Munakata Satoru, Watanabe Taku, Takahashi Tomohiro, Kimuro Shiori, Ishimori Kanae, Hashizume Tsuneo
Scientific Product Assessment Center, Japan Tobacco Inc, 6-2, Umegaoka, Aoba-Ku, Yokohama, Kanagawa, 227-8512, Japan.
Genes Environ. 2023 Apr 12;45(1):14. doi: 10.1186/s41021-023-00269-2.
The use of organotypic human tissue models in genotoxicity has increased as an alternative to animal testing. Genotoxicity is generally examined using a battery of in vitro assays such as Ames and micronucleus (MN) tests that cover gene mutations and structural and numerical chromosome aberrations. At the 7th International Workshop on Genotoxicity Testing, working group members agreed that the skin models have reached an advanced stage of maturity, while further efforts in liver and airway models are needed [Pfuhler et al., Mutat. Res. 850-851 (2020) 503135]. Organotypic human airway model is composed of fully differentiated and functional respiratory epithelium. However, because cell proliferation in organotypic airway models is thought to be less active, assessing their MN-inducing potential is an issue, even in the cytokinesis-blocking approach using cytochalasin B (CB) [Wang et al., Environ. Mol. Mutagen. 62 (2021) 306-318]. Here, we developed a MN test using EpiAirway™ in which epidermal growth factor (EGF) was included as a stimulant of cell division.
By incubating EpiAirway™ tissue with medium containing various concentrations of CB, we found that the percentage of binucleated cells (%BNCs) almost plateaued at 3 μg/mL CB for 72 h incubation. Additionally, we confirmed that EGF stimulation with CB incubation produced an additional increase in %BNCs with a peak at 5 ng/mL EGF. Transepithelial electrical resistance measurement and tissue histology revealed that CB incubation caused the reduced barrier integrity and cyst formation in EpiAirway™. Adenylate kinase assay confirmed that the cytotoxicity increased with each day of culture in the CB incubation period with EGF stimulation. These results indicated that chemical treatment should be conducted prior to CB incubation. Under these experimental conditions, it was confirmed that the frequency of micronucleated cells was dose-dependently increased by apical applications of two clastogens, mitomycin C and methyl methanesulfonate, and an aneugen, colchicine, at the subcytotoxic concentrations assessed in %BNCs.
Well-studied genotoxicants demonstrated capability in an organotypic human airway model as a MN test system. For further utilization, investigations of aerosol exposure, repeating exposure protocol, and metabolic activation are required.
作为动物试验的替代方法，基因毒性研究中器官型人体组织模型的使用有所增加。基因毒性通常使用一系列体外试验进行检测，如Ames试验和微核（MN）试验，这些试验涵盖基因突变以及结构和数量染色体畸变。在第7届基因毒性检测国际研讨会上，工作组成员一致认为皮肤模型已达到成熟的高级阶段，而肝脏和气道模型仍需进一步努力[Pfuhler等人，《突变研究》850 - 851（2020）503135]。器官型人体气道模型由完全分化且功能正常的呼吸道上皮组成。然而，由于器官型气道模型中的细胞增殖被认为不太活跃，即使在使用细胞松弛素B（CB）的胞质分裂阻断方法中，评估其诱导微核的潜力仍是一个问题[Wang等人，《环境分子突变》62（2021）306 - 318]。在此，我们开发了一种使用EpiAirway™的微核试验，其中包含表皮生长因子（EGF）作为细胞分裂的刺激剂。
通过将EpiAirway™组织与含有不同浓度CB的培养基孵育，我们发现对于72小时的孵育，双核细胞百分比（%BNCs）在3μg/mL CB时几乎达到平稳状态。此外，我们证实CB孵育时的EGF刺激使%BNCs进一步增加，在5 ng/mL EGF时达到峰值。跨上皮电阻测量和组织组织学显示，CB孵育导致EpiAirway™中的屏障完整性降低和囊肿形成。腺苷酸激酶测定证实，在EGF刺激下，CB孵育期间细胞毒性随培养天数增加。这些结果表明化学处理应在CB孵育之前进行。在这些实验条件下，已证实通过在%BNCs评估的亚细胞毒性浓度下，向顶端应用两种断裂剂丝裂霉素C和甲基磺酸甲酯以及一种非整倍体秋水仙碱，微核细胞频率呈剂量依赖性增加。
经过充分研究的基因毒性物质在器官型人体气道模型中作为微核试验系统显示出能力。为了进一步应用，需要对气溶胶暴露、重复暴露方案和代谢活化进行研究。
