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Measurement of the intracellular distribution of reduced glutathione in cultured rat hepatocytes using monochlorobimane and confocal laser scanning microscopy

Stevenson, D. and Wokosin, D. and Girkin, J. and Grant, M.H. (2002) Measurement of the intracellular distribution of reduced glutathione in cultured rat hepatocytes using monochlorobimane and confocal laser scanning microscopy. Toxicology in Vitro, 16 (5). pp. 609-619. ISSN 0887-2333

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Abstract

Intracellular reduced glutathione (GSH) plays a key role in protecting cells from toxicity by maintaining intracellular redox status, conjugating with electrophilic xenobiotics and free radicals, and detoxifying reactive peroxides. Several toxic chemicals interact with GSH during their metabolism, and in many cases it would be advantageous to monitor intracellular GSH distribution during that process. We present a novel method to monitor intracellular GSH levels utilising a new laser light source, InGaN laser, for confocal microscopy and fluorescent detection of monochlorobimane (mBCl) binding to GSH. The sensitivity of the method was compared with that obtained using o-phthalaldehyde (OPT) as a fluorochrome. In the presence of a source of glutathione S-transferase (GST), mBCl was specific for GSH, forming a fluorescent conjugate that was retained in hepatocytes for at least 35 min. mBCl was able to detect the GSH depleting effects caused by progressive inhibition of GSH synthesis by increasing concentrations of buthionine sulfoximine. It effectively monitored the rapid effects of menadione and chromium VI metabolism on intracellular GSH levels in the cytosol and nuclear compartments of the cells. The combination of a specific stain, a novel laser light source and confocal microscopy provide a valuable system for mechanistic studies of intracellular GSH distribution in toxicology studies.

Item type: Article
ID code: 6765
Keywords: intracellular reduced glutathione, monochlorobimane, confocal microscopy, InGaN laser source, cultured hepatocytes, lasers, bioengineering, photonics, Bioengineering, Optics. Light, Human anatomy, Toxicology
Subjects: Technology > Engineering (General). Civil engineering (General) > Bioengineering
Science > Physics > Optics. Light
Science > Human anatomy
Department: Faculty of Engineering > Bioengineering
Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences > Physiology and Pharmacology
Faculty of Science > Institute of Photonics
Related URLs:
    Depositing user: Strathprints Administrator
    Date Deposited: 28 Aug 2008
    Last modified: 04 Sep 2014 15:05
    URI: http://strathprints.strath.ac.uk/id/eprint/6765

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