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

D. Stevenson, D. Wokosin, J. Girkin, M.H. Grant

Research output: Contribution to journalArticle

35 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)609-619
Number of pages10
JournalToxicology in Vitro
Volume16
Issue number5
DOIs
Publication statusPublished - 2002

Keywords

  • intracellular reduced glutathione
  • monochlorobimane
  • confocal microscopy
  • InGaN laser source
  • cultured hepatocytes
  • lasers
  • bioengineering
  • photonics

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