Effects Of Benzo[a]pyrene On Energy Metabolism In SH-SY5Y Cells

Objective:The objectives of this study were to:study the effect of benzo[a]pyrene?B[a]P?on the energy metabolism of human neuroblastoma cells?SH-SY5Y?and analyze the relationship with oxidative damage and apoptosis with the exposure of B[a]P.The effect of B[a]P on the energy metabolism of nerve cells provides a basis for studying the mechanism of B[a]P neurotoxicity.Methods:Cell Culture and Exposure Methods:SH-SY5Y cells were purchased from Wuhan Procell and the cells were seeded in 96-well plates/6-well plates at appropriate concentrations according to experimental requirements.The cells were allowed to reach the logarithmic growth phase for experiments.SH-SY5Y cells were treated with different concentrations of B[a]P?0,0.1,1,10?mol/L?for 24 hours.The dose-response relationship of B[a]P to SH-SY5Y cells was examined.The time-effect of B[a]P on SH-SY5Y cells was studied by treatment of cells with 0,1?mol/L B[a]P for 24h,48h and 72 hours,The effect of B[a]P on SH-SY5Y cell damage was observed through the cell recovery experiment.Briefly,the final concentration of 1?mol/L B[a]P was added to cultured cells for 24h,48h,and 72h,respectively,and the cells were checked after 24 hours culture with normal culture medium.Cell damage related indicators detection methods:MTT assay was performed to measure the cell viability.cell malondialdehyde?MDA?assay kit was used for determination of intracellular MDA content;superoxide dismutase?SOD?typing test kit was used for measuring of the intracellular T-SOD,CuZn-SOD and Mn-SOD activity.Intracellular ROS levels was detected with reactive oxygen detection kit.Mitochondrial membrane potential assay kit?JC-1?was chosen to measure cell mitochondrial membrane potential;Annexin V-FITC/PI double staining cell apoptosis detection kit was used for Apoptosis levels measurements.Finally,the oxygen consumption rate?OCR?and extracellular acidification rate?ECAR?were measured using a Seahorse XFp cell energy analyzer.Results:Cytotoxicity of B[a]P on SH-SY5Y cells:The results of cytotoxicity test showed that as the dose increasing,cell viability,Mn-SOD activity,and mitochondrial membrane potential???m?showed a decreasing trend(P_trend<0.01),while intracellular ROS level,MDA content,and early apoptosis showed an upward trend(P_trend<0.01).The T-SOD and CuZn-SOD activity increased at low doses,but decreased at the mid-high doses.The cell viability in 1,10 and 100?mol/L group was significantly lower than that of the control group?P<0.05?;the intracellular MDA content in 1,10?mol/L group was significantly higher than that of the control group?P<0.05?;0.1,1,10?mol/L group intracellular ROS levels,Mn-SOD,T-SOD,CuZn-SOD activity,??m and early apoptosis rate were significantly high comparing with the control group?P<0.05?.With the prolonged exposure time,the time-lapse experiments showed that,T-SOD,CuZn-SOD and Mn-SOD activity,??m showed a downward trend(P_trend<0.01);intracellular ROS levels,MDA content,early withered.The mortality rate showed an upward trend(P_trend<0.01).The Mn-SOD activity and the mitochondrial membrane potential???m?were significantly decreased in the 48h and 72h groups compared with the 24h group?P<0.05?;In the 48h and 72h groups,compared with the 24h group,the intracellular ROS level,MDA content and early apoptosis rate were significantly increased?P<0.05?.The recovery experiments showed that the cell viability,T-SOD,CuZn-SOD,Mn-SOD activity,and??m were significantly increased?P<0.05?,and the ROS level,MDA content,and early apoptosis rate were significantly reduced in the 24 h group compared with that before the recovery?P<0.05?.The effect of B[a]P on cell energy metabolism:Our study showed that:with the increase of B[a]P exposure dose,the cells'basic respiration,ATP-linked respiration,maximal respiration,glycolysis,glycolytic capacity and glycolytic reserve were all decreased(P_trend<0.01).The basic respiration and ATP-linked respiration in each exposure group were significantly lower than those in the control group?P<0.05?;The maximal respiration,spare respiratory capacity,glycolysis,glycolytic capacity and glycolytic reserve in 10?mo1/L group were significantly lower than those in control group?P<0.05?.The proton Leak in 10?mo1/L group was significantly higher than that of control group?P<0.05?.Time-effect experiments showed that with prolonged exposure time,basic respiration,ATP-linked respiration,maximal respiration,spare respiratory capacity and glycolytic capacity were decreased(P_trend<0.01).The ATP-linked respiration,maximal respiration,spare respiratory capacity and glycolytic capacity in the 72-hour exposure group were lower than the 24h group?P<0.05?;The proton Leak in the 72-hour exposure group was significantly higher than that of the 24h group?P<0.05?.The basic respiration,ATP-linked respiration,maximal respiration,spare respiratory capacity,glycolysis and glycolytic reserve in 72-hour exposure group were lower than 24-hour exposure group?P<0.05?.Recovery experiments showed that ATP-linked respiration was significantly increased in the 24 h group compared with that before the recovery?P<0.05?.Antioxidant intervention results:Compared with that B[a]P exposure group,in the addition of antioxidant BHA,basic respiration,ATP-linked respiration,maximal respiration,spare respiratory capacity,glycolysis and??m were significantly increased while the early apoptosis rate and late apoptosis rate were significantly decreased?P<0.05?.Correlation analysis between parameters of cell injury and energy metabolism:MDA content,ROS levels and energy metabolism parameters were negatively correlated;T-SOD activity and basic respiration,ATP-linked respiration,maximal respiration,spare respiratory capacity,glycolysis,glycolytic capacity and glycolytic reserve were positively correlated in this study;CuZn-SOD activity was positively correlated with spare respiratory capacity and glycolytic reserve.Mn-SOD activity was positively correlated with basic respiration,ATP-linked respiration,maximal respiration,spare respiratory capacity,glycolysis and glycolytic capacity.??m,early apoptotic rate,late apoptotic rate,and energy metabolism were negatively correlated.Conclusion:B[a]P leads to a decrease in the survival rate of SH-SY5Y cells,causing lipid peroxidation and an increase in cell apoptosis rate.The effect is dose and time-dependent.Cell injury induced by low-dose and short-term exposure could be recovered at certain degree.B[a]P mainly causes the damage to mitochondrial respiratory function and causes a decrease in glycolysis at high doses,which may be mainly due to cellular oxidative damage.The energy metabolism caused by B[a]P eventually causes apoptosis.