Metabolic Changes Of Di-2-Ethylhexyl Phosphate And Bisphenol A Mixed Exposure During Gestation And Their Relationship With Liver Oxidative Damage In Offspring

ObjectvesThere are many kinds of environmental chemicals,and the body is often exposed to multiple chemicals at the same time.Therefore,the mixed toxicity of environmental chemicals is the focus of people’s attention.Diethylhexyl phthalate(DEHP)and Bisphenol A(BPA)are two common environmental endocrine disruptors that have different effects on the endocrine,reproductive,nervous and immune systems.Studies have shown that the combined exposure of DEHP and BPA has synergistic or antagonistic effects,but the reason is not clear.Both substances are endocrine disruptors with similar toxic targets,so whether the synergistic or antagonistic effects of the two substances interfere with each other in the biotransformation process,that is,the metabolism of one substance affects the metabolism of the other substance,that’s our concern.The liver is an important metabolic detoxification organ of the body,and it is also the organ most vulnerable to damage by foreign chemicals.Whether this damage is caused by maternal changes is also a question we are concerned about.Therefore,this study established the animal model of mixed infection during pregnancy,and used LC-MS/MS to analyze the plasma metabolism profile of maternal rats,and through the identification of metabolites,to clarify the metabolic pathway of DEHP and BPA in maternal body and the correlation between them.In combination with the oxidative injury index of offspring liver,the relationship between maternal metabolic changes of chemicals and offspring injury was discussed,and the possible signal pathways or genes of injury were analyzed by bioinformatics,so as to provide a reference for the toxicological mechanism research of chemical mixing.MethodThere were 90 normal SD rats with specific pathogen-grade(SPF),10 weeks old,including 30 male rats and 60 female rats.According to female:male for 2:1 and cage,the next day morning pregnancy by vaginal smear method,sperm remember as conceived by 0 days.48 pregnant female rats were randomly divided into the control group,BPA group,DEHP group and BPA+DEHP mixed group.The rats were poisoned by gavage with 5ml/kg volume from the 5th day of conception to the 21st day after delivery.The rats were given equal volume corn oil once a day.One female and one male were randomly treated in each litter on the 7th,21st and 56th day after the birth of their offspring.The histopathological changes of the offspring were observed,and the indicators of oxidative damage were detected:malondialdehyde(MDA),hydrogen peroxide(H2O2),superoxide dismutase(SOD),glutathione peroxidase(gsh-px).The plasma metabolic profiles of DEHP,BPA and the pregnant rats infected with combined toxicity were analyzed,and the metabolites of BPA and DEHP were identified.The correlation between DEHP,BPA and their metabolites and oxidative injury of offspring liver was evaluated by bioinformatics analysis.Results1.A total of 8 substances including BPA,DEHP,MEHP,5oxo-mehp,5oh-mehp,bpa-glca,bpa-oh,and glca-bpa-oh were identified from the metabolites in maternal rat blood.There were differences in plasma metabolic profiles between the mixed exposure group and the isolated exposure group,with four different chromatographic peaks.2.On the 7th day after birth,the body weight of the DEHP group was higher than that of the control group,while that of the BPA group was lower than that of the control group.At day 21,the weight of the offspring in the DEHP group was higher than that in the control group and the BPA group,and the difference was significant(P<0.05).At 7 days after birth,the liver organ coefficient of the DEHP group was higher than that of the control group and the BPA group.At day 21,the BPA group was lower than the control group,and the difference was significant(P<0.05).3.Histopathological changes of the liver were observed under light microscope.The lobule structure of the control group was normal,and the liver cells were arranged neatly.On the 7th day after birth,hepatocyte necrosis and fat vacuolar degeneration occurred in the liver of female mice in the DEHP group,and diffuse fat vacuolar degeneration occurred in the liver of female mice in the mixed infection group.At the 21st day,diffuse fat vacuolar degeneration occurred in the liver of the BPA group and DEHP group,especially the severe lobular centricity and hypertrophy of hepatocytes around the portal area.Severe diffuse fat vacuolar degeneration occurred in the liver of the mixed toxicity group.On the 56th day,the offspring of BPA group showed lobular central hepatocyte hypertrophy,hepatic sinus dilatation,hepatic fat vacuolar degeneration and lobular central hepatocyte hypertrophy,and liver granuloma,Kupffer cell proliferation,diffuse fat vacuolar degeneration and liver cell necrosis in the offspring of mixed poisoning group4.Oxidative stress index detection:the activity of gsh-px was higher in the mixed exposure group than in the DEHP group,the BPA group and the control group on the 7th day after birth.On day 21,the BPA group,DEHP group and BPA+DEHP group were higher than the control group.At day 56,the DEHP group was higher than the control group,and the differences were statistically significant(P<0.05).At 7 days after birth,CAT activity in the mixed exposure group was higher than that in the control group and BPA group,lower than that in the DEHP group,and higher than that in the DEHP group and BPA group.On the 21st day,the mixed group was lower than the control group and DEHP group.At day 56,the DEHP group was higher than the control group and BPA group,and the difference was statistically significant(P<0.05).At the 7th day after birth,MDA content in BPA group,DEHP group and mixed exposure group was significantly lower than that in control group(P<0.05).On the 21st day after birth,the content of H202 in the mixed exposure group was higher than that in the control group,lower than that in the DEHP group,and higher than that in the control group and BPA group.At the 56th day,the mixed poisoning group was higher than the control group,the DEHP group and the BPA group,with significant differences(P<0.05).On the 7th day after birth,the activity of SOD in mixed exposure group was lower than that in BPA group and DEHP group,and the activity in mixed exposure group,BPA group and DEHP was higher than that in control group.On the 21st day,the mixed exposure group and BPA group were lower than the control group.At the 56th day,the mixed poisoning group and DEHP group were higher than the control group,and the DEHP group was higher than the BPA group,with significant differences(P<0.05).5.Five different markers of fatty acids including palmitic acid,oxalic acid,glycolic acid,lactic acid and 3-hydroxybutyric acid were detected by plasma metabolism spectrum analysis in the poisoned group.In addition,lipid metabolites such as palmitic acid and glyceryl monopalmitate were changed in the infected group.6.To evaluate the correlation between DEHP and BPA and its metabolites and liver damage,a total of 11 chemical targets were screened.These targets were significantly enriched in the serotoninergic synaptic signaling pathway,insulin sensitivity,Cholesterol storage,MAPK cascade,and phospholipid metabolism(P<0.05),these signaling pathways are associated with chemical-induced liver damageConclusions1.Metabolite detection revealed that DEHP enters the body and is mainly in the form of DEHP prototype and metabolites MEHP,5oxo-MEHP,50H-MEHP.After BPA enters the body,it mainly uses BPA prototype and metabolite BPA-in blood.GlcA,BPA-OH,GlcA-BPA-OH exist in the form.2.Exposure to DEHP and BPA during pregnancy can affect the growth and development of the rat,causing fatty vacuolization,liver cell enlargement,necrosis,etc.;antioxidant enzymes CAT and SOD activity are reduced,and peroxidation product H2O2 is increased,resulting in liver production.Oxidative damage,and mixed exposure can increase or decrease the toxic effect.3.The exposure of BPA and DEHP changes the plasma metabolic profile of the mother,which causes damage to the liver through the disorder of lipid metabolism,which provides a reference for the study of the mechanism of toxicity.