Oxidative actions of hydrogen peroxide in human gingival and oral periosteal fibroblasts: Responses to glutathione and nicotine, relevant to healing in a redox environment()

BACKGROUND: This study aims to validate pro-oxidant actions of nicotine (N), using hydrogen peroxide (H(2)O(2)) and the antioxidant glutathione (G) in an in vitro model of human gingival fibroblasts (HGF) and human oral periosteal fibroblasts (HPF); radiolabelled androgens are used as biomarkers of redox status. Oxidative stress is an important mediator of inflammatory repair. The androgen metabolite 5α-dihydrotestosterone (DHT) is an effective biomarker of oxidative stress and healing. METHODS: 6 Cell-lines of HGF and HPF established in confluent monolayer culture were incubated in Eagle's MEM using 14C-testosterone and 14C-4-androstendione as substrate; in conjunction with effective concentrations of N, G and H(2)O(2) established at N250, G3 μg/ml and 3%H(2)O(2) w/w, 0.5 μl/ml. Combinations of H(2)O(2)G and H(2)O(2)GN were used in order to compare the oxidative effects of N/H(2)O(2) and their responses to glutathione. At 24 h, the medium was solvent extracted, evaporated to dryness and subjected to TLC in a benzene/acetone solvent system 4:1 v/v for the separation of metabolites. The separated metabolites were quantified using a radioisotope scanner. RESULTS: The mean trends of 6 cell-lines for both substrates and each cell type demonstrated that the yield of the main metabolite DHT was significantly reduced by N and H(2)O(2) alone (2-fold, n=6; p<0.01). The inhibition caused by H(2)O(2) was overcome by the antioxidant glutathione in the combination H(2)O(2)G, to values similar to those of controls (n=6; p<0.01). It is relevant that when N was added to this neutralized combination, the decrease in yields of DHT triggered by N were comparable to those induced by H(2)O(2); and retaining the positive effect of G. CONCLUSION: Oxidative stress mediated by H(2)O(2) was overcome by glutathione and recurred when nicotine was added, suggestive of a pro- oxidant role for nicotine. Androgen biomarkers are a sensitive index of oxidative stress which affects wound healing.