Hyperoxia-induced ciliary loss and oxidative damage in an in vitro bovine model: The protective role of antioxidant vitamins E and C

Although elevated oxygen fraction is used in intensive care units around the world, pathological changes in pulmonary tissue have been shown to occur with prolonged exposure to hyperoxia. In this work a bovine bronchus culture model has been successfully used to evaluate the effects of hyperoxia on ciliated epithelium in vitro. Samples were cultured using an air interface method and exposed to normoxia, 21% O2 or hyperoxia, 95% O2. Cilial coverage was assessed using scanning electron microscopy (SEM). Tissue damage (lactate dehydrogenase, LDH, in the medium), lipid peroxidation (thiobarbituric acid reactive substances, TBARS), DNA damage (comet assay), protein oxidation (OxyBlot kit) and antioxidant status (total glutathione) were used to assess whether the hyperoxia caused significant oxidative stress. Hyperoxia caused a time-dependent decline (t½ = 3.4 d compared to 37.1 d under normoxia) in cilial coverage (P < 0.0001). This was associated with a significant increase in the number of cells (2.80 ± 0.27 × 106 compared to 1.97 ± 0.23 × 106 ml−1 after 6 d), many apparently intact, in the medium (P < 0.05); LDH release (1.06 ± 0.29 compared to 0.83 ± 0.36 μmol min−1 g−1 after 6 d; P < 0.001); lipid peroxidation (352 ± 16 versus 247 ± 11 μmol MDA g−1 for hyperoxia and normoxia, respectively); % tail DNA (18.7 ± 2.2 versus 11.1 ± 1.5); protein carbonyls (P < 0.05); and total glutathione (229 ± 20 μmol g−1 versus 189 ± 15 μmol g−1). Vitamins E (10−7 M) and C (10−6 or 10−7 M) alone or in combination (10−7 M and 10−6 M, respectively) had a significant protective effect on the hyperoxia-induced reduction in percentage cilial coverage (P < 0.05). In conclusion, hyperoxia caused damage to cultured bovine bronchial epithelium and denudation of cilia. The antioxidant vitamins E and C significantly protected against hyperoxia-induced cilia loss.

► A new bovine bronchial model for studying hyperoxia-induced cilia loss is presented.
► Hyperoxia-induced cilia loss was associated with increased sloughing of cells.
► Hyperoxia led to higher epithelial glutathione levels, evidence of oxidative stress.
► Hyperoxia led to increased DNA damage (Comet), and lipid peroxidation (TBARS).
► Vitamins C and E partially protected against hyperoxia-induced cilia loss.