| Cadmium(Cd)and bisphenol A(BPA),as typical environmental pollutants,are common contaminants in food,which are mainly absorbed by people in their daily diet,and pose potential threats to human health.Therefore,it is of great significance to systematically study the toxic effects of combined exposure to Cd and BPA.In this paper,zebrafish embryos were selected as model organisms and exposed to different concentrations of Cd and BPA(0,1.03 mg/L Cd+1.35 mg/L BPA,1.54 mg/L Cd+2.03 mg/L BPA,3.09 mg/L Cd+4.06 mg/L BPA)for 96 h.During the exposure period,the growth and development parameters(death rate,malformation rate,hatching rate,heart rate,and autonomous movement frequency)of zebrafish embryos were observed by stereomicroscope.Then,the m RNA expression changes of five candidate internal reference genes(gapdh、ef1α、β-actin、rnf7 and rpl7)in zebrafish embryos exposed to Cd and BPA were detected,and the best internal reference was selected by three commonly used internal reference analysis software(ge Norm,Norm Finder and Best Keeper),which laid a foundation for the subsequent detection and analysis of target genes.Finally,real-time fluorescent quantitative PCR method was used to detect estrogen receptor genes(ERα and ERβ1),antioxidant enzyme genes(SOD,CAT and GPx),inflammatory factors(IL-1β,IL-8,IL-6 and TNF-α)and GABA receptor pathway related genes(gadra1,abat,gat1 and gad1b)m RNA expression level.Enzyme activity kits were applied to measure the activity of antioxidant enzymes(T-SOD and CAT)to reveal the toxic effects of Cd and BPA combined exposure on zebrafish embryos from different aspects.The results are as follows:1.During the combined exposure of Cd and BPA,developmental indicators were significantly affected to varying degrees.The combined exposure of high concentration(3.09 mg/L Cd+4.06 mg/L BPA)of Cd and BPA for 96 h led to the increase in the rates of mortality and deformity of zebrafish embryos(P<0.01),and the occurrence of spinal curvature,yolk sac edema,tail curvature,pericardial swelling and other malformations.The hatching rate and heart rate decreased significantly with the increase of exposure concentration(P<0.01),which showing a concentration-dependent effect.After exposure to medium and high concentrations of Cd and BPA(1.54 mg/ L Cd+2.03 mg/L BPA,3.09 mg/L Cd+4.06 mg/L BPA)for 24 h,the frequency of autonomous movement of zebrafish embryos was decreased significantly(P<0.01).2.The Ct values of the candidate internal reference genes of zebrafish embryos were tested after exposed to Cd and BPA for 96 h.The most stable internal reference gene,ef1α,was selected by three kinds of analysis software and the comprehensive evaluation method in the Cd and BPA combined exposed zebrafish embryos.3.Real-time fluorescent quantitative PCR was used to detect the m RNA expression of estrogen receptor genes(ERα and ERβ1),antioxidant enzyme genes(SOD,CAT and GPx),inflammatory factors(IL-1β,IL-8,IL-6 and TNF-α)and GABA receptor pathway related genes(gadra1,abat,gat1 and gad1b).The activity of T-SOD and CAT was tested by enzyme activity kits.The results showed that the m RNA expression of the estrogen receptor gene ERα in zebrafish embryos was significantly down-regulated(P<0.01)after exposure to Cd and BPA for 96 h.The m RNA expression of antioxidant enzyme genes SOD,CAT,and GPx was significantly up-regulated(P<0.05),and the activity of SOD was significantly increased(P<0.01),while CAT activity was reduced(P < 0.05).The m RNA expression of inflammatory factor-related genes IL-1β,IL-8,IL-6 and TNF-α increased significantly(P<0.05).The transcriptional expression of GABA receptor pathway related genes gadra1 and abat was significantly up-regulated(P<0.05),and m RNA expression of gat1 and gad1 b was significantly down-regulated(P<0.05).In summary,the combined exposure of Cd and BPA caused developmental toxicity and estrogenic effects,disrupted the antioxidant system,led to inflammation,and interfered GABA metabolism pathway,which indicated the toxic effects in the zebrafish embryos. |
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