Toxicity Of Monobutyl Phthalate (MBP) To Gills In Zebrafish (Danio Rerio)

Phthalates(PAEs)are widely used as plasticizers in industrial production and daily life,which main function is to soften plastics and increase the plasticity and ductility of plastics.Its toxic effects have received extensive attention from researchers.Among all PAEs,dibutyl phthalate(DBP)is one of the most widely used PAEs with the highest concentration detected in the environment.When DBP is absorbed by the human body,it can be hydrolyzed to monobutyl phthalate(MBP)at a fast rate.The detection result of PAEs metabolites in the urine of some workers,pregnant women and children shows that the detected content of MBP is the highest.Unfortunately,most of the current researches are limited to the reproductive toxicity and developmental toxicity caused by MBP.It is particularly important to perfect the study of the toxic effects of MBP.Through the activity of key enzymes of energy metabolism and ion transp ort in zebrafish gill tissue;transcription level of related functional genes;changes of mitochondrial membrane potential(MMP);changes of cell morphology and activity to evaluate the effects of MBP exposure on energy metabolism and ion transport in zebrafish gill tissue.Through histopathological examination,cell ultramicrostructure analysis,antioxidant enzyme activity and related functional gene transcription level determination to evaluate the effect of MBP on the antioxidant capacity and inflammatory response of zebrafish gill tissue.Principal component(PCA)analysis and integrated biomarker response(IBR)analysis are used to identify key transcription factors and biomarkers in gill tissue under MBP stress.Finally,according to the IBR score,UV-visible spectroscopy,circular dichroism,fluorescence spectroscopy,molecular docking simulation,thermodynamic fitting and enzymatic kinetic fitting were used to identify the interaction between MBP and the most sensitive biomarker.The main findings of the test are as follows:(1)MBP induces energy metabolism disorder and ion transport imbalance in zebrafish gill tissue.The activity of energy metabolizing enzymes(succinate dehydrogenase(SDH);malate dehydrogenase(MDH)and ion transport enzymes(Na⁺K⁺ATPase;Ca²⁺ATPase;Mg²⁺ATPase)and the transcription level of genes related to energy metabolism and ion transport(ndufs3;sdhb;mt-cytb;atp5a1;atp1b1;atp1b2;atp2b1)were significantly inhibited in the 10 mg L^-1 MBP treatment group.The activity of lactate dehydrogenase(LDH)and the transcription levels of atp2a1and cox1 increased significantly.Ultramicrostructure and MMP results suggest that MBP causes damage to the structure and function of mitochondria and the analysis results of cell morphology and viability indicate that MBP causes part of cell morphological changes and decreased viability.(2)MBP causes oxidative stress in zebrafish gill tissue and induces chronic inflammation.After 96 hours of exposure,in the 10 mg L^-1 MBP treatment group,the content of reactive oxygen species(ROS)and malondialdehyde(MDA)increased significantly and the activity of antioxidant enzymes(superoxide dismutase(SOD);catalase(CAT);glutathione peroxidase(GPX))decreased significantly.Histopathological results showed that MBP induced chronic inflammation in the gill tissue.The transcription level of inflammation-related genes indicates that MBP mainly activates the pro-inflammatory pathway regulated by NF?b.(3)The results of IBR analysis showed that MBP mainly inhibited the activity of CAT in gill tissue.In vitro studies have shown that MBP can spontaneously interact with CAT in a static quenching manner in a class of sites outside CAT catalytic activity domain dom inated by hydrogen bonds.Enzymatic kinetics results show that MBP can affect the catalytic activity of CAT in a non-competitive way...