Localization of γ-Glutamylcysteine Synthetase mRNA Expression in Mouse Brain Following Methylmercury Treatment Using Reverse Transcriptionin SituPCR Amplification

Abstract

In previous studies we reported that prolonged treatment of rats with subtoxic levels of mercury as methymercury hydroxide (MMH) elicited a two- to three-fold increase in renal glutathione (GSH) content and a three- to fourfold increase in the mRNA encoding the catalytically active heavy subunit of γ-glutamylcysteine synthetase (GCS), the rate-limiting enzyme in GSH synthesis. Since methylmercury is a potent neurotoxicant, we investigated the effect of methylmercury treatment on GSH synthesis and the distribution of GCS mRNA expression in the brain. Male C57B1/6 mice were treated for 3 consecutive days with MMH (3 mg/kg/day, ip). GSH levels in whole brains were increased by twofold 24 hr following the first injection and remained elevated two to three times control levels after two subsequent MMH treatments. Concomitantly, whole brain GCS mRNA levels were increased 2.7-fold 24 hr after the third MMH treatment. Reverse transcriptionin situPCR amplification of GCS heavy subunit mRNA in brain slices taken from MMH-treated mice showed that GCS expression was selectively localized to the cerebellum and hippocampal regions and, within these regions, to areas which are known to resist methylmercury toxicity. In contrast, no GCS mRNA expression was found in brain regions which are known to be highly susceptible to mercury toxicity. These findings suggest that resistance to methylmercury toxicity in the brain may reflect the ability of specific neuronal cell types to up-regulate GSH synthesis as a protective response to mercury-mediated cell damage.