The Mechanism Of Antiestrogen Effect Of Semicarbazide On Zebrafish(Danio Rerio)

In recent years, semicarbazide is an emerging pollutant which is mainly found in foodstuffs such as baby food, fruit juice, jam, candied fruit, while in the liquid milk, flour and other food also detected semicarbazide residue. In 2009 September, semicarbazide content in China Dongying Tidal River Estuary and adjacent waters as high as 46.41 μg/L, the average content of semicarbazide in shellfish and other organisms were 3.14-6.46μg/kg, far more than the semicarbazide maximum residue 1μg/kg in animal origin foods limited by European Union. Therefore, semicarbazide as a new type of pollutants not only threatens human food security, has also caused damage to marine ecological environment in China. Researches have found that semicarbazide plays the potential antiestrogen effect in mammals and fish, such as decreased the plasma E2 levels in female rats, inhibited estrogen receptor and vitellogenin gene expression in the liver of female zebrafish, but the mechanism of antiestrogen effect of semicarbazide is not clear. Therefore, this paper design uses the semicarbazide exposure to mature adult zebrafish, measures the content of E2 and T in zebrafish gonads, definites that semicarbazide plays antiestrogen effect in zebrafish whether by decreasing the 17β-estradiol levels; secondly definites the effect of semicarbazide in disturbing gene expression of steroid synthetase and hypothalamus-pituitary-gonadal axis from the perspective of E2 synthesis to explore the mechanism of antiestrogen effect of semicarbazide in zebrafish.Main results are as follows:(1) After 96h/28d exposure to 1μg/L,10μg/L, 100μg/L,1000μg/L semicarbazide in adult female zebrafish, analyses zebrafish gonad content of sex hormone by radioimmunoassay.The results demonstrates that 96h exposure find that E2 levels in ovary decreases significantly at 10μg/L group and the levels of E2 in other three concentration groups had no changes. The 28d exposure results showes that the female E2 levels in ovary is significantly reduced only in 1μg/L group, but has no changes in other three concentration groups. In addition, another doctoral student’s results in our laboratory exposures 1ug/L、10ug/L、100g/L and 1000ug/L semicarbazide to adult female zebrafish 96h/28d,and finds that semicarbazide inhibits E2 levels in plasma. So we think that semicarbazide plays antiestrogen effect mainly by inhibiting the synthesis of E2 in female zebrafish ovary.(2) After 96h/28d exposure to 1μg/L,10μg/L, 100μ,1000μg/L semicarbazide in adult female zebrafish,we analyse the gene expression of key steroidogenic enzyme P450scc, P450c17,3p-HSD, P450aromA using RT-PCR.The results showes that 96h semicarbazide exposure does not affect the expression of 3β-HSD1,P450c17, P450sccmRNA levels in four concentration groups,while the expression of P450aromAmRNA decreases significantly in 1 μg/L,1 Oμg/L,100μg/L concentration groups.28d exposure results showes that compared to the control group, the expression level of P450aromA, P450c17, P450sccmRNA in the ovary has reduced in differently in four concentration groups, whereas no significant changes is found in 3β-HSD1mRNA gene expression level. Therefore, this paper indicates that semicarbazide decreased the E2 levels in ovary through downregulating the gene expression of female ovarian steroidogenic enzymes such as P450aromAmRNA, playing the antiestrogen effect.In addition, E2 and 1μg/L,10μg/L,100μg/L, 1000μg/L semicarbazide combined exposure 96h/28d to adult male zebrafish has showed that 96h semicarbazide combined with E2 exposure decreases steroid synthetase gene P450aromA, P450c17, P450sccmRNA levels compared to E2 alone treatment group. Semicarbazide combined with E2 exposes to male zebrafish 28d showes that P450aromA, P450c17, P450sccmRNA expression levels decreases at different concentration groups compared with E2 alone treatment group. The combined experimental exposure results shows that, semicarbazide plays their antiestrogen effect through inhibiting elevation of gene expression of aromatase by exogenous E2 induced in adult male zebrafish.(3) After 96h/28d exposure to 1μg/L,10μg/L,100μg/L,1000μg/L semicarbazide in adult female zebrafish, we analyses the gene expression of regulatory factors in HPG axis such as FSHfβ, LHβ, sGnRH, P450aromBmRNA. The results showes that 96h/28d esposure at low concentration of semicarbazide (1μg/L,10μg/L) significantly reduces the FSHβ gene expression in female zebrafish brain. Our results indicates that semicarbazide reduces FSHβmRNA expression levels by downregulating GnRHmRNA expression levels in the pituitary of female zebrafish, thereby inhibites the gene expression of aromatase. E2 and 1μg/L,10μg/L,10μg/L,1000μg/L semicarbazide combined exposure to adult male zebrafish 96h found that, E2 and low concentration of semicarbazide (1μg/L,10μg/L) combined exposure significantly decreases the expression of LHfβ, sGnRHmRNA levels compared to E2 alone treatment group; 28d results showes E2 and 1ug/L semicarbazide combined exposure decreases expression levels of FSHβ, LHβ, sGnRHmRNA significantly than E2 alone treatment group in male zebrafish brain. Therefore, the experimental combined exposure results show that, semicarbazide plays their antiestrogen effect through inhibiting elevation of gene expression of sGnRH and FSHfi by exogenous E2 induced in adult male zebrafish.In summary, the mechanism of antiestrogen effect of semicarbazide in female zebrafish might inhibit the expression of initiation factor sGnRH in HPG axis, then results in decreasing expression levels of FSHmRNA in the pituitary, further inhibits the aromatase activity, ultimately inhibits E2 synthesis in ovary, playing the antiestrogen effect.