Burcham Philip C, Raso Albert, Henry Peter J
Pharmacology and Anaesthesiology Unit, School of Medicine & Pharmacology, The University of Western Australia, Nedlands, WA 6009, Australia.
Pharmacology and Anaesthesiology Unit, School of Medicine & Pharmacology, The University of Western Australia, Nedlands, WA 6009, Australia.
Toxicology. 2014 May 7;319:44-52. doi: 10.1016/j.tox.2014.02.010. Epub 2014 Mar 2.
The combustion product acrolein is a key mediator of pulmonary edema in victims of smoke inhalation injury. Since studying acrolein toxicity in conventional in vitro systems is complicated by reactivity with nucleophilic culture media constituents, we explored an exposure system which delivers airborne acrolein directly to lung cell monolayers at the air-liquid interface. Calu-3 lung adenocarcinoma cells were maintained on membrane inserts such that the basal surface was bathed in nucleophile-free media while the upper surface remained in contact with acrolein-containing air. Cells were exposed to airborne acrolein for 30 min before they were allowed to recover in fresh media, with cell sampling at defined time points to allow evaluation of toxicity and protein damage. After prior exposure to acrolein, cell ATP levels remained close to controls for 4h but decreased in an exposure-dependent manner by 24h. A loss of transepithelial electrical resistance and increased permeability to fluorescein isothiocyanate-labeled dextran preceded ATP loss. Use of antibody arrays to monitor protein expression in exposed monolayers identified strong upregulation of phospho-keratin-8 (Ser(73)) as an early consequence of acrolein exposure. These changes were accompanied by chemical damage to keratin-8 and other intermediate filament family members, while acrolein exposure also resulted in controlled ubiquitination of high mass proteins within the intermediate filament extracts. These findings confirm the usefulness of systems allowing delivery of airborne smoke constituents to lung cell monolayers during studies of the molecular basis for acute smoke intoxication injury.
燃烧产物丙烯醛是烟雾吸入性损伤患者肺水肿的关键介质。由于在传统体外系统中研究丙烯醛毒性会因与亲核培养基成分发生反应而变得复杂，我们探索了一种暴露系统，该系统可将空气中的丙烯醛直接输送至气液界面的肺细胞单层。将Calu-3肺腺癌细胞培养在膜插入物上，使基底表面浸泡在无亲核试剂的培养基中，而上表面则与含丙烯醛的空气接触。细胞暴露于空气中的丙烯醛30分钟后，再置于新鲜培养基中恢复，在特定时间点采集细胞样本以评估毒性和蛋白质损伤。预先暴露于丙烯醛后，细胞ATP水平在4小时内仍接近对照组，但在24小时时以暴露依赖的方式下降。跨上皮电阻的丧失以及对异硫氰酸荧光素标记的葡聚糖通透性的增加先于ATP的丧失。使用抗体阵列监测暴露单层中的蛋白质表达发现，磷酸化角蛋白-8（Ser(73)）强烈上调是丙烯醛暴露的早期结果。这些变化伴随着角蛋白-8和其他中间丝家族成员的化学损伤，而丙烯醛暴露还导致中间丝提取物中高质量蛋白质的可控泛素化。这些发现证实了在急性烟雾中毒损伤分子基础研究中，允许将空气中的烟雾成分输送至肺细胞单层的系统的实用性。
