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

• Published in: Toxicology in vitro Vol. 16; no. 5; pp. 609 - 619
• Main Authors: Stevenson, D., Wokosin, D., Girkin, J., Grant, M.H.
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.10.2002; Elsevier Science
• Subjects: Animals; Biological and medical sciences; Biomarkers; Breast Neoplasms; Confocal laser scanning microscopy; Cultured hepatocytes; Fluorescent Dyes - metabolism; General aspects. Methods; Glutathione - metabolism; Hepatocytes - metabolism; Humans; InGaN laser source; Intracellular reduced glutathione; Lasers; Medical sciences; Microscopy, Confocal; Monochlorobimane; o-Phthalaldehyde - metabolism; Pyrazoles - metabolism; Rats; Rats, Sprague-Dawley; Sensitivity and Specificity; Toxicology; Tumor Cells, Cultured; PBS; GSSG; DMSO; GST; Monochlorobimane; mBCl; Intracellular reduced glutathione; DMEM; OPT; TCA; PMMA; InGaN laser source; FBS; Cultured hepatocytes; l-cys; CLSM; GSH; Confocal laser scanning microscopy; BSO; Rat; Liver; Rodentia; Scanning light microscopy; Confocal microscopy; Laser microscope; Vertebrata; Mammalia; Hepatocyte; Investigation method; Fluorescent labelling; Sulfur peptide; Animal; Distribution; Reduced state; Intracellular; Glutathione
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/S0887-2333(02)00042-5

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.