The Reproductive Endocrine Disturbing Effects Of Carbofuran On Zebrafish(Danio Rerio)

Carbofuran is a highly toxic carbamate pesticide with high toxicity. It could cause certain reproductive damages, including reducing the weight of epididymides, seminal vesicle and ventral prostate in rats and decreasing the hatching rate and fertilization ability in earthworm. In order to determine the endocrine disrupting effects of carbofuran on the reproduction of fish and underlying mechanisms, male and female zebrafish(Daio rerio) were exposured to 0.1,1.0,10.0 and 100.0 μg/L carbofuran, and vitellogenin (VTG) protein in the liver, whole-body 17β-estradiol (E2) and testosterone (T) levels and gene expression of VTG, gonadalaromatase (CYP19ala), estrogen receptors (ERs) and androgen receptor (AR) was measured.The results indicated that:(1) Following a 21-day exposure of male zebrafish to 0.1,1.0,10.0 and 100.0 μg/L carbofuran,VTG protein levels in the liver were determined by enzyme linked immunosorbent assay-sandwich technique, whole-body E2 and T levels were examined by radioimmunoassay (RIA) and the gene expression of VTG,CYP19ala and ER was quantified by real-time PCR. Results showed that hepatic VTG protein levels were merely increased in the 0.1 μg/L group, but VTG mRNA levels were increased in all carbofuran-treated groups. Carbofuran increased concentrations of E2 and reduced T levels in the 1.0,10.0 and 100.0 μg/Lgroups, while CYP19ala gene expression was not changed. In addition, there was a significant increase in ERβ2 mRNA levels in the liver, as well as ERa, ERβ1 and ERβ2mRNA levels in the testis.Thus, carbofuran elevated whole-body E2 levels in male zebrafish, perhaps not by affecting the gonadal aromatase, and stimulated ERs transcription,by which VTG was induced, indicating an estrogenic activity in the males. However, the gonadosomatic index(GSI)of male adult zebrafish was not altered at the end of the 21-day carbofuran exposure.(2) Following a 21-day exposure of female zebrafish to 0.1,1.0,10.0 and 100.0 μg/L carbofuran, whole-body E2 and T levels were examined by radioimmunoassay (RIA), the gene expression of CYP19ala, ER, AR and VTG, was quantified by real-time PCR,VTG protein levels in the liver were determined by enzyme linked immunosorbent assay-sandwich technique. Results were not consistent with that observed in the males. In the female zebrafish, whole-body T and E2 levels were decreased. The results indicated that in females, carbofuran showed notestrogenic activity. And gene expressions of CYP19ala in gonad and ERs in the liver were not altered, while there was a significant increase in AR mRNA levels in the gonad.Thus further research would be needed to confirmed such anti-estrogenic effect of carbofuran in female zebrafish. In addition, VTG protein concentrations in the liver were decreased, epatic VTG mRNA levels were merely increased in the 0.1μg/L treatment group. The gonadosomatic index(GSI) of female adult zebrafish was also not altered at the end of the 21-day carbofuran exposureIn conclusion, sex differences were observed in disrupting effects of carbofuran on the reproductive encocrine system of zebrafish. And carbofuran showed estrogenic activity in the male zebrafish, but had potential anti-estrogenic effect on the females.