四溴双酚A对电离辐射诱导的斑马鱼肝脏毒性效应的影响

Effects of tetrabromobisphenol A on ionizing radiation-induced liver toxicity in zebrafish

目的基于斑马鱼模型研究四溴双酚A对电离辐射诱导的肝脏毒性效应的影响,为评估微塑料-放射性双重暴露对水体生物及人类造成的生存和健康风险提供科学依据。方法将4~6月龄健康成年斑马鱼采用3种方式按随机数表法进行分组(每组20条,雌雄各半)。四溴双酚A暴露浓度筛选实验分为4组:对照组和3、30、300μg/L四溴双酚A处理组。双重暴露对肝脏功能影响实验分为5组:对照组、电离辐射组(10、20或30 Gy)和电离辐射+四溴双酚A(60、300和1500μg/L)处理组。四溴双酚A对肝脏辐射毒性效应实验分为3组:对照组、电离辐射组(20 Gy)和电离辐射+四溴双酚A处理组(300μg/L)。检测肝脏中肝功指标、氧化应激标志物、炎症因子及肝脏细胞凋亡情况的改变,并进行非靶向代谢组学检测寻找差异代谢通路和代谢物。结果不同浓度的四溴双酚A暴露导致斑马鱼肝脏ALT和AST活性以剂量依赖方式升高,且300μg/L暴露组与对照组相比具有统计学意义(t=-2.22、-3.20,P<0.05)。20 Gy起电离辐射可诱导明显的肝功下降,此辐射剂量下从300μg/L四溴双酚A浓度起联合暴露可加剧肝脏辐射毒性(与对照组比较,t=-8.18~-4.63,P<0.05;与电离辐射组比较,t=-5.22~-0.30,P<0.05)。电离辐射组与联合暴露组斑马鱼肝脏ALT和AST活性,ROS、MDA和SOD活性,TNF-α、IL-1β、Cox-2、Caspase-8和Caspase-9的mRNA和蛋白表达水平,以及肝细胞凋亡情况较对照组逐级升高(与对照组比较,t=-12.29~-2.88,P<0.05;与电离辐射组比较,t=-4.40~-2.31,P<0.05);D-葡萄糖酸、对甲酚等代谢物含量差异在3组肝组织中持续加大,涉及生物合成、降解及代谢等KEGG途径。结论300μg/L四溴双酚A暴露可加剧电离辐射诱导的斑马鱼肝脏毒性效应,涉及使氧化应激、炎症和凋亡水平进一步升高等机制,且四溴双酚A加重了辐射导致的肝脏代谢紊乱。

Objective To investigate the effects of tetrabromobisphenol A(TBBPA)on ionizing radiation(IR)-induced liver toxicity based on a zebrafish model and provide a scientific basis for assessing microplastic-radiation exposure hazards to the survival and health of aquatic organisms and humans.Methods Healthy adult zebrafish aged 4-6 months were grouped(20 fish each group,sex in half)by random number table method in three different ways.The TBBPA exposure concentration screening experiment was divided into 4 groups:control group and TBBPA(3,30 and 300μg/L)treatment groups.The experiment of effects of double exposure on liver function was divided into 5 groups:control group,IR(10,20 or 30 Gy)groups and IR+TBBPA(60,300 and 1500μg/L)treatment groups.The experiment of effects of TBBPA on hepatic radiation toxicity was divided into 3 groups:control group,IR(20 Gy)group,and IR+TBBPA(300μg/L)group.The changes in liver function indexes,oxidative stress markers,pro-inflammatory cytokines,and liver cell apoptosis were monitored,differential metabolic pathways and metabolites were identified upon untargeted metabolomics assays,and inter-group data were compared by One-way ANOVA test.Results The activities of ALT and AST in zebrafish liver increased in a dose-dependent manner after exposure to TBBPA,and the differences between 300μg/L TBBPA group and control group were statistically significant(t=-2.22,-3.20,P<0.05).IR at a dose of 20 Gy or above induced a significant decline of liver function,and at this radiation dose,combined exposure to 300μg/L or above TBBPA intensified the liver toxicity(compared with the control group,t=-8.18 to-4.63,P<0.05,compared with IR group,t=-5.22 to-0.30,P<0.05).Compared with the control group,the activities of ALT and AST,levels of ROS,MDA and SOD,mRNA and protein expression levels of TNF-α,IL-1β,Cox-2,Caspase-8 and Caspase-9,and cell apoptosis in zebrafish livers of IR and IR+TBBPA groups increased gradually(compared with the control group,t=-12.29 to-2.88,P<0.05,compared with IR group,t=-4.40 to-2.31,P<0.05).The differences in the content of D-gluconic acid,p-cresol and other metabolites in liver tissues were more and more significant among the three groups,involving multiple KEGG pathways such as biosynthesis,degradation and metabolism.Conclusions Exposure to 300μg/L TBBPA can aggravate IR-induced liver toxicity of zebrafish,which involves the mechanism that further elevates the levels of oxidative stress,inflammation,and apoptosis,as well as radiation-induced liver metabolic disorders.