ABSTRACT
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.
Subject(s)
Antifungal Agents/pharmacology , Azoles/pharmacology , Candida albicans/enzymology , Cytochrome P-450 Enzyme Inhibitors , Enzyme Inhibitors/pharmacology , Fungal Proteins/antagonists & inhibitors , Oxidoreductases/antagonists & inhibitors , Azoles/chemistry , Azoles/classification , Dose-Response Relationship, Drug , Enzyme Inhibitors/classification , Food Contamination , Humans , Inhibitory Concentration 50 , Pesticide Residues , Risk Assessment , Species Specificity , Sterol 14-Demethylase , Structure-Activity RelationshipABSTRACT
Azoles are used as fungicides in agriculture or antifungal drugs in medicine. Their therapeutic activity is based on the inhibition of fungal lanosterol-14alpha-demethylase (CYP51). Azoles are also used for the treatment of estrogen-dependent diseases, e.g. in breast cancer therapy. Inhibition of CYP19 (aromatase) is the working principle for tumor therapy, but is an unwanted side effect of azoles used as fungicides or antifungal drugs. The inhibition of recombinant human CYP19 by 21 azoles in use for the three different purposes was investigated using the natural substrate testosterone. Estradiol product formation was measured by a newly developed and fully validated analytical method based on liquid chromatography-tandem mass spectrometry utilizing photospray ionization (APPI). Potency of enzyme inhibition was expressed in terms of IC50 concentrations. The two cytostatic drugs fadrozole and letrozole were the most potent inhibitors. However, azoles used as fungicides, e.g. prochloraz, or as antifungal drugs, e.g. bifonazole, were almost as potent inhibitors of aromatase as the drugs used in tumor therapy. Comparison of plasma concentrations that may be reached in antifungal therapy do not allow for large safety factors for bifonazole and miconazole. The IC50 values were compared to data obtained with other substrates, such as the pseudo-substrate dibenzylfluorescein (DBF). A high correlation was found, indicating that the fluorescence assay with DBF can well be used for potency ranking and screening of chemicals for aromatase inhibition. The data for antifungal drugs show that side effects on steroid hormone synthesis in humans due to inhibition of aromatase should be considered.
Subject(s)
Antifungal Agents/pharmacology , Aromatase Inhibitors/pharmacology , Azoles/pharmacology , Enzyme Inhibitors , Estradiol/metabolism , Fungicides, Industrial/pharmacology , Cholesterol/metabolism , Chromatography, High Pressure Liquid , Chromatography, Liquid , Fluorescent Dyes , Humans , In Vitro Techniques , Indicators and Reagents , Microsomes/enzymology , Spectrum AnalysisABSTRACT
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the direct quantitation of residues of 13 azole fungicides in wine and has been successfully applied to 103 wine samples. The method utilizing 25 transitions is highly sensitive and specific with centrifugation as the only sample work-up step. Precision was better than 14% and accuracy ranged between 80 and 120%. Quantitation limits (LOQs) ranged between 0.25 (penconazole) and 7.5 ng/mL (triadimefon). Since the LOQs achieved are at least four times lower than the maximum residue levels for azole-fungicides in wine prescribed, the method presented here can be conveniently used as a screening assay for azole-residues in wine samples.
Subject(s)
Azoles/analysis , Chromatography, Liquid/methods , Food Contamination/analysis , Fungicides, Industrial/analysis , Mass Spectrometry/methods , Wine/analysis , Centrifugation , Reproducibility of Results , Triazoles/analysisABSTRACT
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.
Subject(s)
Agrochemicals/pharmacology , Antifungal Agents/pharmacology , Aromatase Inhibitors/pharmacology , Aromatase/drug effects , Azoles/pharmacology , Fungicides, Industrial/pharmacology , Agrochemicals/chemistry , Antifungal Agents/chemistry , Antineoplastic Agents, Hormonal/chemistry , Antineoplastic Agents, Hormonal/classification , Antineoplastic Agents, Hormonal/pharmacology , Aromatase Inhibitors/chemistry , Aromatase Inhibitors/classification , Azoles/chemistry , Azoles/classification , Cytochrome P-450 Enzyme System/drug effects , Estrogen Receptor Modulators/chemistry , Estrogen Receptor Modulators/classification , Estrogen Receptor Modulators/pharmacology , Fungal Proteins/drug effects , Fungicides, Industrial/chemistry , Humans , Imidazoles/chemistry , Imidazoles/classification , Imidazoles/pharmacology , Inhibitory Concentration 50 , Logistic Models , Pharmaceutical Preparations/chemistry , Pharmaceutical Preparations/classification , Recombinant Proteins , Triazoles/chemistry , Triazoles/classification , Triazoles/pharmacologyABSTRACT
Azoles affect the steroid balance in all biological systems and may therefore be called endocrine disrupters. Lanosterol 14alpha-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-5alpha-cholesta-8,14,24-triene-3beta-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 <20% (n = 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) >/= 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 microM and 0.057 microM.
