Abstract：
The human in vitro organotypic air-liquid-interface (ALI) airway tissue model is structurally and functionally similar to the human large airway epithelium and, as a result, is being used increasingly for studying the toxicity of inhaled substances. Our previous research demonstrated that DNA damage and mutagenesis can be detected in human airway tissue models under conditions used to assess general and respiratory toxicity endpoints. Expanding upon our previous proof-of-principle study, human airway epithelial tissue models were treated with 6.25-100 µg/mL ethyl methanesulfonate (EMS) for 28 days, followed by a 28-day recovery period. Mutagenesis was evaluated by Duplex Sequencing (DS), and clonal expansion of bronchial-cancer-specific cancer-driver mutations (CDMs) was investigated by CarcSeq to determine if both mutation-based endpoints can be assessed in the same system. Additionally, DNA damage and tissue-specific responses were analyzed during the treatment and following the recovery period. EMS exposure led to time-dependent increases in mutagenesis over the 28-day treatment period, without expansion of clones containing CDMs; the mutation frequencies remained elevated following the recovery. EMS also produced an increase in DNA damage measured by the CometChip and MultiFlow assays and the elevated levels of DNA damage were reduced (but not eliminated) following the recovery period. Cytotoxicity and most tissue-function changes induced by EMS treatment recovered to control levels, the exception being reduced proliferating cell frequency. Our results indicate that general, respiratory-tissue-specific and genotoxicity endpoints increased with repeat EMS dosing; expansion of CDM clones, however, was not detected using this repeat treatment protocol. DISCLAIMER: This article reflects the views of its authors and does not necessarily reflect those of the U.S. Food and Drug Administration. Any mention of commercial products is for clarification only and is not intended as approval, endorsement, or recommendation.
摘要：
人体外器官型气液界面（ALI）气道组织模型在结构和功能上与人大型气道上皮相似，并且，因此,越来越多地用于研究吸入物质的毒性。我们先前的研究表明，在用于评估一般和呼吸道毒性终点的条件下，可以在人气道组织模型中检测到DNA损伤和诱变。在我们之前的原理证明研究基础上，用6.25-100µg/mL甲磺酸乙酯（EMS）治疗人气道上皮组织模型28天，接下来是28天的恢复期。通过双重测序（DS）评估诱变，并且通过CarcSeq研究了支气管癌症特异性癌症驱动突变（CDMs）的克隆扩增，以确定是否可以在同一系统中评估两个基于突变的终点。此外,在治疗期间和恢复期之后分析DNA损伤和组织特异性反应。EMS暴露导致28天治疗期内诱变的时间依赖性增加，不扩增含有CDM的克隆；恢复后突变频率保持升高。EMS还产生了通过CometChip和MultiFlow测定测得的DNA损伤的增加，并且在恢复期后，DNA损伤的升高水平降低（但未消除）。EMS治疗引起的细胞毒性和大多数组织功能变化恢复到对照水平，例外是细胞增殖频率降低。我们的结果表明，总的来说，呼吸组织特异性和遗传毒性终点随着重复EMS给药而增加；CDM克隆的扩增，然而,使用此重复治疗方案未检测到。免责声明：本文反映了作者的观点，不一定反映了美国食品和药物管理局的观点。任何商业产品的提及仅用于澄清，不打算作为批准，背书，或推荐。
