Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals.

Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamylcysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by sq RT-PCR, to improve our knowledge on the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM resulting as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlighted a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, cigclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.