Holme J A, Nyvold H E, Tat V, Arlt V M, Bhargava A, Gutzkow K B, Solhaug A, Låg M, Becher R, Schwarze P E, Ask K, Ekeren L, Øvrevik J
Division of Environmental Medicine, Norwegian Institute of Public Health, N-0403 Oslo, Norway.
Department of Medicine, McMaster University, Hamilton, ON, Canada.
Toxicol Rep. 2014 Jul 27;1:459-473. doi: 10.1016/j.toxrep.2014.07.009. eCollection 2014.
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-HDCFDA-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 P-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.
本研究探讨并表征了两种密切相关的致癌性二硝基芘（DNP），即1,3 - DNP和1,8 - DNP，对人支气管上皮BEAS - 2B细胞和小鼠肝癌Hepa1c1c7细胞的毒性。1,3 - DNP和1,8 - DNP（3 - 30μM）均未在BEAS - 2B细胞中诱导细胞死亡。在Hepa1c1c7细胞中，仅1,3 - DNP（10 - 30μM）在24小时后诱导了凋亡和坏死性细胞死亡的混合情况。通过CM - HDCFDA荧光测量，两种化合物均增加了BEAS - 2B细胞中活性氧（ROS）的水平。甲酰胺嘧啶 - DNA糖基化酶（fpg）修饰的彗星试验揭示了DNA氧化损伤的相应增加。在没有fpg的情况下，彗星试验检测到的DNP诱导的DNA损伤仅在Hepa1c1c7细胞中发现。仅1,8 - DNP通过³²P后标记法检测到形成了DNA加合物。在Hepa1c1c细胞中，1,8 - DNP在比1,3 - DNP更低的浓度下诱导了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，p53转录活性抑制剂pifithrin - α抑制了1,3 - DNP诱导的凋亡。此外，通过CHOP、ATF4和XBP1表达的增加来测量，1,3 - DNP引发了未折叠蛋白反应（UPR）。因此，其他可能与内质网（ER）应激和/或UPR相关的损伤类型可能参与了诱导的凋亡。我们的结果表明，与1,3 - DNP相比，1,8 - DNP更强的致癌效力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能力相结合，可能会增加导致突变的概率。
