Institute of Fundamental Technological Research

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). Methods: 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 Ca2+ 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. Results: 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. Conclusion: 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.

BEAS-2B cells, candidate modified risk tobacco product, cigarette smoke, mitochondrial dynamics, tobacco heating system 2.2.

Bioenergetic failure, oxidative stress, and changes in mitochondrial morphology are common pathologic hallmarks of amyotrophic lateral sclerosis (ALS) in several cellular and animal models. Disturbed mitochondrial physiology has serious consequences for proper functioning of the cell, leading to the chronic mitochondrial stress. Mitochondria, being in the center of cellular metabolism, play a pivotal role in adaptation to stress conditions. We found that mitochondrial dysfunction and adaptation processes differ in primary fibroblasts derived from patients diagnosed with either sporadic or familial forms of ALS. The evaluation of mitochondrial parameters such as the mitochondrial membrane potential, the oxygen consumption rate, the activity and levels of respiratory chain complexes, and the levels of ATP, reactive oxygen species, and Ca2+ show that the bioenergetic properties of mitochondria are different in sporadic ALS, familial ALS, and control groups. Comparative statistical analysis of the data set (with use of principal component analysis and support vector machine) identifies and distinguishes 3 separate groups despite the small number of investigated cell lines and high variability in measured parameters. These findings could be a first step in development of a new tool for predicting sporadic and familial forms of ALS and could contribute to knowledge of its pathophysiology.

amyotrophic lateral sclerosis, neurodegeneration, primary fibroblasts, PCA

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.

adaptation, mitochondria, nicotine, oxidative stress

Mitochondrial dysfunction caused by cigarette smoke is involved in the oxidative stress-induced pathology of airway diseases. Reducing the levels of harmful and potentially harmful constituents by heating rather than combusting tobacco may reduce mitochondrial changes that contribute to oxidative stress and cell damage. We evaluated mitochondrial function and oxidative stress in human bronchial epithelial cells (BEAS 2B) following 1- and 12-week exposures to total particulate matter (TPM) from the aerosol of a candidate modified-risk tobacco product, the Tobacco Heating System 2.2 (THS2.2), in comparison with TPM from the 3R4F reference cigarette. After 1-week exposure, 3R4F TPM had a strong inhibitory effect on mitochondrial basal and maximal oxygen consumption rates compared to TPM from THS2.2. Alterations in oxidative phosphorylation were accompanied by increased mitochondrial superoxide levels and increased levels of oxidatively damaged proteins in cells exposed to 7.5 μg/mL of 3R4F TPM or 150 μg/mL of THS2.2 TPM, while cytosolic levels of reactive oxygen species were not affected. In contrast, the 12-week exposure indicated adaptation of BEAS-2B cells to long-term stress. Together, the findings indicate that 3R4F TPM had a stronger effect on oxidative phosphorylation, gene expression and proteins involved in oxidative stress than TPM from the candidate modified-risk tobacco product THS2.2.

Mitochondria, Mitochondrial respiratory chain, Oxidative stress, BEAS-2B cells, Cigarette, Tobacco heating system