B(a)P和DEHP联合染毒对HepG2细胞CYP1A1和CYP3A4酶活性的影响
Effects of Co-exposure of Di-(2-ethylhexyl) Phthalate and Benzo(a)pyrene on CYP1A1 and CYP3A4 Enzyme Activities in HepG2 Cells
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摘要:
目的 研究邻苯二甲酸二乙基己基酯di(-2-ethylhexyl)phthalate, DEHP和苯并(a)芘benzo (a) pyrene,B(a)P联合染毒人肝癌细胞株(HepG2细胞)对其细胞色素P450酶系1A1(cytochrome 1A1, CYP1A1)和细胞色素P450酶系3A4(CYP3A4)酶活性的影响。 方法 分别用B(a)P 64.0 μmol/L和DEHP 62.5、125.0、250.0、500.0、1 000.0 μmol/L单独染毒和两者联合染毒HepG2细胞48 h和72 h。溶剂对照组为二甲基亚砜(dimethyl sulfoxide, DMSO<1‰)。用CCK8法检测细胞活性;用7-乙氧基异吩噁唑-O-去乙氧基酶和7-乙氧基香豆素O-去乙基酶(EROD和ECOD)实验评价细胞CYP1A1和CYP3A4酶活性;分别用实时荧光定量PCR法和Western blot法检测CYP1A1和CYP3A4基因的mRNA和蛋白质表达水平。 结果 与DEHP单独染毒组相比,联合染毒组细胞的存活率明显下降(P<0.01); EROD和ECOD活性却明显增加(P<0.05, P<0.01);联合染毒组CYP1A1基因仅有mRNA表达增加,但CYP3A4基因在mRNA和蛋白质表达水平均较DEHP单独染毒组明显升高(P<0.01)。 结论 DEHP和B(a)P联合染毒诱导了HepG2细胞CYP1A1和CYP3A4酶活性,并在mRNA和蛋白质水平对CYP1A1和CYP3A4的表达量产生一定影响。 Abstract:Objective Effects of co-exposure of Di-(2-ethylhexyl) phthalate and Benzo(a)pyrene on CYP1A1 and CYP3A4 enzyme activities were investigated in HepG2 cells. Methods HepG2 cells were treated with either 64.0 μmol/L B(a)P alone or DEHP alone (62.5,125.0,250.0,500.0,1 000.0 μmol/L) or DEHP at the indicated concentrations plus 64.0 μmol/L B(a)P or dimethyl sulfoxide (DMSO,solvent control,<1 ‰) for 48 h and 72 h.The cell proliferation was evaluated by CCK8 assay.The activities of CYP 1A-associated deethylation of ethoxyresorufin (EROD) and CYP3A-associated ethoxycoumarin-O-deethylation (ECOD) were measured.Expressions of CYP1A1 and CYP3A4 at mRNA and protein were detected by quantitative real time PCR method and Western blot,respectively. Results The cell viability was decreased significantly in all co-exposure of B(a)P and DEHP groups compared with the controls (P<0.01).However,the EROD and ECOD activities significantly increased,when compared with the DEHP alone treated group (P<0.05,P<0.01).Only up-regualtion of CYP1A1 transcriptional levels were observed in all co-exposure of B(a)P and DEHP groups.However,the levels of mRNA and protein expression of CYP3A4 were increased (P<0.01),when compared with the DEHP alone treated group. Conclusion Co-exposure of DEHP at certain concentrations and B(a)P (64.0μmol/L) induced the increases in the EROD and ECOD activities,as well as the CYP1A1 transcriptional level and the CYP3A4 transcriptional and protein levels.
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