Toxicity of a water-soluble free radical generator AAPH in in vitro studies

Introduction
2,2'-Azobis(2-methylpropionamidine) dihydrochloride (AAPH) is a water-soluble free radical-generating compound, which induces generation of hydroxyl and peroxyl radicals with constant rate in the first hours after dissolving. Because AAPH may be used effectively for oxidative stress or lipid peroxidation in vitro studies, it is necessary to establish cytotoxicity of this compound, which determines its dosage in the experiments.

Methods
Cytotoxicity of AAPH (CAS 2997-92-4) was evaluated on Chinese hamster ovary (CHO-9) as well as human lung adenocarcinoma (A549) cells after 24-hour exposure, using two cell viability assays: the MTT reduction assay, which assesses the metabolic activity of cells, and the NRU (Neutral Red Uptake) assay measuring cell membrane permeability. The IC50 values (i.e. the compound concentration required for a 50% cell viability reduction) were used as the measure for assessing the cytotoxicity of the tested compound.

Results
The IC50 values obtained for AAPH with the CHO-9 cell line were twice lower than those obtained on A549 cell line (CHO-9 cells proved more sensitive to AAPH). The average IC50 values determined for CHO-9 cells in the MTT and NRU assay were 1567 µg/ml (5.78 µM) and 1467 µg/ml (5.41 µM),respectively, whereas the average IC50 values obtained for A549 cells were 3272 µg/ml (12.10 µM) and 3841 µg/ml (14.16 µM) in the MTT and NRU assay, respectively.

Discussion
Significant differences in IC50 values obtained in studies using different cell types indicate that the use and dosage of AAPH as a free radical generator in the study of oxidative stress in vitro should take into consideration the type of cells used in the study.
It seems necessary, to evaluate the cytotoxicity of all compounds on each cell line, which will be applied, prior to the study of oxidative stress, to ensure the use of a proper dose (lower than IC50) of tested compounds for that particular cell line.