An experimental in vitro model for dynamic direct exposure of human cells to airborne contaminants

doi:10.1016/j.toxlet.2006.01.008

Toxicology Letters

Volume 165, Issue 1, 1 August 2006, Pages 1-10

https://doi.org/10.1016/j.toxlet.2006.01.008 Get rights and content

Abstract

The aim of this study was to establish a dynamic in vitro model for direct exposure of human cells to gaseous contaminants to investigate the cellular responses to airborne chemical exposures. Nitrogen dioxide (NO₂) was selected as a model gas compound. Standard test atmospheres were generated (2.5–10 ppm), using a dynamic direct dilution method. Human cells including: A549 pulmonary type II-like epithelial cell lines and skin fibroblasts were grown on porous membranes. Human cells on snapwell inserts were placed in horizontal diffusion chambers and exposed to various airborne concentrations of NO₂ directly at the air/liquid interface for 1 h at 37 °C. Cytotoxicity of the test gas was investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), NRU (neutral red uptake) and ATP (Adenosine triphosphate) assays. Dose-dependent effects of NO₂ were observed in human cells tested which resulted in a significant reduction of cell viability at concentrations normally encountered in workplace environments (p < 0.05). Our findings suggest that the dynamic direct exposure method can be used for in vitro inhalational and dermal toxicity studies and potentially as an advanced technology for biomonitoring of airborne contaminants in future occupational and environmental toxicity assessments.

Introduction

Analyses of air pollution provides evidence that occupational and environmental exposure to airborne contaminants is significantly associated with human health risks ranging from bronchial reactivity to morbidity and mortality due to acute intense or long term low level repeated exposures (Klaassen, 2001, Chauhan and Johnston, 2003, Greenberg et al., 2003, Winder and Stacey, 2004). The general mechanism by which air contaminants induce biological effects is attributed to their oxidative and reactive capacities (Roux et al., 2002). However, further work is still needed to provide evidence for precise mechanisms by which air contaminants produce toxic effects.

The traditional approach of measuring the toxic effects of airborne contaminants relies on whole animal test methods. As well as ethical concerns, heavy reliance on animal data in toxicology is the subject of debate and controversy by the scientific community (Blaauboer, 2002, Bakand et al., 2005a). Although studying the toxic effects of inhaled chemicals is a complex subject, recent studies demonstrate that in vitro methods may have significant potential for assessing the toxicity of airborne contaminants (Chen et al., 1993, Tu et al., 1995, Knebel et al., 1998, Muckter et al., 1998, Diabate et al., 2002, Aufderheide et al., 2003, Ayyagari et al., 2004, Bakand et al., 2005b, Bakand et al., 2006). To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants have been reviewed (Bakand et al., 2005a).

Inhalation is considered the most important means by which humans are exposed to airborne chemicals, especially in the workplace environment (Winder and Stacey, 2004). Therefore, the development of in vitro techniques that are comparable to in vivo environments during inhalation exposures should be encouraged (Lambre et al., 1996). To achieve this, previously we developed a static exposure technique for direct exposure of human cells to vapours of volatile organic compounds (VOCs) at the air/liquid interface using cultured human cells on porous membranes in snapwell inserts (Bakand et al., 2006). The aim of this present study is to establish a dynamic in vitro model for direct exposure of human cells to gaseous contaminants to study the cellular response to airborne chemical exposures.

Nitrogen dioxide (NO₂), a well known indoor and outdoor oxidant gas, was selected as a model compound for gaseous airborne contaminants in this study. NO₂ is a pulmonary toxicant inducing irritation, acute inflammation, pulmonary oedema and pneumonia (Winder, 2004). Epithelial lung cells are the primary target of inhaled NO₂. However, the underlying mechanisms by which NO₂ causes pulmonary epithelial injury and how such interactions link to pulmonary disease are still to be understood (Persinger et al., 2001). Toxicity of NO₂ is probably associated with both oxidative and nonoxidative mechanisms (Schlesinger et al., 2000). NO₂ can produce oxidative injury by the generation of free radicals, or react with polyunsaturated fatty acids in cell membranes (Roux et al., 2002, McDow and Tollerud, 2003). Due to both low water solubility and high reactivity, NO₂ may react with pulmonary cells causing direct cytotoxicity (Tu et al., 1995).

To study the cytotoxicity of NO₂, standard test atmospheres were generated using a dynamic direct dilution method. Human cells including: A549 human pulmonary type II-like epithelial cell lines and skin fibroblasts were grown on porous membranes. The Navicyte horizontal diffusion chamber system (Harvard Apparatus, Inc., USA) was used for dynamic direct exposure of human cells to the test gas. Human cells on snapwell inserts were placed in horizontal diffusion chambers and exposed to various airborne concentrations of NO₂ directly at the air/liquid interface for 1 h at 37 °C. Cytotoxicity of the test gas was investigated using the MTS (Tetrazolium salt, Promega), NRU (neutral red uptake, Sigma) and ATP (Adenosine triphosphate, Promega) in vitro assays.

Section snippets

Chemical compounds

NO₂ (CAS No. 10102-44-0, 50 ppm, balanced in synthetic air) was purchased from Linde Gas Pty Ltd., Australia. The actual concentration of NO₂ was reported as 49.1 ppm (Method No. 81 FT-IR). Synthetic air was purchased from Linde Gas Pty Ltd., Australia. Chemicals and reagents for chemical analysis were obtained from Sigma (USA). In vitro assay reagents were purchased from Promega (USA) and Sigma (USA).

Cell types and culture conditions

Two human cells including epithelial lung carcinoma cell lines (A549, ATCC No. CCL-185) and

Effects of air flow rates on cell viability

Cell viability of human cells exposed to two different flow rates of synthetic air during 1 h exposure time using the MTS assay are presented in Table 2. After 1 h exposure of human cells to 25 ml/min synthetic air no statistically significant difference was observed between cell viability of both cells and incubator control cells. However, increasing the flow rate to 50 ml/min significantly reduced cell viability in both human cells tested (p < 0.05). At 25 ml/min flow rate no significant cell

Discussion

A dynamic in vitro model was established for direct exposure of human cells to gaseous airborne contaminants at the air/liquid interface using cultured cells on porous membranes in conjunction with a horizontal diffusion chamber system. Standard test atmospheres of selected gas model compound, NO₂, were generated at concentrations from 2.5 to 10 ppm using a dynamic direct dilution method. The cytotoxicity of NO₂ at workplace relevant concentrations was investigated in A549 human pulmonary type

Acknowledgments

This research was supported by a postgraduate scholarship (S. Bakand) from Iranian Ministry of Health and Medical Education. The authors would also like to thank Dr. Zhanhe Wu (Westmead Hospital, Sydney) for supplying the human cells and Dr. Paul Thomas (Department of Medicine, Prince of Wales Clinical School) and Dr. Maria Kavallaris (Experimental Therapeutics Program Children's Cancer Institute Australia for Medical Research) for providing of the A549 cell lines.

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An experimental in vitro model for dynamic direct exposure of human cells to airborne contaminants

Abstract

Introduction

Section snippets

Chemical compounds

Cell types and culture conditions

Effects of air flow rates on cell viability

Discussion

Acknowledgments

Toxicol. Lett.

Toxicology

Environ. Toxicol. Pharmacol.

Fundam. Appl. Toxicol.

Biochem. Biophys. Res. Commun.

Toxicol. Lett.

Toxicology

J. Pharmacol. Toxicol. Meth.

Exp. Toxicol. Pathol.

Toxicology

In vitro cytotoxicity testing of airborne formaldehyde collected in serum-free culture media

Toxicol. Ind. Health

Toxicity assessment of industrial chemicals and airborne contaminants: transition from in vivo to in vitro test methods: a review

Inhal. Toxicol.

A novel in vitro exposure technique for toxicity testing of selected volatile organic compounds

J. Environ. Monit.

Neutral red assay for toxicology in vitro

Air pollution and infection in respiratory illness

Br. Med. Bull.