Cytotoxicity of NO 2 gas to cultured human and murine cells in an inverted monolayer exposure system

Abstract

We report the development of an optimised exposure system for the exposure of inverted cell cultures to NO 2, which presents several advantages over conventional, right-side-up exposure systems. Firstly, the cells may be directly exposed to NO 2 in the gas phase for up to 1 h, without the interposition of an aqueous layer. Secondly, the chamber system allows simple and precise control of the gas concentration during the exposure. Finally, the system allows the simultaneous exposure of large numbers of cells under sterile conditions, facilitating further culture of the cells after the exposure period. We report the application of this system to a comparative study of the toxicity of NO 2 in three different cell types involved in the circuit of the inflammatory response, the IC-21 murine macrophage line, the A-549 human pulmonary type II-like epithelial cell line and human umbilical vein endothelial cells. As little as 2 ppm NO 2 for 20 min reduced colony-forming efficiency of HUVE cells and A-549 cells to 35% and 78% of their air controls, respectively. Exposure to 5 ppm NO 2 for 1 h increased lactate dehydrogenase release of HUVE cells, IC-21 macrophages and A-549 cells from 7.9% to 21.6%, 5.7% to 10.9% and 2.0% to 3.4%, respectively, whilst 10 ppm NO 2 for 1 h lowered cellular glutathione in HUVE cells, IC-21 cells and A-549 cells from 35.2 nmol/mg to 23.3 nmol/mg, from 45.0 nmol/mg to 31.0 nmol/mg and from 86.4 nmol/mg to 69.2 nmol/mg, respectively. Of the cell types tested it was shown that HUVE cells and IC-21 cells were equally sensitive to the toxicity of NO 2 whilst A-549 cells displayed considerable resistance, perhaps due to the considerably higher levels of glutathione in this cell line. Further, a comparison of the sensitivity of HUVE cells to NO 2 using several modes of exposure (inverted and right-side-up (either rocked or static)) and the assay of lactate dehydrogenase and [ 3H]deoxyglucose release, revealed that the present inverted exposure technique potentiated the acute cytotoxicity of the gas.