Caenorhabditis elegans之gcs-1基因與砷造成之氧化壓力之探討

Abstract

Gamma-glutamylcysteine synthetase (γ-GCS) 是glutathione (GSH) 合成的速率決定步驟酵素，證明GSH對於C. elegans抵抗砷所造成的氧化壓力扮演重要的角色，另外亦探討在C. elegans中所預測出GCS (h)的同源基因, gcs-1,對於暴露砷時所造成的反應。當C. elegans暴露於三價砷以及五價砷時，gcs-1突變種對於抵抗砷所造成的毒性比野生種線蟲更為敏感。另外，gcs-1突變種C. elegans亦比野生種C. elegans對於砷所造成的毒性反應較快。將gcs-1突變種C. elegans預先暴露於GSH，可明顯提高gcs-1突變種暴露於三價砷與五價砷時的存活率。我們亦探討C. elegans體內的GSH含量與不同砷劑量間的關係。並且利用RNA干擾技術，mRNA表達和基因轉殖C. elegans探討GCS-1的功能。我們亦探討砷對於C. elegans中其他可能和氧化壓力有關基因的表達的關係。研究的結果顯示在C. elegans中GCS-1是合成GSH所必須的，並且GSH的含量對於抵抗砷所造成的氧化壓力扮演著重要的角色，以及在C. elegans中砷會誘導化壓力相關基因mRNA的表達。

Gamma-glutamylcysteine synthetase (γ-GCS) catalyzes the first, rate-limiting step in the biosynthesis of glutathione (GSH). To evaluate the protective role of cellular GSH against arsenic-induced oxidative stress in Caenorhabditis elegans (C. elegans), we examined the effect of the C. elegans ortholog of GCS(h), gcs-1, in response to inorganic arsenic exposure. We have evaluated the responses of wild-type and gcs-1 mutant nematodes to both inorganic arsenite (As(III)) and arsenate (As(V)) ions and found that gcs-1 mutant nematodes are more sensitive to arsenic toxicity than that of wild-type animals. gcs-1 mutant nematodes also showed an earlier response to the exposure of As(III) and As(V) than that of wild-type animals. Pretreatment with GSH significantly raised the survival rate of gcs-1 mutant worms compared to As(III)- or As(V)-treated worms alone. The intracellular GSH level increases in C. elegans exposed to As(III) and gcs-1 expression level is induced by As(III). The functional importantance of GCS-1 in C. elegans exposed to As(III) is investigated by analysis of gcs-1 transcription in transgenic C. elegans and RNAi mediated GCS-1 knock-down worms. The level of mRNA expression of several potential oxidative response gene is response to As(III) exposure was also investigated. Our results show that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress. Furthermore, As(III) is capable of inducing mRNA expression of oxidative stress-related genes.