| Dibutyl phthalate(DBP)is one of the environmental endocrine disruptors.As a plasticizer,it is widely used in various plastic products.Due to its wide application,DBP has been detected in various environmental systems,many studies have fully reported its toxic effects.DBP that enters the environment and enters organisms can be easily hydrolyzed into monobutyl phthalate(MBP).Moreover,the presence of high MBP has been detected in most human bodies.The bioaccumulation of MBP in biological tissues can last for up to 6 months,and may have toxic effects on the organism.At present,the toxic effect and mechanism of MBP are not clear,given that it can exist in the environment and in the organism.Therefore,it is necessary to conduct a comprehensive evaluation of its toxic effects.In this study,the acute semi-lethal test determined that the acute semi-lethal concentration of MBP to zebrafish was 49.85 mg·L-1.Then,the acute exposure concentration was set according to the acute half-lethal concentration,and the effects of oxidative stress,endoplasmic reticulum stress,cell activity,metabolic process,and immune system of MBP on zebrafish after 96hours of exposure were studied to reveal the toxicity mechanism of MBP.The main conclusions are as follows:(1)The toxic effect and mechanism of MBP was preliminarily judged by detecting the influence of MBP on the relevant indexes of the antioxidant system in zebrafish liver.The results showed that in the high concentration(10 mg·L-1)MBP treatment group,the antioxidant Nrf2-Keap1 pathway was insufficient to resist MBP-induced liver toxicity,resulting in the decrease of superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx)activities,and the down-regulation of related gene expression(SOD,CAT,GPx).As a result,the activity of ATPases were inhibited and the imbalance of membrane ion homeostasis was triggered.(2)Through the detection of MBP on the morphology of endoplasmic reticulum in zebrafish liver and the expression level of endoplasmic reticulum stress-related genes,it was found that the exposure of MBP can lead to an increase in the number of endoplasmic reticulum in liver cells and slight swelling.Meanwhile,MBP can stimulate the expression of hspa5,activate the ire-xbp1pathway to cause the occurrence of endoplasmic reticulum stress,and induce the overexpression of chop gene,which may lead to the occurrence of cell apoptosis.(3)Through histopathology,MTT method,Holo Monitor M4 laser holographic cell imaging,flow cytometry,transmission electron microscope,and detection of related gene expression levels,the liver morphology,liver cell activity and cell death mode were determined after MBP exposure.The results showed that exposure to high concentrations of MBP can cause massive degeneration of liver cells,producing vacuoles of different sizes,accompanied by portal vein congestion and sinusoids.At the same time,the activity of hepatocytes also decreased with the increase of exposure concentration.The cell morphology changed,the thickness decreased,the degree of irregularity decreased,and the degree of roughness increased,eventually leading to cell death.The expression level of apoptosis related genes(p53,bax,cas3)increased with the increase of exposure concentration,and the early apoptosis rate of 63.6%was detected by flow cytometry at the highest MBP exposure concentration,which confirmed that one of the ways of cell death was apoptosis.In addition,autophagy was another way for MBP to induce cell death.The overexpression of autophagy genes(atg5,lc3)and the discovery of autophagosomes in hepatocytes confirmed this result.(4)The detection of physiological and biochemical indicators and gene expression levels related to lipid metabolism in zebrafish liver revealed the effect of MBP on lipid metabolism.The results showed that MBP can significantly increase the content of triglycerides(TG)and total cholesterol(T-CHO)in zebrafish liver,leading to the accumulation of lipid droplets in the liver.The reason for the increase in TG content was that MBP can up-regulate the expression levels of acc1,fasn,and srebp1 to promote TG synthesis.At the same time,the excessive increase of TG led to a significant increase in the activity of lipoprotein lipase(LPL)and liver lipase(HL)related to TG degradation,as well as an increase in the expression of related genes acox1 and ppar?.This was an adaptive response that compensates for liver lipid accumulation.Besides,the up-regulation of TG storage gene ppar?and insulin gene(prl,ide)expression levels was another reason for the increase in TG content.Changes in various indicators ultimately affected the disorder of lipid metabolism.(5)Through the detection of physiological and biochemical indicators related to glucose metabolism,changes in gene expression levels and liver PAS staining,the effect of MBP was revealed on the glucose metabolism process of zebrafish liver.The results of PAS staining indicated that MBP exposure caused the accumulation of glycogen in the liver.The main reason for the accumulation of glycogen was that the enzyme activity and substance content related to glycolysis(lactate dehydrogenase LDH,pyruvate)and aerobic metabolism(succinate dehydrogenase SDH)were significantly reduced,resulting in the failure of glycogen decomposition to provide energy for the body.However,the pentose phosphate pathway and gluconeogenesis were activated as a compensation mechanism for energy supply,which in turn led to an increase in glycogen synthesis,and a disorder in the glucose metabolism process.(6)MBP exposure can significantly reduce zebrafish epidermal mucus lysozyme,alkaline phosphatase(AKP),acid phosphatase(ACP)activity,and antibacterial ability,which led to immunosuppression.Further analysis of the molecular mechanism of MBP affecting genes related to the Toll-like receptor pathway in the kidney revealed that MBP induced inflammatory responses(il-1?,il-6)through the TLRs/TRIF,My D88/NF-?B pathway,and eventually had a negative impact on non-specific immune responses.In addition,MBP can activate the expression of rags,thereby activating specific immune responses.The activation of rags can induce B cells to produce immunoglobulin(Ig)in response to antigen invasion,and MBP mainly relied on Ig Z.In short,MBP can activate the non-specific and specific immune response of zebrafish,leading to abnormal immune function,and ultimately leading to kidney damage.In summary,combined with relevant data analysis,MBP exposure can lead to the inhibition of the antioxidant Nrf2-Keap1 pathway in zebrafish liver,which in turn triggered changes in cell redox state,membrane damage,and imbalances in ion homeostasis.The disorder of the antioxidant system can further lead to the activation of the ire-xbp1 pathway of endoplasmic reticulum stress.The two influenced each other and can cause liver cell apoptosis and autophagy.The ire-xbp1 pathway activated by endoplasmic reticulum stress can promote lipid synthesis and cause the accumulation of lipid droplets.The gene ppar?related to lipid storage affected the level of insulin,which can also further affect the glucose metabolism process,that is,glycolysis and aerobic respiration were inhibited.And the pentose phosphate pathway was activated as a compensation mechanism to alleviate glycogen accumulation.The abnormal supply of energy and the death of excessive cells will eventually severely damage the zebrafish liver.On the other hand,MBP exposure can induce inflammation through TLRs/TRIF,My D88/NF-?B pathway,and ultimately negatively affected the non-specific immune response of zebrafish.It can also activate rags to generate Ig in response to antigen invasion,thereby disrupting the specific immune response of zebrafish,and the disorder of immune function ultimately led to kidney damage.This study verified the toxic effects of MBP on zebrafish stress,metabolism and immunity,and provided a theoretical basis for revealing the toxic mechanism of MBP to organisms. |
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