Effect Of Phthalates On Lipid Metabolism In Adolescent Students And Adolescent Rats As Well As Its Mechanisms

Phthalates(PAEs)are typical environmental endocrine disruptors and are widely used as plasticizers in the production of plastic products.PAEs can enter the body through a variety of ways,and have toxic effects on various tissues and organs.With the increasing obesity rate of children and adolescents worldwide,PAEs have gradually entered people’s vision.Studies have found that the exposure of PAEs can affect the body’s lipid metabolism,promote adipocyte differentiation and lipid deposition,and associated with the occurrence of obesity.In this study,the levels of PAEs metabolites in the urine of adolescent students were analyzed,and the relationship between the levels of PAEs metabolites and lipid metabolism of adolescent students were explored.The effects of PAEs-DEHP exposure with the highest estimated daily intake on lipid metabolism in adolescent rats were determined by animal experiments.In combination with the previous study of this research group,the mediating effect of JAK-STAT-PPARγ pathway on the association between PAEs exposure and plasma lipid level was analyzed.This study can provide scientific basis for the prevention and treatment of adolescent obesity and lipid metabolism disorders.Part I Urinary levels of phthalate metabolites and their association with lifestyle behaviors in Chinese adolescents and young adultsObjective:This study was undertaken to assess phthalate exposure and determine the associations between lifestyle behaviors and phthalate metabolite levels in Chinese adolescents and young adults.Methods:The cluster random sampling method was used to select freshmen aged 16-19 in a college in Jilin Province as the research objects after informed consent,a total of 463 cases.General demographic data and lifestyle data were collected through questionnaire survey.Blood and urine were collected from the subjects.The creatinine oxidase method was used to detect the urine creatinine level of the subjects.Seven high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)were used to detect seven PAEs metabolites in the urine of the subjects(include five DEHP metabolites,MEHP,MEOHP,MEHHP,MECPP and MCMHP;DEP metabolites MEP;DBP metabolites MBP).IBM SPSS24.0 was used for statistic analysis,and the pharmacokinetic formulas were used to calculation of estimated daily intake of DEHP,DEP and DBP,and to assess the risk of PAEs exposure.Independent sample t test and Mann-Whitney test and Multiple linear regression model was used to analyze the association between PAEs metabolite levels and lifestyle behaviors.The inspection level was set to α=0.05.Results:1.Except for the detection rates of MEOHP and MEHHP,which were 99.4% and 98.7%,respectively,the detection rates of the other five PAEs metabolites were 100%.2.The median concentration of MBP was 42.80 μg/L,followed by ΣDEHPm(34.56 μg/L)and MEP(27.50 μg/L).The median concentration of DEHP metabolites was MECPP(13.00 μg/L),MCMHP(7.58 μg/L),MEOHP(6.04 μg/L),MEHHP(4.78 μg/L),and MEHP(3.18 μg/L),and the the trends of creatinine-adjustment levels were the same.3.DEHP was the highest median daily intake of PAEs(2.41 μg/kg·bw/d),followed by DBP(2.18 μg/kg·bw/d)and DEP(1.35 μg/kg·bw/d).The median estimated daily intakes of DEHP,DBP and DEP based on the creatinine model were 1.50,1.33,and 0.82 μg/kg·bw/d,which were lower than the volume model.4.Of the 463 subjects,12(2.6%)of the estimated daily intake of DBP exceeded 10 μg/kg·bw/d(TDI reference limit),and 13(2.8%)showed a cumulative risk of anti-androgens due to PAEs exposure.In the creatinine model,the estimated daily intake of DBP in three(0.6%)subjects exceeded 10 μg/kg·bw/d,and five(1.1%)subjects showed a cumulative risk of antiandrogens.5.The use of plastic food packaging was significantly associated with higher levels of urine DEHP metabolites MEHP,MEHHP,MECPP,and ΣDEHPm(P<0.05);Longer periods of physical exercise were significantly associated with lower MEOHP levels(P<0.05);Regular fast food consumption was significantly associated with higher levels of MBP(P<0.05);The increase in the frequency of skincare cosmetics and make-up was significantly associated with higher levels of MEP(P<0.05).Conclusions:1.Chinese adolescent students have extensive exposure to DEP,DBP and DEHP.2.DEHP was the highest estimated daily intake of PAEs in adolescent students.3.About 3% of adolescent students had a cumulative risk of anti-androgenic effects.4.The levels of PAEs metabolites in adolescent students’ urine were influenced by lifestyle behaviors.Part II Association between urine PAEs metabolites and lipid metabolism in adolescent studentsObjective:To clarify the correlation between the levels of PAEs metabolites in the urine of adolescent students and lipid metabolism,and to explore its underlying mechanism.Methods:463 adolescent students were selected as the subject,measured the height,weight,waist and hip circumference of the subject,and calculated the BMI andWHR.Automatic biochemical analyzer was used to analyze the levels of TC,TG,HDL-C and LDL-C in the plasma of subjects;Real-time PCR was used to measure the expression levels of lipid metabolism-related genes SREBP-2,SR-BI,LDLR,NR1H3,and ABCA1 m RNA in peripheral blood leukocytes of the subjects;Multivariate linear regression model was used to analyze the correlation between the levels of PAEs metabolites in urine and physical indicators,plasma lipids,and m RNA levels of lipid metabolism-related genes in peripheral blood leukocytes.The inspection level was set to α=0.05.Results:1.There was a significant positive correlation between WHR and MECPP and ΣDEHP levels in urine(P<0.05).2.MBP,MECPP,MCMHP,and ΣDEHP in the urine of the study subjects were significantly associated with HDL-C(P<0.05);Except for MCMHP,all DEHP metabolites were significantly positively correlated with TC/HDL-C(P<0.05);All DEHP metabolites were significantly positively correlated with LDL-C/HDL-C(P<0.05).3.The expression levels of SREBP-2,NR1H3,and LDLR m RNA in peripheralblood leukocytes were significantly positively correlated with the levels of urineMCMHP(P<0.05),and the m RNA expression levels of the SR-BI were significantlypositively correlated with MECPP and MCMHP(P<0.05).Conclusions:1.PAEs exposure may promote the occurrence of adolescent obesity.2.PAEs exposure may interfere with plasma lipid levels in adolescent students.3.PAEs exposure may interfere with the expression of SREBP-2,NR1H3,SR-BI and LDLR,and affect lipid metabolism.Part III Effects of DEHP exposure on lipid metabolism in adolescent ratsObjective:To determine the effect of the highest daily estimated PAEs-DEHP exposure on lipid metabolism in adolescent rats,and to investigate the effect of DEHP on lipid metabolism in rats.Methods:80 healthy and clean Wistar rats were selected,21 days old,half male and half female.After adaptive breeding,the rats were ingested with the highest daily intake of adolescent students PAEs-DEHP.The experimental animals are divided into 4 groups: control group(corn oil),low-dose DEHP group(5 mg/kg/d),middle-dose DEHP group(50 mg/kg/d)and high-dose DEHP group(500 mg/kg/d)which were administered with gastric administration once a day in the morning for 8 weeks.After the exposure,the rats were sacrificed after anesthetized,and blood and liver tissues were collected.The levels of TC,TG,HDL-C and LDL-C in serum and liver of rats were measured with automatic biochemical analyzer and biochemical kit.Real-time PCR was used to analyze the m RNA expression levels of lipid metabolism related genes SREBP-2,CYP7A1,SR-BI,LDLR,NR1H3 and ABCA1 in rat liver.Western Blot was used to measure the expression level of SREBP-2,CYP7A1,SR-BI,LDLR,NR1H3 and ABCA1 protein.IBM SPSS 24.0 was used for statistical analysis.One-way ANOVA was used to compare multiple groups,LSD test was used for comparison between two groups.The inspection level was set to α=0.05.Results:1.The serum TC levels of rats in the high-dose DEHP group were significantly higher than those in the control group and low-dose DEHP group(P<0.05),and the HDL-C levels in the high-dose DEHP group were significantly higher than those in other groups(P<0.05),the LDL-C levels in high-dose DEHP group were significantly higher than those in the control group(P<0.05).2.The levels of TC in liver tissue of rats in the high-dose DEHP group were significantly higher than those in other groups(P<0.05),the HDL-C levels in thecontrol group and low-dose DEHP group were significantly lower than those in the middle-dose and high-dose DEHP group(P<0.05).3.The levels of SREBP-2 m RNA in liver of rats exposed to middle and high doses of DEHP were significantly higher than those of low-dose group and control group(P<0.05).The levels of CYP7A1 and LDLR m RNA in the liver of rats in all DEHP groups were significantly lower than those in the control group(P<0.05).The m RNA levels of SR-BI in the liver of rats in the low-dose and middle-dose DEHP group were significantly lower than those in the control group(P<0.05),and the SR-BI m RNA levels in high-dose DEHP group were significantly lower than those in the control group and middle-dose DEHP group(P<0.05).The levels of NRIH3 m RNA in the liver of rats in the low-dose and high-dose DEHP group were significantly lower than those in the control group(P<0.05).The levels of ABCA1 m RNA in livers of rats exposed to low and high doses of DEHP were significantly lower than those in the control and middle-dose DEHP groups(P<0.05).4.The levels of SREBP-2 protein in the liver of rats exposed to middle and high doses of DEHP were significantly higher than those in the control group(P<0.05).The levels of CYP7A1 and NR1H3 protein in the liver of rats in the high-dose DEHP group were significantly lower than those in other groups(P<0.05),and the levels of the middle-dose DEHP group were significantly lower than those of the control group(P<0.05).The levels of SR-BI protein in the high-dose DEHP group were significantly lower than those in the control group(P<0.05).The ABCA1 protein levels in liver of rats in high-dose DEHP group were significantly lower than those in the low-dose DEHP group(P<0.05).Conclusions:1.DEHP exposure could interfere with lipid levels in serum and liver in adolescent rats.2.DEHP exposure could change the expression of lipid metabolism-related genes SREBP-2,CYP7A1,SR-BI,NR1H3,LDLR and ABCA1 genes in the liver of adolescent rats.3.DEHP exposure could affect the metabolism of cholesterol and lipoproteins in adolescent rats,interfere with the reverse transport of cholesterol,and affect lipid metabolism.Part IV Mediating effects of JAK-STAT-PPARγ pathway on PAEs exposure and plasma lipid levels in adolescent studentsObjective:To determine the effect of PAEs exposure on genes related to JAK-STAT-PPARγ pathway in peripheral blood cells of adolescent students,and to explore the mediating effect of JAK-STAT-PPARγ pathway on PAEs exposure and plasma lipid levels in adolescent students.Methods:Adolescent students were selected as subjects.The m RNA levels of JAK-STAT-PPARγ pathway-related genes in subjects’ peripheral blood leukocytes were measured by Real-time PCR.Multiple linear regression models were used to analyze the association between the levels of urinry PAEs metabolites and the m RNA levels of JAK-STAT-PPARγ pathway-related genes in the peripheral blood leukocytes of subjects;Mediation analysis was used to explore the mediating effect of the JAK-STAT-PPARγ pathway on PAEs exposure and plasma lipid levels in adolescent students.The inspection level was set to α=0.05.Results:1.There was a significant positive association between the level of DEHP metabolites in the urine of the study subjects and the m RNA level of the JAK2(P<0.05);The level of MBP and DEHP metabolites in subjects’ urine were significantly positively associated with the m RNA level of the JAK3(P<0.05);The levels of MEOHP and MEHHP were significantly associated with the m RNA level of the STAT5b(P<0.05);The levels of MEOHP and MCMHP were significantly associated with the m RNA level of the PPARγ(P<0.05).2.The m RNA expression level of JAK3 in peripheral blood leukocytes of the subjects had a mediating effect in the association between MEHP,MEOHP,MEHHP,MECPP,and ΣDEHP and TC/HDL-C,and the mediating ratio ranged from 15.71%-17.18%,The expression level of JAK3 m RNA also has a mediating effect in the association between DEHP metabolites and LDL-C/HDL-C,the mediating ratio ranged from 12.69% to 20.86%;The expression level of STAT5 b m RNA has a mediating effect on the association of MEOHP,MEHHP and ΣDEHP with TC/HDL-C,and the mediating ratio ranged from 6.59% to 10.76%;The m RNA expression level of PPARγ mediated the association between MEOHP and TC/HDL-C,with a mediating ratio of 14.87%,the PPARγ also has mediated the association of MEOHP and MCMHP with LDL-C/HDL-C,and the mediating ratio was 15.06% and 18.12%,respectively.Conclusions:1.PAEs exposure up-regulates the expression of JAK2,JAK3,STAT5 b and PPARγ genes in peripheral blood cells of adolescent students.2.The m RNA levels of JAK3,STAT5 b and PPARγ genes have significant mediating effects in the association of PAEs exposure with TC/HDL-C and LDL-C/HDL-C.3.PAEs may interfere with the plasma lipid level of adolescent students through the JAK-STAT-PPARγ pathway and affect lipid metabolism.