Inhibition of fungal lanosterol-14 alpha-demethylase (CYP51) is the working principle of the antifungal activity of azoles used in agriculture and medicine. Inhibition of human CYP51 may result in endocrine disruption since follicular fluid-meiosis activating steroid (FF-MAS), the direct product of lanosterol demethylation, is involved in the control of meiosis. To investigate the specificity of antifungal agents for the fungal enzyme, assays to determine inhibitory potencies of 13 agricultural fungicides and 6 antimycotic drugs were established. FF-MAS product formation was measured by LC-MS/MS analysis in the incubations using lanosterol as substrate. Recombinant human enzyme (hCYP51) was available from BD Gentest. CYP51 of Candida albicans (cCYP51) was co-expressed with Candida tropicalis oxidoreductase in the baculovirus system. IC(50) values of 13 fungicides for cCYP51 ranged about six-fold (0.059-0.35 microM); for hCYP51 the range was about 30-fold (1.3-37.2 microM). The most favourable IC(50) ratio human to Candida was observed for imazalil (440-fold), while the specificity of epoxiconazole and tebuconazole for cCYP51 was only by a factor of 10. For the antimycotic drugs, the range of IC(50) values for cCYP51 was similar to those of fungicides (0.039-0.30 microM). For the inhibition of hCYP51, IC(50) values split into two classes: the newer drugs fluconazole and itraconazole showed little inhibition (> or = 30 microM) while the older drugs were even more potent than the agricultural fungicides, with miconazole being the most potent (0.057 microM). No correlation was seen between the IC(50) values determined for the two enzymes, indicating that a housekeeping gene can show significant diversity if inhibition is concerned. Our data indicate that fungicide residues in food are unlikely to exert a relevant inhibition of CYP51 in humans whereas systemic use of some antimycotic drugs, e.g. ketoconazole or miconazole, should be carefully considered regarding disturbance of human steroid biosynthesis.
Azoles (imidazoles and triazoles) are used as antifungal agents in agriculture and in medicine, and also for antiestrogen therapy, e.g., for breast cancer treatment. Antifungal activity is based on inhibition of fungal CYP51 (lanosterol 14alpha-demethylase), and estrogen biosynthesis reduction is due to azole inhibition of CYP19 (aromatase). Inhibition of aromatase by antifungal agents is usually an unwanted side effect and may cause endocrine disruption. A fluorimetric assay based on human recombinant CYP19 enzyme with dibenzylfluorescein as a substrate was used to compare the inhibitory potency of 22 azole compounds. Dose responses were established and duplicate datasets were analyzed with a nonlinear mixed-effects model with cumulative normal distribution for the logarithm of concentration. IC50 values (50% inhibitory concentration) of 13 fungicides used in agriculture ranged more than 700-fold, starting from 0.047 microM. The potency of seven human drugs spanned more than 7000-fold, starting from 0.019 microM. Most potent fungicides included prochloraz, flusilazole, and imazalil, and most potent medicinal antifungals were bifonazole, miconazole, and clotrimazole. These in vitro data indicate that the top-ranking azoles used as antifungal agents or drugs are as potent inhibitors of aromatase as are antiestrogen therapeutics used to treat breast cancer. These putative effects of azole agents and drugs on steroid biosynthesis and sex hormone balance should be considered when used in human subjects and also in wildlife exposed to azole fungicides used in agriculture.
Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14␣-demethylase (CYP51) is an enzyme inhibited by azoles. Only few data have been reported showing their inhibitory potency since an assay in an in vitro system is not available so far. In the present work an inhibition assay using human recombinant CYP51, coexpressed with human P450 oxido-reductase by the baculovirus/insect cell expression system, and LC-MS/MS as analytical method is described. Atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI) sources were used with a triple quadrupole mass spectrometer to compare quantitation of lanosterol (substrate) and 4,4-dimethyl-5␣-cholesta-8,14,24-triene-3-ol (FF-MAS) (product of CYP51) with d 6 -2,2,3,4,4,6-cholesterol (d 6 -cholesterol) as internal standard. Optimization of analytical parameters resulted in a LC-APPI-MS/MS method with a LOQ of 10 pg on column for FF-MAS. The sensitivity of the method (LOD 0.5 ng/ml) makes it possible to analyze supernatants of inhibition experiments after precipitation of proteins by isopropanol without any sample enrichment. The coefficient of variation of the analytical method was Ͻ20% (n ϭ 5) for FF-MAS, lanosterol and d 6 -cholesterol. The external calibration curve was linear from 1 to 10,000 ng/ml with R 2 Ն 0.999 and an accuracy of 94 -115%. Compared with APCI, APPI provides a ten-to 500-fold increase in sensitivity for the analytes in this study. IC 50 values of epoxiconazole and miconazole-two widely used azole fungicides used in agriculture and in human medicine, respectively-were 1.95 M and 0.057 M. (J Am Soc Mass Spectrom 2004, 15, 1216 -1221
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