engleski

Interaction of ABC transport proteins with toxic metals at the level of gene and transport activity in the PLHC-1 fish cell line

The aim of this study was to investigate the interaction of four toxic metals with ABC transport proteins in piscine cell line PLHC-1. 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 qRT-PCR. Using the fluorescent model substrates calcein-AM and monochlorbimane we measured interaction of metals with the transport activity of ABC transporters. P-glycoprotein (P- gp) activity was measured in PLHC-1/dox (P-gp overexpressing cells) while activity and interactions of metals with MRPs was measured in PLHC-1/wt cells. After 24 h exposure, abcc2-4 genes were dose-dependently up-regulated by all metals, while abcb1 and abcc1 were less affected. Up-regulation of abcc2 was more pronounced, with up to 8-fold increase in expression. Abcc3 and abcc4 were moderately inducible by HgCl2 with 3.3- fold and 2.2-fold, respectively. All metals caused a significant inhibition of both P-gp (2.9 - 4- fold vs. controls) and MRP (1.3 - 1.8-fold) transport activities. Modulation of ABC genes and transport activities was further investigated in PLHC-1/wt cells exposed to 1 µM HgCl2 for 72 h and in Hg resistant cells selected by long term cultivation of PLHC-1/wt cells in increasing concentrations of HgCl2. Exposure to HgCl2 for 72 h induced MRP genes expression and efflux activity. The long term cultivation of PLHC-1/wt cells in HgCl2, did not cause prolonged up- regulation of the tested abc genes but resulted in higher MRP transport activities as determined by the increased sensitivity of these cells to MK571 (MRP specific inhibitor). Results of the present study indicated specific interaction of metals with selected ABC transport proteins. Modulation of ABC transporters takes place at both transcriptional and functional level. An active involvement of efflux pumps in Hg clearance in fish is suggested.

ABC transporters ; metals ; PLHC-1 ; qRT-PCR

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