The Research Of Anti-estrogenic Effects Of Semicarbazide On Zebrafish(Danio Rerio)

Semicarbazide （SEM） has been known as the characteristic metabolite ofnitrofurans drugs （e.g. nitrofurazone）, and it is also found that SEM can be producedby decomposition of azodicarbonamide blowing agents. Due to the use of nitrofuransdrugs and azodicarbonamide, SEM was present in animal-derived and glass jarpackaged food. Experiment in rat shows that SEM has a moderate acute toxicity.Besides, a variety of toxic effects such as latent carcinogenicity, neurotoxicity andanti-estrogenic effect of SEM have also been reported. According to the EuropeanUnion （EU）, the maximum residue limit （MRL） of SEM in the animal-derived food is1.0μg/kg. However, SEM in the export aquatic products （e.g.Tilapia, eels, etc.） fromChina has been detected many times exceeding EU MRL, which causes seriouslyinfluence on the quality of export aquatic products. A recent report has demonstratedthat SEM was detected in coastal waters adjacent to the Chaohe River estuary, wherethe average concentration of SEM in shellfish was6.46μg/kg, much higher than theEU MRL, and that in seawater reached46.41μg/L. Accordingly, as an emergingmarine contaminant, SEM is threatening the marine ecological environment of China.Despite of the fact, little research related to the toxicity of SEM on aquatic organismshas been conducted. Therefore, acute toxicity experiment of SEM on zebrafish wasperformed and anti-estrogenic test was conducted, aiming to provide scientific basisand support for assessment of marine ecological risks and aquatic food safety.Main results are as follows:（1） Acute toxicity experiment results show that96h LC₅₀of SEM for the maturezebrafish is26.29mg/L, with95%confidence interval being24.42²7.78mg/L.According to The Guidelines for the hazard evaluation of new chemical substances（HJT154-2004）, SEM is of moderate acute toxicity.（2） Test for the estrogenic effect: biomarkers including vtg-1, ERα, ERβ mRNAin male zebrafish are used to determine whether SEM can exert an estrogenic effect. After a96-hour and28-day exposure to a range of concentrations of semicarbazide（1.0、10.0、100.0、1000.0μg/L）, the expression of vtg-1、ERα、ERβ mRNA in malezebrafish does not show a significant increase, which indicates that SEM cannotinduce expression of liver vtg-1and ERs in male zebrefish during the exposure period.This result shows that SEM does not show an estrogenic effect.（3） Test for anti-estrogenic effect: E₂in female zebrafish can induce theexpression of VTG in liver via the mediation of ERs, while anti-estrogen canantagonize such induction. In this paper, after exposure to SEM （1.0、10.0、100.0、1000.0μg/L） for96hours and28days, the variation of vtg-1and ERs mRNA wasdetected. After96-hour exposure, the level of vtg-1mRNA decreased graduallycompared to the control group, while after28days, this level dropped remarkably aswell as the gonad index in all treated groups. This result illustrates that SEM caninhibit the induction of vtg-1mRNA in livers of female zebrafish, showing ananti-estrogenic effect. The test for ERs level demonstrates that the level of ERα andERβ mRNA fell significantly with ERαmRNA showing a dose-dependent decreaseafter96-hour exposure. Further decrease in the levels of ERs was detected after a28-day exposure. The result of ERs illustrates that SEM can inhibit the estrogenicactivity of E₂by inhibiting the expression of ERs.（4） Test for anti-estrogenic activity: male zebrafish were exposed to aconcentration of E₂（500ng/L） and SEM （0,1.0,10.0,100.0,1000.0μg/L respectively）combination. After96hours, compared to the E₂only group, the level of vtg-1in the1000μg/L SEM and500ng/L E₂group shows a significant decrease while it shows asimilar level to the control group, which suggests that the anti-estrogenic activity of1000μg/L SEM can totally antagonize the estrogenic activity of500ng/L E₂. Theantagonizing ability of SEM against E₂is1/2000, showing a weak anti-estrogenicactivity. On the other hand, the levels of both vtg-1and ERα mRNA in the othergroups （i.e.500ng/L E₂with SEM of1.0μg/L,10.0μg/L,100.0μg/L respectively） arehigher than that in the control group, even higher than the E₂only group. The possibleexplanation to this might be a compensation action in male zebrafish during ashort-time exposure. However, after28days, vtg-1, ERα, ERβ mRNA in all joint exposure groups drop significantly, approaching to the control group. This result isanother evidence for the anti-estrogenic activity of SEM.In conclusion, SEM is a chemical substance with moderate acute toxicity, withits96h LC₅₀for the mature zebrafish being26.29mg/L. SEM does not show anestrogenic effect while it can exert a weak anti-estrogenic activity, with itsantagonizing ability of SEM （96h） against E₂is1/2000. One mechanism of this maybe that SEM can inhibit the expression of ERs, which accordingly inhibit theestrogenic activity of E₂.