This is an investigation of potential oxidative effects of AGE in response to the antioxidant glutathione in cultured human gingival fibroblasts, using radiolabelled steroids as substrates and their product 5 alpha dihydrotestosterone (DHT) as a marker of wound healing.
Confluent monolayer cultures of human gingival fibroblasts were incubated with 14C-Testosterone and 14C-4-androstenedione respectively in Eagles Minimum essential medium with serial concentrations of AGE at 10–200 micrograms/ml. The optimal effective concentrations of 50 and 100 micrograms/ml were used subsequently, alone and in combination with an effective concentration of 3 micrograms/ml of glutathione (G3), established previously. At the end of a 24 h incubation period, the medium was solvent extracted for metabolites, evaporated to dryness and subjected to thin layer chromatography in a benzene acetone solvent system 4:1 for their separation and quantified using a radioisotope scanner.
The optimal inhibitory effect of AGE on DHT synthesis was 25–50% at 50 and 100 micrograms/ml (n=4; p<0.01) and 30% in a further study in response to AGE 50 and 100 micrograms/ml (n=6; p<0.01); this was overcome by G3 resulting in 60% increases over yields in response to AGE and 30% greater than controls (n=6; p<0.01). G3 alone resulted in yields of DHT similar to those of controls. When 14C-4-A was used as substrate, there was an approximately 50% reduction in the yields of DHT at AGE 50/100 (n=6; p<0.05). In the combined incubation with G3, there were 45% & 62% increases over AGE 50/AGE 100 alone (n=6; p<0.05). Yields of diols showed an inverse relationship in response to AGE and the combinations.
Effective concentrations of AGE seem to have significant inhibitory effects on DHT synthesis, overcome by the antioxidant glutathione suggestive of an oxidative mechanism.
07 - 09 Nov 2005
Society for Endocrinology