Study On The Mechanism Of 2-Ethylhexyl Diphenyl Phosphate Induced Lung Toxicity In Chicks

2-Ethylhexyl Diphenyl Phosphate(EHDPHP)is a common organophosphorus flame retardant and plasticizer,which can improve the toughness and weather resistance of products,and is widely used in food packaging,flame retardants,and other fields.EHDPHP is frequently detected in the atmosphere,water,soil,food,and organisms,causing inevitable environmental pollution and potential harm to organisms.EHDPHP has reproductive toxicity,neurotoxicity,developmental toxicity,hepatotoxicity,incredibly high toxicity to fish and aquatic organisms and may have bioaccumulation.However,there are few toxicity studies for EHDPHP and almost no studies on avian lung toxicity.This study aimed to explore the mechanism of subacute toxicity of EHDPHP on chicks,provide a reference for the rational use of EHDPHP in human life,and provide a scientific basis for the application of organophosphorus flame retardants.In this study,90 healthy male chicks were randomly divided into five groups,18 in each group: Con group(Blank control group),Vcon group(Solvent control group),D800 group(800 mg/kg EHDPHP group),D1600group(1600 mg/kg EHDPHP group),and D3200 group(3200 mg/kg EHDPHP group)were continuously exposed to 42 days by gavage.On the 14 th,28th,and 42 nd days of exposure,dissect,and weigh the lungs of chicks,and observe the morphological,microstructural,and ultrastructural changes of the lungs: The lung tissues of chicks were selected to detect the activities of peroxidase(GSH-Px)and superoxide dismutase(SOD),the contents of malondialdehyde(MDA)and lactate dehydrogenase(LDH)in the lung supernatant of chicks with kits.The levels of interleukin-6(IL-6),interleukin-18(IL-18),interleukin-1β(IL-1β),and tumor necrosis factor-α(TNF-α)in lung supernatant were detected by ELISA kits.The m RNA expression levels of IL-6,TNF-α,nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3),apoptosis-related spot-like protein(ASC),nuclear factor-κB(NF-κB),caspase-1,IL-18,and IL-1β in lung tissues were detected by q PCR.The protein expression of NLRP3,ASC,NF-κB,Pro-Caspase-1,and Caspase-1 in lung tissue was detected by Western Blot.The results showed that the body weight of chicks in the D800,D1600,and D3200 groups was significantly lower than that in the Con group from 19 days of age(P<0.05).Compared to the Con group,the chicks in the EHDPHP exposure group had disordered fur,depressed spirits,and poor appetite.The histopathological results showed that on the 14 th day,the lung organ coefficients of chicks in the exposed group were significantly increased(P<0.05),the number of lung chambers in the D1600 group was decreased,and inflammatory cells in the D3200 group were increased.On the 28 th day,lung organ coefficients in the D3200 group were significantly increased(P<0.05),lung lobular structure was deformed,and the number of lung chambers was decreased in all exposed groups,and obvious inflammatory cell masses appeared in D3200 group.On the 42 nd day,the lung organ coefficients in the exposed group were significantly increased(P<0.05),the pulmonary lobular space was unclear,and inflammatory cell infiltration appeared in all of them.The inflammatory cell mass was more in the D3200 group.The electron microscopy results showed that the cells in the lungs of D3200 group chicks were ruptured,and the alveolar cavities were filled with impurities.Other results showed that on the 14 th day,compared with the Con group,the contents of MDA,TNF-α and LDH in the exposed group were significantly increased(P<0.05),the activity of SOD in D1600 and D3200 groups decreased significantly(P<0.05).The contents of IL-6,IL-1β,IL-18,and the expression of p NF-κB/NF-κB protein in the exposed group were significantly increased(P< 0.05).The m RNA expression levels of IL-6,TNF-α,NF-κB,NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in the exposed group were significantly increased(P<0.05),and there are related to the dose.On the 28 th day,compared with the Con group,the activities of GSH-Px and SOD in the lung tissue of the exposed group chicks decreased(P<0.05),while MDA,IL-6,TNF in the exposed group were also reduced-α,IL-1β,the levels of IL-18 and LDH significantly increased(P<0.05).The m RNA expression levels of IL-6,TNF-α,NF-κB,NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in the exposure group were significantly increased(P<0.05).The protein expression levels of NLRP3,ASC,p NF-κB / NF-κB,Pro-Caspase-1,and Caspasoe-1 in the lung tissue of chicks in the exposure group were significantly increased(P<0.05),which was correlated with the dose.On the 42 nd day,compared with the Con group,the activity of GSH-Px and SOD in the lung tissue of the exposed group chicks decreased(P<0.05),the contents of MDA,IL-6,TNF-α,IL-1β,IL-18,and LDH in exposure group were significantly increased(P<0.05).The m RNA expression levels of IL-6,TNF-α,NF-κB,NLRP3,ASC,Caspase-1,IL-1β,and IL-18 in the exposed group were significantly increased(P<0.05).The protein expressions of NLRP3,ASC,p NF-κB/NF-κB,Pro-Caspase-1,and Caspasoe-1 in the lungs of exposed group were significantly increased(P<0.05)and were correlated with the dose.The research results indicate that:(1)EHDPHP exposure affects the weight of chicks,hinders development,and significantly reduces growth performance.The lung structure of chicks is disordered,with blurry boundaries of lung lobules,an increase in inflammatory cells,and the appearance of inflammatory cell clusters.Ultrastructure showed cell rupture,nuclear deformation,and an alveolar cavity filled with impurities.(2)EHDPHP exposure increases the content of MDA and the content LDH in the lung tissue of chicks,reduces the activity of SOD and GSH-Px,induces oxidative stress in the lung tissue,and subsequently damages the lung tissue of chicks.(3)EHDPHP exposure increased the expression of NF-κB m RNA and protein in lung tissue,and the levels of inflammatory cytokines IL-6 and TNF-α,IL-6 and TNF-α m RNA levels were significantly increased,which caused the release of inflammatory cytokines in the lung of chicks,resulting in lung injury.(4)EHDPHP exposure led to increased expression levels of regulatory factors related to pyrodeath(NLRP3,ASC,NF-κB,Pro-Caspase-1,Caspase-1,IL-18,and IL-1β)in chick lung tissue,indicating that EHDPHP induced pyrodeath in chick lung.That is one of the mechanisms by which EHDPHP induces lung damage in chicks.