title: Oxidative actions of hydrogen peroxide in human gingival and oral periosteal fibroblasts: responses to glutathione and nicotine, relevant to healing in a redox environment.
abstract: BACKGROUND
  This 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.
  
  
  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 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.
  
  
  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 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.
  
  
  CONCLUSIONS
  Oxidative 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.
authors.affiliation: King's College London Dental Institute, Guy's Dental Hospital, London SE1 9RT, UK.
authors.forename: Federico
authors.initials: F
authors.lastname: Tinti
authors.affiliation: King's College London Dental Institute, Guy's Dental Hospital, London SE1 9RT, UK.
authors.forename: Mena
authors.initials: M
authors.lastname: Soory
mesh_terms: D043582:5-alpha-Dihydroprogesterone / Q000494:pharmacology; D000328:Adult; D000735:Androstenedione / Q000032:analysis / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000737:chemistry; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005881:Gingiva / Q000166:cytology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity*; D008875:Middle Aged; D009538:Nicotine / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects*; D010521:Periosteum / Q000166:cytology; D013379:Substrate Specificity; D013739:Testosterone / Q000032:analysis / Q000378:metabolism
chemical_list: D002250:Carbon Radioisotopes; D013739:Testosterone; D000735:Androstenedione; D009538:Nicotine; D043582:5-alpha-Dihydroprogesterone; D006861:Hydrogen Peroxide; D005978:Glutathione
keywords: Glutathione; Hydrogen peroxide; Nicotine; Oxidative stress; Redox markers; Wound healing