| Objective:The clockla knockout(clockla-/-)zebrafish embryo was used as a model to investigate its role and underlying mechanism in Bisphenol A-induced neurodevelopmental toxicity in zebrafish embryo.Method:1.Determination of the median lethal dose of BPA The mortality of zebrafish embryos at 72 h,96 h and 120 h of BPA treatment was counted under concentrations of 0,2.5,5,and 10 mg/L.The median lethal dose(LC50)at each time point was calculated according to the karner’s method,and the exposure time and final concentration of subsequent experiments was determined.2.Luciferase reporter gene assay Plasmids containing or not containing the ERE sequence of the zebrafish clockl a promoter were constructed.Cells were treated with 1 μM BPA or E2 for 12 h after co-transfection with ER.After 24 h,the cells were lysed to determine the fluorescence value.3.Assays for zebrafish malformation rate,behavioral changes and cellular apoptosisHealthy fertilized WT and clockla-/-embryos were selected under a microscope and grouped randomly.These embryos were treated with 0,1,5,and 25 μM BPA at 6 hpf(6 h post fertilization).After 96 h,larvae were observed under microscope and the mortality and deformity rate were recorded.Then larvae were washed and placed in a 96 well-plate and allowed to adapt for 20 min before monitoring the swimming speed and number of movements under the microscope.The lighting interval was 20min light followed by a 20min dark,and repeated for 140 min to record free swimming distance and time.After the exposure,live larvae were stained with acridine orange(5 g/L)for 20 min and washed twice with PBS.The distribution of apoptotic/necrotic cells were identified using a fluorescence microscope.4.Determination of related gene expressionAfter 96 h of BPA treatment,the zebrafishes were collected into one EP tube,washed twice with DEPC water,and the RNA was extracted with Trizol to detect gene expression by RT-PCR.Results:1.Similar to estradiol,BPA can activates clockla transcription by binding to the ERE sequence in the clockla promoter region.2.The effects of BPA on zebrafish embryos depend on its concentration:in the middle and high concentration groups,the deformity rate increased and the behavior activity decreased.At the high concentration group,the number of apoptotic cells increased.All these changes were more pronounced in clockla-l-zebrafish than those in WT zebrafish.3.BPA exposure resulted in alterations in the expression of genes involved in the PI3k/AKT-Nrf2-ARE pathway.In the WT zebrafish,the PI3k,AKT1,Nrf2,and Nqo-1 genes expression increased at 1 μM,and Nrf2 and Keapl genes expression increased at 5 μM.But in the clockla knockout zebrafish,only Nrf2 showed a reduced gene expression at 25 μM.Conclusions:1.As an estrogen-like environmental pollutant,BPA can activate the clockla gene expression in vivo.2.EPA produced more pronounced neurodevelopmental toxicity in clockla-l-zebrafish than those in WT zebrafish,indicating a protective role of the clockla gene in BPA-induced toxicity in zebrafish embryos.3.The protective role of clockla might be mediated via the Pl3k/AKT-Nrf2-ARE pathway. |
