Toxicological Evaluation of Azole Fungicides in Agriculture and Food Chemistry Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Eva-Regina Trösken aus Frankfurt am Main Würzburg 2005 Eingereicht am:……………………………….. Bei der Fakultät für Chemie und Pharmazie 1. Gutachter……………………………………. 2. Gutachter…………………………………….. der Dissertation 1. Prüfer……………………………………. 2. Prüfer……………………………………. 3. Prüfer……………………………………. des Öffentlichen Promotionskolloquiums Tag des Öffentlichen Promotionskolloquiums:…………………….. Doktorurkunde ausgehändigt am:…………………………………… To my dear family Table of Contents I Table of Contents Table of Contents .........................................................................................................I 1 Introduction and Background ...............................................................................1 1.1 Agricultural Fungicides..................................................................................1 1.1.1 Azole Compounds..................................................................................1 1.1.2 Toxicological Profiles of Azole Fungicides .............................................6 1.1.3 Workplace Exposure of Agricultural Workers.........................................7 1.1.4 Pesticide Residues in and on Food........................................................8 1.2 Azoles as Medical Agents .............................................................................9 1.2.1 Antifungal Agents...................................................................................9 1.2.2 Azole Compounds................................................................................10 1.2.2.1 Toxicokinetics ...............................................................................10 1.2.2.2 Adverse Drug Reactions...............................................................11 1.2.3 Azole Compounds in Chemotherapy ...................................................13 2 Objectives ..........................................................................................................15 3 Inhibition of CYP19 (aromatase) ........................................................................17 3.1 Dibenzylfluorescein as Substrate for CYP19 ..............................................17 3.1.1 Introduction ..........................................................................................17 3.1.2 Materials and Methods.........................................................................17 3.1.2.1 Chemicals and Reagents..............................................................17 3.1.2.2 Inhibition Assay.............................................................................18 3.1.2.3 Data Analysis................................................................................19 3.1.3 Results.................................................................................................20 3.1.4 Discussion ...........................................................................................22 3.1.4.1 Literature IC50 Values ...................................................................23 3.1.4.2 IC50 Values in this Manuscript.......................................................23 3.1.4.3 Toxicological Evaluation ...............................................................24 3.2 Testosterone as Substrate for CYP19.........................................................25 3.2.1 Introduction ..........................................................................................25 3.2.2 Materials and Methods.........................................................................25 3.2.2.1 Chemicals and Reagents..............................................................25 3.2.2.2 Inhibition Assay.............................................................................26 3.2.2.3 Optimisation of Mass Spectrometry ..............................................27 3.2.2.4 HPLC ............................................................................................27 3.2.2.5 MS Parameters.............................................................................28 3.2.2.6 Method Validation .........................................................................29 3.2.2.7 Data Analysis................................................................................32 3.2.3 Results and Discussion........................................................................32 3.2.3.1 List of IC50 Values.........................................................................32 3.2.3.2 Comparison of Assays..................................................................34 3.2.3.3 Comparison with Literature Data ..................................................36 3.2.3.4 Confounding Factors ....................................................................37 3.2.3.5 IC50 Values vs. Exposure to Fungicides........................................37 3.2.3.6 IC50 Values vs. Exposure to Antifungal Drugs ..............................38 3.2.4 Conclusions .........................................................................................38 4 Inhibition of CYP51 (Lanosterol-14α-demethylase)............................................40 4.1 Analytical Method to Detect Lanosterol.......................................................40 4.1.1 Introduction ..........................................................................................40 4.1.2 Materials and Methods.........................................................................41 4.1.2.1 Chemicals and Reagents..............................................................41 II Table of Contents 4.1.2.2 Enzyme Inhibition Assay .............................................................. 42 4.1.2.3 Optimisation of Mass Spectrometry Methods............................... 42 4.1.2.4 Quantitation.................................................................................. 43 4.1.2.5 Data Evaluation............................................................................ 44 4.1.3 Results ................................................................................................ 44 4.1.3.1 Characterization of CYP51........................................................... 44 4.1.3.2 Identification and Quantitation of FF-MAS.................................... 44 4.1.3.3 Inhibition of CYP51 by Azoles ...................................................... 46 4.1.4 Discussion........................................................................................... 47 4.1.4.1 Enzyme Assay ............................................................................. 47 4.1.4.2 Steroid Analysis ........................................................................... 47 4.1.4.3 Liquid-Chromatography Mass Spectrometry................................ 47 4.1.4.4 Fragmentation .............................................................................. 48 4.1.4.5 Photospray Ionisation................................................................... 48 4.1.4.6 Analytical Method......................................................................... 51 4.2 Production of Candida albicans CYP51...................................................... 52 4.2.1 Introduction.......................................................................................... 52 4.2.1.1 Baculovirus Protein Expression System....................................... 52 4.2.2 Materials and Methods ........................................................................ 54 4.2.2.1 Apparatus..................................................................................... 54 4.2.2.2 Chemicals .................................................................................... 55 4.2.2.3 Buffers and Bacterial-Sera ........................................................... 55 4.2.2.4 Cellculture-Media and Sera.......................................................... 55 4.2.2.5 Other Materials............................................................................. 55 4.2.3 Procedures.......................................................................................... 56 4.2.3.1 Agarose Gel Electrophoresis........................................................ 56 4.2.3.2 Transformations ........................................................................... 56 4.2.3.3 DNA Isolation from Bacterial Cells ............................................... 56 4.2.3.4 Restriction Analysis...................................................................... 57 4.2.3.5 DNA Purification from Agarosegels.............................................. 57 4.2.3.6 DNA Dephosphorylation............................................................... 57 4.2.3.7 DNA Ligation ................................................................................ 57 4.2.3.8 Cloning and Protein Expression Procedure.................................. 58 4.2.4 Vector DNA Amplification .................................................................... 61 4.2.5 Construction of the pFastBac™ Dual - Candida albicans CYP51 - Candida tropicalis Oxidoreductase Vector......................................................... 61 4.2.5.1 Construction of pFastBac™ Dual - Candida albicans CYP51 ...... 61 4.2.5.2 Ligation of CYP51 and pFastBac™ Dual Vector .......................... 62 4.2.5.3 Amplification of Candida tropicalis Oxidoreductase DNA by PCR 62 4.2.5.4 Topo Cloning of the Candida tropicalis Oxidoreductase............... 63 4.2.5.5