Effects of cadmium exposition on sea urchin development by SSH and RT-qPCR techniques

Abstract

Cadmium (Cd) is a toxic heavy metal contaminating coastal environments. It has been suggested that the mechanisms of acute Cd toxicity involve the depletion of glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of ROS. Cd-increased ROS in turn produces lipid peroxidation, and results in DNA damage. However, little is known about direct evidence and mechanism for Cd-generated radicals until recently. In order to study the early defense strategies activated by P. lividus 30 hours embryos, in response to exposition to sub-lethal doses of Cd (100μM), we analyzed the induced transcriptome comparing it to that of control embryos by Suppression Subtractive Hybridization (SSH) technique. By this technique, we isolated five metallothionein (MT) cDNA and we analyzed their levels by RT-qPCR at different Cd concentrations. Two MT (MT7 and MT8) isogenes appeared to be constitutively expressed and upregulated upon high Cd concentration treatment (100μM). Three isogenes (MT4/MT6) are not transcribed in control embryos and they are specifically activated in response to low doses of Cd (0.1-1 μM). Moreover, in Cd upregulated group, there were: 10 genes related to intracellular signaling pathway components and transcription factors, 9 to oxidative, reductive and conjugative biotransformation, 7 to RNA maturation and protein synthesis, 6 to metal detoxification (including 5 MTs), 5 conserved hypothetical proteins with unknown functions and 16 unidentified sequences. In Cd down-regulated group, there were: 4 genes related to transcription regulation, 2 to oxidative phosphorilation, 1 to protein synthesis, 3 other hypothetical proteins and 4 unidentified sequences. We analyzed cDNA levels for 20 genes by RT-qPCR. Preliminary results show significant upregulation for apolipoprotein D, molybdopterin oxidoreductase, splicing coactivator subunit and ER lumen protein retaining receptor, TGFβ family receptor and TGFβ-activated kinase1