⁷ etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).

Free radicals are beneficial in low amounts ³. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation ⁸. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise ^{9, 10}. Several publications have also demonstrated that ROS are involved in insulin sensitivity ^{11, 12}.

Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Protocol

1. Detection of ROS using carboxy-H₂DCFDA

Carboxy-H₂DCFDA is non-fluorescent but in the presence of ROS, when this reagent is oxidized, it becomes green fluorescent.

1. Immediately prior to use, prepare a fresh stock solution of carboxy-H₂DCFDA in sterile dimethylsulfoxide (DMSO) or 100% ethanol. Avoid multiple thaw/freeze cycles of your dye.
2. Wash the cells with HEPES buffered salt solution (HBSS) or phosphate-buffered saline (PBS) to remove traces of the original medium.
3. Load the cells with the dye (and the control dye, cf note section). In this assay we used Jurkat, a human leu....

Discussion

Several pathological situations such as inflammatory diseases, cancers, ischemia/reperfusion, and also treatments such as radiation or chemotherapy (i.e cisplatin) induce ROS overproduction. Thus, detecting and measuring ROS levels is important in many basic, pre-clinical and clinical studies. However, ROS have very short half lives and could be complicated to detect. Here, we propose simple tests that are routinely used and widely accepted for the detection of free radical production in mammalian cells.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
5-(and-6)-carboxy-2’,7’-dichlorofluorescein diacetate (carboxy-DCFDA)	Molecular Probes, Life Technologies	C369	control
carboxy-H2DCFDA	Molecular Probes, Life Technologies	C400
Sulfanilamide	Sigma-Aldrich	S9251-100G
N-1-napthylethylenediamine dihydrochloride	Sigma-Aldrich	N9125-10G
Nitrite standard	Sigma-Aldrich	237213-100G
GSH/GSSG-Glo Assay	Promega Corp.	V6612	To quantify oxidized, reduced or oxidized/reduced glutathione