Xenobiotic metabolism can induce the generation of
protein radicals, which are believed to play an important role in the toxicity of chemicals and drugs. It is therefore important to identify chemical structures capable of inducing macromolecular
free radical formation in living cells. In this study, we evaluated the ability of four structurally related environmental chemicals,
aniline,
nitrosobenzene,
N,N-dimethylaniline, 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 assays, and morphological changes were observed using phase contrast microscopy.
Protein free radicals were detected by immuno-spin trapping using in-cell western experiments and confocal microscopy to determine the subcellular locale of
free radical generation. DMNA induced
free radical generation,
lactate dehydrogenase release, and morphological changes in HepG2 cells, whereas
aniline,
nitrosobenzene,
N,N-dimethylaniline 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 on subsequent incubation with DMNA, whereas preincubation with
apocynin and
dimethyl sulfoxide had no effect. These results suggest that DMNA is metabolized to reactive
free radicals capable of generating
protein radicals which may play a critical role in DMNA toxicity. We propose that the captodative effect, the combined action of the electron-releasing
dimethylamine substituent, and the electron-withdrawing nitroso substituent, leads to a thermodynamically stabilized radical, facilitating enhanced
protein radical formation by DMNA.