Benchmark Dose Modeling Approaches for Volatile Organic Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System

Abstract

Inhalation is the most relevant route of volatile organic chemical (VOC) exposure; however, due to unique challenges posed by their chemical properties and poor solubility in aqueous solutions, in vitro chemical safety testing is predominantly performed using direct application dosing/submerged exposures. To address the difficulties in screening toxic effects of VOCs, our cell culture exposure system permits cells to be exposed to multiple concentrations at air-liquid interface (ALI) in a 24-well format. ALI exposure methods permit direct chemical-to-cell interaction with the test article at physiological conditions. In the present study, BEAS-2B and primary normal human bronchial epithelial cells (pHBEC) are used to assess gene expression, cytotoxicity, and cell viability responses to a variety of volatile chemicals including acrolein, formaldehyde, 1,3-butadiene, acetaldehyde, 1-bromopropane, carbon tetrachloride, dichloromethane, and trichloroethylene. BEAS-2B cells were exposed to all the test agents, whereas pHBECs were only exposed to the latter 4 listed above. The VOC concentrations tested elicited only slight cell viability changes in both cell types. Gene expression changes were analyzed using benchmark dose (BMD) modeling. The BMD for the most sensitive gene set was within one order of magnitude of the threshold-limit value reported by the American Conference of Governmental Industrial Hygienists, and the most sensitive gene sets impacted by exposure correlate to known adverse health effects recorded in epidemiologic and in vivo exposure studies. Overall, our study outlines a novel in vitro approach for evaluating molecular-based points-of-departure in human airway epithelial cell exposure to volatile chemicals.

Keywords: in vitro; VOC; benchmark dose; cell culture exposure system; inhalation; transcriptomics.

Figure 1.

Schematic of CCES dilution manifold and exposure plates. A) Dynamic headspace generator design used for chemical generation and transport to the exposure manifold. B) The cell culture exposure system (CCES) includes a diffusion humidifier designed to regulate relative humidity levels in the dilution air before being passed through a dilution manifold. Each diluted chemical concentration is transported to a sampling port which allows real-time concentration analysis before being delivered to four wells containing inserts (n=4 per dose). For visual clarity, only tubing for the first three doses are shown. A separate control plate receives only humidified dilution air for the duration of the exposure. Both the VOC and sham exposure plates are enclosed in a warmed, humidified enclosure, while the incubator control remains in the 37°C 5% CO₂ cell culture incubator.

Figure 1.

Schematic of CCES dilution manifold and exposure plates. A) Dynamic headspace generator design used for chemical generation and transport to the exposure manifold. B) The cell culture exposure system (CCES) includes a diffusion humidifier designed to regulate relative humidity levels in the dilution air before being passed through a dilution manifold. Each diluted chemical concentration is transported to a sampling port which allows real-time concentration analysis before being delivered to four wells containing inserts (n=4 per dose). For visual clarity, only tubing for the first three doses are shown. A separate control plate receives only humidified dilution air for the duration of the exposure. Both the VOC and sham exposure plates are enclosed in a warmed, humidified enclosure, while the incubator control remains in the 37°C 5% CO₂ cell culture incubator.

Figure 2.

Dose response of each respective chemical as measured by LDH release presented as % Change in Cytotoxicity compared to the filtered air control and 100% lysed control. BEAS-2B and pHBEC cells exposed to chemicals A. Acrolein, B. 1,3-Butadiene, C. Acetaldehyde, D. Formaldehyde, E. 1-Bromopropane, F. Dichloromethane, G. Trichloroethylene, H. Carbon Tetrachloride. Mean±SEM, n=3, *p≤0.05.

Figure 3.

Dose response of each respective chemical as measured by CellTiter-Glo ATP availability and presented as % Cell Viability compared to the filtered air control. BEAS-2B and pHBEC cells exposed to chemicals A. Acrolein, B. 1,3-Butadiene, C. Acetaldehyde, D. Formaldehyde, E. 1-Bromopropane, F. Dichloromethane, G. Trichloroethylene, H. Carbon Tetrachloride. Mean±SEM, n=3, *p≤0.05.

Figure 4:

Bar plot of Gene and Gene Set level accumulation as a function of VOC test concentration for each cell type and chemical combination tested. Each bar represents the number of genes or gene sets that had a BMD less than or equal to the test concentration. Light grey and black shaded bars represent genes or gene sets (respectively) which meet all criteria and were analyzed while the dark grey portion of the bar represents those gene targets which were not annotated in the MSigDB gene set collection and did not contribute to gene set level BMDs. Sample size n=3 per test condition.

Figure 5.

Accumulation plots for the best BMD values for enriched gene sets for BEAS-2B cells (black squares) and pHBEC cells (grey circles). Each point represents the Median BMD for a gene set. Black dashed lines indicate the range of concentrations tested and the dotted line indicates the ACGIH TLV.