engleski

Gene expression and functional analysis of toxicologically important ABC transporters in rainbow trout (Oncorhynchus mykiss) tissues and cell lines

The important role of ABC transporters in tissue defense is reflected by their tissue distribution. Numerous studies revealed the highest expression of transporters from the ABCB and ABCC subfamily in important physiological and/or pharmacological barriers of mammals. On the contrary, detailed studies of ABC transporter genes expression and function in aquatic organisms are scarce. Consequently, the main goals of our study were: (1) gene expression analysis of ABC transporters (abcb1, abcb11, abcc1-2 and abcg2) implicated in disposition of various xenobiotics in rainbow trout tissues (brain, gills, liver, kidney, gonads, distal and proximal intestine) ; (2) gene expression and functional analysis of target ABC transporters in rainbow trout cell lines originating from different tissues (liver: RTL-W1, R1 ; hepatoma: RTH-149 ; gonad: RTG-2 ; gill: RTgill- W1 ; gut: RTgut and brain: RTbrain) ; and (3) obtaining the first insights into transcriptional regulation of target ABC transporters in liver cell line (R1) after 24 h exposure to various xenobiotics. Gene expression of target ABC transporters was measured using relative qPCR with SYBR green. Expression of target genes was normalized to the housekeeping gene (18S rRNA) using Q-Gene application. Functional assays in trout cell lines were preformed using various model fluorescent substrates and inhibitors. Most of the analyzed genes showed similar tissue expression pattern as the ones found in mammals. The lowest expression of all analyzed ABC genes was found in gills, despite their direct contact with external environment and possible role as barrier tissue. Compared to the corresponding tissues, cell lines exhibited marked down regulation of abcb1, abcb11 and abcg2, while expression pattern of most abcc transporters was same or relatively higher than in tissues. Low ABC transporters activity measured by functional assay was found in all cell lines. In accordance with the gene expression data, the highest functional activity was measured for abcc transporters using Calcein-AM and MK571 as model substrate and inhibitor, respectively. However, significant differences in ABC transporters activities among different cell lines were not observed. Short term exposure of R1 cell line to various xenobiotics revealed high induction potential of abcb1 and relatively low induction potential for all measured abcc transporters.

ABC transporters ; expression ; functional analysis ; rainbow trout ; tissues ; cell lines

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