Molecular Toxicological Effect Of Acrylamide On Cardiac Developmental Toxicity And Activation Mechanism Of Notch Signaling Pathway In Zebrafish Embryos

In 2002,Swedish scientists firstly discovered a large amount of acrylamide in carbohydrate-rich foods,which aroused world-wide concerns.Acrylamide and its primary metabolite,glycidamide,can break through the blood-placenta barrier and enter the fetal body from their mother's body through conjugating with hemoglobin.Several large maternal-child cohort studies in Europe have clearly defined the developmental toxicity of acrylamide.Our previous human studies showed that acrylamide exposure is significantly positively associated with the risk of cardiovascular diseases(CVDs)as well as CVD-caused mortality.The heart is the first organ to form and function during vertebrate embryogenesis.In this study,we used zebrafish embryos to systematically study the cardiac developmental toxicity of acrylamide and its potential mechanism.The main results are as follows:First,acute exposure to acrylamide can casue deformity and death in zebrafish embryos.The Lethal Concentration 50(LC₅₀)of newly fertilized zebrafish embryos exposed to acrylamide for72 and 96 hours were 2.7 mM and 2.2 mM,respectively.The major malformation of embryos exposed to acrylamide was pericardial edema,slow heartbeat,cardiac linearization,shorten body and collapsed swimming bladder.The detection of redox state,such as reactive oxygen species(ROS)levels,superoxide dismutase(SOD)activity,glutathione(GSH)content and glutathione S-transferase(GST)activity showed that 2.0 mM exposed groups showed significant oxidative stress while lower than 2.0 mM exposed group showed no significant difference compared with the control group,suggesting that oxidative damage may be the toxicity mechanism of high dose acrylamide exposure.In addition,the staining of reactive oxygen probe and Oil Red O both showed significant positive signal in the heart region,suggesting that the heart is likely to be the key target organ for acrylamide developmental toxicity.Second,acute exposure to acrylamide can induce cardiac developmental toxicity in zebrafish embryos.We found that the 2.0 mM acrylamide-exposed embryos had significantly larger distance between cardiac sinus venosus and bulbus arteriosus,reduced ventricular cross-sectional area and ventricular volume,as well as decreased heart rate,ventricular shortening fraction and red blood cell flow rate compared with the embryos in control group.The results of whole embryo in situ hybridization(WISH)and real-time quantitative PCR(q PCR)showed that acrylamide exposure resulted in the failed process of the heart looping and chamber ballooning,and abnormal expression of genes myl7,vmhc and myh6 coding myocardial proteins during early heart development;the partial or complete loss of regional restriction expression of genes bmp4,notch1b and tbx2b during atrioventricular canal(AVC)differentiation;the ectopic expression of genes has2,klf2a and nfatc1 during the development of atrioventricular valve precursor;the significantly overexpressed cardiac disease-related markers nppa and nppb while down-regulated tnnc1a.These results indicated that acute exposure to acrylamide might suppress the chamber myocardium development,AVC differentiation and valve development.In addition,the decreased expression of cardiac progenitor marker nkx2.5 in the anterior lateral plate mesoderm(ALPM)heart also suggested that acrylamide might interfere with the differentiation of cardiac precursor at the beginning of cardiogenesis.Third,acute exposure to acrylamide can inhibit cell proliferation and differentiation during the heart development.Acrylamide can significantly reduce the number of cardiomyocytes and endocardial cells.Using proliferation markers PH3 immunofluorescence and TUNEL apoptosis staining,we found that acrylamide did not affect the level of cardiomyocyte apoptosis in the developing heart,but significantly inhibited its mitotic proliferation,which led to a significant reduction in myocardial numbers.Acrylamide did not affect the specific differentiation of cardiac ventricular cardiomyocytes,but significantly inhibited the transformation of endocardial cells from epithelial cells to mesenchymal cells at the AVC region.In addition,acrylamide exposure induced an increase in the physical spacing between the early developmental myocardium and the endocardium,resulting in a reduced crosstalk.We also found myocardium and endocardium obviously thickened with abnormal accumulation of cardiomyocytes due to the inability of the cell migrate during cardiac maturation.Fourth,subchronic exposure to acrylamide had no significant effect on morphology and function during early development of the heart,but significantly affected morphogenesis and contractile function during cardiac maturation.Using the Notch signal reporter transgenic fish line Tg(TP1:d2GFP),we found that 0.5 mM and 1.0 mM acrylamide exposure caused the loss of transient dynamic activity of the endocardial Notch signal in early cardiac development.It continues to be highly expressed during the early trabeculation.In addition,the Notch signal in the myocardium showed high expression in the late trabeculation,while the trabecular buds excessively proliferated in the early stage and accumulated in the ventricular wall in the late stage,which could not be distinguished.After partial inhibition of endocardial Notch signal by Notch signal inhibitor DAPT,the trabeculae generated from the ventricular wall could extend to the interior of the chamber,and the 3D structure of the ventricle was fully expanded.This result imply that acrylamide exposure caused a partial loss of endocardial activity during the early cardiac development and the embryos was unable to achieve restricted expression of Notch signaling,then leading to excessive trabeculation during the cardiac maturation.However,the high activation of Notch signal in myocardium at the later stage of maturation might be a stress response.Fifth,subchronic exposure to acrylamide led to trabecular accumulation in ventricular wall.The potential reasons are as follows:after exposure to 1.0 mM acrylamide,(1)the genes in Nrg/Erb B signaling,the downstream of Notch signaling,erbb2 and nrg2a significantly up-regulated,which might be the reason of trabecular hyperplasia;(2)the over-aggregation protein N-cadherin regulated by the Erbb2 greatly increased the adhesion between cardiomyocytes and inhibited the migration of trabecular cardiomyocytes,which might be the reason of accumulated trabeculae in the ventricular wall;and(3)the abnormal structure of mitochondria and myofibril required for energy metabolism and contractile function of cardiomyocytes may be the possible reasons for decreased contractile function during cardiac maturation.Also,we found that crucial specific transcription factors in the early development of the heart were reactivated during ventricular maturation.It suggests that acrylamide Subchronic exposure may have a unique regenerative stress mechanism in the zebrafish heart.In summary,we used zebrafish embryos as a model to systematically study the effects of acute and subchronic exposure to acrylamide on various stages of cardiac development and its potential toxicity from the veiw of toxicological effects,mechanisms of cell biology and molecular biology.In addition,this study provides a foundation for improving the toxicological effects evaluation system for food processing induced pollutants,and promotes the development of evidence-based and traceability research on food safety hazards for our country.