Buehrlen Michael, Harréus Ulrich A, Gamarra Fernando, Hagen Rudolf, Kleinsasser Norbert H
Department of Internal Medicine, Munich-Neuperlach Hospital, Munich, Federal Republic of Germany.
Toxicol Lett. 2007 Mar 8;169(2):152-61. doi: 10.1016/j.toxlet.2007.01.005. Epub 2007 Jan 19.
Three-dimensional mini organ cultures of human inferior nasal turbinate epithelia have proved to be a useful tool in genotoxicology studies. They allow repetitive or chronic exposure of cells to xenobiotics in a well-preserved organ-specific mucosal architecture for an extended period of time. It is the aim of the present study to concurrently monitor cumulative genotoxic and apoptotic effects of sodium dichromate, N-nitrosodiethylamine (NDEA) and N-methyl-N-nitro-N-nitroso-guanidine (MNNG). Mini organs were raised by separating fresh specimens of human inferior nasal turbinates (n=11) into 1 mm3 sized pieces and culturing them on multiwell plates with bronchial epithelial basal medium for 6 days. Aliquots of the mini organs were subsequently exposed to sodium dichromate (1.0 mM, 1h), NDEA (50 mM, 1h) or MNNG (0.07 mM, 1h) on days 7, 9 and 11 versus a single exposure on day 11 only. DNA fragmentation and apoptotic events were assessed on day 11 using the alkaline single cell microgel electrophoresis assay (comet assay) and the annexin V-affinity assay. Significant DNA fragmentation could be demonstrated after a single exposure of the mini organs to sodium dichromate. Following three subsequent incubations, there was a further increase in the genetic damage observed, accompanied by an increase in the rate of apoptotic cells. In contrast, after single and triple incubation with NDEA there was neither an increase in genetic damage nor in the fraction of apoptotic cells detectable. Repetitive exposure to MNNG resulted in an accumulation of DNA damage without an observable increase in apoptosis. The results verify the need to assess apoptosis in genotoxicology research and to investigate cumulative effects of xenobiotics. Three-dimensional mini organ cultures of human upper aerodigestive tract epithelia have shown to be well-suited for improving the ability to distinguish between cumulative genotoxic and apoptotic effects.
人下鼻甲上皮的三维微型器官培养已被证明是遗传毒理学研究中的一种有用工具。它们能使细胞在保存完好的器官特异性黏膜结构中长时间反复或长期接触外源化学物。本研究的目的是同时监测重铬酸钠、N-亚硝基二乙胺（NDEA）和N-甲基-N-硝基-N-亚硝基胍（MNNG）的累积遗传毒性和凋亡效应。通过将人下鼻甲的新鲜标本（n = 11）分离成1立方毫米大小的碎片，并在多孔板中用支气管上皮基础培养基培养6天来培养微型器官。随后，在第7天、第9天和第11天，将微型器官的等分试样分别暴露于重铬酸钠（1.0 mM，1小时）、NDEA（50 mM，1小时）或MNNG（0.07 mM，1小时），而对照组仅在第11天进行单次暴露。在第11天，使用碱性单细胞微凝胶电泳试验（彗星试验）和膜联蛋白V亲和试验评估DNA片段化和凋亡事件。微型器官单次暴露于重铬酸钠后可显示出明显的DNA片段化。在随后进行三次孵育后，观察到的遗传损伤进一步增加，同时凋亡细胞的比例也增加。相比之下，在单次和三次孵育NDEA后，遗传损伤和可检测到的凋亡细胞比例均未增加。重复暴露于MNNG导致DNA损伤的积累，但凋亡未见明显增加。这些结果证实了在遗传毒理学研究中评估凋亡以及研究外源化学物累积效应的必要性。人上呼吸道和消化道上皮的三维微型器官培养已显示非常适合提高区分累积遗传毒性和凋亡效应的能力。
