title: Free radical generation from an aniline derivative in HepG2 cells: a possible captodative effect.
abstract: 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.
authors.affiliation: Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: yuya.h@basic.med.tokushima-u.ac.jp.
authors.forename: Yuya
authors.initials: Y
authors.lastname: Horinouchi
authors.affiliation: Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: summersfa@niehs.nih.gov.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: Summers
authors.affiliation: Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: ehrensh1@niehs.nih.gov.
authors.forename: Marilyn
authors.initials: M
authors.lastname: Ehrenshaft
authors.affiliation: Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA. Electronic address: mason4@niehs.nih.gov.
authors.forename: Ronald P
authors.initials: RP
authors.lastname: Mason
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology*; D015153:Blotting, Western; D002273:Carcinogens / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects*; D004578:Electron Spin Resonance Spectroscopy; D016166:Free Radical Scavengers / Q000494:pharmacology*; D005609:Free Radicals / Q000378:metabolism*; D056945:Hep G2 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018613:Microscopy, Confocal; D009603:Nitroso Compounds / Q000494:pharmacology*; D019097:Spin Trapping
chemical_list: D000814:Aniline Compounds; D002273:Carcinogens; D016166:Free Radical Scavengers; D005609:Free Radicals; D009603:Nitroso Compounds; C008651:4-nitrosodimethylaniline; C015157:N,N-dimethylaniline; D007770:L-Lactate Dehydrogenase; C023650:aniline; C012456:nitrosobenzene
keywords: Captodative effect; Confocal microscopy; Cytotoxicity; Environmental chemicals; Immuno-spin trapping; In-cell western