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Investigating free radical generation in HepG2 cells using immuno-spin trapping.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Horinouchi, Yuya Summers, Fiona A. Ehrenshaft, Marilyn Kawazoe, Kazuyoshi Tsuchiya, Koichiro Tamaki, Toshiaki Mason, Ronald P.
Description	Author Affiliation: Horinouchi Y ( Tokushima University Hospital (Tokushima), Department of Pharmacy, Japan. Electronic address: yuya.h@basic.med.tokushima-u.ac.jp.); Summers FA ( National Institute of Environmental Health Sciences, National Institutes of Health (NC), Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, USA.); Ehrenshaft M ( National Institute of Environmental Health Sciences, National Institutes of Health (NC), Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, USA.); Kawazoe K ( Tokushima University Hospital (Tokushima), Department of Pharmacy, Japan.); Tsuchiya K ( Institute of Health Biosciences, The University of Tokushima Graduate School (Tokushima), Department of Medical Pharmacology, Japan.); Tamaki T ( Institute of Health Biosciences, The University of Tokushima Graduate School (Tokushima), Department of Pharmacology, Japan.); Mason RP ( National Institute of Environmental Health Sciences, National Institutes of Health (NC), Free Radical Metabolism Group, Laboratory of Toxicology and Pharmacology, USA.)
Abstract	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.
File Format	HTM / HTML
ISSN	08915849
Issue Number	Suppl 1
Journal	Free Radical Biology and Medicine
Volume Number	75
e-ISSN	18734596
Language	English
Publisher	Elsevier
Publisher Date	2014-10-01
Publisher Place	United States
Access Restriction	One Nation One Subscription (ONOS)
Content Type	Text
Resource Type	Article
Subject	Physiology (medical) Biochemistry

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