Identification And Significance Of BPDE-SLC3A2 Protein Adducts In SH-SY5Y Cells And Mouse Brain Tissue Exposed To BPDE

Objective:Benzo[a]pyrene(B[a]P)is neurotoxic and can cause behavioral impairments such as learning and memory in exposed populations and animals.Its metabolite is 7,8-dihydroxy-9,10-epoxybenzo[a]Pyrene(BPDE)can form adducts with intracellular proteins and play a role in its neurotoxicity.In this paper,the drug affinity response target stability method(DARTS)was used to screen BPDE-protein adducts in human neuroblastoma cells(SH-SY5Y)exposed to BPDE,and to establish BPDE-SLC3A2 protein adducts in cells and animal brain tissues.Quantitative detection methods were used to preliminarily explore the relationship between BPDE-SLC3A2 protein adducts and cytotoxicity and neurobehavioral damage in mouse,providing a basis for the study of the mechanism of B[a]P neurotoxicity.Methods:Infect SH-SY5Y cells with 1.00 μmol/L BPDE and DMSO(final concentration<0.1%)for 24 hours,extract protein,and screen and identify BPDE adduct by DARTS method(including SDS-PAGE gel electrophoresis,LC-MS/MS analysis)protein.The thermolysin digestion method and the cell thermal analysis(CETSA)method are used for verification,and the identified proteins are analyzed by bioinformatics.In addition,SH-SY5Y cells were divided into solvent control group(DMSO,final concentration<0.1%)and 0.02,0.03125,0.0625,0.125,0.25,0.375,0.50,0.75,1.00 μmol/L,1.25(μmol/L)BPDE Group,24h exposure,CCK-8 method to detect cell viability.BPDE-SLC3A2 protein adduct was detected by thermolysin digestion method and CETSA method,and the protein expression levels of SLC3A2 and SLC7A11 were detected by Western blot.At the same time,50μmol/L deferoxamine(DFO)and 10μmol/L lipid ROS scavenger(Fer-1)were added to the solvent control group,0.25,0.50 and 1.00(μmol/L)BPDE group for 24h intervention,CCK-8 method Detect cell viability.Twenty four 12 week old Kunming mice were randomly divided into solvent(olive oil)control group,0.8,2.0 and 5.0(mg/kg·bw)B[a]P groups,with 6 mice in each group.The mice were intraperitoneally exposed once every other day for 4 weeks.Open field test and Morris water maze test were used to observe the neurobehavioral changes of mice.The protein of hippocampus was extracted.The protein adduct level of BPDE-SLC3A2 was detected by thermophilic protease digestion and CETSA method.The protein expression levels of SLC3A2 and SLC7A11 were detected by Western blot.Results:The results of SDS-PAGE gel electrophoresis showed that after digestion with thermolysin,there was a difference band visible to the naked eye between the BPDE-exposed group and the DMSO solvent control group.A total of 264 adduct proteins were identified by the DARTS method.According to the standard of multiple of difference>2.0 times and P value<0.05(including proteins that only appeared in one group),190 proteins with significant differences were determined to be formed with BPDE.The target protein of the adduct.GO analysis shows that biological processes mainly involve cellular reactions and regulation.Among them,the classification with the largest number of proteins is mainly related to metabolic processes such as organic nitrogen compounds,proteins,small molecules,and organics;cell components are mainly related to cell vesicle transport.The classification with the largest number of proteins annotated mainly involves cytoplasm,intracellular organelles,vesicles and exosomes;molecular functions mainly involve intermolecular binding,and the classification with the largest number of proteins annotated mainly involves nucleotide binding and phosphate nucleus Glycoside binding,anion binding,same protein binding and enzyme binding,etc.KEGG analysis shows that the classification involving the largest number of proteins is metabolism and genetic information processing.The top 10 KEGG signaling pathways with the most significant P values are proteasome,fructose and mannose metabolism,alanine,aspartic acid and glutamate metabolism,Epstein-Barr virus infection,carbon metabolism,and cysteine and methionine metabolism.Signal pathways such as metabolism,amino sugar and nucleotide sugar metabolism,fatty acid metabolism,spliceosome,phenylalanine,tyrosine and tryptophan biosynthesis.SH-SY5Y cells were infected with different doses of BPDE,and the results showed that as the dose increased,the cell survival rate gradually decreased.The results of thermolysin digestion method and CETSA method both showed that BPDE-SLC3A2 protein adduct increased with increasing dose(P<0.05),while the expression level of SLC3A2 and SLC7A11 protein decreased with increasing dose.(P<0.05).After intervention with DFO and Fer-1,the cell survival rate of 1.00μmol/LBPDE+DFO and BPDE+Fer-1 group was significantly higher than that of 1.00μmol/LBPDE group(P<0.05);the results of correlation analysis showed,BPDE-SLC3A2 protein adduct was negatively correlated with SLC7A11 protein expression level and cell survival rate(Thermolysin digestion method:r=-0.9032,P<0.0001;r=-0.8153,P<0.0001.CETSA method:r=-0.7618,P<0.001;r=-0.7055,P<0.001).BPDE-SLC3A2 protein adduct was positively correlated with the dose of BPDE(Thermolysin digestion method:r=0.8579,P<0.0001;CETSA method:r=0.8622,P<0.0001).The body weight of mice in B[a]P group was lower than that in control group from the third week.The results of open field test showed that there was no significant difference in the total movement distance among the groups(P>0.05).The times of entering the central area in the 2.0 and 5.0 mg/kg·bw B[a]P groups were lower than those in the solvent control group(P<0.05),and the movement distance in the peripheral area was higher than that in the solvent control group(P<0.05);The retention time of 5.0 mg/kg·bw B[a]P group was lower than that of solvent control group(P<0.05).The results of Morris water maze showed that the escape latency of 2.0,5.0 mg/kg·bw B[a]P groups on the 2nd,3rd,4th and 5th day was significantly higher than that of the solvent control group and 0.8 mg/kg·bw B[a]P group(P<0.05),the first time to reach the platform of the 5.0 mg/kg·bw B[a]P group was longer than that of the solvent control group(P<0.05),and the times of crossing the platform and plateau stagnation time of the 5.0 mg/kg·bw B[a]P group were less than those of the solvent control group(P<0.05).The results of thermophilic protease digestion and CETSA showed that the content of BPDE-SLC3A2 protein adduct in 0.8,2.0 and 5.0 mg/kg·bw B[a]P groups was significantly higher than that in solvent control group(P<0.05),and the content of BPDE-SLC3A2 protein adduct in 2.0 and 5.0 mg/kg·bw B[a]P groups was higher than that in 0.8 mg/kg bw B[a]P group(P<0.05).Compared with the solvent control group,there was no significant difference in the expression of SLC3A2 protein in B[a]P group(P>0.05),but the expression of SLC7A11 protein in B[a]P group was decreased(P<0.05).Correlation analysis showed that BPDE-SLC3A2 protein adduct level in mice brain tissue was negatively correlated with SLC7A11 protein expression level in mice brain tissue(Thermolysin digestion method:r=-0.5229,P<0.05;CETSA method:r=-0.5935,P<0.05),and it was positively correlated with the movement distance of the surrounding area(Thermolysin digestion method:r=0.7989,P<0.001;CETSA method:r=0.868,P<0.001),and it was negatively correlated with the central dead time(Thermolysin digestion method:r=-0.886,P<0.001;CETSA method:r=-0.9536,P<0.001).It was positively correlated with the average escape latency and the first time to reach the platform(Thermolysin digestion method:r=0.822,P<0.001;r=0.6495,P<0.001;CETSA method:r=0.9103,P<0.001;r=0.7413,P<0.001),and negatively correlated with plateau quadrant dead time(Thermolysin digestion method:r=-0.615,P<0.05;CETSA method:r=-0.677,P<0.001).There was a positive correlation between BPDE-SLC3A2 protein adduct and the dose of B[a]P(Thermolysin digestion method:r=0.9862,P<0.0001;CETSA method:r=0.9754,P<0.0001).Conclusion:DARTS method was used to screen and identify 190 BPDE adduct proteins in SH-SY5Y cells infected with BPDE.Established thermolysin digestion method,CETSA method BPDE-SLC3A2 adduct protein relative dose detection method,and showed that the level of BPDE-SLC3A2 adduct protein is correlated with BPDE exposure dose.The level of BPDE-SLC3A2 adduct protein is negatively correlated with the expression level of SLC7A11 protein,which may be involved in cell death and neurobehavioral damage mechanisms in exposed mice.