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Characterization of zebrafish Abcc4 as an efflux transporter of organochlorine pesticides.

Lu X, Long Y, Lin L, Sun R, Zhong S, Cui Z - PLoS ONE (2014)

Bottom Line: DDT and lindane were able to induce the expression of abcc4 gene 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 mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides.Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, China.

ABSTRACT
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. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification of organochlorine pesticides. Here, we demonstrated the roles of zebrafish Abcc4 in cellular efflux of DDT and lindane. Zebrafish abcc4 was maternally expressed in the oocytes and its transcripts were detected in the lens, pancreas, gills, liver, intestine and bladder of developing embryos and in adult tissues examined. DDT and lindane were able to induce the expression of abcc4 gene 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 mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides. Moreover, glutathione (GSH) was involved in the efflux of cellular pesticides and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

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GSH is involved in the efflux of DDT and lindane in zebrafish embryos.(A–B) Intracellular GSH contents in zebrafish embryos exposed to different concentrations of DDT or lindane from 96 to 120 hpf. (C–D) Contents of DDT or lindane in embryos after treatment with GSH at indicated concentrations for 24 hours. Embryos were exposed to medium containing 5 µg/L DDT or lindane and simultaneously to 0.1–5 µM GSH from 96 to 120 hpf. (E–F) Contents of DDT or lindane in embryos after treatment with BSO, an inhibitor of GSH biosynthesis, at indicated concentrations for 24 hours. Embryos were treated with medium containing 5 µg/L DDT or lindane and simultaneously with 1–25 µM BSO from 96 to 120 hpf. (G–H) ATPase activities as shown by Pi levels in 96-hpf embryos after exposure to DDT or lindane at indicated concentrations. Values are expressed as means ± standard deviations (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.
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pone-0111664-g008: GSH is involved in the efflux of DDT and lindane in zebrafish embryos.(A–B) Intracellular GSH contents in zebrafish embryos exposed to different concentrations of DDT or lindane from 96 to 120 hpf. (C–D) Contents of DDT or lindane in embryos after treatment with GSH at indicated concentrations for 24 hours. Embryos were exposed to medium containing 5 µg/L DDT or lindane and simultaneously to 0.1–5 µM GSH from 96 to 120 hpf. (E–F) Contents of DDT or lindane in embryos after treatment with BSO, an inhibitor of GSH biosynthesis, at indicated concentrations for 24 hours. Embryos were treated with medium containing 5 µg/L DDT or lindane and simultaneously with 1–25 µM BSO from 96 to 120 hpf. (G–H) ATPase activities as shown by Pi levels in 96-hpf embryos after exposure to DDT or lindane at indicated concentrations. Values are expressed as means ± standard deviations (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.

Mentions: Previous studies have shown that the organochlorine pesticides such as DDT or lindane are able to induce the activation of GSTs [41], [42] and GSTs can detoxify insecticides by promoting their reductive dehydrochlorination or by conjugation reactions with reduced GSH, to produce water-soluble metabolites that are more readily excreted [21]. So, we analyzed GSH levels in developing embryos to investigate mechanisms underlying organochlorine pesticide detoxification. As shown in Fig. 8A–B, treatments of embryos at 96 hpf with 0.05–5 µg/L DDT or 0.01–1 µg/L lindane for 24 hours were able to significantly increase GSH levels in a dose-dependent manner. However, the accumulation of DDT or lindane in embryos was markedly decreased with the increase of GSH concentrations (0.1–5 mM) in culture medium (Fig.8C–D). In addition, the accumulation of DDT or lindane in embryos was significantly increased by treatments with 5 and 25 µM of BSO, an inhibitor of GSH biosynthesis (Fig.8E–F). These data suggest GSH is involved in the detoxification of DDT and lindane probably through conjugation with organochlorine pesticides.


Characterization of zebrafish Abcc4 as an efflux transporter of organochlorine pesticides.

Lu X, Long Y, Lin L, Sun R, Zhong S, Cui Z - PLoS ONE (2014)

GSH is involved in the efflux of DDT and lindane in zebrafish embryos.(A–B) Intracellular GSH contents in zebrafish embryos exposed to different concentrations of DDT or lindane from 96 to 120 hpf. (C–D) Contents of DDT or lindane in embryos after treatment with GSH at indicated concentrations for 24 hours. Embryos were exposed to medium containing 5 µg/L DDT or lindane and simultaneously to 0.1–5 µM GSH from 96 to 120 hpf. (E–F) Contents of DDT or lindane in embryos after treatment with BSO, an inhibitor of GSH biosynthesis, at indicated concentrations for 24 hours. Embryos were treated with medium containing 5 µg/L DDT or lindane and simultaneously with 1–25 µM BSO from 96 to 120 hpf. (G–H) ATPase activities as shown by Pi levels in 96-hpf embryos after exposure to DDT or lindane at indicated concentrations. Values are expressed as means ± standard deviations (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257548&req=5

pone-0111664-g008: GSH is involved in the efflux of DDT and lindane in zebrafish embryos.(A–B) Intracellular GSH contents in zebrafish embryos exposed to different concentrations of DDT or lindane from 96 to 120 hpf. (C–D) Contents of DDT or lindane in embryos after treatment with GSH at indicated concentrations for 24 hours. Embryos were exposed to medium containing 5 µg/L DDT or lindane and simultaneously to 0.1–5 µM GSH from 96 to 120 hpf. (E–F) Contents of DDT or lindane in embryos after treatment with BSO, an inhibitor of GSH biosynthesis, at indicated concentrations for 24 hours. Embryos were treated with medium containing 5 µg/L DDT or lindane and simultaneously with 1–25 µM BSO from 96 to 120 hpf. (G–H) ATPase activities as shown by Pi levels in 96-hpf embryos after exposure to DDT or lindane at indicated concentrations. Values are expressed as means ± standard deviations (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.
Mentions: Previous studies have shown that the organochlorine pesticides such as DDT or lindane are able to induce the activation of GSTs [41], [42] and GSTs can detoxify insecticides by promoting their reductive dehydrochlorination or by conjugation reactions with reduced GSH, to produce water-soluble metabolites that are more readily excreted [21]. So, we analyzed GSH levels in developing embryos to investigate mechanisms underlying organochlorine pesticide detoxification. As shown in Fig. 8A–B, treatments of embryos at 96 hpf with 0.05–5 µg/L DDT or 0.01–1 µg/L lindane for 24 hours were able to significantly increase GSH levels in a dose-dependent manner. However, the accumulation of DDT or lindane in embryos was markedly decreased with the increase of GSH concentrations (0.1–5 mM) in culture medium (Fig.8C–D). In addition, the accumulation of DDT or lindane in embryos was significantly increased by treatments with 5 and 25 µM of BSO, an inhibitor of GSH biosynthesis (Fig.8E–F). These data suggest GSH is involved in the detoxification of DDT and lindane probably through conjugation with organochlorine pesticides.

Bottom Line: DDT and lindane were able to induce the expression of abcc4 gene 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 mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides.Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, School of Basic Medical Science, Wuhan University, Wuhan, Hubei, China.

ABSTRACT
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. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification of organochlorine pesticides. Here, we demonstrated the roles of zebrafish Abcc4 in cellular efflux of DDT and lindane. Zebrafish abcc4 was maternally expressed in the oocytes and its transcripts were detected in the lens, pancreas, gills, liver, intestine and bladder of developing embryos and in adult tissues examined. DDT and lindane were able to induce the expression of abcc4 gene 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 mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides. Moreover, glutathione (GSH) was involved in the efflux of cellular pesticides and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

Show MeSH
Related in: MedlinePlus