engleski

INVOLVEMENT OF ABC TRANSPORT PROTEINS IN RESPONSE TO TOXIC HEAVY METALS IN THE PLHC-1 FISH CELL LINE

The role of ABC transporters in elimination of xenobiotics is well recognized. However, their interaction with heavy metals is not fully investigated in aquatic animals. The aim of this study was to investigate the potential involvement of ABC transport proteins in the cellular response to toxic heavy metals, using PLHC-1 fish cell line as in vitro model. Cells were exposed for 24 h to 0.01-1 µM of CdCl2, HgCl2, As2O3, or K2Cr2O7 and the expression of a series of ABC genes (abcb1, abcc1-4) was determined using qPCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC tansporters. P-glycoprotein (Pgp) activity was measured in PLHC-1/dox (Pgp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. Finally, by long term cultivation of PLHC-1/wt cells in increasing concentrations of CdCl2 and HgCl2 we tried to develop haevy metal resistant PLHC-1 cells. After 24 h exposure, abcc1-4 genes were dose-dependently up-regulated by all metals, while abcb1 was less affected. Up-regulation of abcc2 was more pronounced, with up to 10-fold increase in expression after exposure to HgCl2. Abcc3 and abcc4 were moderately inducible by HgCl2 with 3.4-fold and 2.8-fold induction, respectively. All metals caused a significant inhibition of both Pgp (2.5 – 4-fold vs. controls) and MRP (1.5 - 1.8-fold) transport activities with IC50 values below 1 µM. The long term cultivation of PLHC-1/wt cells in HgCl2, resulted in higher transport activities of MRPs, as determined by the increased sensitivity of these cells to MK571 (an MRP specific inhibitor). In conclusion, our results indicate that: (1) specific fish ABC transporters may contribute to cellular defence against toxic heavy metals, with a dominant role of MRP2 in metal excretion, and (2) modulation of ABC transporters by metals take place at both transcriptional and functional level.

ABC transporters ; metals ; PLHC-1

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