DEHP与BaP联合诱导Changliver细胞线粒体介导细胞凋亡作用

Increased ROS levels and mitochondria-mediated apoptosis in Chang liver cells treated with DEHP plus BaP

目的研究邻苯二甲酸二(2-乙基)己酯(DEHP)与苯并(a)芘(BaP)联合染毒对Chang liver细胞的毒性。方法 DEHP(62.5、125、250、500和1 000μmol/L)与BaP(64μmol/L)单独或联合染毒Chang liver细胞24 h,检测细胞活力、细胞内活性氧(ROS)含量、线粒体膜电位(MMP)、细胞凋亡率和Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤因子2(Bcl-2)以及半胱氨酸蛋白酶-3(caspase-3)蛋白表达量。结果联合染毒各组细胞存活率均低于DEHP单独染毒组(t=6.22、4.64、6.64、5.84、7.29,P<0.01),但胞内ROS水平均升高(t=4.57、2.23、2.39、2.22、2.16,P<0.05或P<0.01);≥250μmol/L DEHP与BaP联合染毒组的MMP分别为(80.12±6.41)%、(69.92±5.56)%和(53.76±1.88)%,均低于BaP单独染毒组的(165.93±5.09)%(t=10.92、12.51、14.62,P<0.01);细胞凋亡率分别为(7.73±1.91)%、(11.00±3.04)%和(14.20±3.96)%,均高于BaP单独染毒组的(1.55±0.21)%(t=3.96、6.06、8.11,P<0.01);此外,活化caspase-3的高表达和升高的Bax/Bcl-2比值也被检出。结论较高浓度DEHP与BaP联合染毒Chang liver细胞促发ROS水平升高和线粒体膜电位降低,并导致了细胞凋亡;Bax、Bcl-2和活化caspase-3参与了此调控过程。

Objective To investigate effects of di （2-ethylhexyl） phthalate （DEHP） plus benzo （a） pyrene （BaP） on Chang liver cells. Methods Chang liver cells were treated with BaP（64 μmol/L） alone or plus DEHP at the concentrations of 0,62. 5,125,250,500, and 1 000 μmol/L for 24 hours. The indicators including cell viability, level of intracellular reactive oxygen species （ ROS ）, mitochondrial membrane potential （ MMP ）, apoptosis percentage, and the expres- sions of Bax,Bcl-2 and activiated caspase-3 proteins were determined. Results Decrease in cell viability in all DEHP and BaP treatment groups and increased ROS generation in all DEHP plus BaP treatment groups were found compared with those of the DEHP treated groups （P 〈 0. 05 or P 〈 0. 01 for all）. The decreases in MMP （80. 12 ± 6.41%, 69. 92 ---5.56% ,and 53.76 ± 1.88% ） in the DEHP treatment（ 〉1250 p, mol/L） plus BaP treatment （64 p, mol/L） groups were observed compared with that of the BaP treatment group（ 165.93 ± 5.09% ,P 〈 0. 01 ）. The increases in percentage of apoptotic Chang liver cells in the DEHP plus BaP treatment groups（7.73 ± 1.91% ,11.00 ±3.04% ,and 14. 20 ±3.96% ） were observed compared with that of the BaP treament group（ 1.55 ±0. 21% ,P 〈0. 01 ）. Furthermore,in the DEHP plus BaP treatment groups,the high active caspase-3 expression and high Bax/Bcl-2 ratio were observed. Conclusion The find- ings suggest that the co-treatment of DEHP plus BaP induces the reduction of cell viability and the ROS generation and mediate cell apoptosis ,which associates with high Bax/Bcl-2 ratio and active caspase-3 expression.