Comparative in vitro cytotoxicity assessment of selected gaseous compounds in human alveolar epithelial cells.

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. However, the precise mechanisms that derive such effects are not always understood. Although inhalation studies are technologically complicated, correct hazard characterisation is essential for comparable risk assessment of inhaled materials. The aim of this study was to investigate the comparative in vitro cytotoxicity of selected gaseous contaminants in human lung cells. The cytotoxicity of nitrogen dioxide (NO(2)), sulphur dioxide (SO(2)) and ammonia (NH(3)) was investigated in A549- human pulmonary type II-like epithelial cell lines cultured on porous membranes in Snapwell inserts. A dynamic direct exposure method was established by utilizing the horizontal diffusion chamber system (Harvard Apparatus Inc, USA) for delivery of test atmospheres. Test atmospheres were generated using a dynamic direct dilution method and the concentration monitored by appropriate analytical methods. A diversified battery of in vitro assays including the MTS (tetrazolium salt; Promega), NRU (neutral red uptake; Sigma) and ATP (adenosine triphosphate; Promega) assays was implemented. Airborne IC(50) (50% inhibitory concentration) values were calculated based on the most sensitive assay for each test gas including NO(2) (IC(50)=11+/-3.54 ppm; NRU)>SO(2) (IC(50)=48+/-2.83 ppm; ATP)> and NH(3) (IC(50)=199+/-1.41 ppm; MTS). However, all in vitro assays revealed similar toxicity ranking for selected gaseous contaminants. Identical toxicity ranking was achieved using both in vitro and published in vivo data. This comparison suggests that results of in vitro methods are comparable to in vivo data and may provide greater sensitivity for respiratory toxicity studies of gaseous contaminants.

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

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 degrees 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.

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

Exposure to vapours of volatile chemicals is a major occupational and environmental health concern. Toxicity testing of volatile organic compounds (VOCs) has always faced significant technological problems due to their high volatility and/or low solubility. The aim of this study was to develop a practical and reproducible in vitro exposure technique for toxicity testing of VOCs. Standard test atmospheres of xylene and toluene were generated in glass chambers using a static method. Human cells including: A549-lung derived cell lines, HepG2-liver derived cell lines and skin fibroblasts, were grown in porous membranes and exposed to various airborne concentrations of selected VOCs directly at the air/liquid interface for 1 h at 37 degrees C. Cytotoxicity of test chemicals was investigated using the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and NRU (neutral red uptake) assays following 24 h incubation. Airborne IC(50) (50% inhibitory concentration) values were determined using dose response curves for xylene (IC(50)=5350+/- 328 ppm, NRU; IC(50)=5750+/- 433 ppm, MTS in skin fibroblast) and toluene (IC(50)=0 500+/- 527 ppm, NRU; IC(50)=11,200 +/- 1,044 ppm, MTS in skin fibroblast). Our findings suggest that static direct exposure at the air/liquid interface is a practical and reproducible technique for toxicity testing of VOCs. Further, this technique can be used for inhalational and dermal toxicity studies of volatile chemicals in vitro as the exposure pattern in vivo is closely simulated by this method.

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

Exposure to occupational and environmental contaminants is a major contributor to human health problems. Inhalation of gases, vapors, aerosols, and mixtures of these can cause a wide range of adverse health effects, ranging from simple irritation to systemic diseases. Despite significant achievements in the risk assessment of chemicals, the toxicological database, particularly for industrial chemicals, remains limited. Considering there are approximately 80,000 chemicals in commerce, and an extremely large number of chemical mixtures, in vivo testing of this large number is unachievable from both economical and practical perspectives. While in vitro methods are capable of rapidly providing toxicity information, regulatory agencies in general are still cautious about the replacement of whole-animal methods with new in vitro techniques. 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. In this review, current toxicity test methods for risk evaluation of industrial chemicals and airborne contaminants are presented. To evaluate the potential applications of in vitro methods for studying respiratory toxicity, more recent models developed for toxicity testing of airborne contaminants are discussed.

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

The purpose of this study was to identify a suitable sampling model for on-site toxicity assessment of soluble air contaminants such as formaldehyde, a well known industrial and indoor air contaminant. The in vitro cytotoxicity of formaldehyde, the selected model for soluble air contaminants, was studied using the MTS (tetrazolium salt) assay in two carcinoma cell lines, A549 epithelial lung and HepG2 hepatocarcinoma, and in skin fibroblasts. The cytotoxic effects of airborne formaldehyde were evaluated using test atmospheres in concentrations below 10 ppm (12.3 mg/m3), generated by a dynamic diffusion method and bubbled (0.3 L/min) through serum-free culture media for one or four hours. Human cells were treated with formaldehyde air samples, and cell viability was determined after four hours incubation. In parallel, the concentration of airborne formaldehyde was monitored, using the 3500 NIOSH method. Cell viability of the HepG2 cells exposed to formaldehyde air samples (8.75 ppm x 4 h) was reduced to less than 50% (31.6 +/- 1.24%). The HepG2 cell lines were found to be more sensitive (IC50 = 103.79 +/- 23.55 mg/L) to formaldehyde than both A549 cell lines (IC50= 198.36 +/- 9.54 mg/L) and skin fibroblasts (IC50 = 196.68 +/- 36.73 mg/L) (P < 0.01). An average of 96.8% was determined for collection efficiency of formaldehyde in serum-free culture media. The results of this study suggest that absorption of soluble air contaminants, such as formaldehyde, in serum-free culture media can be used as a suitable sampling model for on-site toxicity assessments.