Citation:

Winters, B., M. Angrish, A. Wallace, J. Pleil, AND M. Madden. Metabolomics Approach for Toxicity Screening of Volatile Substances. Society of Toxicology, San Antonio, TX, March 11 - 15, 2018.

Impact/Purpose:

We primarily propose to establish and validate a HTS with in vitro assays mainly dependent on use of the detection of gas phase biomarkers rather than markers in liquid (i.e. media, buffers) or the cells themselves. This will be implemented by providing cells with a substrate (i.e. probe) which, when catabolized (presumably by a cytochrome P450 (CYP450) enzyme), will produce a gaseous metabolite. Production of the metabolite (preferably at steady state) will be the endpoint used to examine the toxicity of test substances such as a pharmaceutical agent.

Description:

In 2007 the National Research Council envisioned the need for inexpensive, high throughput, cell based toxicity testing methods relevant to human health. High Throughput Screening (HTS) in vitro screening approaches have addressed these problems by using robotics. However, the challenge is that many of these chemicals are volatile and not amenable to HTS robotic liquid handling applications. We assembled an in vitro cell culture apparatus capable of screening volatile chemicals for toxicity with potential for miniaturization for higher throughput. BEAS-2B lung cells were grown in an enclosed culture apparatus under air-liquid interface (ALI) conditions, and exposed to an array of xenobiotics in 5% CO2. Use of ALI conditions allows direct contact of cells with a gas xenobiotic, as well as release of endogenous gaseous molecules without interference by medium on the apical surface. To identify potential xenobiotic-induced perturbations in cell homeostasis, we monitored for alterations of endogenously-produced gaseous molecules in air directly above the cells, termed “headspace”. Alterations in specific endogenously-produced gaseous molecules in headspace is indicative of xenobiotic-induced perturbations of specific cellular processes. Additionally, endogenously produced volatile organic compounds (VOCs) may be monitored in a nonspecific, discovery manner to determine whether cell processes are perturbed by xenobiotic exposure, potentially producing specific patterns of changes. Preliminary results indicate the novel cell culture system is capable of detecting gaseous products of specific enzyme pathways such as CYP2A6. Numerous VOCs were detected in the headspace of cells grown in the novel cell culture apparatus. The system is being optimized to minimize artifact volatiles using different materials. We believe our novel cell culture apparatus, once refined and validated, will allow for screening of both volatile and non-volatile xenobiotics by measuring cellular responses detected as alterations in gaseous molecules released by cells. [This abstract may not reflect official US EPA policy.]

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