Endocrine Abstracts

The aim of this investigation is to establish the effect of alkaline phosphatase on androgen metabolism in human gingival fibroblasts (obtained local Ethical Committee approval) and its possible mediation by the alkaline phosphatase inhibitor levamisole and nicotine, using radiolabelled testosterone as substrate. Monolayer cultures of fibroblasts were established in Eagle's MEM. Duplicate incubations were performed in multiwell plates with 14C-testosterone as substrate, in the presence or absence of testing agents as follows: serial concentrations of nicotine (N100, 500 & 1000 microgram / ml), an optimal concentration of alkaline phosphatase ( AP 3 units / ml, as previously established) and an optimal concentration of the alkaline phosphatase inhibitor levamisole (L30 micrograms / ml). Combined incubations of AP with N100, 500 and 1000 were also performed followed by further combinations of APN with L30, in order to confirm specific inhibition by the alkaline phosphatase inhibitor levamisole. At the end of a 24h incubation period, the medium was solvent extracted with ethyl acetate for metabolites, evaporated to dryness, subjected to thin layer chromatography for separation of metabolites and quantified using a radioisotope scanner. The substrate testosterone was metabolised mainly to 5alpha dihydrotestosterone (DHT) and 4-androstenedione. Nicotine caused progressive inhibition of the synthesis of DHT and 4-A from testosterone with increasing concentrations, of up to 3-fold (n=4; p<0.01). AP stimulated the yields of DHT and 4-A by 30-33% (n=4; p<0.01), while L caused significant inhibition of 33% - 2.6-fold (n=4; p<0.01). Nicotine reduced the stimulatory effects of alkaline phosphatase by 1.7-3.4-fold, with further reduction in combination with levamisole of 1.6-2-fold for yields of DHT and 28% for 4-A (n=4; p<0.01). These results indicate that nicotine impairs the turnover of physiologically active androgen metabolites which may be instrumental in affecting matrix synthetic capacity in fibroblasts. The stimulatory effect of alkaline phosphatase on androgen metabolism and its inhibition by levamisole in combination with nicotine, reinforce the relevance of alkaline phosphatase in this mechanism.