Examining the Effects of Volatile Agents Using Cell Based Gaseous Metabolite Production
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Many agents (e.g., pollutants, pharmaceuticals, additives) requiring toxicity testing are gaseous and cannot be screened adequately in standard cell exposure systems. Submerged cell culture systems impede delivery of gases to cells due to media interference. Gas-media component reactions could form toxic products. We are using an exposure system and cell line (BEAS-2B human airway epithelial cell line) that can tolerate a lack of medium on the apical cell side for potential screening of gaseous agents minimizing the confounding effects of media. The system also can examine the conversion of an exogenously added volatile substrate to a gaseous metabolite. Monitoring production of a gas phase metabolite can be utilized to screen metabolism perturbations induced by an agent of interest (i.e., “probe molecule approach” to toxicity testing). Cells were incubated with 1 or 2% ethanol (EtOH) for up to 6 hr without the loss of cell viability (using released LDH activity and trypan blue dye exclusion). In a 37oC flow through system (with air plus 5% CO2 inflow), EtOH was added apart from the cell culture dishes. Acetaldehyde (C2) was collected onto dinitrophenylhydrazine packed cartridges on the outflow line. C2 was eluted from the cartridges and analyzed by LC\UV\MS. With EtOH exposures alone, increased cell C2 production was observed compared to vehicle controls (0% EtOH). The effects of pharmaceutical and flavoring agents on EtOH-induced C2 production were then examined. Cells pretreated with disulfiram (to inhibit C2 catabolism) and exposed to EtOH had increased C2 compared to untreated cells. Co-incubation of cells with EtOH and an e-cigarette flavoring component, cinnamaldehyde, decreased C2 production suggesting effects on alcohol dehydrogenase and/or other enzymes. These findings show the utility of this exposure system for measuring effects of different classes of agents on the metabolism by an important enzyme pathway. The use of this flow through gas system shows promise for 1) the ability to expose cells to volatile substances and preserve cell viability for several hours, and 2) capture a gas phase metabolite for use in probe molecule approaches for screening the induction of toxicity by gaseous agents. The use of in vitro substrate metabolism for chemical toxicity screening may reflect in vivo metabolism responses induced by exogenous chemicals. [This abstract may not reflect official US EPA policy.]