Modulation of the effects of glucose on fibroblasts by glutathione

Objectives: A hyperglycaemic environment can impair healing responses in fibroblasts due to superoxide effects. Physiologically active androgen metabolites are biomarkers of repair potential. This is an investigation of the modulation of a hyperglycaemic response in fibroblasts by the antioxidant glutathione using steroid hormone biomarkers of healing. Methods: Confluent monolayer cultures of human gingiva fibroblasts derived from four patients were established in 24 well multiwell plates and incubated in Eagle’s MEM for 24h with serial concentrations of glucose at 500-3000 micrograms / ml. Two radiolabelled androgen substrates 14C-testosterone and 14C-4-androstenedione were used. The optimal inhibitory concentration of glucose was established (G1000) and used for subsequent experiments, alone and in combination with serial concentrations of the antioxidant glutathione at 1-4 microrograms / ml, for each substrate. The medium was then solvent extracted for steriod metabolites, evaporated to dryness and subjected to thin layer chromatography in a benzene acetone solvent system 4:1 v/v for separation of formed metabolites. The metabolites were quantified, using a radioisotope scanner. Results: The main metabolites formed in response to glucose were 5-alpha dihydrotestosterone (DHT), 4-androstenedione or testosterone from each substrate. There were 2-3-fold reductions in the yields of DHT (n=4; p< 0.01 Wilcoxon signed rank statistic for paired observations) in response to G1000 for the two substrates used. In combined incubations using glutathione and two substrates, inhibition of 1.5-2-fold caused by glucose was overcome in combination with glutathione reaching control values or 25% greater than controls at G1-4 (n=4; p<0.01). The other weaker metabolites showed an inverse relationship. Conclusion: Glucose had a significant inhibitory effect on the yields of the androgen biomarker DHT, which was overcome by the antioxidant glutathione. This was suggestive of a possible oxidative effect of glucose on fibroblasts affecting biomarkers of healing, with possible implications on healing in uncontrolled diabetics. 298 words.