In Vitro Effects of Hyperoxia on Alveolar Type II Pneumocytes: Inhibition of Glutathione Synthesis Increases Hyperoxic Cell Injury

Abstract

An in vitro model of alveolar epithelial oxidant injury was developed based on exposure to hyperoxia of cultured guinea pig type II pneumocytes using a biphasic cell culture system in aerobiosis. The present study investigates the roles of intracellular antioxidant enzymes and of glutathione in providing protection against hyperoxia. A 2-day type II cell culture in normoxia was associated with a significant decrease in protein, catalase, and Cu-Zn SOD cell content, whereas ATP cell content, Mn-SOD, and glutathione peroxidase (GPx) activities did not change and glutathione cell content significantly increased. Exposure of type II cells to hyperoxia did not induce significant changes in cell content in protein, SOD, catalase, GPx, or glutathione cell content when compared to control cells (exposed to normoxia). With ATP cell content expressed as a cell injury index (CII), type II cell injury was found to increase with increasing O2 concentrations. Indeed, a 2-day 50% O2 and 95% O2 exposure resulted in a CII of -7.5 +/- 6.2% and 17.9 +/- 5.9%, respectively, LDH release by type II cells was not significantly increased after hypoxic exposure. Cell injury effects of hyperoxia did not correlate with the endogenous antioxidant enzyme activities (SOD, Mn-SOD, catalase). In marked contrast, there was a significant correlation between the CII and total glutathione content of type II cells (p < .01). This correlation was largely due to the close relationship between CII and reduced glutathione. Hyperoxic induced cell injury (as demonstrated by CII > 0) was clearly associated with significantly lower intracellular glutathione level when compared to experiments without hyperoxia induced cell injury (CII < 0). In addition, in the presence of buthionine sulfoximine (BSO), the ability of type II cells to synthetize new glutathione was severely impaired, whereas ATP cell content and cell antioxidant enzyme activities did not change. As a consequence, the reduction of intracellular glutathione significantly increased the susceptibility of cells to hyperoxia injury (p < .05). The results strongly support the hypothesis that the regulation of glutathione levels is an important mechanism in protecting hyperoxia-induced type II cell injury.

Thiol regulation of the production of TNF-α, IL-6 and IL-8 by human alveolar macrophages

“…In other culture wells, 0.5 mM BSO was added, a concentration previously shown to be optimal for GSH inhibition [20]; BSO had no cytotoxic effect at this concentration as defined by lactate dehydrogenase (LDH) release (data not shown). The cultures treated with BSO, GSH and NAC were either activated simultaneously or pre-incubated at 378C (for 3 and 24 h with BSO; for 24 h with GSH and NAC) before the stimulation.…”

Comparative in vitro toxicity of cadmium and lead on redox cycling in type II pneumocytes

“…The presence of glutathione is a prerequisite for the integrity of cell membranes. 9 Cadmium and lead caused permanent damage to the cell membranes, with disturbance of the structural oligosaccharide components in the membranes of type II cells. There is increasing evidence that oxygen radical formation catalysed directly or indirectly by cadmium or lead is a key factor underlying cytotoxic and cell activation responses.…”

“…6 As is known, type II cells synthesize and secrete the alveolar surfactant, replace the more easily damaged type I alveolar cells, take part in glutathione synthesis 7,8 and maintain the redox stability (superoxide dismutase) of these cells. 9 In spite of this, one of the main sites where xenobiotics exert their effect is the alveolar epithelium. There are no comprehensive data on lead and cadmium toxicity in type II pneumocytes 10 but recently much interest has been focused on developing in vitro toxicity tests and evaluating their usefulness in predicting toxicities observed in vivo.…”

The effect of stone-wool on rat lungs and on the primary culture of rat alveolar macrophages and type II pneumocytes

“…(ii) The presence of stone-wool particles was detected by light and scanning electron microscopy and the particles in the lungs were examined by energy dispersive x-ray analysis (EDXA) comparing their composition with that of natural stone-wool particles (Brody, 1984). (iii) The effect of stone-wool on some enzymes, the activity of gamma-glutamyl transpeptidase (surface marker of type II cells -Hook, 1978;Klaveren et al, 1997), GSH-Px and GSH-Rd were determined (Kelly, 2002;Rahman and McNee, 2000;Aerts et al, 1992;Weller et al, 1997). Further, cytoplasmic Cu,Zn/SOD activity (Holley et al, 1992;Marks-Konczalik et al, 1998) and the activity of alkaline phosphatase (Richards et al, 1987) were measured.…”