Abstract
Benzalkonium chloride (BAC), an antimicrobial agent in inhalable medications and household sprays, has been reported to be toxic to pulmonary organs. Although cell membrane damage has been considered as the main cytotoxic mechanism of BAC, its concentration- and time-dependent cellular effects on lung epithelium have not been fully understood. In the present study, human lung epithelial (H358) cells were exposed to 0.2–40 μg/mL of BAC for 30 min or 21 days. Cell membranes were rapidly disrupted by 30 min exposure, but 24 h incubation of BAC (4–40 μg/mL) predominantly caused apoptosis rather than necrosis. BAC (2–4 μg/mL) induced mitochondrial depolarization, which may be associated with increased expression of pro-apoptotic proteins (caspase-3, PARP, Bax, p53, and p21), and decreased levels of the anti-apoptotic protein Bcl-2. The protein expression levels of IRE1α, BiP, CHOP, and p-JNK were also elevated by BAC (2–4 μg/mL) suggesting the possible involvement of endoplasmic reticulum stress in inducing apoptosis. Long-term (7–21 days) incubation with BAC (0.2–0.6 μg/mL) did not affect cell viability but led to epithelial-mesenchymal transition (EMT) as shown by the decrease of E-cadherin and the increase of N-cadherin, fibronectin, and vimentin, caused by the upregulation of EMT transcription factors, such as Snail, Slug, Twist1, Zeb1, and Zeb2. Therefore, we conclude that apoptosis could be an important mechanism of acute BAC cytotoxicity in lung epithelial cells, and chronic exposure to BAC even at sub-lethal doses can promote pulmonary EMT.
Highlights
Benzalkonium chloride (BAC), a mixture of n-alkylbenzyldimethylammonium chlorides, is an antimicrobial agent used in agricultural, medical, and household products as a disinfectant, Toxics 2020, 8, 17; doi:10.3390/toxics8010017 www.mdpi.com/journal/toxicsToxics 2020, 8, 17 an antiseptic, and a preservative, respectively [1]
The absorbance of the converted dye was measured at 540 nm using the MULTISKAN GO reader (Thermo Scientific, Sunnyvale, CA, USA), and the results were expressed as a percentage of viable BAC-treated cells normalized to the percentage of untreated cells
Activity in a dose- and time-dependent manner (Figure 2E). These results suggest that apoptosis, increased caspase-3 activity in a dose- and time-dependent manner (Figure 2E). These results suggest rather than necrosis, might be the major cell death pathway induced by BAC in lung epithelial cells
Summary
Benzalkonium chloride (BAC), a mixture of n-alkylbenzyldimethylammonium chlorides, is an antimicrobial agent used in agricultural, medical, and household products as a disinfectant, Toxics 2020, 8, 17; doi:10.3390/toxics8010017 www.mdpi.com/journal/toxicsToxics 2020, 8, 17 an antiseptic, and a preservative, respectively [1]. The pulmonary effects of BAC have been evaluated, since this preservative is found in inhalable medications such as nasal sprays, nebulizers, and inhalers for patients with nasal congestion, allergic rhinitis, asthma, and chronic obstructive pulmonary diseases (COPD) [6,7]. Animal studies have demonstrated that BAC inhalation induces pulmonary irritation, inflammation, and damages the blood-air barrier in rodents [11,12,13,14,15]. Several mechanisms of BAC cytotoxicity, such as cell membrane disruption, oxidative stress, and DNA damage have been proposed in in vitro studies using human lung cell lines [14,16]. The exact cellular events in BAC-induced lung injury have not been fully understood
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