An Alternative Gas-phase In Vitro Exposure System for Toxicity Testing: The Interaction Between Nitrous Oxide and A549 Cells

Abstract

An original in vitro approach was adopted to expose cells to volatile agents. The anaesthetic nitrous oxide (N₂O) was chosen as the model agent, and type II pneumocyte-like cells (A549 cells) were used as the target to represent the lungs. A time-lapse microscopy station was equipped with a manual gas mixer that allowed the generation of a mixture of N₂O/air/CO₂ in the gas phase, to provide a uniform distribution of the volatile agent. The dissolution of N₂O in the culture medium was monitored by gas chro-matography–electron capture detection. Biochemical alterations, in terms of homocysteine accumulation, demonstrated that intracellular methionine synthase had been inactivated by N₂O absorbed by the cells, a process that also occurs in vivo. Toll-like receptors, which are key molecules in inflammatory lung diseases, were also investigated at the molecular level. Our experiments indicated that biochemical and molecular alterations occurred in the cells, even under conditions where neither morphologic changes nor consistent alterations in cell proliferation were evident. This in vitro exposure system can be efficiently adopted for looking at the repeat-dose effects of volatile agents on respiratory tissues. Moreover, it could be of further benefit for identifying the wide range of specific cell targets, and for monitoring relevant endpoints in the cellular and molecular processes that occur during exposure to volatile compounds.