Fraser, Gillian Mary; (1992) Studies of the content and distribution of glutathione and glutathione S-transferases in cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The Glutathione S-transferases (GSTs; EC 2.5.1.18) are a multigene family of dimeric isoenzymes which catalyse a variety of reactions utilizing the tripeptide glutathione (GSH; γ-Glu-Cys-Gly). Many of these, such as the conjugation of GSH to xenobiotic and endogenous electrophiles and the reduction of organic hydroperoxides, are involved in cellular detoxification and the GSTs are of particular interest for their anti-carcinogenic action. This thesis is concerned with the application of a variety of techniques to investigate the content and intracellular distribution of GSH and GSTs in rat liver, in cell lines derived from rat liver (IAR20 & IAR6.1) and mouse fibroblasts (Balb 3T3), and in two human tumour cell lines (HeLa & MCF7). Of particular interest was the controversial presence of the GSTs in the nucleus, which may be linked to their ability to protect against chemical carcinogenesis. The GSH and GST isoenzyme contents of these five cell lines were determined and the subcellular distribution of the GSTs was investigated using immunocytochemistry. Some evidence for a nuclear localization was obtained. However, analysis of the intracellular distribution of GSTs in rat liver using subcellular fractionation was inconclusive. Monochlorobimane (MCB), which undergoes GST-catalysed conjugation to GSH to yield a fluorescent product (MB-SG), was tested in conjunction with flow cytometry as a possible means of measuring cellular GSH. The GST enzyme kinetics were first analysed and considerable isoenzyme specificity towards MCB was observed. It was predicted that the kinetics of MCB conjugation in the above cell lines would vary according to their GSH and GST isoenzyme contents, and this was confirmed using flow cytometry. The implications of this for the use of MCB to measure cellular GSH are discussed. MCB-stained cell lines and primary rat hepatocytes were examined by fluorescence microscopy. Fluorescence was observed over the whole cell, but was often brighter over the nucleus. However, when the MB-SG conjugate was microinjected into the cytoplasm of primary rat hepatocytes, it diffused rapidly through the cell and into the nucleus, producing a very similar pattern of fluorescence to that observed using MCB. Therefore, MCB-staining may not indicate the true intracellular distribution of GSH and the GSTs.

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