Progress in assessing air pollutant risks from in vitro exposures: matching ozone dose and effect in human airway cells

Abstract

In vitro exposures to air pollutants could, in theory, facilitate a rapid and detailed assessment of molecular mechanisms of toxicity. However, it is difficult to ensure that the dose of a gaseous pollutant to cells in tissue culture is similar to that of the same cells during in vivo exposure of a living person. The goal of the present study was to compare the dose and effect of O3 in airway cells of humans exposed in vivo to that of human cells exposed in vitro. Ten subjects breathed labeled O3 ((18)O3, 0.3 ppm, 2 h) while exercising intermittently. Bronchial brush biopsies and lung lavage fluids were collected 1 h post exposure for in vivo data whereas in vitro data were obtained from primary cultures of human bronchial epithelial cells exposed to 0.25-1.0 ppm (18)O3 for 2 h. The O3 dose to the cells was defined as the level of (18)O incorporation and the O3 effect as the fold increase in expression of inflammatory marker genes (IL-8 and COX-2). Dose and effect in cells removed from in vivo exposed subjects were lower than in cells exposed to the same (18)O3 concentration in vitro suggesting upper airway O3 scrubbing in vivo. Cells collected by lavage as well as previous studies in monkeys show that cells deeper in the lung receive a higher O3 dose than cells in the bronchus. We conclude that the methods used herein show promise for replicating and comparing the in vivo dose and effect of O3 in an in vitro system.

Keywords: bronchoalveolar lavage; epithelial cells; extrapolation; in vivo versus in vitro dose; ozone.

FIG. 1.

Flow chart of the experimental design. The study involved both an in vivo and an in vitro exposure to ¹⁸O₃ of human epithelial cells. Bronchoalveolar lavage cells and constituents were also measured for comparison with the epithelial cells. Biological effects of O₃ consisted of measurements of IL-8 and COX-2 gene expression in cells harvested after exposure to unlabeled O₃. Cells were sampled 1 h after in vivo exposures and immediately following the in vitro exposures.

FIG. 2.

In vitro and in vivo dose comparison: ¹⁸O incorporation into bronchial brush biopsies collected from human subjects exposed for 2 h to 0.3 ppm ¹⁸O₃ while exercising intermittently or human primary epithelial cells exposed in vitro to a range of ¹⁸O₃ concentrations (mean ± SE). Standard error bars are within the data point for the in vivo data (N = 10 human subjects for in vivo data and 5 culture wells derived from two replicate in vitro exposures to each ¹⁸O₃ concentration for in vitro data). Each mean shown is significantly elevated (* denotes p < 0.05) relative to natural background ¹⁸O (zero ¹⁸O₃).

FIG. 3.

In vitro and in vivo biological effects comparison of O₃ exposures under conditions similar to the dose comparison in Figure 2. Shown are the fold increases in expression of IL-8 or COX-2 genes (means ± SE) in bronchial brush biopsies obtained from nine human subjects exposed in vivo to O₃ compared with their response following exposure to clean air. Also shown are fold increases of primary epithelial cells exposed in vitro to each O₃ concentration (means ± SE for three subjects and three replicates per subject) compared with their response following exposure to clean air. Asterisks denote significance (p < 0.05).

FIG. 4.

Comparison of ¹⁸O incorporation into epithelial cells (shown in Fig. 2) and BALF constituents of the same subjects following exposure to ¹⁸O₃ (0.3 ppm, 2 h with intermittent exercise). Means ± SE are shown for 10 subjects. All ¹⁸O₃ exposed tissues had significantly elevated ¹⁸O when compared with the natural abundance ¹⁸O concentration of blood plasma.