Cadmium-induced Cytotoxicity in a Zebrafish Liver Cell-line ZFL

The cadmium ion (Cd2+) is one of the major metal ions to cause toxic effects in aquatic organisms and their associated ecosystem; however, its toxic effects on the hepatic system are not very well characterized in a systematic manner. In this study, a zebrafish liver cell line - ZFL was used as a model to investigate the mechanism of Cd2+ - induced cytotoxicity on hepatocytes. Hepatocyte has a significant role in chemical metabolism and detoxification and thus a very important organ worthy of detail studies. Cd is a common contaminant which is well known to cause cell necrosis and apoptosis; but the role of Cd as an inhibitor of apoptosis was also observed. Although Cd2+ is not a redox-active metal ion, it was speculated that Cd2+ caused damage to cell and the molecular mechanism might be related to oxidative stress by the induction of ROS. Many reports demonstrated that excessive reactive oxygen species (ROS) levels lead to tissue damages and thus reduce the activities of the antioxidant defense enzymes. Antioxidant levels and related enzyme activity and gene expression were detected in ZFL cells exposed to Cd2+; the intracellular level of reactive oxygen species was however decreased by the administrations of Cd2+. A proteomic approach was further used to investigate the protein profiles related to sub-lethal exposure of Cd2+. 77 differentially expressed proteins were detected by two-dimensional gel electrophoresis after Cd2+ exposure; 43 of them were further identified by MALDI-TOF-MS. The proteins that responded to Cd2+ in ZFL cells were related to stress response, transporters, regulation of transcription, redox homeostasis, or some signaling pathways, with half of these proteins having metal ion binding capabilities.;Because Cd2+ has estrogenic activity and able to affect detoxification enzymes such as the cytochrome P450IA1, we also studied the effects of Cd2+ on VTG and CYPIA1 gene expression related to Cd2+ toxicity in ZFL cell. VTG and CYPIA1 mRNA levels were measured using quantitative real-time PCR.;Cellular responses to Cd using various metabolic programs, including genes involved in the cell cycle, gene repair and death and genes induce cell apoptosis to as a clearance for oxdatively damaged cells. In this study, we detect apoptosis related genes (c-jun, p53, bax), DNA repair related genes (gadd, rad51), estrogenic effect related genes (vtg1, ERalpha, ERbeta1, ERbeta2). Gene expression profile showed more sensitive in low dose group than high dose group for two time-points. And the profile of gene expression was reduced first then rised especially in low dose exposure group. We speculate that our results indicating Cd-induced apoptosis, DNA-damage response, induction of estrogenic pathways in ZFL cells might be associated with potential Cd cytotoxicity after exposure.;Since Cd2+ may also affect other metal transporters and Cd2+ transporter was recently found to be Zn2+ transporter, we also studed the relationship of Cd2+ and other metal transporters such as divalent metal transporters, Cu2+ transporters and Zn2+ transporters. Our results showed that almost all zinc transporter showed reduced expression in different degrees. For the copper transporter, ATP7A, ATP7B showed the same increased expression trend; but Ctr1 expression showed opposite decreased trend. While DMT1, MDR1 showed significant induced expression after 24h Cd exposure instead of 6h exposure. Therefore, Cd2+ might induce more DMT1 and MDR1 to help with Cd 2+ efflux. This also might explain decreased Cd concentration in ZFL cell after Cd2+ exposures.;Finally, luciferase reporter systems (3ERE-luc, 3XRE4-luc, 3MREd-luc) were constructed to investigate Cd cytotoxicity effect on respective signaling pathway. From the results, ERE, MREd and XRE4 activities all showed significant inhibition in ZFL cells after Cd administration. Putting these studies together, these results also indicated that Cd2+ cytotoxicity might not mainly affect cell via ER, MTF-1 and AhR pathway. Though these in-vitro reporter systems might be limited by the fact that they do not exactly represent the in vivo situation because many biological processes.;In summary, this study revealed an extraordinary oxidative stress response; extraordinary estrogen signaling pathway response, AhR pathway and metal ion response element active transcription regulated by Cd in ZFL cell. The ZFL cell line is very special from other models. Cd2+ may exert its toxicity by through special pathway besides common path such as oxidative stress, estrogenic effect, etc.