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The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Antioxidant enzymes are known to protect living organisms against the oxidative stress risk, also induced by metals. In the present study, we describe the purification and molecular characterization of two Cu,Zn superoxide dismutases (SODs), referred to as Ci-SODa and Ci-SODb, from Ciona intestinalis, a basal chordate widely distributed in temperate shallow seawater. The putative amino acid sequences were compared with Cu,Zn SODs from other metazoans and phylogenetic analyses indicate that the two putative Ci-SODs are more related to invertebrate SODs than vertebrate ones. Both phylogenetic and preliminary homology modeling analyses suggest that Ci-SODa and Ci-SODb are extracellular and intracellular isoform, respectively. The mRNA of the two Cu,Zn SODs was localized in hemocytes and in ovarian follicular cells, as revealed by in situ hybridization. The time course of SOD mRNA levels in the presence of three different metals showed upregulation of ci-soda and inhibition of ci-sodb. Spectrophotometric analysis confirms the presence of SOD activity in Ciona tissues. Our in silico analyses of the ci-soda promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE), suggesting that the transcription of these genes directly depends on metals. These data emphasize the importance of complex metal regulation of ci-soda and ci-sodb transcription, as components of an efficient detoxification pathway allowing the survival of C. intestinalis in continued, elevated presence of metals in the environment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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-glutamyl-cysteine 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 semiquantitativ RT-PCR to improve our knowledge about 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 generally, 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 being 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 highlight 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, ci-gclm 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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The present article reports on the characterization of the urochordate metallothionein (MT) gene, CiMT-1, from the solitary ascidian Ciona intestinalis. The predicted protein is shorter than other known deuterostome MTs, having only 39 amino acids. The gene has the same tripartite structure as vertebrate MTs, with some features resembling those of echinoderm MTs. The promoter region shows the canonical cis-acting elements recognized by transcription factors that respond to metal, ROS, and cytokines. Unusual sequences, described in fish and echinoderms, are also present. In situ hybridization suggests that only a population of hemocytes involved in immune responses, i.e. granular amebocytes, express CiMT-1 mRNA. These observations support the idea that urochordates perform detoxification through hemocytes, and that MTs may play important roles in inflammatory humoral responses in tunicates. The reported data offer new clues for better understanding the evolution of these multivalent proteins from non-vertebrate to vertebrate chordates and reinforce their functions in detoxification and immunity., (Copyright © 2010 Wiley-Liss, Inc., A Wiley Company.)