The use of human gingival fibroblasts in culture for studying the effects of phenytoin on testosterone metabolism

Abstract

In initial experiments, monolayer cultures of human gingival fibroblasts from healthy male and female subjects were incubated for various time intervals with [4- 14C]-testosterone. This was rapidly taken up by the cells to reach 1.8 fmol/50,000 cells by 2 h. At 6, 12 and 24 h, the values were considerably lower (0.1–0.2 fmol/50,000 cells). In order to maintain a sufficient intracellular concentration of testosterone, unlabelled testosterone was incubated in the presence of [ 14C]-testosterone. This gave optimum yields of metabolites, which were separated by thindashlayer chromatography and provisionally identified by comparison of their mobilities with those of authentic steroids. Final characterization of 5α-dihydrotestosterone was achieved by combined capillary gas chromatography-mass spectrometry. The metabolites of testosterone were 5α-dihydrotestosterone (5α-DHT), 4-androstenedione, 5α-androstanedione and 5α-androstanediols, but the quantities formed varied with different cell lines. A similar pattern of metabolites was noted for minced human gingival tissue. Low concentrations of phenytoin generally increased the production of 5α-DHT and 4-androstendione but there were marked variations between individual cell lines with regard to the magnitude of stimulation. Higher concentrations of phenytoin generally caused inhibition of steroid formation but the concentration required for this again varied with different cell lines. Thus human gingival fibroblasts in culture provide a suitable model for the study of testosterone metabolism and of the effects of drugs such as phenytoin. Variation in these effects may be reflected in individual susceptibility to phenytoindashinduced gingival overgrowth.