Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems

Abstract

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray‐form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1‐10 μg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 μg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC‐promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N‐acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 μg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 μg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 μg/kg) and TEG (1000 μg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF‐α), and interleukin 6 (IL‐6) in BALF. These results suggest that TEG can potentiate BAC‐induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray‐form products containing this chemical combination is potentially harmful to humans.

Concentration- and Time-Dependent Effects of Benzalkonium Chloride in Human Lung Epithelial Cells: Necrosis, Apoptosis, or Epithelial Mesenchymal Transition

“…In addition, p53 arrests cell cycle via inducing p21 expression thereby allowing cells to recover, but, triggers apoptosis under the severe stress conditions [35,44]. It has been reported that BAC induces DNA damage and oxidative stress in human lung epithelial cells [14,16], suggesting that these cellular stress events promoted by BAC could be possible reasons for the increased expression of p53 ( Figure 3C). PARP catalyzes the transfer reaction of ADP-ribose for DNA repair, and this enzyme can be cleaved and inactivated by caspase-3 [45].…”

“…As a result, hydrophilic voids are created, phospholipids are solubilized, and finally the membrane structure is destroyed [2,4]. In alveolar and bronchial epithelial cells, BAC (1-100 μg/mL) treatment has been shown to induce acute cytotoxicity with cell membrane disruptions [14,16]. Inhalation of BAC damages airway epithelium resulting in the release of proteins and LDH to the bronchioalveolar spaces in rodents [11,12,15].…”

“…Especially, 17 in 28 asthmatic patients showed at least 20% decrease of 1-s forced expiratory volume (PD 20 FEV 1 ) after the inhalation of anti-asthma respirator solutions containing BAC (0.35 to 5.55 µmol) [10]. 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].…”

“…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]. Nevertheless, the exact cellular events in BAC-induced lung injury have not been fully understood.…”

Acute toxicity of benzalkonium chloride in Balb/c mice following intratracheal instillation and oral administration

“…Thus, serious bronchoconstriction may be one of the major factors inducing death in mice immediately post-treatment. Although inflammatory response and oxidative stress were known to be induced by BAC [14,15], they were not associated with fatal outcomes. Effects of BAC on bronchoconstriction will be further investigated considering the possibility of neurotoxicity, which may interfere with respiration or cardiac function.…”