Walczak Jarosław, Malińska Dominika, Drabik Karolina, Michalska Bernadeta, Prill Monika, Johne Stephanie, Luettich Karsta, Szymański Jędrzej, Peitsch Manuel C, Hoeng Julia, Duszyński Jerzy, Więckowski Mariusz R, van der Toorn Marco, Szczepanowska Joanna
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland.
Cell Physiol Biochem. 2020 Mar 11;54(2):230-251. doi: 10.33594/000000216.
BACKGROUND/AIMS: Adverse effects of cigarette smoke on health are widely known. Heating rather than combusting tobacco is one of strategies to reduce the formation of toxicants. The sensitive nature of mitochondrial dynamics makes the mitochondria an early indicator of cellular stress. For this reason, we studied the morphology and dynamics of the mitochondrial network in human bronchial epithelial cells (BEAS-2B) exposed to total particulate matter (TPM) generated from 3R4F reference cigarette smoke and from aerosol from a new candidate modified risk tobacco product, the Tobacco Heating System (THS 2.2).
Cells were subjected to short (1 week) and chronic (12 weeks) exposure to a low (7.5 µg/mL) concentration of 3R4F TPM and low (7.5 µg/mL), medium (37.5 µg/mL), and high (150 µg/mL) concentrations of TPM from THS 2.2. Confocal microscopy was applied to assess cellular and mitochondrial morphology. Cytosolic Ca levels, mitochondrial membrane potential and mitochondrial mass were measured with appropriate fluorescent probes on laser scanning cytometer. The levels of proteins regulating mitochondrial dynamics and biogenesis were determined by Western blot.
In BEAS-2B cells exposed for one week to the low concentration of 3R4F TPM and the high concentration of THS 2.2 TPM we observed clear changes in cell morphology, mitochondrial network fragmentation, altered levels of mitochondrial fusion and fission proteins and decreased biogenesis markers. Also cellular proliferation was slowed down. Upon chronic exposure (12 weeks) many parameters were affected in the opposite way comparing to short exposure. We observed strong increase of NRF2 protein level, reorganization of mitochondrial network and activation of the mitochondrial biogenesis process.
Comparison of the effects of TPMs from 3R4F and from THS 2.2 revealed, that similar extent of alterations in mitochondrial dynamics and biogenesis is observed at 7.5 µg/mL of 3R4F TPM and 150 µg/mL of THS 2.2 TPM. 7 days exposure to the investigated components of cigarette smoke evoke mitochondrial stress, while upon chronic, 12 weeks exposure the hallmarks of cellular adaptation to the stressor were visible. The results also suggest that mitochondrial stress signaling is involved in the process of cellular adaptation under conditions of chronic stress caused by 3R4F and high concentration of THS 2.2.
背景/目的：香烟烟雾对健康的不良影响广为人知。加热而非燃烧烟草是减少有毒物质形成的策略之一。线粒体动力学的敏感特性使线粒体成为细胞应激的早期指标。因此，我们研究了暴露于3R4F参考香烟烟雾产生的总颗粒物（TPM）以及新型候选改良风险烟草产品烟草加热系统（THS 2.2）产生的气溶胶中的人支气管上皮细胞（BEAS-2B）线粒体网络的形态和动力学。
细胞分别短期（1周）和长期（12周）暴露于低浓度（7.5μg/mL）的3R4F TPM以及低浓度（7.5μg/mL）、中等浓度（37.5μg/mL）和高浓度（150μg/mL）的THS 2.2 TPM。应用共聚焦显微镜评估细胞和线粒体形态。使用合适的荧光探针在激光扫描细胞仪上测量细胞质钙水平、线粒体膜电位和线粒体质量。通过蛋白质印迹法测定调节线粒体动力学和生物发生的蛋白质水平。
在短期暴露于低浓度3R4F TPM和高浓度THS 2.2 TPM 1周的BEAS-2B细胞中，我们观察到细胞形态、线粒体网络碎片化、线粒体融合和裂变蛋白水平改变以及生物发生标志物减少等明显变化。细胞增殖也减缓。长期暴露（12周）后，与短期暴露相比，许多参数受到相反的影响。我们观察到NRF2蛋白水平显著增加、线粒体网络重组以及线粒体生物发生过程激活。
比较3R4F和THS 2.2的TPM的影响发现，在7.5μg/mL的3R4F TPM和150μg/mL的THS 2.2 TPM下，线粒体动力学和生物发生的改变程度相似。暴露于所研究的香烟烟雾成分7天会引发线粒体应激，而长期暴露12周后，细胞对应激源的适应特征明显可见。结果还表明，线粒体应激信号参与了在3R4F和高浓度THS 2.2引起的慢性应激条件下的细胞适应过程。
