Study On The Roles Of Zebrafish Abcb4 And Abcc4 Genes In Preventing Accumulation Of Toxicants

Toxicants such as heavy metal, microcystins (MCs) and persistent organic pollutants (POPs) are substantially accumulated in the aquatic environment due to environment contamination, which can directly or indirectly pose a serious threat to aquatic organisms and cause long-term health effects on humans through drinking contaminated water and the food chain. ABCB1 and ABCC4 belong to two members of ATP-binding cassette (ABC) transporters that mediate multi-xenobiotic resistance (MXR). They have been reported function in toxicants excretion, tissue defense and signal transduction. Previous studies have demonstrated that zebrafish abcb4 and abcc4 genes are mammalian ABCB1 and ABCC4 orthologs respectively, which also function in recognize and transport a wide variety of xenobiotics. Currently, little is known about their roles in MC-LR (Microcystin-LR) and POPs such as DDT and lindane detoxification. Here, we cloned the cDNA sequence of zebrafish abcb4 and abcc4 genes. Abcb4-or its dominant negative mutant Abcb4-G1177D-, and Abcc4- or its dominant negative mutant Abcc4-G1188D-overexpressing LLC-PK1 cells were obtained. Moreover, we had developed a stable Abcc4-overexpressing transgenic zebrafish line. These models can be utilized to dissect zebrafish Abcb4 and Abcc4 roles in toxicants detoxification and its mechanisms. The results are listed as follows:1. In this study, we demonstrated the roles of zebrafish Abcb4 in cellular efflux of MC-LR in LLC-PK1 cells and developing embryos. Transcripts of zebrafish abcb4 were detected in all of adult tissues examined, with the highest expression in intestine. MC-LR was able to induce the expression of abcb4 gene in 96-hpf embryos and overexpression of this gene significantly decreased the cytotoxicity and accumulation of MC-LR in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcb4-G1177D mutant abolished its transporter function but not substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to this cyanobacterial toxin by reason of competitive inhibition of endogenous Abcb4. Moreover, ATPase activity in developing embryos can be induced by MC-LR. Thus, zebrafish Abcb4 plays crucial roles in cellular efflux of MC-LR and is a potential molecular marker for the monitoring of cyanobacteria contamination in the aquatic environment.2. DDT and lindane are highly toxic organochlorine pesticides and posing adverse effects on the environment and public health due to their frequent usage in developing countries. Here, we demonstrated zebrafish Abcc4 functions in cellular efflux of DDT and lindane in LLC-PK1 cells and developing embryos. Our results show that DDT or lindane were able to induce the expression of abcc4 mRNA and overexpression of Abcc4 significantly decreased the cytotoxicity and accumulation of DDT and lindane in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcc4-G1188D which can inhibit endogenous Abcc4 sensitized LLC-PKl cells and developing embryos to toxic pesticides. Moreover, cellular GSH content and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 may be an essential factor responsible for cellular detoxification and efflux of organochlorine pesticides.3. Additionally, transgenic technology combined with sleeping beauty transposon system, which can effectively improve the efficiency of transgene integration, are used to develop abcc4 transgenic zebrafish. An abcc4 transgenic construction was cloned the fish gene (also called "the all fish" constructs). It includes a strong promoter and a polyadenylation signal sequence from carp ?-actin gene, zebrafish abcc4 coding sequence which was introduced a Flag-tagged at the carboxyl terminus for transgene expression analysis. The positive transgene rate of the abcc4 transgenic zebrafish at PO generation is 22.1%, and three PO positive founders were detected to successfully transferred the abcc4 gene to its F1 offspring. After individually mating three sexually mature abcc4 positive fish with wild-type fish, it was found that the positive rates of the F1 generation are 3.75%,47.22% and 52.08% respectively. Analysis of qPCR and Western Blotting have demonstrated two F1 positive fish can successfully express Flag-Abcc4. A homozygous F3 fish was obtained by the cross of two heterozygous F2 fish.Overall, zebrafish abcb4 or abcc4 can be induced by MC-LR or organochlorine pesticides such as DDT and lindane respectively, and exert their functions, which called MXR, in detoxification and excretion of these toxicants in both LLC-PK1 cells and zebrafish embryos. And furthermore, we have obtained the stably express zebrafish Abcb4 and Abcc4 LLC-PKl cell lines and developed an abcc4 transgenic zebrafish, which provide ideal models for further investigation of zebrafish Abcb4 and Abcc4 functions in preventing accumulation of toxicants and its mechanisms.