Combined α-tocopherol and ascorbic acid protects against smoke-induced lung squamous metaplasia in ferrets

Many epidemiological studies show the benefit of fruits and vegetables on reducing risk of lung cancer, the leading cause of cancer death in the United States. Previously, we demonstrated that cigarette smoke exposure (SM)-induced lung lesions in ferrets were prevented by a combination of low dose of β-carotene, α-tocopherol (AT), and ascorbic acid (AA). However, the role of a combination of AT and AA alone in the protective effect on lung carcinogenesis remains to be examined. In the present study, we investigated whether the combined AT (equivalent to ∼100 mg/day in the human) and AA (equivalent to ∼210 mg/day) supplementation prevents against SM (equivalent to 1.5 packs of cigarettes/day) induced lung squamous metaplasia in ferrets. Ferrets were treated for 6 weeks in the following three groups (9 ferrets/group): (i) Control (no SM, no AT + AA), (ii) SM alone, and (iii) SM + AT + AA. Results showed that SM significantly decreased concentrations of retinoic acid, AT, and reduced form of AA, not total AA, retinol and retinyl palmitate, in the lungs of ferrets. Combined AT + AA treatment partially restored the lowered concentrations of AT, reduced AA and retinoic acid in the lungs of SM-exposed ferrets to the levels in the control group. Furthermore, the combined AT + AA supplementation prevented SM-induced squamous metaplasia [0 positive/9 total ferrets (0%) vs. 5/8 (62%); p < 0.05] and cyclin D1 expression (p < 0.05) in the ferret lungs, in which both were positively correlated with expression of c-Jun expression. Although there were no significant differences in lung microsomal malondialdehyde (MDA) levels among the three groups, we found a positive correlation between MDA levels and cyclin D1, as well as c-Jun expressions in the lungs of ferrets. These data indicate that the combination of antioxidant AT + AA alone exerts protective effects against SM-induced lung lesions through inhibiting cyclin D1 expression and partially restoring retinoic acid levels to normal.