1-硝基芘和1,2-萘醌的联合细胞毒性和致DNA损伤

Combined Effects of 1-Nitropyrene and 1,2-Naphthoquinone on Cytotoxicity and DNA Damage in A549 Cells

以人肺上皮细胞A549为研究对象,运用MTT方法检测1-硝基芘(1-NP)处理后A549的细胞存活率;测定细胞培养液中乳酸脱氢酶(LDH)的漏出率,评价细胞膜损伤;运用彗星实验检测DNA损伤;通过荧光探针的方法测定细胞内产生的活性氧自由基(reactive oxygen species,ROS).通过1,2-萘醌(1,2-NQ)预先染毒24 h,再使用1-NP染毒24 h的方法,评估1-NP和1,2-NQ对A549的联合细胞毒性和DNA损伤.结果表明,1-NP对A549暴露24 h和48 h的半致死浓度(LC50)分别为5.2μmol·L-1和2.8μmol·L-1.LC50随着染毒时间的增加而降低,提示暴露时间越长1-NP的细胞毒性越强.A549在1、2、3和4μmol·L-1浓度的1-NP染毒下,DNA损伤显著增强,ROS水平不断升高,呈现剂量-效应关系(P<0.05);但LDH漏出率无显著变化.1,2-NQ(5μmol·L-1)预染毒A549细胞24 h,能明显减弱1-NP造成的DNA损伤和ROS升高.结果说明,1,2-NQ预处理可能通过抑制1-NP暴露产生的ROS,从而降低A549的DNA的损伤.

Using human lung epithelial A549 cells, viability was measured by MTT assay after treated with 1-nitropyrene (1-NP);lactate dehydrogenase (LDH) leakage was determined to evaluate the cellular membrane injury; DNA damage was detected with comet assay; reactive oxygen species (ROS) generation was measured with fluorescent probe. The combined toxic effects of 1-NP and 1,2-naphthoquinone (1,2-NQ) on A549 were also evaluated. 1-NP caused a significantly concentration-dependent and time-dependent viability decrease. The LC50 for 24 h and 48 h were 5. 2 μmol·L - 1 and 2. 8 μmol·L - 1 , respectively. DNA damage and intracellular ROS levels were also increased significantly through a dose-dependent manner after exposure to 1-NP. The LDH leakage were not significantly changed. Compared with the groups treated with 1-NP alone, the viability and LDH leakage was not changed significantly in combined-treated groups with 1-NP and 1,2-NQ. However, the DNA damage and ROS levels were significantly reduced in the combined-treated groups compared with the groups treated with 1-NP alone. These results suggest that 1-NP may mediated the genotoxic and cytotoxic effects through ROS generation, and pretreatment with 1,2-NQ, may inhibit the ROS generation induced by 1-NP, and thereby reducing the DNA damage in A549 cells.