基于斑马鱼幼鱼的塑化剂肝毒性危害识别研究

Hazard identification of phthalates based on hepatotoxicity of zebrafish larvae

目的 采用塑化剂邻苯二甲酸二乙基己酯(DEHP)和邻苯二甲酸二丁酯(DBP)作为阳性对照,建立斑马鱼幼鱼脂肪肝毒性风险评估模型进行塑化剂邻苯二甲酸丁酯苯甲酯(BBP)肝毒性危害识别。方法 分析DEHP和DBP暴露后斑马鱼肝脏脂肪信号强度及过氧化物酶体增殖物激活受体(PPAR)靶基因CD36的表达,初步构建斑马鱼幼鱼肝毒性模型;基于此模型对BBP进行肝毒性评价,此外采用肝脏表达绿色荧光转基因斑马鱼幼鱼进行肝密度分析、甘油三酯(TG)定量检测以及成鱼肝脏病理检查进一步验证模型的可靠性,应用基准剂量法(BMD)推导关键效应点。结果 DEHP、DBP表现出极显著诱发脂肪肝风险(均P<0.01),显著上调CD36相对表达量(P<0.001,P<0.05),提示造模成功。BBP(0.000 012 5%、0.000 025%、0.000 05%)诱发斑马鱼幼鱼显著的脂质沉积(P<0.05,P<0.001,P<0.001)、显著影响密度(P<0.05,P<0.01,P<0.01)、显著增加TG蓄积(P<0.05,P<0.05,P<0.01)、上调CD36基因表达。此外,BBP(≥0.000 012 5%)诱发斑马鱼成鱼肝脏部位脂质空泡形成、空泡间隙和数量减少,提示BBP具有明显的肝毒性。关键效应为肝脏脂肪信号强度(S),基准剂量下限(BMDL)值为0.013 mg/L。结论 本研究成功构建了一种通过肝脏脂肪信号强度来识别塑化剂肝毒性的斑马鱼幼鱼模型,结合基准剂量法进一步为塑化剂肝毒性危害识别提供科学依据。

Objective The risk assessment model of fatty liver accumulation in zebrafish larvae was established to identify the hepatotoxicity hazards of plasticizer butyl benzyl phthalate(BBP)using Di(2-ethylhexyl)phthalate(DEHP)and dibutyl phthalate(DBP)as positive control samples. This model was used to identify hepatotoxicity of butyl benzyl phthalate(BBP).Methods The fatty liver signal intensity and expression of peroxisome proliferator activated receptor(PPAR)target gene CD36 in zebrafish liver after DEHP and DBP exposure were analyzed,and the hepatotoxicity model of zebrafish larvae was preliminarily constructed,which was used to evaluated the hepatotoxicity of BBP. In addition,liver density analysis,TG quantitative detection of zebrafish larvae and liver pathological examination of adult zebrafish were used to further verify the reliability of the model. The benchmark dose method(BMD)was used to derive the point of departure.Results DEHP and DBP showed significant risk of fatty liver induction(both P<0. 01),and the relative expression level of CD36 was significantly higher(P<0. 001 and P<0. 05),indicating the success of modeling. BBP(0. 000 012 5%,0. 000 025%,0. 000 05%) induced significant lipid deposition(P<0. 05,P<0. 001,P<0. 001),significantly affected liver parenchyma(P<0. 05,P<0. 01,P<0. 01),increased TG accumulation significantly(P<0. 05,P<0. 05,P<0. 01),and up-regulated CD36 gene expression. Furthermore,BBP(≥0. 000 012 5%)induced the formation of lipid vacuoles in the liver of adult zebrafish,as well as the reduction of vacuolar space and number,suggesting that BBP had significant hepatotoxicity. The key effect was fatty liver signal intensity(S)and the benchmark dose lower-bound confidence limit(BMDL)value was 0. 013 mg/L.Conclusion This study successfully established a model of liver fat signal intensity to identify the hepatotoxicity of phthalates in zebrafish larvae,and preliminarily clarified the PPAR target gene CD36 involved in hepatotoxicity,and further provided a scientific basis for the risk assessment of phthalates combined with the BMD.