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Toxicity Screening of Volatile Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System
Citation:
Zavala-Mendez, J., R. Collins, L. Dailey, N. Hanley, A. Ledbetter, Todd Krantz, P. Evansky, S. McCullough, J. Harrill, AND M. Higuchi. Toxicity Screening of Volatile Chemicals Using a Novel Air-Liquid Interface In Vitro Exposure System. NC Society of Toxicology Fall Meeting, RTP, NC, October 30, 2017.
Impact/Purpose:
The objective of this study is to evaluate the capability of the transcriptomic data to identify concentration-dependent changes in mechanism/mode-of-action for volatile chemicals and evaluate the ability of the transcriptomic data to group chemicals by similar bioactivity profiles for potential grouping and read across applications.
Description:
Traditional in vitro dosing methods require, for example, the addition of particulate matter (PM), PM extracts, or chemicals in dimethyl sulfoxide (DMSO) or water into cell culture medium. However, about 10% of chemicals nominated for study in the U.S Environmental Protection Agency’s (EPA) Toxic Substances Control Act (TSCA) chemical substance inventory are insoluble in DMSO, water, or are volatile, thus their toxicity cannot be adequately tested using traditional in vitro dosing methods. To circumvent the difficulties in screening for volatile/insoluble chemicals, we developed a new cell culture exposure system (CCES) that permits cells to be exposed at an air-liquid interface (ALI) in both a 6-well and 24-well format. The ALI method permits a direct pollutant-to-cell interaction in which the test substance is in its natural state, thus providing a more realistic exposure scenario (Figure 1). We validated the CCES by exposing primary normal human bronchial epithelial (NHBE) cells to 0.3 ppm of acrolein at EPA’s Inhalation Toxicology Facility (ITF) located in Research Triangle Park, NC and comparing our results to that of cells exposed using the standard incubator exposure chamber system housed at EPA’s Human Studies Facility (HSF) located in Chapel Hill, NC. The HSF chambers system, widely used since 1991, served as our benchmark and we showed that the CCES provided similar biological responses (Figure 2). The key advantage of the CCES is that by accommodating the multi-well plates, we minimize the handling of Transwell inserts and improve turnaround times between back-to-back exposures because no maintenance or cleaning is needed between testing. Additionally, this novel system is capable of testing 6 different chemical concentrations simultaneously to generate concentration-response curves. Our on-going pilot study, in collaboration with the National Center for Computational Toxicology (NCCT) and the Chemical Safety for Sustainability (CSS) research programs at EPA, uses the BEAS-2B cell line and primary normal human bronchial epithelial (NHBE) cells to assess the toxicity of 10 chemicals in the TSCA work plan; 1,3-butadiene, acrolein, and formaldehyde have been tested to date. Using the setup shown in Figure 3, we exposed cells for 2 h to 6 concentrations in half-log dilutions, plus an air (vehicle) control. Cell viability is assessed 4 h post-exposure via the CellTiter-Glo Assay (Figure 4). Cell lysates are also collected 4 h post-exposure for whole transcriptome targeted RNA-Sequencing (i.e. BioSpyder TempO-Seq). The objective of this study is to evaluate the capability of the transcriptomic data to identify concentration-dependent changes in mechanism/mode-of-action for volatile chemicals and evaluate the ability of the transcriptomic data to group chemicals by similar bioactivity profiles for potential grouping and read across applications.