这项研究探索和表征了两种密切相关的致癌二硝基苯(DNP)的毒性,1,3-DNP和1,8-DNP,人支气管上皮BEAS-2B细胞和小鼠肝癌Hepa1c1c7细胞。在BEAS-2B细胞中,1,3-DNP和1,8-DNP(3-30μM)均不诱导细胞死亡。在Hepa1c1c7细胞中,仅1,3-DNP(10-30μM)在24小时后诱导凋亡和坏死细胞死亡的混合物。通过CM-H2DCFDA荧光测量,两种化合物都增加了BEAS-2B中活性氧(ROS)的水平。通过甲酰氨基嘧啶-DNA糖基化酶(fpg)修饰的彗星测定法揭示了对DNA的氧化损伤的相应增加。没有fpg,彗星试验检测到的DNP诱导的DNA损伤仅在Hepa1c1c7细胞中发现。通过32P后标记测量,只有1,8-DNP形成DNA加合物。在Hepa1c1c细胞中,1,8-DNP在低于1,3-DNP的浓度下诱导H2AX(γH2AX)和p53的磷酸化,并且DNA损伤/DNA损伤反应(DR)与诱导的细胞毒性之间没有直接相关性。另一方面,1,3-DNP诱导的细胞凋亡被吡虫啉-α抑制,p53转录活性的抑制剂。此外,1,3-DNP触发了未折叠的蛋白质反应(UPR),通过CHOP的表达增加来衡量,ATF4和XBP1。因此,可能与内质网(ER)应激和/或UPR相关的其他类型的损伤可能参与诱导的细胞凋亡。我们的结果表明,与1,3-DNP相比,1,8-DNP更强的致癌作用与其更高的遗传毒性作用有关。这与其较低的诱导细胞死亡的效力组合可以增加引起突变的可能性。
This study explores and characterizes the toxicity of two closely related carcinogenic dinitro-pyrenes (DNPs), 1,3-DNP and 1,8-DNP, in human bronchial epithelial BEAS-2B cells and mouse hepatoma Hepa1c1c7 cells. Neither 1,3-DNP nor 1,8-DNP (3-30 μM) induced cell death in BEAS-2B cells. In Hepa1c1c7 cells only 1,3-DNP (10-30 μM) induced a mixture of apoptotic and necrotic cell death after 24 h. Both compounds increased the level of reactive oxygen species (ROS) in BEAS-2B as measured by CM-H2DCFDA-fluorescence. A corresponding increase in oxidative damage to DNA was revealed by the formamidopyrimidine-DNA glycosylase (fpg)-modified comet assay. Without fpg, DNP-induced DNA damage detected by the comet assay was only found in Hepa1c1c7 cells. Only 1,8-DNP formed DNA adduct measured by 32P-postlabelling. In Hepa1c1c cells, 1,8-DNP induced phosphorylation of H2AX (γH2AX) and p53 at a lower concentration than 1,3-DNP and there was no direct correlation between DNA damage/DNA damage response (DR) and induced cytotoxicity. On the other hand, 1,3-DNP-induced apoptosis was inhibited by pifithrin-α, an inhibitor of p53 transcriptional activity. Furthermore, 1,3-DNP triggered an unfolded protein response (UPR), as measured by an increased expression of CHOP, ATF4 and XBP1. Thus, other types of damage possibly linked to endoplasmic reticulum (ER)-stress and/or UPR could be involved in the induced apoptosis. Our results suggest that the stronger carcinogenic potency of 1,8-DNP compared to 1,3-DNP is linked to its higher genotoxic effects. This in combination with its lower potency to induce cell death may increase the probability of causing mutations.