Elsevier

Toxicology in Vitro

Volume 59, September 2019, Pages 221-227
Toxicology in Vitro

Effects of benzalkonium chloride on cell viability, inflammatory response, and oxidative stress of human alveolar epithelial cells cultured in a dynamic culture condition

https://doi.org/10.1016/j.tiv.2019.04.027Get rights and content

Highlights

  • Dynamic culture device used in this study more closely mimics lung conditions than that produced by a conventional monolayer culture method.

  • Screening toxic substances using a dynamic culture method can reduce the effects of cost and ethical issues on researchers.

Abstract

Recently, the importance of inhalation toxicity assessment increased due to recent humidifier disinfectant-associated deaths in children. Benzalkonium chloride (BAC) is currently used as a cationic surfactant and germicide in food industry processing lines and as a hand sanitizer. Animal models are mainly used as a method of evaluating the inhalation toxicity of a hazardous substance, but that approach requires considerable amounts of time and cost. As a replacement for animal experiments, in vitro cell culture can be used to assess toxicity. However, such culture does not reflect the natural microenvironment of the lung, particularly its dynamic nature. In this study, we simulated normal breathing levels (tidal volume 10%, 0.2 Hz) through surface elongation of an elastic membrane in a dynamic culture system. The low-cost dynamic system provided easy control of breathing rate during lung cell culture. We assessed the toxicity using different concentrations of BAC (0, 2, 5, 10, 20, and 40 μg/mL) under static and dynamic culture conditions. Following 24 h of exposure to BAC, cellular metabolic activity, cell membrane integrity, interleukin-8 (IL-8) and reactive oxygen species (ROS) levels, and the total amount of protein in cells were analyzed. Our results showed that significant differences in cellular metabolic activity, as well as IL-8 and ROS profiles, between static and dynamic cell growth conditions, following BAC exposure.

Introduction

The toxicity of chemical disinfectants has received great attention in South Korea since the Korea Center for Disease Control and Prevention (KCDC) reported that some lung injuries with unknown causes were associated with humidifier disinfectants (Korea Centers for Disease Control and Prevention, 2011). Since then, many toxicological studies of chemicals used in household disinfectants have been reported, but most of them were focused on specific chemicals, such as polyhexamethylene guanidine phosphate (PHMG), oligo (2-(2-ethoxy) ethoxyethyl) guanidinium (PGH), didecyldimethylammonium chloride (DDAC), 2-methyl-4-isothiazolin-3-one (MIT), and 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT) (Kim and Paek, 2016; Lee et al., 2012; Park et al., 2017). Benzalkonium chloride (BAC), a quaternary ammonium compound, has been used, frequently and for a long time, as a disinfectant (McPherson and Wood, 1949). Many available pharmaceutical and personal care products, including ophthalmic solutions, nasal sprays, nebulizer, hand sanitizers, shampoos, and even cosmetics, contains BAC as a preservative (Liebert, 1989; Marple et al., 2004; McPherson and Wood, 1949). The extensive list of usages of BAC is a result of its multiple functions as a biocide, a cationic surfactant, and as a phase transfer agent (Maximilian and Peter, 2013).

However, adverse effects of BAC on eyes, skin, and respiratory system have been reported (Basketter et al., 2004; Cha et al., 2004; Debbasch et al., 2000; Larsen et al., 2012; Lee et al., 2000; Swiercz et al., 2013). Most of the reports on adverse effects were based on animal test or in vitro cell test results; however, both of those experimental methods have issues or limitations. Although animal experiments have been widely used to test the safety of drug candidates or specific chemicals, important ethical issues concerning animal research have arisen recently (Festing and Wilkinson, 2007; Ghasemi and Dehpour, 2009; Monamy, 2017). Further, the results of animal testing are not perfectly predictive of human results due to the significant differences between human and nonhuman animals, such as anatomy, metabolism, and physiologic and pharmacologic properties related to genetic variations (Dayan, 1991; Langley, 2009; Shanks et al., 2009). In the case of in vitro studies, most experiments have been performed on two-dimensional (2D) culture surfaces, such as those in glass dishes and polystyrene flasks, but 2D monolayer culture does not completely reflect the microenvironments of actual tissues (Hoarau-Vechot et al., 2018; Imamura et al., 2015). In particular, regarding the investigation of inhalation toxicity of BAC, the dynamic microenvironment of the lungs, which repeatedly contracts and expands, must be considered, since the mechanical stretching of the lungs influences cell physiological characteristics, such as proliferation, differentiation, and gene expression (Liu et al., 1995; Liu et al., 1993).

In this study, we designed a cell culture model that is able to apply cyclic stretching to a flexible membrane on which cells can be cultured. The model is composed of a flexible culture plate and a 6-place loading post attached to a voice coil actuator. Since human adults take about 15 breaths each minute and use approximately 10% of their total lung capacity (Stocks and Quanjer, 1995), we operated our culture model to induce a 1% surface elongation of the flexible membrane at 0.2 Hz, corresponding to normal breathing conditions (Tschumperlin and Margulies, 1998). We investigated the effects of BAC under that cyclic stretching condition on cell metabolic activity, cell membrane integrity, pro-inflammatory response, and oxidative stress. Under the cyclic stretching condition, BAC produced higher toxic effects on cells in most assays than those produced under a static condition.

Section snippets

Cyclic stretch inducing cell culture model

The cell culture model used to induce cyclic stretching consisted of an acrylic frame (15 cm × 25.4 cm × 8.5 cm, width × length × height), a 6-place loading post, a flexible culture plate (BF-3001C, FlexCell International, Hillsborough, NC, USA), a voice coil actuator (VCA; BEI Kimco, Vista, CA, USA), and a digital servo driver (Pluto, Ingenia, Motion Control, Barcelona, Spain) (Fig. 1). This model was powered by a direct current (DC) supply and was controlled by software (MotionLab, Ingenia)

Effect of cyclic stretching on viable cell count and cell metabolic activity

The cell counting assay and the EZ-Cytox cell viability assay were used to investigate the effects of cyclic stretching on cell viability and metabolic activity (Fig. 2). The applied cyclic stretching condition in this study did not induce differences in viable cell numbers compared to the static control (Fig. 2A); however, cyclic stretching culture increased metabolic activity levels over that in static culture by 14.5%, 24.8%, and 48.7% after 6, 12, and 24 h, respectively (all p < .001) (Fig.

Discussion

A need for replacement of animal-based experimentation is emerging, because indiscriminate animal experimentation has created many problems, both ethical and cost related. Recently, many efforts have been made to replace animal experiments with in vitro experiments (Höfer et al., 2004). However, in vitro experiments using single cells do not fully simulate the conditions in the human body. Our tissues are heavily influenced by the surrounding environments. Further, the tissues of our body are

Acknowledgments

This study was funded by the Korea Ministry of Environment (MOE), as Environmental Health Action Program (2016001360005).

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