Metabolomics Study Of The Effect Of Different Doses Of Bisphenol A On Lipid Metabolism In Rats

ObjectiveBisphenol A（BPA）is one of the most common Environmental Endocrine Disruptors（EEDs）.It can affect the metabolism of substances in the body,leading to insulin resistance and lipid disorder.Then produce diabetes,obesity and other diseases related to metabolic disorders.Studies have confirmed that BPA has a non-monotonic dose effect on lipid metabolism,low dose of BPA（50 μg/kg/d）exposure can induce liver cholesterol synthesis,while high dose of BPA（5 mg/kg/d）exposure has no significant effect on liver total cholesterol content and its synthesis.The differences in the effects of BPA on lipid metabolism at different doses need further investigation.In this study,adolescent SD rats were exposed to BPA with different doses.Estradiol（E2）was used as the control.Non-targeted metabolomics and lipidomics methods were used to investigate the signaling pathway and key target factors of BPA’s influence on lipid metabolism in rats,and to explore the molecular mechanism of BPA’s influence on lipid metabolism.To determine whether there is Hormesis in the process of lipid metabolism affected by low dose BPA,and to provide toxicological basis for preventing and controlling the effects of exogenous chemicals on body function.Methods1.Forty 3-week-old healthy male SD rats without specific pathogen grade（SPF）were randomly divided into solvent control group,BPA 0.5 μg/kg group,BPA 5 mg/kg group and E2 50 μg/kg group,with 10 rats in each group.A volume of 5 m L/kg was used for continuous gavage for 28 days.Blood from abdominal aorta was collected and serum was separated for reserve.Liver was extracted,weighed and organ coefficient was calculated,and pathological changes were observed by HE staining.2.Ultra-high performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）was used to detect the non-targeted metabolomics of rat serum.Proteowizard and R-XCMS software were used for data processing and analysis,and the differential compounds were identified using an online database based on their mass spectrum information.Biomarker enrichment and metabolic pathway analysis were performed to reveal the changes and associations between different dose groups.3.The serum lipidomics of rats was detected by UPLC-MS/MS technology,and the obtained data were processed by multivariate statistical analysis technology to observe the changes in lipid metabolism between BPA-exposed group,E2 group and solvent control group.Lipid Search database was used to identify potentially significant differentially expressed lipids after BPA exposure,and R language was used to conduct correlation analysis.To explore the differences of potential lipid metabolites in serum of rats exposed to different doses of BPA and their correlations,and to obtain a deeper understanding.Results1.Effects of BPA on general toxicity in ratsAfter 4 weeks of exposure,the body weight of each group increased,and there was no significant difference compared with the control group.The liver weight and organ coefficient of all exposed groups were increased,and the liver weight and organ coefficient of BPA 0.5 μg/kg group were significantly higher than those of control group（p<0.05）,but there was no significant difference among other groups.HE staining showed that there were obvious pathological changes in the liver of rats in the BPA 0.5 μg/kg group,including irregular morphology of liver cells,degeneration of fatty vacuoles,and enlargement of hepatic cord space.The liver of rats with BPA of 5mg/kg also showed diffuse lipovacuolar degeneration and a small amount of inflammatory cell infiltration.The liver of E2 group showed a small amount of fatty vacuolar degeneration.2.Non-targeted metabolomics analysis of rat serum2.1 Identification results and trend of primary metabolitesNon-target metabolomics analysis was performed to search for differential metabolites from the list of primary substances in the sample with preset thresholds of p<0.05 and VIP>1.Under the positive ion mode,a total of 6065 metabolites were detected and analyzed,among which 1831 were changed,accounting for 30.2%,among which 1064 were up-regulated and 767 were down-regulated.Under the negative ion mode,a total of 7197 metabolites were detected and analyzed,and 2072 metabolites were changed,accounting for 28.2%,among which 895 metabolites were up-regulated and 1177 metabolites were down-regulated.2.2 Expression of different metabolites of BPA in different dose groupsIn the positive ion mode,compared with the control group,there were 301 metabolites in the 0.5 μg/kg BPA group,of which 146 were up-regulated and 155 were down-regulated.There were 471 kinds of BPA in the 5mg/kg group,among which 213 kinds were up-regulated and 258 kinds were down-regulated.Compared with E2 group,there were 532 metabolites in the 0.5 μg/kg BPA group,among which 357 were up-regulated and 175 were down-regulated.There were 527 types of BPA in the 5mg/kg group,of which 348 were up-regulated and 179 were down-regulated.In the negative ion mode,compared with the control group,there were 388 metabolites in the 0.5 μg/kg BPA group,of which 161 were up-regulated and 227 were down-regulated.There were 559 types of BPA in the 5mg/kg group,of which 275 were up-regulated and 284 down-regulated.Compared with E2 group,there were 504 metabolites in the 0.5 μg/kg BPA group,among which 211 were up-regulated and 293 were down-regulated.In the 5mg/kg group,there were 621 kinds of BPA,among which248 kinds were up-regulated and 373 kinds were down-regulated.2.3 Differential analysis of metabolic pathwaysCompared with the solvent control group,BPA 0.5 μg/kg and BPA 5 mg/kg groups showed the same pathway of effects on amino acid metabolism,cancer and lipid metabolism.BPA 0.5 μg/kg group had specific effects on m TOR signaling pathway,glucagon signaling pathway and cholesterol metabolism pathway,while BPA 5 mg/kg group had specific effects on iron death,insulin resistance,glycolysis/gluconeogenesis,pentose phosphate pathway and so on.The E2 group only had specific effects on drug metabolism,biotin metabolism,ascorbic acid and uronic acid metabolism.33 of the 38 metabolic pathways affected by E2 group were the same as those in the BPA 5 mg/kg dose group,and sixteen of them coincided with that in the BPA 0.5 μg/kg dose group.3.Serum lipidomics analysis of rats3.1 Analysis of lipid types and different lipidsAfter screening,we found 1314 kinds of lipid compounds,including glycerol phospholipids,triglycerides,glycolipids,sphingolipids,sterols and fatty acylates,including 113 kinds of differentially expressed lipids,accounting for 8.6%.Among the differentially expressed lipids,53 kinds of glycerol phospholipids accounted for 46.9%,26 kinds of glycerol esters accounted for 23.0%,34 kinds of sterols,sphingolipids,glycolipids and fatty acylates accounted for 30.0%.3.2 Different lipid expression types of BPA in each dose groupThere was no significant difference in triglyceride content between BPA 0.5 μg/kg group and BPA 5 mg/kg group,but E2 group was lower.Glycerol phospholipids BPA0.5 μg/kg group was significantly lower than BPA 5 mg/kg group and E2 group.The content of glycolipids was higher only in BPA 5 mg/kg group.The content of sphingoid BPA in 5 mg/kg group and E2 group was significantly higher than that in BPA 0.5 μg/kg group,and the content of sterol E2 group was significantly higher than that in BPA5mg/kg group,but BPA 0.5 μg/kg group was not detected.3.3 Different lipid specific expressions of BPA in each dose groupThe comparison between BPA 0.5 μg/kg group and BPA 5 mg/kg group showed that there were 19 and 30 different differential lipids between the two groups,and there were 7 common differential lipids.They are TG（18:1:18:3₂2:6）,TG（18:1:20:4₂0:4）,TG（18:4:16:0₁8:2）,PC（16:18:18:2）,PC（40:4）,PI（37:0）and SPH（d17:1）,mainly glycerol triolipids and glycerol phospholipids.It can be used as a biomarker of the effect of BPA on lipid metabolism.Compared with the control group,there were 2kinds of the same differential lipids in BPA 0.5 μg/kg group and E2 group,and 7 kinds of the same differential lipids in BPA 5 mg/kg group and E2 group,indicating that BPA5 mg/kg group has more overlapping pathways with E2 in terms of lipid metabolism.Conclusions1.Low dose of BPA（0.5 μg/kg）can affect the metabolism of endogenoussubstances in rats,under the same conditions,and the effect of 5 mg/kg BPA on bodymetabolism showed a non-monotonic linear relationship.2.5 mg/kg BPA and E2 were similar in terms of primary metabolites andmetabolic pathways.3.Different doses of BPA have different effects on lipid metabolism.Low dosesof BPA may be mainly through m TOR signaling pathway,glucagon signalingpathway and cholesterol metabolism pathway,while high doses of BPA may haveeffects on ferroptosis,pentose phosphate pathway,anti-insulin signaling,glycolysis/gluconeogenesis.4.Triglyceride and glycerophospholipid can be used as candidate biomarkers for the effects of BPA on lipid metabolism in the body.Glycerophospholipid is a sensitive candidate marker for high dose BPA,but no sensitive candidate has been found for low dose BPA on lipid metabolism.