Limits...
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 are highly toxic organochlorine pesticides and posing adverse effects on the environment and public health due to their frequent usage in developing countries.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.

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

Zebrafish Abcc4 plays crucial roles in cellular detoxification of DDT and lindane.(A) Death rates of 96-hpf embryos expressing GFP, Abcc4 or Abcc4-G1188D were monitored after treatment with 100 µg/L DDT or lindane. (B) Embryos injected with pT2/CMV2-Abcc4 or pT2/CMV2-GFP were exposed to 100 µg/L DDT or lindane from 24 to 96 hpf. Eighty embryos at 96-hpf stages were pooled for total RNA extraction and subjected to real-time PCR analysis. (C and D) Survival rates of LLC-PK1 cells expressing Abcc4 or Abcc4-G1188D, transfected empty vectors (CTRL) and treated with 25 µM MK571 were determined with MTT assays after exposure to DDT or lindane at indicated concentrations for 24 hours. Values are expressed as mean ± standard deviation (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4257548&req=5

pone-0111664-g003: Zebrafish Abcc4 plays crucial roles in cellular detoxification of DDT and lindane.(A) Death rates of 96-hpf embryos expressing GFP, Abcc4 or Abcc4-G1188D were monitored after treatment with 100 µg/L DDT or lindane. (B) Embryos injected with pT2/CMV2-Abcc4 or pT2/CMV2-GFP were exposed to 100 µg/L DDT or lindane from 24 to 96 hpf. Eighty embryos at 96-hpf stages were pooled for total RNA extraction and subjected to real-time PCR analysis. (C and D) Survival rates of LLC-PK1 cells expressing Abcc4 or Abcc4-G1188D, transfected empty vectors (CTRL) and treated with 25 µM MK571 were determined with MTT assays after exposure to DDT or lindane at indicated concentrations for 24 hours. Values are expressed as mean ± standard deviation (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.

Mentions: To address the physiological roles of elevated Abcc4 expression, we examined the effects of Abcc4 and its dominant negative form on the survival of developing embryos exposed to organochlorine pesticides. The dominant negative form of zebrafish Abcc4 (Abcc4-G1188D) was generated by site-specific PCR to abolish its ATP hydrolysis and transport function without effects on the substrate binding activity as described previously [38], [39]. In this study, LC50 values were defined as organochlorine pesticide concentrations that killed 50% of embryos at 96 hpf and LC50 values for DDT and lindane were 93.25 µg/L with 95% confidence interval (89.38 to 98.73 µg/L) and 98.87 µg/L with 95% confidence interval (95.34 to 103.58 µg/L), respectively. Therefore, acute toxicity assays were performed with embryos exposed to 100 µg/L of DDT or lindane. In addition, developing embryos injected with 150 pg/embryo of pT2/CMV-Abcc4 or pT2/CMV-Abcc4-G1188D showed no abnormal phenotypes at 96 hpf, implying that overexpressed Abcc4 or Abcc4-G1188D has little side effect on developing embryos. To reduce experimental variations, injected embryos without abnormal phenotypes at 24 hpf were selected for subsequent toxicity assays. As shown in Fig.3A, the death rate of Abcc4-expressing embryos at 96 hpf was significantly lower than that of GFP-expressing embryos (p<0.05), while the death rate of Abcc4-G1188D-expressing embryos was significantly higher than that of GFP-expressing embryos (p<0.05). These data suggest that zebrafish Abcc4 is able to protect developing embryos from toxic effects of DDT and lindane.


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)

Zebrafish Abcc4 plays crucial roles in cellular detoxification of DDT and lindane.(A) Death rates of 96-hpf embryos expressing GFP, Abcc4 or Abcc4-G1188D were monitored after treatment with 100 µg/L DDT or lindane. (B) Embryos injected with pT2/CMV2-Abcc4 or pT2/CMV2-GFP were exposed to 100 µg/L DDT or lindane from 24 to 96 hpf. Eighty embryos at 96-hpf stages were pooled for total RNA extraction and subjected to real-time PCR analysis. (C and D) Survival rates of LLC-PK1 cells expressing Abcc4 or Abcc4-G1188D, transfected empty vectors (CTRL) and treated with 25 µM MK571 were determined with MTT assays after exposure to DDT or lindane at indicated concentrations for 24 hours. Values are expressed as mean ± standard deviation (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-g003: Zebrafish Abcc4 plays crucial roles in cellular detoxification of DDT and lindane.(A) Death rates of 96-hpf embryos expressing GFP, Abcc4 or Abcc4-G1188D were monitored after treatment with 100 µg/L DDT or lindane. (B) Embryos injected with pT2/CMV2-Abcc4 or pT2/CMV2-GFP were exposed to 100 µg/L DDT or lindane from 24 to 96 hpf. Eighty embryos at 96-hpf stages were pooled for total RNA extraction and subjected to real-time PCR analysis. (C and D) Survival rates of LLC-PK1 cells expressing Abcc4 or Abcc4-G1188D, transfected empty vectors (CTRL) and treated with 25 µM MK571 were determined with MTT assays after exposure to DDT or lindane at indicated concentrations for 24 hours. Values are expressed as mean ± standard deviation (n = 3). Significant differences are indicated by *p<0.05 and **p<0.01.
Mentions: To address the physiological roles of elevated Abcc4 expression, we examined the effects of Abcc4 and its dominant negative form on the survival of developing embryos exposed to organochlorine pesticides. The dominant negative form of zebrafish Abcc4 (Abcc4-G1188D) was generated by site-specific PCR to abolish its ATP hydrolysis and transport function without effects on the substrate binding activity as described previously [38], [39]. In this study, LC50 values were defined as organochlorine pesticide concentrations that killed 50% of embryos at 96 hpf and LC50 values for DDT and lindane were 93.25 µg/L with 95% confidence interval (89.38 to 98.73 µg/L) and 98.87 µg/L with 95% confidence interval (95.34 to 103.58 µg/L), respectively. Therefore, acute toxicity assays were performed with embryos exposed to 100 µg/L of DDT or lindane. In addition, developing embryos injected with 150 pg/embryo of pT2/CMV-Abcc4 or pT2/CMV-Abcc4-G1188D showed no abnormal phenotypes at 96 hpf, implying that overexpressed Abcc4 or Abcc4-G1188D has little side effect on developing embryos. To reduce experimental variations, injected embryos without abnormal phenotypes at 24 hpf were selected for subsequent toxicity assays. As shown in Fig.3A, the death rate of Abcc4-expressing embryos at 96 hpf was significantly lower than that of GFP-expressing embryos (p<0.05), while the death rate of Abcc4-G1188D-expressing embryos was significantly higher than that of GFP-expressing embryos (p<0.05). These data suggest that zebrafish Abcc4 is able to protect developing embryos from toxic effects of DDT and lindane.

Bottom Line: 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.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.

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