Oxidative stress can induce the generation of
free radicals, which are believed to play an important role in both physiological and
pathological processes and a number of diseases such as
cancer. Therefore, it is important to identify chemicals which are capable of inducing oxidative stress. In this study, we evaluated the ability of four environmental chemicals,
aniline,
nitrosobenzene (NB),
N,N-dimethylaniline (DMA) and
N,N-dimethyl-4-nitrosoaniline (DMNA), to induce
free radicals and cellular damage in the
hepatoma cell line HepG2. Cytotoxicity was assessed using
lactate dehydrogenase (LDH) assays and morphological changes were observed using phase contrast microscopy.
Free radicals were detected by immuno-spin trapping (IST) in in-cell western experiments or in confocal microscopy experiments to determine the subcellular localization of
free radical generation. DMNA induced
free radical generation, LDH release and morphological changes in HepG2 cells whereas
aniline, NB and DMA did not. Confocal microscopy showed that DMNA induced
free radical generation mainly in the cytosol. Preincubation of HepG2 cells with
N-acetylcysteine and
2,2'-dipyridyl significantly prevented
free radical generation upon subsequent incubation with DMNA, whereas preincubation with
apocynin and
dimethyl sulfoxide did not. These results suggest that DMNA induces oxidative stress and that
reactive oxygen species, metals and
free radical generation play a critical role in DMNA-induced cytotoxicity.