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The oxidative effect of nicotine was investigated using androgen biomarkers of redox status and wound healing in fibroblasts; using the antioxidant glutathione for confirmation of responses. Cultures of human gingival (HGF) and periosteal fibroblasts (HPF) were incubated with substrates 14C-testosterone/14C-4-androstenedione in the presence or absence of serial concentrations of nicotine (N100-500), glutathione (G1–5) and their combinations, in medium. At 24 h the medium was solvent extracted for metabolites, separated by TLC and quantified using radioisotope scanning. Nicotine caused significant inhibition in yields of the physiologically active metabolite 5α-dihydrotestosterone (DHT) in HGF and HPF, overcome to varying degrees by the anti-oxidant glutathione (n = 6; p<0.01, one way ANOVA); this is suggestive of moderation of an oxidative mechanism induced by nicotine. Down-regulation of 5α-reductase activity by nicotine resulting in reduced yields of DHT was overcome by glutathione. Overcoming oxidative stress in a redox environment is applicable to treatment outcome.

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BackgroundThis study aims to validate pro-oxidant actions of nicotine (N), using hydrogen peroxide (H2O2) 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.Methods6 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 H2O2 established at N250, G3 μg/ml and 3%H2O2 w/w, 0.5 μl/ml. Combinations of H2O2G and H2O2GN were used in order to compare the oxidative effects of N/H2O2 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.ResultsThe 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 H2O2 alone (2-fold, n=6; p<0.01). The inhibition caused by H2O2 was overcome by the antioxidant glutathione in the combination H2O2G, 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 H2O2; and retaining the positive effect of G.ConclusionOxidative stress mediated by H2O2 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.

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