Neurotoxic Effects And Mechanism Research Of DBP Exposure In Zebrafish

Phthalate esters（PAEs）are widely used as plasticizers in personal care products,medical devices,toys,building materials,electronic products,food packaging,and other fields.Dibutyl phthalate（DBP）,an important member of the PAEs family,is commonly detected in environmental media and is potentially neurotoxic,and it’s ecological risks and health hazards have been of high concern,but the neurotoxic effects of DBP exposure on aquatic organisms have rarely been reported.In this study,zebrafish（Danio rerio）embryos are used as the model,behavior,biochemical analysis,and transcriptomics were combined,and the LC₅₀of DBP on zebrafish embryos was determined to be 535.5μg/L by acute semi-lethal test,and the exposure concentration（0,5,10μg/L）was set according to LC₅₀,and to study the effects of DBP on behavioral response,neuroinflammatory response,autophagy,energy metabolism and gene expression levels of zebrafish embryos after 72 h acute exposure,to decipher the neurotoxic effects and the molecular mechanisms involved of DBP exposure on zebrafish embryos.The following results are obtained:（1）DBP caused early neurological abnormalities in zebrafish larvae by inducing neuroinflammatory response and autophagy,which further affected zebrafish behavior.When exposed to 72 hpf,the motor behavior（average speed,movement distances,and locomotor activity）as well as tropism of zebrafish larvae in DBP-treatment groups were significantly inhibited.Activation of microglia,reactive oxygen species content increased significantly,dysregulation of antioxidant enzymes（catalase,glutathione peroxidase,and superoxide dismutase）activity,and the occurrence of neuron cell apoptosis indicated the occurrence of neuroinflammation in zebrafish larvae.In addition,the observation of autophagosomes in neuron cells suggested that DBP promoted the autophagy response by inducing inflammation.（2）DBP caused neurotoxicity in zebrafish larvae by inducing changes in the astrocyte-neuron lactate shuttle system.After exposure to 72 hpf,the activities of hexokinase and lactate dehydrogenase,as well as the contents of lactic acid and pyruvate were significantly decreased in5 and 10μg/L DBP-treatment groups,suggesting that DBP can reduce lactic acid production in astrocytes by inhibiting aerobic glycolysis.The transcript levels of lactate transporter protein-related genes（MCT-1,MCT-2,and MCT-4）were significantly down-regulated in the DBP-treated groups suggesting that DBP affects lactate transfer and uptake in neurons and astrocytes.In addition,the neuronal ultrastructure and ATP content were significantly reduced suggesting that DBP caused impairment of neuronal mitochondrial structure and function resulting in inadequate neuronal energy supply.（3）Transcriptome sequencing results showed that DBP produced neurotoxicity through neuroinflammation,cell death,and energy metabolism.Gene Ontology enrichment analysis of differentially expressed genes showed that DBP had significant effects on the transcription of genes related to locomotor behavior,central nervous system development,mitochondrial transport,metabolic processes,endoplasmic reticulum,and lysosomes in larvae,probably by affecting the transcription of genes related to glycolysis/gluconeogenesis,autophagy,TNF,JAK-STAT,and MAPK signaling pathways.