Research On Cardiotoxicity And Neurotoxicity Of Triphenyl Phosphate In Zebrafish

Triphenyl phosphate(TPhP)is a type of organophosphate flame retardant,commonly used as a substitute for brominated flame retardants in electronic equipment,plastic products,and furniture materials.However,it is easily emitted due to the physical addition method used,and thus has now been widely detected in various environmental media.As an important fate of TPhP,the evaluation of its pollution level and the health status of aquatic organisms in water environment has become hot scientific issues in environmental science and ecotoxicology research.Numerous studies have reported that TPhP can cause various toxicity effects on aquatic organisms,such as metabolic toxicity,developmental toxicity,reproductive toxicity,etc.Among them,studies on cardiotoxicity and neurotoxicity are mainly acute toxicity,and the focus is mainly on developmental toxicity during the embryonic period.However,there are few reports on the subchronic/chronic toxicity after long-term exposure,and there is also a lack of systematic studies on toxicity effects and mechanisms in adult fish.In this study,adult zebrafish were used as test organisms to explore the cardiotoxicity and neurotoxicity of TPhP after long-term exposure at the individual,tissue and molecular levels,and investigated the cellular and molecular mechanisms underlying the toxic effects.The main research work included: 1)measuring ventricular volume,heart rate and cardiac output by dynamic microscopic imaging;2)analyzing the individual swimming ability,social preference behavior,learning and memory ability using behavioral technology;3)observing the thickness of the ventricular dense layer and the apoptosis of cardiomyocyte through histological staining technology;4)observing the ultrastructure of cardiomyocytes and intercalated discs in the myocardium and neurons in the brain with the help of transmission electron microscopy technology;5)quantifying the expression levels of apoptosis-related and myocardial regeneration-related,as well as nerve regeneration-related genes based on fluorescence quantitative PCR.The main results were as follows:1)After prolonged exposure to TPhP,the ventricular volume of zebrafish decreased and the thickness of the ventricular dense layer decreased.In the high concentration group,even necrotic fibers,decreased numbers of mitochondria with scattered arrangements,and increased interval between the intercalated discs were observed.At the same time,the heart rate,cardiac output and ejection fraction decreased.It is suggested that TPhP can damage the morphological structure of zebrafish heart and affect its normal physiological function.2)After 20 d of TPhP treatment,the number of apoptotic cardiomyocytes increased,and the expression of apoptosis-related genes caspase8 and caspase3 simultaneously up-regulated,while the expression of caspase9 began to up-regulate after 40 d of exposure,indicating that TPhP may lead to the occurrence of cell apoptosis through death receptor and mitochondrial-mediated pathways.After 60 d of exposure,the differences in expression levels of the above genes disappeared between groups,suggesting that apoptosis is no longer the main pathway leading to cardiomyocyte death with the accumulation of TPhP.3)After 20 d of TPhP treatment,the expression of cardiomyocyte regeneration-related genes nkx2.5,gata4 and tbx5 up-regulated,and gata4 in the medium concentration group was significantly different from that in the control group.However,after 40 and 60 d of exposure,the expression of these genes showed a trend of down-regulation,with gata4 and tbx5 significantly down-regulated after 40 d and nkx2.5 significantly down-regulated after 60 d.These suggest that TPhP-induced myocardial injury can activate the cardiac self-repair process in the short term,but this process can be inhibited with the accumulation of TPhP.4)The behavioral results showed that there was no significant difference in either individual swimming ability or learning and memory ability compared to the control group after 20 d of exposure.However,after 40 d of exposure,the average speed significantly decreased,the memory latency significantly prolonged,and the cumulative time in the reward area significantly decreased,suggesting that TPhP exposure can reduce the individual locomotor level and damage the learning and memory ability.5)The results of transmission electron microscopy showed that the neuronal structure in zebrafish brain tissue was damaged,characterized by nuclear condensation,disappearance of mitochondrial cristae,and mitochondrial vacuolization.The results of ACh content,ACh E activity and ache expression level in the brain showed that at different exposure times,the activity of ACh E decreased,the expression level of ache down-regulated,and the content of ACh increased,showing a dose-dependent effect.This indicates that TPhP not only can destroy the ultrastructure of neurons,but also can cause the accumulation of ACh in the synaptic cleft.6)With the prolongation of exposure time,the expressions of the nerve regeneration-related genes gap43 and syn2 a showed a trend of up-regulation first and then down-regulation.Both genes significantly up-regulated after 40 d of exposure,and the expression of gap43 significantly down-regulated after 60 d of exposure,indicating that TPhP-induced neuronal injury can activate its self-repair process within a short period,but this process can be inhibited with its accumulation.In summary,long-term exposure to TPhP can cause cardiomyocyte apoptosis through the death receptor and mitochondrial pathways,further damaging the cardiac structure,affecting its normal physiological function in zebrafish.It also can alter the neuronal structure and normal synaptic transmission,reducing the individual movement level and learning and memory ability.With the accumulation of TPhP in zebrafish,the regenerative or repair capacity of cardiac myocytes and neurons can be inhibited.