Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells

Abstract

Murine macrophage tumor cells infected with Leishmania mexicana were exposed to the antimycotic drug ketoconazole and to [2- 14C]mevaonate, then the amastigotes were isolated, collected, purified, and their free sterols were analyzed by chromatographic and mass spectrometric methods. Control amastigotes contained as products of de novo biosynthesis C 28 4-desmethyl sterols (episterol, 5-dehydroepisterol), C 29 4-desmethyl sterols (stigmasta-7,24(28)-dien-3β-ol, stigmasta-5,7,24(28)-trien-3β-ol), 4-methyl sterols (4α, 14α-dimethylzymosterol, obtusifoliol) and a 4,4-dimethyl sterol (lanosterol). Present also were macrophage sterols (cholesterol, desmosterol) and a putative product of the C-24 alkylation of demosterol by amastigotes (24-methylenecholesterol). Amastigotes from macrophages exposed to ketoconazole showed notable changes in the proportions, concentrations and specific activities of their free sterols; increased for 4α,14α-dimethylzymosterol and decreased for the endogenous C 28 and C 29 4-desmethyl sterols. Such changes were observed at a ketoconazole concentration as low as 0.01 μg ml −1. By contrast, uninfected macrophages accumulated only small amounts of lanosterol of high specific activity at a ketoconazole concentration of 10 μg ml −1. The ketoconazole-induced alterations in amastigote sterols parallel those previously reported in fungi and L. m. mexicana promastigotes, and suggest a biochemical mechanism for the anti-leishmanial activity of the drug in which changes in sterol composition are linked to disturbances of cell membrane structure and function, and hence to cytotoxicity.