Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defense, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic; Biomedical Research Center, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec Kralove, Czech Republic.
Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defense, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic.
Chem Biol Interact. 2015 Jul 25;237:31-7. doi: 10.1016/j.cbi.2015.05.009. Epub 2015 May 15.
Sulfur mustard is a highly toxic chemical warfare agent with devastating impact on intoxicated tissues. DNA cross-links are probably the most toxic DNA lesions induced in the cell by sulfur mustard. The comet assay is a very sensitive method for measuring DNA damage. In the present study using the A-549 lung cell line, the comet assay protocol was optimized for indirect detection of DNA cross-links induced by sulfur mustard. The method is based on the additional treatment of the assayed cells containing cross-links with the chemical mutagen, styrene oxide. Alkali-labile adducts of styrene oxide cause DNA breaks leading to the formation of comets. A significant dose-dependent reduction of DNA migration of the comet's tail was found after exposing cells to sulfur mustard, indicative of the amount of sulfur mustard induced cross-links. The remarkable decrease of % tail DNA could be observed as early as 5min following exposure to sulfur mustard and the maximal effect was found after 30min, when DNA migration was reduced to the minimum. Sulfur mustard preincubated in culture medium without cells lost its ability to induce cross-links and had a half-life of about 15min. Pre-incubation longer than 30min does not lead to a significant increase in cross-links when applied to cells. However, the amount of cross-links is decreased during further incubation due to repair. The current modification of the comet assay provides a useful tool for detecting DNA cross-links induced by sulfur mustard and could be used for detection of other DNA cross-linking agents such as chemotherapeutic drugs.
芥子气是一种具有毁灭性影响的高毒性化学战剂，会对中毒组织造成伤害。DNA 交联可能是芥子气在细胞中诱导的最具毒性的 DNA 损伤。彗星试验是一种非常灵敏的测量 DNA 损伤的方法。在本研究中，我们使用 A-549 肺细胞系，优化了彗星试验方案，以间接检测芥子气诱导的 DNA 交联。该方法基于用化学诱变剂苯氧化物对含有交联的检测细胞进行额外处理。苯氧化物的碱不稳定加合物会导致 DNA 断裂，从而形成彗星。暴露于芥子气后，发现细胞中的 DNA 迁移明显呈剂量依赖性减少，这表明芥子气诱导的交联量。暴露于芥子气 5 分钟后即可观察到 %尾 DNA 的显著下降，而 30 分钟后观察到最大效应，此时 DNA 迁移减少到最低水平。未与细胞孵育的芥子气在培养基中失去了诱导交联的能力，半衰期约为 15 分钟。孵育超过 30 分钟不会导致交联显著增加，当应用于细胞时。然而，由于修复，交联的数量会在进一步孵育过程中减少。彗星试验的当前改进为检测芥子气诱导的 DNA 交联提供了有用的工具，并可用于检测其他 DNA 交联剂，如化疗药物。
