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In vitro hepatic metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5-pentabromodiphenyl… Erratico, Claudio 2013

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  IN VITRO HEPATIC METABOLISM OF 2,2',4,4'-TETRABROMODIPHENYL ETHER (BDE-47) AND 2,2',4,4',5-PENTABROMODIPHENYL ETHER (BDE-99)  by Claudio Erratico  B.Sc., Universita’degli Studi Milano–Bicocca, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  Doctor of Philosophy  in THE FACULTY OF GRADUATE STUDIES (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  June 2013  © Claudio Erratico, 2013  ii  Abstract  Polybrominated diphenyl ethers (PBDEs) are flame retardants that were added to many consumer products and have emerged as persistent and bioaccumulative environmental contaminants. Penta-BDE was a commercial PBDE mixture that was used extensively in North America. 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) and 2,2',4,4',5- pentabromodiphenyl ether (BDE-99) are the dominant congeners in the Penta-BDE mixture and occur at similar levels in the mixture and in air, dust, and sediments. In contrast, the concentration of BDE-99 is 10-fold lower than that of BDE-47 in most wildlife and human samples, which could be due to more extensive metabolism of BDE-99 than BDE-47 by hepatic cytochrome P450 (CYP) enzymes. To investigate this hypothesis, a liquid chromatography-mass spectrometry based assay was developed and validated to characterize the biotransformation of BDE-99 by liver microsomes. Rat liver microsomes were obtained from animals treated with dexamethasone, phenobarbital, 3-methylcholanthrene or corn oil. Up to six hydroxylated metabolites of BDE-99 were formed by different rat liver microsomal preparations. The major metabolite, 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90), was formed at 2.7 pmol/min/mg protein by liver microsomes obtained from corn oil treated rats. CYP3A1, CYP1A1, and CYP2A2 were the most active rat recombinant CYP enzymes. Incubating BDE-99 with human liver microsomes resulted in the formation of 10 hydroxylated metabolites. The major metabolites were 2,4,5- tribromophenol (2,4,5-TBP), 5ʹ-hydroxy-2,2ʹ,4,4ʹ,5-pentabromodiphenyl ether (5'-OH-BDE- 99) and 4'-hydroxy-2,2',4,5,5'-pentabromodiphenyl ether (4'-OH-BDE-101) and their rates of formation ranged between 25 and 45 pmol/min/mg protein. Incubating BDE-47 with iii  human liver microsomes resulted in the formation of 9 hydroxylated metabolites. The major metabolites were 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47) and 6- hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47) and their rates of formation were 23 and 27 pmol/min/mg protein, respectively. CYP2B6 was the major human CYP enzyme responsible for the formation of all the hydroxylated metabolites of BDE-47 and BDE-99. In conclusion, BDE-99 underwent more extensive oxidative metabolism by human than rat liver microsomes and was biotransformed into a different set of hydroxylated metabolites by human than rat liver microsomes. Metabolism of BDE-47 and BDE-99 by human liver microsomes proceeds at similar pace, which suggests that oxidative metabolism does not explain the difference in BDE-47 and BDE-99 blood concentrations in humans.   iv  Preface  The Animal Care Committee at the University of British Columbia examined and approved the use of animals for the experimental project described in this thesis. The project, titled “Biotransformation of Organochlorine Compounds in Biota” was completed under the Animal Care Certificate A06-0146 issued on May 1, 2006 and renewed on May 1, 2010 as Certificate A10-0225.  The research work presented in my thesis was presented in posters and oral presentations at several international conferences (including annual meetings of Society of Toxicology, Society of Environmental Toxicology and Chemistry, Dioxin, and Brominated Flame Retardants research network) and was subsequently published in international peer- reviewed scientific journals as follows: 1. Erratico, C., Szeitz, A., Bandiera, S.M., 2010. Development and validation of a novel liquid chromatography-mass spectrometry method for the quantitation of hydroxylated metabolites of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99) in rat liver microsomes. J. Chromatogr. B 878 (19): 1562-1568. Results of this study are presented in Section 3.1. 2. Erratico, C., Moffatt S., Bandiera, S.M., 2011. Comparative oxidative metabolism of BDE-47 and BDE-99 in rat liver microsomes.  Toxicol. Sci. 123(1): 37-47. Results of this study are presented in Section 3.2. 3. Erratico, C., Szeitz, A., Bandiera, S.M., 2012. Oxidative metabolism of BDE-99 by human liver microsomes: predominant role of CYP2B6. Toxicol. Sci. 192(2): 280- 292. Results of this study are presented in Section 3.3. v  4. Erratico, C., Szeitz, A., Bandiera, S.M., 2013. Biotransformation of BDE-47 by human liver microsomes: identification of P450 2B6 as the major enzyme involved. Chem. Res. Toxicol. 26: 721-731. Results of this study are presented in Section 3.4.  The author was responsible for formulation of the research and experimental hypothesis, experimental design, sample preparation, data acquisition, analysis, and interpretation, and for manuscript preparation throughout the project. Mr. Andras Szeitz provided analytical support in the first part of the project.   vi  Table of Contents  Abstract .................................................................................................................................. ii  Preface ................................................................................................................................... iv  Table of Contents .................................................................................................................. vi  List of Tables ......................................................................................................................... xi  List of Figures ...................................................................................................................... xv  List of Abbreviations ......................................................................................................... xxii  Acknowledgements ........................................................................................................... xxvi  1.  Introduction .................................................................................................................... 1  1.1  Flame Retardants ..................................................................................................... 1  1.2 Polybrominated Diphenyl Ethers ................................................................................... 2  1.2.1 Physicochemical Properties of PBDEs ................................................................... 5  1.2.2 Synthesis of PBDEs ................................................................................................ 5  1.2.3 Formulations, Market Demand, and Presence of PBDEs in Market Products ....... 8  1.2.4 Regulation of PBDEs Manufacture and Use ........................................................ 14  1.2.5 Emission Sources of PBDEs ................................................................................. 15  1.2.6 Persistency of PBDEs in the Environment ........................................................... 16  1.2.7 PBDE Concentrations in Human Samples ........................................................... 17  1.2.8 Routes of PBDEs Exposure for Humans .............................................................. 19  1.3 BDE-47 and BDE-99: Two Major PBDE Congeners ................................................. 21  1.3.1 Physicochemical Properties and Environmental Distribution of BDE-47 and BDE-99 ................................................................................................................ 21  vii  1.3.2 Bioaccumulation of BDE-47 and BDE-99 ........................................................... 22  1.4 Biotransformation ........................................................................................................ 24  1.5 CYP Enzymes .............................................................................................................. 25  1.5.1 Structure of CYP Enzymes ................................................................................... 27  1.5.2 CYP Catalytic Cycle ............................................................................................. 31  1.5.3 Nomenclature and Classification of CYP Enzymes ............................................. 34  1.5.4 Occurrence and Distribution of CYP Enzymes in Humans and Rats ................... 35  1.5.5 CYP1 Family ........................................................................................................ 42  1.5.6 CYP2 Family ........................................................................................................ 43  1.5.7 CYP3 Family ........................................................................................................ 50  1.6 Oxidative Metabolism of PBDE Mixtures in Rodents ................................................ 52  1.7 Metabolism of BDE-47 in Mammals .......................................................................... 53  1.8 Metabolism of BDE-99 in Mammals .......................................................................... 57  1.9 Analytical Methods for Analysis of Hydroxylated PBDEs ......................................... 63  1.10 Rationale .................................................................................................................... 65  1.11 Research Hypothesis and Specific Aims ................................................................... 66  2.  Materials and Methods ................................................................................................ 68  2.1 Chemicals and Reagents .............................................................................................. 68  2.2 Rat Treatment with Prototypical CYP Inducers and Preparation of Hepatic Microsomes ........................................................................................................... 70  2.3 Determination of Total Protein and Cytochrome P450 Content, Marker Activities for CYP1A, 2B, and 3A Enzymes in Rat Hepatic Microsomes ............................ 71  2.4 Preparation of Metabolite Standard Stock Solutions for Method Validation .............. 72  viii  2.5 Preparation of Calibration Standard and Quality Control Samples ............................. 73  2.6 Ultra Performance Liquid Chromatography/Mass Spectrometry Conditions ............. 75  2.7 Assay Validation Using the UPLC/MS Method .......................................................... 76  2.8 Biotransformation Assays for BDE-47 and BDE-99 ................................................... 79  2.9 Ultra High Performance (UHP)LC/MS/MS Methods for Analysis of the Hydroxylated Metabolites of BDE-47 and BDE-99 ............................................. 83  2.10 Quality Control .......................................................................................................... 85  2.11 Bupropion Hydroxylation Assay ............................................................................... 86  2.12 Antibody Inhibition Experiments .............................................................................. 88  2.13 Data Analysis ............................................................................................................. 90  3.  Results ........................................................................................................................... 92  Part 1: Development and Validation of a New UPLC/MS-based Assay to Characterize the Oxidative Metabolism of BDE-99 .................................................................. 92  3.1 Optimization of UPLC/MS Parameters ....................................................................... 93  3.2 Choice of the Internal Standard ................................................................................... 94  3.3 Optimization of Sample Preparation ........................................................................... 94  3.4 Assay Validation .......................................................................................................... 95  Part 2: Oxidative Metabolism of BDE-99 by Rat Liver Microsomes and Rat Recombinant CYP Enzymes ............................................................................... 104  3.5 Characterization of Rat Hepatic Microsomes ............................................................ 104  3.6 Identification of the Hydroxylated Metabolites of BDE-99 Formed by Rat Liver Microsomes ......................................................................................................... 106  ix  3.7 Kinetic Analysis of the Hydroxylated Metabolites of BDE-99 Formation by Liver Microsomes from DEX- and Corn Oil-treated Rats ........................................... 112  3.8 Effects of CYP Inducers on BDE-99 Biotransformation .......................................... 114  3.9 Oxidative Biotransformation of BDE-99 by Rat Recombinant CYP Enzymes ........ 116  Part 3: Oxidative Metabolism of BDE-99 by Human Liver Microsomes and Recombinant CYP Enzymes ............................................................................... 123  3.10 Identification of the Hydroxylated Metabolites of BDE-99 Formed by Human Liver Microsomes ............................................................................................... 123  3.11 Kinetic Analysis of the Hydroxylated Metabolites of BDE-99 Formation by Human Liver Microsomes ................................................................................... 131  3.12 Identification of CYP2B6 as the Major CYP Enzyme Involved in the Oxidative Biotransformation of BDE-99 ............................................................................. 134  3.13 Kinetic Analysis of the Hydroxylated Metabolites of BDE-99 Formation by Recombinant CYP2B6 ........................................................................................ 142  3.14 Investigation of the Mechanism of Formation of Secondary Metabolites of BDE- 99 by Human Liver Microsomes ......................................................................... 151  Part 4: Oxidative Metabolism of BDE-47 by Human Liver Microsomes and Recombinant CYP Enzymes ............................................................................... 154  3.15 Identification of the Hydroxylated Metabolites of BDE-47 Formed by Human Liver Microsomes ............................................................................................... 154  3.16 Kinetic Analysis of Hydroxylated Metabolites of BDE-47 Formation by Human Liver Microsomes ............................................................................................... 159  x  3.17 Identification of CYP2B6 as the Major CYP Enzyme Involved in the Oxidative Biotransformation of BDE-47 ............................................................................. 163  3.18 Kinetic Analysis of the Hydroxylated Metabolites of BDE-47 Formation by Recombinant CYP2B6 ........................................................................................ 169  3.19 Investigation of the Mechanism of Formation of Secondary Metabolites of BDE- 47 by Human Liver Microsomes ......................................................................... 177  4.  Discussion ................................................................................................................... 181  4.1 Validation of a Novel Assay for Metabolism of BDE-99 by Liver Microsomes ...... 181  4.2 Oxidative Metabolism of BDE-99 by Rat Liver Microsomes and Rat Recombinant CYP Enzymes ..................................................................................................... 185  4.3 Comparison of Oxidative Metabolism of BDE-47 and BDE-99 by Rat Liver Microsomes ......................................................................................................... 188  4.4 Comparison of Oxidative Metabolism of BDE-47 and BDE-99 by Human Liver Microsomes ......................................................................................................... 189  4.5 Comparison of Oxidative Metabolism of BDE-47 and BDE-99 by Rat and Human Liver Microsomes ............................................................................................... 203  5. Concluding Remarks ..................................................................................................... 213  5.1 Conclusions ................................................................................................................ 213  5.2 Strengths and Weaknesses of the Present Study ........................................................ 215  References ........................................................................................................................... 217  Appendix 1- Metabolism of BDE-99 by Human Liver Microsomes ............................. 274  Appendix 2 - Metabolism of BDE-47 by Human Liver Microsomes ............................ 283   xi  List of Tables  Table 1.1  Global Production of Brominated Flame Retardants Between 1989 and 2001 ...... 3  Table 1.2  Brief History of PBDEs .......................................................................................... 4  Table 1.3  Physicochemical Properties of Selected PBDE Congeners .................................... 7  Table 1.4   Relative Abundance of PBDE Congeners Present in the Three Commercial PBDE Mixtures .................................................................................................... 10  Table 1.5   Continent-Specific Market Demand (in tons) for Penta-, Octa-, and Deca- BDE Commercial Mixtures in 1999 and 2001 .................................................... 12  Table 1.6   Major Products Containing Penta-, Octa-, and Deca-BDE Commercial Mixtures ............................................................................................................... 13  Table 1.7   Human CYP Enzymes Ordered According to the Major Classes of Substrates Metabolized ........................................................................................ 36  Table 1.8  Total CYP Content in Various Human and Rat Organs ....................................... 37  Table 1.9   Presence of CYP1, CYP2, and CYP3 Enzymes in Various Organs of Humans and Rats ................................................................................................. 39  Table 1.10 Major Drug Metabolizing CYP Families in Human and Rats ............................. 41  Table 3.1   Relative Retention Time, Capacity Factor, and Resolution Values for The Validated UPLC/MS-based assay ........................................................................ 96  Table 3.2  LOQ Values of Hydroxylated Metabolites of BDE-99 Using the UPLC/MS- based Assay ......................................................................................................... 98  Table 3.3  Inter-Day and Intra-Day Precision (%RSD) and Accuracy (%Dev) Values ........ 99  Table 3.4  Calibration Curve Parameter Values Determined Using CS Samples ............... 101  xii  Table 3.5  Recovery Values Determined Using QC-Low and QC-High Samples .............. 103  Table 3.6   Effect of Treatment with CYP Inducers on Total Protein Content, Total CYP Content, and CYP Marker Activities in Pooled Rat Hepatic Microsomes (n=6) .............................................................................................. 105  Table 3.7 Effect of Treatment with CYP Inducers on Rates of Formation of Hydroxylated Metabolites of BDE-99 by Rat Hepatic Microsomes ................. 117  Table 3.8   Rates of Formation of Hydroxylated Metabolites of BDE-99 by Single Donor Human Liver Microsome ....................................................................... 127  Table 3.9   Apparent Vmax, Km, and Ki Values for the Formation of the Hydroxylated Metabolites of BDE-99 by Pooled Human Liver Microsomes ......................... 135  Table 3.10 Correlation Analysis of BDE-99 Hydroxylated Metabolite Formation and CYP Marker Activities Using Single Donor Human Liver Microsomes .......... 141  Table 3.11 Values of Vmax, Km, and Ki for the Formation of the Hydroxylated Metabolites of BDE-99 by Human Recombinant CYP2B6 .............................. 150  Table 3.12 Rates of Formation of Metabolites of BDE-47 by Single Donor Human Liver Microsomes .............................................................................................. 158  Table 3.13 Apparent Vmax, Km, and Ki Values for the Formation of the Hydroxylated Metabolites of BDE-47 by Human Liver Microsomes ...................................... 164  Table 3.14 Correlation Analysis of Formation of Hydroxylated Metabolites of BDE-47 and CYP Marker Activities Using Single Donor Human Liver Microsomes ... 168  Table 3.15 Values of Vmax, Km, and Ki for the Formation of the Hydroxylated Metabolites of BDE-47 by Human Recombinant CYP2B6 .............................. 176 xiii  Table 4.1   Structures and Mechanisms For the Formation of Hydroxylated Metabolites of BDE-99 by Pooled Human Liver Microsomes…………………..………....190 Table 4.2   Structures and Mechanisms For the Formation of Hydroxylated Metabolites of BDE-47 by Pooled Human Liver Microsomes…………………..………....193 Table 4.3   Comparison of BDE-99 Hydroxylated Metabolite Formation by Human and Rat Liver Microsomes………………………………………….……………...204 Table 4.4   Comparison of BDE-47 Hydroxylated Metabolite Formation by Human and Rat Liver Microsomes……………………………………………..……..…....205 Table A1.1 Cross Reactivity of Mouse Anti-Human CYP2B6 Antibody…........................276 Table A1.2 Retention Time (RT), Precursor and Product Ion (Q1/Q3) Transitions, Declustering Potential (DP), and Collision Energy (CE) Values of the Hydroxylated BDE-99 Metabolites and Internal Standard (IS) Using the Modified UPLC/MS/MS-based assay…………………………..……..…........277 Table A1.3 % Relative Standard Deviation (%RSD), Percent Deviation (%Dev), and Signal- To-Noise (S/N) Ratio Values of the Limit of Quantification for the Analytes of Interest……………………………………………..……..…............................278 Table A1.4 Inter-day Precision (%RSD) and Accuracy (%Dev) Values Determined With QC Samples at Low, Mid, and High Concentrations (n=8) Using the Modified UPLC/MS/MS-based Assay……………………………………..……..….......279 Table A1.5 Marker Activity Values for Major CYP and UGT Enzymes in the Single Donor Human Liver Microsomes………………………………………..……..…......280 Table A2.1 Retention Time (RT), Precursor and Product Ion (Q1/Q3) Transitions, Declustering Potential (DP), and Collision Energy (CE) Values of the xiv  Hydroxylated BDE-47 Metabolites and Internal Standard (IS) Using the Modified UPLC/MS/MS-based assay…………………………………….…...286 Table A2.2 % Relative Standard Deviation (%RSD), Percent Deviation (%Dev), and Signal- to-Noise (S/N) Ratio Values of the Limit of Quantification for the Analytes of Interest………….…………………………………..……..…...........................287 Table A2.3 Inter-day %RSD and %Dev Values Determined With QC Samples at Low, Mid, and High Concentrations (n=8) .……………………….………..……..….......288                xv  List of Figures  Figure 1.1   Chemical structure of PBDEs (A) and PCBs (B). ................................................ 6  Figure 1.2   Chemical structure and substitution pattern of the most commonly produced PBDEs. ................................................................................................. 9  Figure 1.3  Absorbance spectra of CYP enzymes. ................................................................ 26  Figure 1.4  Structure of the heme prosthetic group of CYP enzymes. .................................. 28  Figure 1.5  Electron transfer and substrate oxidation by CYP enzymes. .............................. 30  Figure 1.6  Major steps of the CYP catalytic cycle. .............................................................. 32  Figure 1.7  Hydroxylated metabolites of BDE-47 produced in vivo by rats treated with BDE-47 (A) and in vitro by rat liver microsomes (B). ...................................... 56  Figure 1.8   Hydroxylated metabolites of BDE-47 formed by human hepatic preparations (A) and hydroxylated PBDEs detected in human plasma and serum samples (B). ............................................................................................. 58  Figure 1.9   Hydroxylated metabolites of BDE-99 produced by rats and mice treated with BDE-99 (A) and by rat hepatocytes (B). .................................................... 60  Figure 1.10 Hydroxylated metabolites of BDE-99 formed by human hepatic preparations (A) and hydroxylated PBDEs detected in human plasma and serum samples (B). ............................................................................................. 62  Figure 3.1   Representative chromatograms of rat liver microsomes spiked with methanol or authentic standards at the LOQ values. .......................................... 97  Figure 3.2   Rates of BDE-99 metabolite formation in liver microsomes from corn oil-, DEX-, MC-, and PB-treated rats. ..................................................................... 107  xvi  Figure 3.3   Effect of incubation time on formation of hydroxylated metabolites of BDE-99 (mean ± SD) by liver microsomes from DEX-treated rats. ............... 109  Figure 3.4   Effect of total liver microsomal protein concentration on rates of BDE-99 metabolite formation (mean ± SD) by liver microsomes from DEX-treated rats. ................................................................................................................... 110  Figure 3.5   Effect of incubation time (A) and total liver microsomal protein (B) on formation of hydroxylated metabolites of BDE-99 (mean ± SD) by liver microsomes from corn oil-treated rats. ............................................................ 111  Figure 3.6   Enzyme kinetic profiles for the formation of hydroxylated metabolites of BDE-99 by liver microsomes from DEX-treated rats. ..................................... 113  Figure 3.7   Enzyme kinetic profiles of 4-OH-BDE-90, 5'-OH-BDE-99 and 6'-OH- BDE-99 formation by hepatic microsomes from corn oil-treated rats. ............ 115  Figure 3.8   Scheme showing the chemical structures of metabolites produced incubating BDE-99 with the four rat liver microsomal preparations used. ..... 118  Figure 3.9   Effect of incubation time on formation of hydroxylated metabolites of BDE-99 by rat recombinant CYP3A1. ............................................................. 120  Figure 3.10 Effect of recombinant CYP3A1 concentration on rates of formation of hydroxylated metabolites of BDE-99. .............................................................. 121  Figure 3.11 Rates of formation of hydroxylated metabolites of BDE-99 by a panel of rat recombinant CYP enzymes. ............................................................................. 122  Figure 3.12 Scheme showing the chemical structures of hydroxylated metabolites formed following the incubation of human liver microsomes with BDE-99. General structures for M1-M4 are also shown. ................................................ 124  xvii  Figure 3.13 Representative UHPLC/MS/MS chromatograms of 2,4,5-TBP (A), mono- OH-pentabrominated-PBDE (B), and di-OH-pentabrominated-PBDE (M4) (C) metabolites produced by pooled human liver microsomes. ....................... 126  Figure 3.14 Effect of incubation time on formation of the identified (A) and unidentified (B) hydroxylated metabolites of BDE-99 produced by pooled human liver microsomes. ................................................................................. 129  Figure 3.15 Effect of total protein concentration on formation of the identified (A) and unidentified (B) hydroxylated metabolites of BDE-99 produced by pooled human liver microsomes. ................................................................................. 130  Figure 3.16 Enzyme kinetic profiles for the formation of the major identified and unidentified hydroxylated metabolites of BDE-99 by pooled human liver microsomes. ..................................................................................................... 132  Figure 3.17 Enzyme kinetic profiles for the formation of the minor identified and unidentified hydroxylated metabolites of BDE-99 by pooled human liver microsomes. ..................................................................................................... 133  Figure 3.18 Rates of formation of 2,4,5-TBP, 5'-OH-BDE-99, 4'-OH-BDE-101, and M1following incubation of BDE-99 with a panel of human recombinant CYP enzymes. .................................................................................................. 136  Figure 3.19 Effect of rabbit anti-rat CYP2B1 IgG and mouse anti-human CYP2B6 ascites on the formation of the major identified and unidentified metabolites of BDE-99 by human liver microsomes. ...................................... 138  xviii  Figure 3.20 Effect of rabbit anti-rat CYP2B1 IgG on the formation of the minor identified and unidentified metabolites of BDE-99 by human liver microsomes. ..................................................................................................... 139  Figure 3.21 Effect of mouse anti-human CYP2B6 ascites on the formation of the minor identified and unidentified metabolites of BDE-99 by human liver microsomes. ..................................................................................................... 140  Figure 3.22 Effect of incubation time on formation of 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 (A) and of 4-OH-BDE-90, 6'-OH-BDE-99, and 2-OH- BDE-123 (B) by recombinant CYP2B6. .......................................................... 143  Figure 3.23 Effect of incubation time on formation of M1 and M2 (A) and of M3 and M4 (B) by recombinant CYP2B6. ................................................................... 144  Figure 3.24 Effect of recombinant CYP2B6 concentration on rates of formation of 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 (A) and of 4-OH-BDE- 90, 6'-OH-BDE-99, and 2-OH-BDE-123 (B). ................................................. 145  Figure 3.25 Effect of recombinant CYP2B6 concentration on rates of formation of M1 and M2 (A) and of M3 and M4 (B). ................................................................. 146  Figure 3.26 Enzyme kinetic profiles for the formation of the major identified and unidentified hydroxylated metabolites of BDE-99 by human recombinant CYP2B6. .......................................................................................................... 148  Figure 3.27 Enzyme kinetic profiles for the formation of the minor identified and unidentified hydroxylated metabolites of BDE-99 by human recombinant CYP2B6. .......................................................................................................... 149  xix  Figure 3.28 Effect of anti-epoxide hydrolase IgG on the formation of the unidentified di-OH-pentabrominated-PBDE metabolite of BDE-99 by pooled human liver microsomes. ............................................................................................. 152  Figure 3.29 Scheme showing possible pathways for the formation of 2,4,5-TBP and M4 from the three major primary hydroxylated metabolites (namely 5'-OH- BDE-99, 6'-OH-BDE-99, and 4'-OH-BDE-101) of BDE-99. .......................... 153  Figure 3.30 Scheme showing the chemical structures of the hydroxylated metabolites formed following the incubation of pooled human liver microsomes with BDE-47. General structures for M1 and M2 are also shown. .......................... 155  Figure 3.31 Representative UHPLC/MS/MS chromatograms of mono-OH- tetrabrominated-PBDE (A), di-OH-tetrabrominated-PBDE (B), mono-OH- tribrominated-PBDE (C), and 2,4-DBP (D) metabolites of BDE-47 produced by pooled human liver microsomes. ................................................ 157  Figure 3.32 Effect of incubation time on formation of 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49 (A), of 2,4,-DBP, 4'-OH-BDE-17, and 2'-OH-BDE-28 (B), and of M1 and M2 (C) by pooled human liver microsomes. ..................................................................................................... 160  Figure 3.33 Effect of total protein concentration on rates of formation of 2,4-DBP, 4- OH-BDE-42, and 6-OH-BDE-47 (A), of 4'-OH-BDE-17, 2'-OH-BDE-28, 5-OH-BDE-47, and 4'-OH-BDE-49 (B), and of M1 and M2 (C) by pooled human liver microsomes. ................................................................................. 161  Figure 3.34 Enzyme kinetic profiles for the formation of the hydroxylated metabolites of BDE-47 by pooled human liver microsomes. .............................................. 162  xx  Figure 3.35 Rates of formation of 5-OH-BDE-47, 6-OH-BDE-47, 4-OH-BDE-42, 4'- OH-BDE-49, M1 and M2 following incubation of BDE-47 with a panel of human recombinant CYP enzymes. ................................................................. 165  Figure 3.36 Effect of mouse anti-human CYP2B6 ascites on the formation of 5-OH- BDE-47 (A), 6-OH-BDE-47 (B), 4-OH-BDE-42 (C), 4'-OH-BDE-49 (D), M1 (D), and M2 (F) metabolites of BDE-47 by pooled human liver microsomes. ..................................................................................................... 167  Figure 3.37 Effect of incubation time on formation of 2'-OH-BDE-28 and 4'-OH-BDE- 49 (A) and of 4'-OH-BDE-17, 5-OH-BDE-47, and 6-OH-BDE-47 (B) by recombinant CYP2B6. ..................................................................................... 170  Figure 3.38 Effect of incubation time on formation of M1 and M2 (A) and of 2,4-DBP and 4-OH-BDE-42 (B) by recombinant CYP2B6. .......................................... 171  Figure 3.39 Effect of recombinant CYP2B6 concentration on rates of formation of 6- OH-BDE-47 and M1 (A) and of 5-OH-BDE-47 and 4'-OH-BDE-49 (B). ...... 172  Figure 3.40 Effect of recombinant CYP2B6 concentration on rates of formation of 4'- OH-BDE-17, 2'-OH-BDE-28, and 4-OH-BDE-42 (A), and of 2,4-DBP and M2 (B). ............................................................................................................. 173  Figure 3.41 Enzyme kinetic profiles for the formation of hydroxylated metabolites of BDE-47 by recombinant CYP2B6. .................................................................. 175  Figure 3.42 Effect of anti-epoxide hydrolase IgG on the formation of the unidentified di-OH-tetrabrominated-PBDE metabolite of BDE-47 produced by pooled human liver microsomes. ................................................................................. 178  xxi  Figure 3.43 Formation of 2,4-DBP as possible secondary metabolite of BDE-47 by pooled human liver microsomes. ..................................................................... 180   Figure A1.1 The specificity of inhibition by mouse anti-CYP2B6 monoclonal antibody. Data provided by BD Biosciences. .................................................. 281 Figure A1.2 Correlation between CYP2B6 and CYP2C19 marker activities for single donor human liver microsomal samples. Data provided by BD Biosciences. . 282     xxii  List of Abbreviations  %Dev Percent deviation %RSD Percent relative standard deviation 2,4-DBP 2,4-dibromophenol 2,4,5-TBP 2,4,5-tribromophenol 4'-OH-CB-50 4'-Hydroxy-2,2',4,6'-tetrachlorobiphenyl 4-OH-CB-121 4-hydroxy-2',3,4',5,6'-pentachlorobiphenyl AICc Akaike Information Criterion corrected for small number of samples BOQD 7-Benzyloxyquinoline O-dealkylation BROD Benzyloxyresorufin O-dealkylase CS Calibration standard CYP  Cytochrome P450 Deca-BDE  Commercial deca PBDE mixture DEX Dexamethasone EROD Ethoxyresorufin O-deethylase IgG Immunoglobulin G IS Internal standard K' Capacity factor Ki Inhibitory constant of the substrate-inhibition model Km Michaelis-Menten constant Koa Octanol-air partition coefficient Kow Octanol-water partition coefficient xxiii  LOQ Limit of quantification M1,2,3,4 Unidentified metabolites no.12,3,4 MAB Monoconal antibody MC 3-methylcholanthrene MS Mass spectrometry NADPH Nicotinamide adenine dinucleotide phosphate NIH Shift U.S. National Institute of Health shift Octa-BDE Commercial octa PBDE mixture Penta-BDE Commercial penta PBDE mixture PB Phenobarbital PBDEs Polybrominated diphenyl ethers PCBs Polychlorinated biphenyls QCs Quality control sample R Resolution R2 Coefficient of determination RH Substrate in the reduced state ROH Hydroxylated metabolite RRT Relative retention time S/N Signal to noise ratio SST System suitability test sample Sw Water solubility T3 3,3'5-triiodothyronine T4 3,3',5,5'-tetraiodothyronine xxiv  UHPLC Ultra High Performance liquid chromatography UPLC Ultra performance liquid chromatography VL Vapour pressure Vmax Maximum velocity  Polybrominated Diphenyl Ethers (PBDEs) BDE-17 4'-Hydroxy-2,2',4-tribromodiphenyl ether BDE-28 2'-Hydroxy-2,4,4'-tribromodiphenyl ether BDE-47 2,2',4,4'-Tetrabromodiphenyl ether BDE-99 2,2,4,4,5-Pentabromodiphenyl ether BDE-100 2,2,4,4,6-Pentabromodiphenyl ether BDE-153 2,2,4,4,5,5-Hexabromodiphenyl ether BDE-154 2,2,4,4,5,6-Hexabromodiphenyl ether BDE-183 2,2',3,4,4',5',6heptabromodiphenyl ether BDE-203 2,2',3,4,4',5,5',6,6'-octabromodiphenyl ether BDE-209 2,2,3,3,4,4,5,5,6,6-Decabromodiphenyl ether  Hydroxylated polybrominated diphenyl ethers (OH-BDEs) 4'-OH-BDE-17 4'-Hydroxy-2,2',4-tribromodiphenyl ether 2'-OH-BDE-28 2'-Hydroxy-2,4,4'-tribromodiphenyl ether 4-OH-BDE-42 4-Hydroxy-2,2',3,4'-tetrabromodiphenyl ether 3-OH-BDE-47 3-Hydroxy-2,2',4,4'-tetrabromodiphenyl ether 5-OH-BDE-47 5-Hydroxy-2,2',4,4'-tetrabromodiphenyl ether xxv  6-OH-BDE-47 6-Hydroxy-2,2',4,4'-tetrabromodiphenyl ether 4'-OH-BDE-49 4'-Hydroxy-2,2',4,5'-tetrabromodiphenyl ether 4-OH-BDE-90 4-Hydroxy-2,2',3,4',5-pentabromodiphenyl ether 5'-OH-BDE-99 5ʹ-hydroxy-2,2 ʹ,4,4 ʹ,5-pentabromodiphenyl ether 6ʹ-OH-BDE-99 6ʹ-hydroxy-2,2 ʹ,4,4 ʹ,5-pentabromodiphenyl ether 4ʹ-OH-BDE-101 4'-hydroxy-2,2',4,5,5'-pentabromodiphenyl ether 2-OH-BDE-123 2-Hydroxy-2ʹ,3,4,4ʹ,5-pentabromodiphenyl ether  xxvi  Acknowledgements  I would like to thank my supervisor Dr. Stelvio Bandiera for his guidance, expertise in the field, and kindness. I would also like to thank my committee members Dr. Thomas Chang, Dr. Marc Levine, Dr. Wayne Riggs, and Dr. Anthony Farrell for the time they dedicated to critically monitoring my progress for almost 5 years. A special thank you to András Szeitz for his help with the LC/MS. His operational knowledge of the LC/MS was invaluable during this project. I would also like to acknowledge Dr. Eugene Hrycay for his suggestions and help. Also thank you to past and present graduate students in Dr. Bandiera’s laboratory: Ravindranath Gilibili, Si Zhang, Subrata Deb, Anand Deo, and Sarah Moffatt. Thank you for bringing your team spirit to the laboratory. The author is also thankful to The University of British Columbia for the 1-year Graduate Student Fellowship and the subsequent 4-year Graduate Student Fellowship in support of my PhD project.   xxvii              Dedicated to those who have always believed in me  1  1. Introduction    1.1 Flame Retardants Advances in polymer science over the last 50 years led to the introduction of a wide variety of polymers used in clothing, furniture, electronics, vehicles, and computers. Because most of these polymers are petroleum-based, the risk of fire is present. Therefore, the addition of chemicals to consumer and industrial products that are capable of retarding the development and spread of fire has become increasingly important. This group of chemicals is usually referred to as flame retardants. Their ultimate effect is to provide more time to escape fires. It has been estimated that the use of flame retardants helped save 4,000 fire deaths in the US in the year 2000 and reduced fire deaths in Europe by 20% for the decade between 1994 and 2004 (BSEF 2004). Fire is the result of a chemical reaction between oxygen and fuel. To prevent a fire from developing or spreading, the chemical reaction between oxygen, fuel, and a heat source has to be prevented or interrupted, which is exactly what flame retardants are designed to do. In particular, halogenated flame retardants capture the free radicals produced during the combustion process that are essential for flame propagation. The most effective halogenated flame retardants are brominated compounds because they release bromine atoms at temperatures at which most organic matter burns and because bromine atoms have high trapping efficiency for free radicals (Alaee et al., 2003). 2  Flame retardants are divided into four major groups: inorganic, phosphorous- containing, nitrogen-containing, and halogenated flame retardants (Segev et al., 2009). The group of inorganic flame retardants contains metal hydroxide (i.e. aluminium and magnesium hydroxide) and elemental phosphorus (Alaee et al., 2003). The most common phosphorous-containing  flame retardants are phosphate esters (Covaci et al., 2003; van deer Ven and de Boer, 2012) and the most common nitrogen-containing flame retardants are melamine or melamine derivatives (Segev et al., 2009). Halogenated flame retardants contain bromine or chlorine. More than 75 different compounds are used as brominated flame retardants, which are usually subdivided into five classes. The five classes are polybrominated biphenyls, polybrominated diphenyl ethers (PBDEs), tetrabromo bisphenol- A, brominated cyclododecanes, and phthalate derivatives (Alaee et al., 2003). Global production data for brominated flame retardants between the years 1989 and 2001are reported in Table 1.1 (Janssen 2005; Law et al., 2006; Wang et al., 2007).   1.2 Polybrominated Diphenyl Ethers Brominated flame retardants became the most widely useed class of flame retardants in 1978. At that time, PBDEs represented approximately 40% of the global market for flame retardants (Vonderheide et al., 2008). Due to the presence of an increasing amount of plastic in consumer products, PBDEs have been added to a large number of everyday products. As a consequence, PBDEs found their way into our homes, vehicles, and offices. A brief timeline of PBDEs production, detection in the environment, and phase out is reported in Table 1.2. 3  Table 1.1 Global Production of Brominated Flame Retardants Between 1989 and 2001a Geographical area 1989b 1994b 1999b 2001c Europe 28.0 32.5 30.9 29.5 Asia 28.7 38.5 113.9 118 United States 50.0 65.0 58.7 53.9 Total 106.7 136 203.5 201.4  a Data are reported as 1,000 metric tons b Data derived from Alaee et al., 2003 c Data derived from Bromine Sscience and Environmental Forum (BSEF, 2004)               4  Table 1.2 Brief History of PBDEsa Year PBDE related events 1960 First patent issued for PBDEs as flame retardants. 1965 Manufacturing of commercial products containing PBDE starts. 1979 PCB manufacturing is banned from the U.S. Presence of Deca-BDE is first detected in the environment. 1987 PBDEs are first suggested as ubiquitous contaminants. 1990 PBDEs are found in human adipose tissues. 1998 Retrospective analysis of archived human milk samples shows a remarkable increase of PBDE levels over the years. 1999 Global production of PBDEs is estimated to be 70,000 tons. 2004 Penta- and Octa-BDE commercial mixtures are banned in Europe and voluntarily phased out in North America. 2006 Deca-BDE is classified as “toxic substance” under the Canadian Environmental Act. 2008 The use of Deca-BDE was banned in the states of Maine and Washington (U.S.) and in the European Union (in electric and electronic equipment only). 2013 Phasing out of PBDEs in the U.S.  a modified from Vonderheide et al., 2008      5  1.2.1 Physicochemical Properties of PBDEs The general chemical formula of PBDEs is C12 H(10-x) Br(x) O, where the number of bromine and hydrogen atoms add up to ten. There are 209 possible PBDE congeners, each containing one to ten bromine atoms attached to a diphenyl ether nucleus (Hutzinger et al., 1976). The structural similarity of PBDEs and polychlorinated biphenyls (PCBs; Figure 1.1) led to a common nomenclature system for PCBs and PBDEs developed by the International Union of Pure and Applied Chemistry. This nomenclature is based on the number and position of the halogen atoms on the rings and assigns a number between 1 and 209 to all possible congeners (Ballschmiter and Zell, 1980). The major physicochemical properties of selected PBDEs are summarized in Table 1.3. PBDEs are very lipophilic compounds. Their octanol-water partition coefficient (Kow) values span four orders of magnitude (approximately 107 to 1011), with the more brominated PBDEs having larger Kow values. The volatility of PBDEs is inversely related to the number of bromine atoms. Therefore, lower brominated congeners tend to escape into the gaseous phase more readily than more highly brominated congeners.   1.2.2 Synthesis of PBDEs PBDEs are synthesized by progressive bromination of a diphenyl ether in the presence of a Freidel-Craft catalyst, such as AlBr3 or FeBr3 (Alaee et al., 2003). Theoretically, 209 congeners can be formed. However, due to the presence of oxygen and the steric hindrance produced by the bromine atoms, the bromination of diphenyl ether is rather position specific. 6    Figure 1.1 Chemical structure of PBDEs (A) and PCBs (B).         7  Table 1.3 Physicochemical Properties of Selected PBDE Congenersa,b  BDE-47 BDE-99 BDE-153 BDE-209 Substitution pattern 2,2,4,4 2,2,4,4,5 2,2,4,4,5,5 2,2,3,3,4,4, 5,5,6,6 Molecular mass (g/mol) 485.8 564.7 643.6 959.2 Sw (mol/L) 3.04x10-8 7.74x10-9 7.82x 10-11 Sw (mg/L) 1.47x10-2 4.37x10-3 4.37x10-3 Log Kow 6.78 7.39 8.05 11.15 Log Koa 9.41 10.44 11.89 Melting point (°C) 80.5 92.3  300 Log VL -2.28 -2.87 -3.46 -5.8  a References: Ballschmiter and Zell, 1980; Kuramochi et al., 2007; Palm et al., 2002 ; Wania and Dugani, 2003; Yang et al., 2003  b Sw,Water solubility at 25°C; Kow, octanol–water partition coefficient; Koa,octanol-air partition coefficient. VL, vapour pressure at 25°C       8  The most commonly formed tri-brominated-PBDEs are 2,2ʹ,4-tribromodiphenyl ether (BDE-17) and 2,4,4ʹ-tribromodiphenyl ether (BDE-28). This initial selectivity in the bromination of the diphenyl ether limits the number and substitution pattern of the congeners formed by further bromination. Therefore, the main congeners formed from further bromination of BDE-17 and BDE-28 are 2,2',4,4'-tetrabromodiphenyl ether (BDE- 47), 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), 2,2',4,4',5,5'-hexabromodiphenyl ether (BDE-153), 2,2',4,4',5,6'- hexabromodiphenyl ether (BDE-154), 2,2',3,4,4',5',6-heptabromodiphenyl ether (BDE-183), 2,2',3,4,4',5,5',6-octabromodiphenyl ether (BDE-203), and 2,2',3,3',4,4',5,5',6,6'- decabromodiphenyl ether (BDE-209) (Alaee et al., 2003). The chemical structure of selected PBDEs is reported in Figure 1.2.   1.2.3 Formulations, Market Demand, and Presence of PBDEs in Market Products PBDEs were produced commercially in the U.S., United Kingdom, Holland, and France (BSEF 2004) as mixtures containing diverse congeners with different levels of bromination. The three commercial mixtures were named according to the average number of bromines per congener present in the mixture (LaGuardia et al., 2006). These mixtures are Penta-, Octa-, and Deca-BDE. The relative abundance of tetrabrominated to decabrominated PBDE congeners in each mixture is reported in Table 1.4. Two Penta-BDE mixtures, DE-71 and Bromkal 70-5DE, were produced. Both the penta-BDE formulations contained mainly BDE-47, -99, -100, -153, and -154. Six minor congeners (i.e. BDE-17, -28, -66, -85, -138, and -183) were also present in the Penta-BDE formulations (LaGuardia 9    Figure 1.2 Chemical structure and substitution pattern of the most commonly produced PBDEs. 10  Table 1.4 Relative Abundance of PBDE Congeners Present in the Three Commercial PBDE Mixturesa  Penta-BDE Octa-BDE Deca-BDE Tetrabrominated BDEs          BDE-47 38-43% 38-43% N.D.b N.D. Pentabrominated BDEs          BDE-85          BDE-99          BDE-100 55-65% 2-3% 45-49% 7.8-13% N.D. N.D. Hexabrominated BDEs          BDE-153          BDE-154          BDE-155 8-11% 5.3-5.4% 2.7-4.5% 0.2-0.7% 0.15-10% 0.1-8.7% 0.04-1.1% N.D. N.D. Heptabrominated BDEs          BDE-180          BDE-183 0.1-0.3% N.D. 0.1-0.3% 13-44% N.D.-1.7% 13-42% N.D.  Octabrominated BDEs          BDE-196          BDE-197          BDE-203  N.D. N.D. N.D. 18-41% 3.1-10.5% 10.5-22.2% 4.4-8.1% N.D.-0.6% N.D.-0.5% N.D.-0.03% N.D.-0.07% Nonabrominated BDEs          BDE-206          BDE-207  N.D. N.D. 13-19% 1.4-7.7% 11.2-11.5% 2.5-9% 2.2-5.1% 0.2-4.1% Decabrominated BDEs BDE-209  N.D.  1.3-50%  92-97%  a data from LaGuardia et al., 2006 b N.D. = not detected 11  et al., 2006; Sjodin et al., 1998). The Octa-BDE formulation contained mainly BDE-153, - 183, -196, -197, -203, and -207, while the Deca-BDE formulation contained BDE-209, with three congeners (BDE-206, -207, and -208) present only at trace levels (<1% v/v each; LaGuardia et al., 2006). The most recent publicly available data about market demands of PBDEs in America, Europe, and Asia are summarized in Table 1.5. According to a recent study by the Freedonia group (World Flame retardants # 2709, 2011), the worldwide demand for flame retardant additives will rise by 6.1% annually from 1.7 million tons in 2009 to 2.2 million tons in 2014. The increase in demand for flame retardants is mainly due to the Asia-Pacific region, with the Chinese market alone increasing almost 10% per year. Electrical, electronic, and motor vehicle products will likely fuel the demand the most (Additives for polymers, 2011; Wang et al., 2007). The three commercial PBDE mixtures were preferentially added to different categories of commercial products. Penta-BDE was mainly added to polyurethane foam, textiles, upholstery, epoxy resins, and paints, while the higher brominated mixtures (i.e. Octa- and Deca-BDE) were preferentially added to products operating at high temperatures such as high impact polystyrene used in TV sets, computer cabinets, and stereos (Table 1.6).      12  Table 1.5 Continent-Specific Market Demand (in tons) for Penta-, Octa-, and Deca- BDE Commercial Mixtures in 1999 and 2001a Commercial mixture Americas Europe  Asia Other Total world wide    1999 Deca-BDE 24,300 7,500  23,000  54,800 Octa-BDE 1,375 450  2,000  3,825 Penta-BDE 8,290 210  25,000  8,500 Total 33,965 8,160  50,000  67,125    2001 Deca-BDE 24,500 7,600  23,000 1,050 56,150 Octa-BDE 1,500 610  1,500 180 3,790 Penta-BDE 7,100 150  150 100 7,500 Total 33,100 8,360  24,650 1,330 67,440  a data from Hites 2004          13  Table 1.6 Major Products Containing Penta-, Octa-, and Deca-BDE Commercial Mixturesa Resin and Polymers Final Product Penta- PBDE Octa- PBDE Deca- PBDE Acrylonitrile-butadiene styrene Molded parts for TVs, computer casings, hairdryers and cars  X Epoxy-Resin Circuit boards and protective coating for computers and electronics X  X Phenolic Resins Paper laminates and glass prepegs for printed circuit boards X  X Polyacrylonitrile Lighting panels and housing of electrical appliances  X X Polystyrene & high impact polystyrene Television cabinets, electrical housing for electrical appliances   X Textiles Coatings on carpets, automotive seating, furniture and tents X  X Unstaturated Polyester Electrical equipment,  circuit boards and construction panels X  X  a data from Rahman et al., 2001   14  1.2.4 Regulation of PBDEs Manufacture and Use Commercial mixtures of PBDEs have undergone a series of restrictions, legislative bans, and voluntarily phase out in Europe and the U.S. while they are still unregulated in Asian countries. The European Union banned the production of the Penta-BDE and Octa- BDE mixtures as of August 2004 to reduce the health and environmental risk due to collection, treatment, recycling, and disposal of electrical and electronic equipment containing the two PBDE mixtures (European Union Directive 2002/95/EC and 2003/11/EC). Penta-BDE and the Octa-BDE mixtures were voluntarily phased out in the U.S. in 2004 after an assessment by the U.S. Environmental Protection Agency found that the alternative flame retardant (namely Firemaster 550) had a favorable environmental profile compared to the Penta-BDE mixture. In addition, Firemaster 550 is highly effective in retarding the spread of fire and meets the California flame retardant standards (U.S. EPA, 2008; Great Lakes Chemical Corporation, 2005). However, an official production ban by the U.S. government has not been made as yet and individual states are acting independently in this regard. Canada is presently conducting risk assessments of the three PBDE mixtures. Recently, the Canadian Ministry of the Environment published a regulation prohibiting the manufacture of PBDEs, as well as the usage, selling or importing of products containing PBDEs (Government of Canada, 2013). At an international level, the Parties of the Stockholm Convention for persistent organic pollutants took the decision to list commercial Penta-BDE and Octa-BDE as persistent organic pollutants in May 2009 (C.N.524.2009.TREATIES-4, 2009). The Deca-BDE mixture can no longer be used in electronic and electric equipment in the European Union as of July, 1st 2008 (European Court of Justice) and was banned in 15  Norway as of April, 1st 2008, but is still unregulated in the many Asian countries (i.e. China, Japan and Korea). U.S. producers and importers are committed to ending the production, import and sale of the Deca-BDE mixture by the end of 2013 (U.S. Environmental Protection Agency DecaBDE phase-out initiative 2012). The Deca-BDE mixture is listed as toxic under the Canadian Environmental Protection Act (1999) but there is no restriction on its use (BSEF 2009).   1.2.5 Emission Sources of PBDEs Although legislative actions have been taken to discontinue the production of PBDEs in Europe and North America, many sources of PBDEs are still present. First, the demand and usage of brominated flame retardants and PBDEs in Asia, and particularly in China and in South Korea, is expected to increase until the year 2014 (Additives to polymers 2011; Wang et al., 2007), suggesting that PBDEs might still be produced in those countries. Therefore, the most obvious source of PBDEs is discharges from factories producing PBDEs (Sellstrom et al., 1995, 1998). Second, commercial products currently in use and those produced before the legislative ban represent ongoing sources of PBDEs. Because PBDEs are simply blended with and not chemically bound to plastic and textile polymer materials (Alaee et al., 2003; Janssen, 2005), they can leach out of the polymer material with time (Hale et al., 2002). In support, high concentrations of PBDEs have been measured in dust collected from the inside of TV sets, computer cabinets, and clothes dryer lint (Schecter et al., 2009; Stapleton et al., 2005; Tamade et al., 2002). Third, landfill discharge of items containing PBDEs continue to be a source of PBDEs, which end up in air, soil, and 16  ultimately in the food chain (Chen et al., 2009; Eens et al., 2013; Odusanya et al., 2009). Fourth, photolysis and microbial degradation of BDE-209 were shown to generate a number of lesser brominated BDE congeners (Ahn et al., 2006; Soderstrom et al., 2004; Torarz et al., 2008).  As a result, PBDEs have been detected in a number of abiotic media (Hale et al., 2006) and in biological samples from around the globe (Hites, 2004), showing that PBDEs have become global environmental pollutants.   1.2.6 Persistency of PBDEs in the Environment The tendency of an environmental contaminant to resist physical, biological, and chemical degradation is called “persistence”. The persistence of a substance in a given medium is defined by its half-life in that medium. According to the Canadian Environmental Protection Act (1999), a pollutant is considered persistent if its half-life values are equal to or longer than 2 days in air, 182 days in water and soil, or 365 days in sediment. In addition, a pollutant is considered persistent in air if it is subject to long-range transport (Canadian Environmental Protection Act, 1999). Laboratory studies revealed that the half-life values of PBDE congeners in organic solvents are inversely related to the number of bromine atoms present in the congener and are longer than 2 days for all of the tetrabrominated-PBDE and pentabrominated-PBDE congeners assessed (Eriksson et al., 2004). Software-based model calculations of persistence resulted in half-life values for BDE-47, BDE-99 and BDE-209 ranging between 10 and 300 days in air, approximately 150 days in water and soil, and 600 days in sediments (Palm et al., 2002). A number of field-based investigations provided evidence for the presence of PBDEs in Arctic abiotic samples such as sediment and air (de 17  Wit et al., 2006). Considering the distance between arctic regions and the known sources of PBDEs, the long-range transport of PBDEs in the lower atmosphere seems to be incontrovertible.   1.2.7 PBDE Concentrations in Human Samples PBDEs have been measured in several human tissue samples and large differences in total PBDE concentrations have been reported. For example, PBDEs have been detected in liver (4.5 to 18 ng/g lipid weight), adipose tissue (1.36 to 9,630 ng/g lipid weight), milk (0.1 to 1,914 ng/g lipid weight), placenta (0.35 to 9.89 ng/g lipid weight), and blood (1.5 to 580 ng/g lipid weight) samples collected world-wide from the general population (Frederiksen et al., 2009). Several studies also showed the presence of PBDEs in umbilical cord blood, often at levels comparable with those of matching maternal blood samples (0.5 to 460 ng/g lipid weight) (Bi et al., 2006; Gomara et al., 2007 a,b; Guvenius et al., 2003; Mazdai et al., 2003; Weiss et al., 2004). Occupational exposure to PBDEs is also important. The total PBDE concentration in the serum of workers dismantling electronic products has been found to be 10 to 20 times greater than that of people with no occupational exposure to PBDEs (Bi et al., 2007; Qiu et al., 2009). Moreover, higher levels of total PBDEs were also measured in blood samples from children living in an electronic waste recycling region compared to those living in a region with no electronic waste recycling activity (Athanasiadou et al., 2008; Shen et al., 2010). This difference between occupationally and non-occupationally exposed individuals shows the impact of electronic waste recycling activities on PBDE blood levels in humans. 18  Time-trends and geographical differences exist in total PBDE concentrations in human samples. Total PBDE concentrations increased exponentially in human blood, milk, and adipose samples between 1970 and 2000, with a doubling time of approximately 5 years (Hites, 2004). In addition, total PBDE concentrations in human samples from North America are often ten times larger than in samples from Europe (Frederiksen et al., 2009; Hites, 2004; Mazdai et al., 2003). For example, the total median serum PBDE concentration in women from California (2008-2009) and Denmark (2007) was 85.5 and 1.8 ng/g lipid, respectively (Frederiksen et al., 2010; Zota et al., 2011). The most likely explanations for this large difference in PBDE human concentrations between North America and Europe are the more extensive use of the Penta-BDE mixture in North America compared to Europe (Table 1.5) and the earlier ban on PBDEs manufacture in some European countries (http://www.nrdc.org/breastmilk/pbde.asp). Differences in PBDE profiles have been noted in human samples. For example, BDE-47, BDE-100, and BDE-153 are the main congeners identified in human samples from North America and Sweden (Hites, 2004; Mazdai et al., 2003). BDE-99, the main congener of the Penta-BDE formulation (LaGuardia et al., 2006), is often present at levels lower than those of BDE-47 in human samples, possibly due to a lower intake of BDE-99 or a more rapid metabolism of BDE-99. In addition, a shift in congener composition from maternal to umbilical cord blood has been observed in most of the studies presently available. For example, BDE-47 and BDE-209 represent a larger percentage of the total PBDE content in umbilical cord blood than in matching maternal blood (Bi et al., 2006; Gomara et al., 2007 a; Guvenius et al., 2003; Weiss et al., 2004).  19  1.2.8 Routes of PBDEs Exposure for Humans For humans, the two main routes of exposure to PBDEs are diet and dust ingestion (Dirtu and Covaci, 2010; Jones-Otazo et al., 2005). Total PBDE concentrations in U.S. food are 10 to 20 times larger than in food from Spain and Japan (Bocio et al., 2003; Schecter et al., 2004), contributing the a greater exposure for North Americans than Europeans and Japanese to PBDEs. In Europe and North America, the highest PBDE concentrations were found in oils and fats, fish and shellfish, and then in meat and dairy products. Fruits and vegetables had much lower PBDE concentrations (Bocio et al., 2003; Schecter et al., 2008). The proportion of each kind of food in the diet is also an important determinant of the total daily intake of PBDEs. In recent years, there has been increasing concern about PBDEs in indoor dust. In general, PBDE concentrations are 1.5 to 20 times greater in indoor than outdoor environments (Besis and Samara, 2012; Butt et al., 2004; Harrad et al., 2010a). Concentrations of total PBDEs in dust are nearly ten times larger in the U.S. than in Europe. Total PBDE concentrations in dust usually range between 1 and 10 µg/g for homes in the U.S., Canada, and the United Kingdom, and between 27 and 510 ng/g for homes in many other European countries (Besis and Samara, 2012). BDE-209 is the dominant congener in many dust samples. BDE-47 and BDE-99 are present at similar concentrations in dust from North America (1,100-1,220 ng/g dry weight and 1,700-1,800 ng/g dry weight, respectively) and Europe (17-543 ng/g dry weight and 14-643 ng/g dry weight, respectively) (Johnson et al., 2010; Stapleton et al., 2005; Vorkamp et al., 2011; Wilford et al., 2005). The importance of diet and dust ingestion on the total daily intake of PBDEs depends on life stage and shows geographical trends. For infants, at least 90% of the total PBDE 20  intake is via human milk, especially in North America because of the higher PBDE levels in North American breast milk (Schecter et al., 2003; Park et al., 2011; She et al., 2007) compared to European breast milk (Jaraczewska et al., 2006; Roosens et al., 2010). For toddlers (12-24 months of age) and young children, most of the total daily intake of PBDEs is due to the ingestion of dust (Besis and Samara, 2012; Dirtu and Covaci, 2010) at home and in daycare centers and primary schools (Harrad et al., 2010b). For adults, the major PBDE intake is from the diet in many European countries, Japan, and Australia; ingestion of car dust in China; ingestion of home dust in the U.S. and the United Kingdom; and a mix of ingestion of home dust and diet in Canada (Besis and Samara, 2012). The high PBDE concentrations in house dust and the large number of hours spent indoors by North Americans are contributing factors in making dust ingestion the most important exposure route for humans in North America. Different concentrations of total PBDEs in food and dust samples in different countries result in differences in estimated total daily intake of PBDEs across countries. The estimated daily intake of total PBDEs (ng/day) for adults in the U.S. and the United Kingdom (480 and 450 ng/day, respectively) is twice that in China (240 ng/day) and 5 to 7 times larger than that in Canada, Australia, Japan and other European countries (ranging between 70 and 100 ng/day; Besis and Samara 2012). Similarly, the estimated daily intake of total PBDEs (ng/day) for toddlers (12-24 months of age) is 5 to 6 times larger in the U.S. and the United Kingdom (400 and 340 ng/day, respectively) than that in Canada and China (65 and 75 ng/day, respectively), and 8 to 40 times larger than that in Australia, Japan and other European countries (ranging between 12 and 50 ng/day; Besis and Samara 2012). Based on hepatotoxicity, embryotoxicity, and thyroid effects reported in animal studies, a 21  lowest-observed-adverse-effect level value of 70 mg/day for total PBDEs was suggested (Darnerud et al., 2001). Therefore, current knowledge regarding human exposure to PBDEs suggests that a safety margin of between 7 x 104 and 7 x 105 exists for PBDE-mediated toxicity in humans.   1.3 BDE-47 and BDE-99: Two Major PBDE Congeners 1.3.1 Physicochemical Properties and Environmental Distribution of BDE-47 and BDE-99 BDE-47 and BDE-99 are the two main congeners present in the two commercial Penta-BDE mixtures, DE-71 and Bromkal 70-5DE. In these two mixtures, BDE-47 represents 38.2 and 42.8% and BDE-99 represents 44.8 and 48.6% of the total PBDE content, respectively (LaGuardia et al., 2006). The substitution pattern and the most relevant physicochemical properties of BDE-47 and BDE-99 are summarized in Table 1.3. The physicochemical properties of BDE-47 and BDE-99 dictate their environmental distribution in abiotic media. Due to their low vapour pressure values and because they are major congeners of the Penta-BDE mixtures, BDE-47 and BDE-99 are the two dominant PBDE congeners detected in the air, both in the vapour state and in the particulate fraction, and in surface films (Butt et al., 2004; Farrar et al., 2004; Wilford et al., 2005). Due to their high lipophilicity (i.e. log Kow values of 6.78 and 7.39, respectively) and because they are major congeners of the Penta-BDE mixtures, BDE-47 and BDE-99 are also the two dominant PBDE congeners detected in indoor dust (Stapleton et al., 2005; Wilford et al., 2005), land 22  and agricultural soils (Hassanin et al., 2004; Matschenko et al., 2002), and in effluent, sludge, and sediment samples (Hites, 2004; North, 2004).   1.3.2 Bioaccumulation of BDE-47 and BDE-99 Although BDE-47 and BDE-99 are present at similar levels in many abiotic media (Butt et al., 2004; Hassanin et al., 2004; North, 2004; Stapleton et al., 2005; Wilford et al., 2005) and in some fish species (Johnson and Olson, 2001; Rayne et al., 2003; Viganò et al., 2008), levels of BDE-99 are lower than those of BDE-47 in many species of freshwater fish (Hale et al., 2001; Viganò et al., 2008), birds (Allchin et al., 2000; Law et al., 2002), saltwater fish, and marine mammals (Ikonomou et al., 2002a,b, 2006). These data suggest that BDE-99 bioaccumulates less than BDE-47 in several wildlife species. In addition, the lower biomagnification potential of BDE-99 compared to that of BDE-47 was shown across food webs (Boon et al., 2002; Kelly et al., 2008; Voorspoels et al., 2003). In humans, levels of BDE-47 are 3 to 10 times larger than those of BDE-99, as reported in plasma and serum samples collected from the general population in the U.S. (Qiu et al., 2009; Stapleton et al., 2011; Zota et al., 2011), Europe (Guvenius et al., 2003; Meijer et al., 2008; Roosens et al., 2010) and Asian countries (Bi et al., 2006; Kawashiro et al., 2008). This difference was also reported in adults and children occupationally exposed to PBDEs (Athanasiadou et al., 2008; Lee et al., 2007; Stapleton et al., 2008). The lower bioavailability or bioaccessibility of BDE-99 compared to BDE-47, or a higher excretion of BDE-99 compared to BDE-47 can explain the lower bioaccumulation of BDE-99 in wildlife species. In laboratory experiments involving fish and rats, the in vivo 23  uptake efficiency of BDE-99 from diet and dust was shown to be lower than that of BDE-47 (60% vs 90%; Burreau et al., 1997; Hakk et al., 2002; Huwe et al., 2008). An in vitro study of the bioaccessibility of PBDEs present in indoor dust using a colon extended model of the human gastrointestinal tract showed similar bioaccessibility values for BDE-47 and BDE-99 in stomach (36±4.3% and 26±3.6%, respectively), intestine (38±4.1% and 29±3.3%, respectively), and colon (38±3.8% and 28±3.5%, respectively)  (Abdallah et al., 2012).  In addition, cumulative excretion of BDE-99 was not larger than that of BDE-47 in mice treated with BDE-47 or BDE-99  at a single dosage of 1mg/kg or in rats fed dust containing equal amounts of BDE-47 and BDE-99 (Huwe et al., 2008; Staskal et al., 2006). Collectively, these studies suggest that there are minimal differences in bioaccessibility or excretion rates for BDE-47 and BDE-99 and the difference cannot explain the larger levels of BDE-47 compared to BDE-99 that have been measured in mammals and other species (Hites, 2004). The different biomagnification potential of BDE-47 and BDE-99 might be due to diverse diets and different trophic positions of the wildlife species in which concentrations of BDE-47 and BDE-99 were determined (Boon et al., 2002; Kelly et al., 2008; Voorspoels et al., 2003). Different trophic positions and diets might be responsible for exposing wildlife species to different loads of BDE-47 and BDE-99. Alternatively, inter-species differences in the biotransformation of BDE-47 and BDE-99 might offer an explanation for the different bioaccumulation and biomagnification abilities of BDE-47 and BDE-99.   24  1.4 Biotransformation Lipophilic compounds such as PBDEs are poorly excreted from animals due to their affinity with lipophilic tissues and their poor solubility in urine and feces. Therefore, lipophilic endogenous and exogenous compounds have to be biotransformed into more hydrophilic compounds to be eliminated from the body more easily. In general, biotransformation of lipophilic compounds is a 2-step process involving phase-I and phase- II reactions. In a majority of cases, phase-I reactions first oxidize the lipophilic compound by introducing or exposing a small functional group such as –OH, -NH, -SH, or -COOH. The major family of enzymes catalyzing phase-I reactions is the cytochrome P450 (CYP) family. In phase-II reactions, the phase-I metabolite undergoes conjugation with a large hydrophilic moiety such as glucuronic acid or sulfate to generate metabolites that are more water soluble. Therefore, phase-I enzymes serve to produce substrates (i.e. oxidized phase-I metabolites) for phase-II enzymes because, most of the time, the parent compounds are not suitable substrates for phase-II metabolism. The major enzymes catalyzing phase-II reactions are uridine diphosphate glucuronic acid transferase and sulfate transferase (Parkinson and Ogilvie 2008). Biotransformation can be a key determinant of the bioaccumulation and toxicity of xenobiotics. Biotransformation can decrease the fraction of the xenobiotic dose that reaches the blood circulation (i.e. xenobiotic bioavailability) and, therefore, the xenobiotic concentration in blood. The concentration of xenobiotic in blood is a critical factor in the effects produced in the body and its bioaccumulation in organs and tissues. In addition, xenobiotic metabolism can also contribute to toxicity because of the metabolites produced. Xenobiotic metabolism catalyzed by CYP enzymes can produce metabolites with decreased 25  or increased pharmacological activity or toxicity compared to the parent compound, resulting in detoxification or bioactivation of the parent compound, respectively. For example, phenobarbital, a widely used sedative drug, is metabolized to para-hydroxy- phenobarbital, which has decreased sedative activity (Williams, 2002). In contrast, hydroxycumyl alcohol, a metabolite of bisphenol-A produced by human liver microsomal CYP enzymes, was shown to have 100-fold greater estrogenic activity than bisphenol-A when measured as luciferase-based reporter assay of estrogen receptor transcriptional activity in human breast cancer cells (Nakamura et al., 2011). In recent years, increasing attention has been paid by regulatory agencies to adverse drug reactions due to the formation of reactive metabolites and screening of drug metabolite toxicity has become a routine practice in the pharmaceutical industry (Evans et al., 2004).   1.5 CYP Enzymes CYP enzymes are a superfamily of enzymes involved in oxidative reactions. The name cytochrome P450 describes the ability of these enzymes to produce a characteristic absorbance spectrum with a maximum at 450 nm when chemically reduced and bound to carbon monoxide (Figure 1.3). CYP enzymes are present in a wide variety of organisms such as bacteria, fungi, plants and animals (Parkinson and Ogilvie, 2008). In mammals, CYP enzymes are present in many organs, including liver, kidneys, adrenal glands, intestine, brain, gonads and lungs. Liver is the organ with the highest total CYP content. Within the cell, CYP enzymes are predominantly localized in the smooth endoplasmic reticulum 26    Figure 1.3 Absorbance spectra of CYP enzymes. Liver microsomes were prepared from rats treated with 3-methylcholanthrene, phenobarbital, or from untreated rats. Rat liver microsomes were diluted to 1 mg/mL using 0.1 M phosphate buffer. The CYP heme group was reduced with a small amount of sodium dithionite. The diluted sample was divided into two spectrophotometric cuvettes and the content of one cuvette was bubbled for 30 sec with carbon monoxide. The difference spectrum depicted in the figure was obtained scanning the absorbance of the cuvettes between 380 and 510 nm (taken from Hasler et al., 1999).      27  (Hrycay and Bandiera 2008), although a subset of CYP enzymes are present in the mitochondria (Omura, 2006). The presence of CYP enzymes in organisms ranging from bacteria to humans together with the finding that CYP enzymes have been conserved throughout evolution (Lewis, 1996) suggest that CYP enzymes play a vital role in the cell. Such a role might be the biotransformation of steroids, a key group of endogenous compounds regulating sexual maturation in vertebrates (Guengerich, 1990, 1991). Alternatively, CYP enzymes might play a key role in protecting organisms against the possible toxic effects of lipophilic exogenous compounds to which organisms are exposed to during their entire lifetime.   1.5.1 Structure of CYP Enzymes CYP enzymes are composed of a heme prosthetic group and the apoprotein. In the heme prosthetic group, the iron atom is coordinated with the four N atoms of the protoporphyrin ring and it is an essential part of the catalytic site of CYP enzymes (Figure 1.4). The iron atom of the heme prosthetic group can exist in two oxidative states, Fe2+ (reduced) or Fe3+ (oxidized). The reduced iron atom (Fe2+) of the CYP heme prosthetic group has a strong affinity for binding molecular oxygen and carbon monoxide (Lewis, 1996). The central iron atom of the heme prosthetic group is noncovalently bound to a sulfur atom of a conserved cysteine residue situated in proximity to the C terminus of the apoprotein. The apoprotein is a single polypeptide chain composed of approximately 500 amino acids (Figure 1.4). 28    Figure 1.4 Structure of the heme prosthetic group of CYP enzymes. The iron atom is coordinated to the protoporphyrin (IX) ring of the heme prosthetic group, to the conserved cysteine (cys) residue of the C terminus of the apoprotein and, when in the oxidized state, to a molecule of water.            S 29  The N terminus of the apoprotein anchors the CYP enzyme to the lipid membrane of the smooth endoplasmic reticulum. Most of the CYP enzyme, including the active site, is exposed to the cytoplasmic side of the endoplasmic reticulum. The lipophilic substrate binding site is located on the apoprotein and in close proximity to the heme group. A substrate binds to the binding site via multiple non-covalent interactions. Individual CYP enzymes can bind several substrates (Hrycay and Bandiera, 2008). Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent CYP oxidoreductase is an enzyme that is essential for CYP activity. It is also located on the surface of the smooth endoplasmic reticulum and forms a complex with each CYP enzyme, allowing the transport of electrons from NADPH to the heme prosthetic group of the CYP enzyme (Lewis, 1996). A schematic representation of the electron transfer chain ultimately leading to the substrate oxidation is presented in Figure 1.5.      30   Figure 1.5 Electron transfer and substrate oxidation by CYP enzymes. Electrons donated by NADPH and transferred through a series of oxidation-reduction reactions by NADPH-dependent CYP oxidoreductase and CYP enzyme are used to reduce molecular oxygen into water and to oxidize the substrate. FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; RH, reduced substrate; ROH, oxidized product.            31  1.5.2 CYP Catalytic Cycle CYP enzymes use molecular oxygen and NADPH to oxidize a variety of substrates, RH. The products of the reaction are the oxidized metabolite (ROH), NADP+, and water (Hasler et al., 1999; Hrycay and Bandiera, 2008; Lewis, 1996), as shown in the following general equation:  ܴܪ	 ൅ 	2ܰܣܦܲܪ	 ൅	ܱଶ 	 ܥܻܲܥܻܲ	ܴ݁݀ݑܿݐܽݏ݁ ൐ 	ܴܱܪ	 ൅ 	2ܰܣܦܲ ା 	൅	ܪଶܱ  The oxidation of the substrate occurs through a series of steps that are referred to as the CYP reaction cycle (Figure 1.6). In the resting state, the heme iron atom is in the oxidized state (Fe3+) and is coordinated to a molecule of water (Lewis, 1996). Substrates bind to the substrate binding site, which is in close proximity to the heme moiety, via non- covalent forces and displace the water molecule coordinated to the heme iron. Displacement of the water molecule is believed to cause a change in the spin state of the heme iron and to lower the CYP redox potential favoring the transfer of an electron from NADPH to the CYP enzyme, which results in the reduction of the heme iron from Fe3+ to Fe2+. Molecular oxygen binds to the heme iron of the reduced CYP (Fe2+)-substrate complex forming a ternary complex of CYP (Fe2+)-substrate-dioxygen. Addition of a second electron to the ternary complex reduces the molecule of oxygen bound to the heme group and forms a reactive oxygen species. The second electron can be donated either by NADPH or cytochrome b5. Protonation and cleavage of the O–O bond result in the transfer of the reactive oxygen to the substrate generating the oxidized metabolite (i.e. ROH) and water. 32   Figure 1.6 Major steps of the CYP catalytic cycle. Aadapted from Hrycay and Bandiera (2008).    33  The hydroxylated metabolite is then released from the CYP enzyme, allowing the CYP enzyme to return to its initial state and repeat the catalytic cycle (Hasler et al., 1999; Hrycay and Bandiera, 2008; Lewis, 1996). Most CYP-catalyzed reactions are oxidative in nature (Hrycay and Bandiera, 2008). Depending on the chemical structure of the substrate, CYP enzymes can catalyze aliphatic and aromatic hydroxylation, N- and S- oxidation, N-, S-, and O-dealkylation, deamination, and epoxide formation. A summary of the mechanisms through which these kinds of CYP enzyme mediated oxidative reactions occur is available in the literature (Guengerich, 2001; Parkinson and Ogilvie, 2008). CYP enzymes can also catalyze reductive reactions. Under normal aerobic conditions, binding of the substrate to the CYP enzyme favors reduction of the heme iron (Fe3+ to Fe2+), which has a high affinity for molecular oxygen. Under anaerobic conditions, the CYP catalytic cycle is interrupted at this point. Therefore, the heme iron atom is in the reduced state but cannot bind molecular oxygen because oxygen is not available and, therefore, oxygen is not reduced. As a consequence, the CYP-Fe2+ complex donates electrons to the bound substrate, reducing the substrate instead of oxidizing it. Anaerobic conditions occur much less frequently than aerobic conditions (Guengerich, 2001). Consequently, reductive reactions catalyzed by CYP enzymes occur much less frequently than oxidative reactions in mammals (Guengerich, 2001, 2005). It must be emphasized that a single CYP enzyme can be involved in several types of reactions with different substrates. As an example, CYP3A4 can catalyze 6β-hydroxylation of testosterone, N-demethylation of imatinib, and N-oxidation of voriconazole (Murayama et al., 2007; Nebot et al., 2010; Yamazaki and Shimada, 1997). This evidence suggests that the 34  metabolic reactions catalyzed by the same CYP enzyme depend on the structure of the substrate rather than the CYP enzyme involved. In general, there is no relationship between CYP enzymes and their abilities to catalyze specific types of reactions. CYP enzymes do not show high substrate specificity, poorly matching the “key and lock” enzyme model. It is also important to note that the metabolism of the same substrate can be catalyzed by several CYP enzymes. For examples, acetaminophen can be metabolized by CYP1A2, CYP2E1, and CYP3A4 (Laine et al., 2009). This evidence suggests that the binding abilities and catalytic properties of several CYP enzymes towards one specific substrate can overlap, depending on the specific substrate considered.   1.5.3 Nomenclature and Classification of CYP Enzymes To date, approximately 400 CYP enzymes have been discovered in various species including bacteria, fungi, plants, birds, fish, and mammals (Lewis, 1996; Nelson et al., 1996, 2004). Individual CYP enzymes are encoded by distinct genes. CYP enzymes are named and categorized into families and subfamilies according to their amino acid sequence and date of discovery (Nebert and Gonzalez, 1987). The nomenclature of CYP enzymes starts with the CYP abbreviation followed by an Arabic number (i.e. 1, 2, 3) which denotes the family, a capitol letter defining the subfamily (i.e. A, B, C), and a numeral denoting the individual enzyme (Nelson et al., 1996). For example, CYP3A4 is a member of family 3, subfamily A, and it is identified as enzyme number 4 within this subfamily. According to this system, CYP enzymes belonging to the same family share at least 40% of their amino acid sequence and CYP enzymes belonging to the same subfamily share at least 55% of their amino acid 35  sequence. CYP enzymes belonging to the same subfamily that differ by one amino acid and are encoded by the same gene are polymorphic variants. To date, 57 CYP enzymes have been identified in humans and classified into 18 families and 43 subfamilies (Guengerich et al., 2005; Hasler et al., 1999). There are 83 CYP identified enzymes in rats and 103 in mice (Lewis, 1996; Nebert and Gonzalez, 1987; Nelson et al., 2004). CYP enzymes can be also grouped according to the major classes of substrates metabolized (Table 1.7). In general, CYP enzymes belonging to families 1,2, and 3 are mainly involved in the biotransformation of xenobiotics (including drugs and environmental pollutants). CYP enzymes belonging to family 4 are mainly responsible for the oxidation of fatty acids and eicosanoids. CYP enzymes belonging to families 7, 8, 11, 17, 19, 21, 27, and 54 play important roles in the biosynthesis of endogenous compounds, mainly hormones and bile acids, and CYP enzymes belonging to the families 24, 26, and 27 are involved in metabolism of vitamins. The metabolic role of some CYP enzymes, which were identified recently and classified based on gene sequence information into various CYP subfamilies, has not been discovered yet. These CYP enzymes are referred to as orphan CYP enzymes.   1.5.4 Occurrence and Distribution of CYP Enzymes in Humans and Rats CYP enzymes are expressed in almost all organs in humans and other mammals. The concentration of CYP enzymes is highest in liver in mammals (Table 1.8). Typically, the total CYP content ranges between 0.8 and 1.1 and between 0.3 and 0.6 nmol/mg of microsomal protein in rat and human liver, respectively (Hrycay and Bandiera, 2008). 36  Table 1.7 Human CYP Enzymes Ordered According to the Major Classes of Substrates Metabolizeda Steroids Xenobiotics Fatty acids Eicosanoids Vitamins Unknown CYP7A1 CYP1A1 CYP2J2 CYP4F2 CYP24 CYP2A7 CYP7B1 CYP1A2 CYP4A11 CYP4F3 CYP26A1 CYP2R1 CYP8B1 CYP2A6 CYP4B1 CYP4F8 CYP261 CYP2S1 CYP11A1 CYP2A13 CYP4F12 CYP5A1 CYP27B1 CYP2U1 CYP11B1 CYP2B6  CYP8A1  CYP2W1 CYP11B2 CYP2C8    CYP3A43 CYP17 CYP2C9    CYP4A22 CYP19 CYP2C19    CYP4F11 CYP21A2 CYP2D6    CYP4F22 CYP27A1 CYP2E1    CYP4V2 CYP39 CYP2F1    CYP4X1 CYP46 CYP3A4    CYP4Z1 CYP54 CYP3A5    CYP20  CYP3A7    CYP26C1      CYP27C1  a Compiled from Guengerich (2005)    37  Table 1.8 Total CYP Content in Various Human and Rat Organsa Organ Human (nmol/mg microsomal protein) Rat (nmol/mg microsomal protein) Liver 0.30-0.60 0.8-1.1 Adrenal Gland 0.23-0.54 0.5 Small Intestine 0.03-0.21 0.05-0.2 Kidney 0.03 0.05-0.2 Lung 0.01 0.035-0.05 Brain 0.10 0.025-0.05 Testis 0.005 0.07-0.12 Skin Not Determined 0.05 Mammary Gland <0.001 0.001-0.003  a  taken from Hrycay and Bandiera (2008)              38  Although the total CYP content in adrenal glands is approximately half of that in liver, the much larger size of liver makes this organ the major site of CYP enzymes catalyzed metabolism in mammalian species. Although many CYP enzymes are constitutively expressed in the liver, other CYP enzymes are preferentially expressed in extra-hepatic tissues (Table 1.9). For example, CYP1A1 is constitutively expressed in the lungs (Shimada et al., 1997) and placenta (Hakkola et al., 1996) and CYP1B1 is constitutively expressed in adrenal cortex, bronchi, breast, ovary and placenta (Downie et al., 2013; Hakkola et al., 1997; Sanderson et al., 2001). CYP1A1 and CYP1B1proteins are normally present only at trace levels in human liver (Chang et al., 2003; Hakkola et al., 1997). In addition, CYP2A13 is predominantly expressed in nasal mucosa, lungs and trachea (Ding and Kaminsky, 2003). In general, extra- hepatic CYP enzymes contribute much less than hepatic CYP enzymes to overall xenobiotic metabolism although they contribute to organ-specific metabolism of xenobiotics. CYP enzymes belonging to the CYP1A, CYP2C, and CYP3A subfamilies represent 45 to 80% of the total CYP content in rodent and human liver (Bandiera, 2001; Guengerich, 2005; Lewis, 1996). CYP1A2, CYP2C9, and CYP3A4 are the three major CYP enzymes representing 15%, 20%, and 30% of the total mean CYP content of human liver microsomes and are responsible for the metabolism of more than half of the currently marketed drugs (Guengerich, 2005; Lewis, 1996). In rat liver, the major CYP enzyme subfamily is CYP2C and CYP2C11 is the predominant CYP enzyme, comprising up to 50% of the total CYP content in rat liver microsomes (Nedelcheva and Gut, 1994). This thesis will focus on the major xenobiotic metabolizing CYP families (CYP1, CYP2, and CYP3) present in humans and rats (Table 1.10). 39  Table 1.9 Presence of CYP1, CYP2, and CYP3 Enzymes in Various Organs of Humans and Ratsa Organ CYP enzymes expressed (mRNA or protein)  Human Rat Liver CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP4A11 CYP1A2, CYP1B1, CYP2A1, CYP2A2, CYP2B1, CYP2B2, CYP2C6, CYP2C11, CYP2C12, CYP2C13, CYP2D2, CYP2D3, CYP2D4, CYP2D18, CYP2E1, CYP3A2, CYP3A9, CYP3A18 Small intestine CYP1A1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5 CYP1A1, CYP2A3, CYP2B1, CYP2B2, CYP2C6, CYP2C11, CYP2D2, CYP2D3, CYP2D4, CYP2D18, CYP3A9, CYP3A18, CYP3A62 Nasal mucosa CYP2A6, CYP2A13, CYP2B6 CYP2A3, CYP2E1 Trachea CYP2A6, CYP2A13, CYP2B6, CYP3A  Lung CYP1A1, CYP1A2, CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2D6, CYP2E1, CYP2F1, CYP3A4, CYP3A5 CYP1A1, CYP1B1, CYP2A3, CYP2B1, CYP2B2, CYP2E1 Stomach CYP1A1, CYP1A2, CYP3A4 Colon CYP1A1, CYP1A2, CYP1B1, CYP3A4, CYP3A5     40  Organ CYP enzymes expressed (mRNA or protein)  Human Rat Kidney CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A5 CYP2C11, CYP2C23, CYP2D2, CYP2D3, CYP2D4, CYP2D18, CYP2E1 Skin CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5  Brain CYP1A1, CYP2C8, CYP2B6, CYP2D6, CYP2E1 CYP2C13, CYP2D4, CYP2D18 Mammary gland CYP1A1, CYP1B1, CYP2C8, CYP2D6, CYP3A4, CYP3A5  Placenta CYP1A1, CYP1B1,CYP2E1, CYP3A4, CYP3A5, CYP4B1 CYP1A1 Adrenal gland CYP1A1, CYP1B1,CYP2C8, CYP2C9 CYP2D4, CYP2D18 Testis CYP1A1,CYP1B1,CYP2A1,CYP2 C9, CYP2E1 CYP2D4, CYP2D18  a Compiled from Hrycay and Bandiera (2008), Klose et al. (1999), Martignoni et al. (2006), and Wang et al. (2002)       41  Table 1.10 Major Drug Metabolizing CYP Families in Human and Ratsa Family Subfamily Human Rat CYP1 A CYP1A1, CYP1A2 CYP1A1, CYP1A2  B CYP1B1 CYP1B1 CYP2 A CYP2A6, CYP2A7, CYP2A13 CYP2A1, CYP2A2, CYP2A3  B CYP2B6 CYP2B1, CYP2B2, CYP2B3  C CYP2C8, CYP2C9, CYP2C19 CYP2C6, CYP2C7, CYP2C11, CYP2C12, CYP2C13, CYP2C22, CYP2C23  D CYP2D6 CYP2D1, CYP2D2, CYP2D3  E CYP2E1 CYP2E1 CYP3 A CYP3A4, CYP3A5, CYP3A7, CYP3A43 CYP3A1, CYP3A2, CYP3A9, CYP3A18, CYP3A62  a adapted from Martignoni et al. (2006)            42  1.5.5 CYP1 Family The CYP family 1 consists of two subfamilies, CYP1A and CYP1B. The mammalian CYP1A subfamily contains two enzymes, CYP1A1 and CYP1A2. The CYP1B subfamily contains one enzyme, CYP1B1. CYP1A1 and CYP1A2 enzymes are highly conserved across mammalian species, as shown by the 83% and 80% amino acid sequence similarity for human and rats, respectively (Mugford and Kedderis, 1998). Constitutive levels of CYP1A1 are very low and often undetectable in many organs. However, after induction, CYP1A1 is expressed in liver, kidney, stomach, brain, and small intestine (Nebert et al., 2004). CYP1A2 is usually undetectable in extra-hepatic tissues but in the liver, the constitutive level of CYP1A2 accounts for 12 to 14% of the total hepatic CYP content in humans (Shimada et al., 1994). CYP1A1 and CYP1A2 metabolize planar molecules, showing distinct but overlapping substrate specificities. For example, CYP1A1 preferentially metabolizes polycyclic aromatic hydrocarbons, whereas CYP1A2 preferentially metabolizes polyaromatic and heterocyclic amines (Nedelcheva and Gut, 1994). Marker activities for CYP1 enzymes include ethoxyresorufin and methoxyresorufin O-dealkylation. Ethoxyresorufin O-dealkylation is more specific for CYP1A1 and methoxyresorufin O- dealkylation for CYP1A2 (Burke et al., 1985; Nerurkar et al., 1993). Levels of CYP1A1 and CYP1A2 are inducible by activation of the aryl hydrocarbon receptor (Nebert et al., 2004). Hepatic levels of CYP1A1 and CYP1A2 mRNA were induced 1,200- and 20-fold, respectively, in rats treated with β-naphthoflavone (80 mg/kg/day for 3 days) compared to control rats (Martignoni et al., 2004). CYP1A1 and CYP1A2 mRNA levels were induced 500- and 5-fold, respectively, in rat liver slices 43  cultured with 50 µM β-naphthoflavone for 24 h compared to the control group (Martignoni et al., 2004). CYP1B1 is constitutively expressed in extra-hepatic tissues, including adrenal cortex, bronchi, breast, ovary and placenta (Downie et al., 2013; Hakkola et al., 1997; Sanderson et al., 2001). CYP1B1 shows overlapping substrate specificity with CYP1A enzymes. For example, CYP1B1 is involved in the metabolism of N-heterocyclic amines and polycyclic aromatic hydrocarbons as benzo(a)pyrene (Harrigan et al., 2006; Nebert et al., 2004). Regulation of CYP1B1 is mediated by the aryl hydrocarbon receptor, as suggested by the 400-fold induction of CYP1B1 mRNA levels in liver of rats treated with 10 mg/kg of benzo(a)pyrene (Harrigan et al., 2006).   1.5.6 CYP2 Family The CYP2 family is the largest family of human and rat CYP enzymes in terms of number of enzymes identified. The mammalian CYP2family contains several subfamiliesincluding CYP2A, 2B, 2C, 2D, and 2E. Expression of CYP2A and 2B enzymes is mainly extra-hepatic, whereas expression of CYP2C, 2D, and 2E enzymes is mainly hepatic (Lewis, 1996). Enzymes of the CYP2 family are involved in the metabolism of chemicals with non-planar structures, including several drugs, environmental pollutants and some steroid hormones. The human CYP2A subfamily includes CYP2A6, CYP2A7 and CYP2A13 enzymes. CYP2A6 accounts for approximately 4% of the total liver CYP content (Martignoni et al., 2006) and is involved in the metabolism of xenobiotics including coumarin, nicotine, and 44  aflatoxin B1 (Guengerich, 1997; Honkakoski and Negishi, 1997). CYP2A13 is predominantly expressed in tissues of the human respiratory tract, including tracheal, lungs and nasal mucosa (Ding and Kaminsky, 2003) and is involved in the metabolism of aflatoxin B1 and nicotine (He et al., 2006; von Weymarn et al., 2006). The mechanisms of CYP2A enzyme regulation are not well understood. The rat CYP2A subfamily includes CYP2A1, CYP2A2 and CYP2A3 enzymes. Rat CYP2A1 and CYP2A2 show sex-dependent expression. CYP2A1 and CYP2A2 are predominantly expressed in female and male rat liver, respectively. CYP2A3 is an extra- hepatic enzyme that is constitutively expressed in lung, intestine and nasal epithelium (Haduch et al., 2005; Lewis, 1996). Endogenous steroids are substrates for rat CYP2A enzymes. For example, CYP2A1 catalyzes 7α-hydroxylation of testosterone and CYP2A2 catalyzes 7α- and 15α-hydroxylation of testosterone (Honkakoski and Negishi, 1997; Lewis and Lake, 1995). In addition, CYP2A1 catalyzes 3-hydroxylation of coumarin, a medicial plant product, and is used as marker activity of CYP2A1 (Lake et al., 1989). CYP2A1 is weakly induced by phenobarbital and CYP2A3 is induced by 3-methylcholanthrene (Honkakoski and Negishi, 1997), suggesting that induction of CYP2A enzymes might involve different nuclear receptors, including the constitutive androstane receptor and the aryl hydrocarbon receptor. CYP2B6 is the only enzyme belonging to the subfamily 2B in humans. CYP2B6 is expressed in the liver and in extra-hepatic tissues including brain, kidney, intestine and skin (Ding and Kaminsky, 2003; Gervot et al., 1999; Janmohamed et al., 2001) but not in fetal liver samples at 11 and 24 weeks of gestation (Maenpaa et al., 1993; Hakkola et al., 1994). CYP2B6 protein content was reported to range between 0.5 and 70 pmol/mg protein (Code 45  et al., 1997; Ekins et al., 1998). CYP2B6 activity varied from 10- to 25-fold using S- mephenytoin N-demehtylation as CYP2B6 marker activity (Ekins et al., 1998; Lamba et al., 2003) and up to 80-fold using bupropion hydroxylation as CYP2B6 marker activity (Faucette et al., 2000) in single donor human liver microsomes. CYP2B6 is a highly polymorphic enzyme with 37 alleles and more than 100 single nucleotide polymorphic variants (Lang et al., 2001; http://www.cypalleles.ki.se/cyp2b6.htm). A growing number of drugs, environmental pollutants and endogenous steroids with diverse chemical structures have been shown to be metabolized by CYP2B6 (Ekins and Wrighton, 1999; Hodgson and Rose, 2007; Wang and Tompkins, 2008). For example, CYP2B6 is involved in the metabolism of drugs, including cyclophosphamide, bupropion, efavirenz, nicotine and tamoxifen (Chang et al., 1993; Faucette et al., 2000; Hesse et al., 2000; Nakajima et al., 1996; Styles et al., 1994; Ward et al., 2003). CYP2B6 is also involved in the biotransformation of environmental pollutants such as organophosphorus insecticides (Buratti et al., 2003, 2005; Ellison 2012; Foxemberg et al., 2007; Sams et al., 2004; Tang et al., 2001), organohalogenated pollutants (Ariyoshi et al. 1995; Hu and Kupfer 2002a,b; Lee et al., 2006; Stresser and Kupfer, 1999) and polycyclic aromatic hydrocarbons (Cho et al., 2006; Shou et al., 1994a,b, 1996b; Yamazaki et al., 1994). In addition, CYP2B6 was shown to be involved in the metabolism of steroid hormones, including estrone and 17β-estradiol (Imaoka et al., 1996; Shou et al., 1997). Formation of 4-hydroxy-bupropion is a well established marker activity for CYP2B6 in human liver microsomes (Faucette et al., 2000; Hesse et al., 2000). CYP2B6 expression is inducible by xenobiotics, alcohol consumption and smoking. For example, CYP2B6 mRNA and protein expression and CYP2B6 catalytic activity 46  (measured as bupropion 4-hydroxylation) were induced in human primary hepatocytes treated with phenobarbital, dexamethasone or rifampicin, suggesting that the constitutive androstane receptor and the pregnane X receptor are involved in the regulation of CYP2B6 expression (Goodwin et al., 2001; Faucette et al., 2007; Pascussi et al., 2000; Wang et al., 2003). CYP2B6 mRNA levels were induced 3- to 5-fold in human hepatocytes exposed to cigarette smoke extract (Washio et al., 2011). In vivo, CYP2B6 protein level was induced in human brain regions by smoking (2.2- to 3.3-fold) and alcohol consumption (3.5- to 5.3- fold) (Mikyss et al., 2003). The rat CYP2B subfamily consists of CYP2B1, CYP2B2, and CYP2B3 enzymes. CYP2B1 and CYP2B2 are constitutively expressed in the liver and in extra-hepatic tissues including small intestine and lungs (Lake et al., 2003; Lindell et al., 2003). Basal levels of CYP2B enzymes are higher in male than in female rat liver (Schuetz et al., 1990). Most CYP2B substrates are hydrophobic xenobiotics with non-planar molecular structures (Lewis, 1996). For example, PCBs (Kaminsky et al., 1981; Kennedy et al., 1981), dichlorodiphenyltrichloroethane, lindane (Lewis, 1996), the antitumor drug cyclophosphamide (Ruzicka and Ruenitz, 1992), and testosterone (Rosenbrock et al., 1999) are substrates of rat CYP2B enzymes. Pentoxyresorufin and benzyloxyresorufin O- dealkylation are commonly used as marker activities for rat CYP2B enzymes (Burke et al., 1985; Nerurkar et al., 1993). Recently, bupropion 4-hydroxylation was shown to be selectively catalyzed by CYP2B1 (Pektong et al., 2012), suggesting overlapping substrate specificity between CYP2B6 and CYP2B1. CYP2B1 and CYP2B2 are highly inducible enzymes. CYP2B1 and CYP2B2 mRNA and protein levels are increased by phenobarbital and dexamethasone (Cui et al., 2005; Lewis, 1996; Shinohara et al., 1997; Soucek and Gut, 47  1992), suggesting the involvement of the constitutive androstane receptor and of the pregnane X receptor in the regulation of CYP2B1 and CYP2B2 expression. Little is known about the regulation and activity of CYP2B3. The human CYP2C subfamily consists of CYP2C8, CYP2C9, and CYP2C19 enzymes. All CYP2C enzymes are predominantly expressed in the liver. Some CYP2C enzymes are also expressed in extra-hepatic tissues, including adrenal gland, prostate, brain, uterus, and mammary glands (Klose et al., 1999). CYP2C enzymes are involved in the metabolism of approximately 20% of clinically used drugs (Goldstein, 2001). For example, CYP2C19 metabolizes (S)-mephenytoin, omeprazole, and other proton pump inhibitors (Andersson et al., 1992), the antidepressant imipramide (Skjelbo et al., 1991), and some barbiturates (Adedoyin et al., 1994). CYP2C9 is involved in the metabolism of the diabetic agent tolbutamide, the anticonvulsant phenytoin, the anticoagulant warfarin, and anti- inflammatory drugs such as ibuprofen and diclofenac (Goldstein and De Morais, 1994). A smaller number of drugs are metabolized by CYP2C8 (Rahman et al., 1994). All CYP2C enzymes exhibit genetic polymorphism, some of which have clinical implications. For example, genetic variants of CYP2C19 lead to a poor metabolizer phenotype. These individuals exhibit reduced rates of omeprazole and diazepam metabolism, which can result in larger circulating levels of these drugs and increased likelihood of side effects (Goldstein, 2001). CYP2C enzymes are inducible. Protein levels of CYP2C8, CYP2C9, and CYP2C19 were increased in human hepatocytes cultured with phenobarbital or rifampin (Gerbal- Chaloin et al., 2001), suggesting the involvement of constitutive androstane receptor and pregnane X receptor in regulation of CYP2C8, CYP2C9, and CYP2C19 enzymes. 48  The rat CYP2C subfamily comprises seven enzymes, namely CYP2C6, CYP2C7, CYP2C11, CYP2C12, CYP2C13, CYP2C22, and CYP2C23. CYP2C enzymes are predominantly expressed in the liver although some CYP2C enzymes are also expressed in extra-hepatic tissues. For example, CYP2C11 is expressed in kidney and small intestine (Zhang et al., 1999; Zhao et al., 2003), CYP2C13 is expressed in the brain (Riedl et al., 2000), and CYP2C6 is expressed in the small intestine (Zhang et al., 1999). The CYP2C subfamily is the most abundant CYP subfamily in rat liver (Guengerich, 2005). Some CYP2C enzymes show sex-specific expression. CYP2C7 and CYP2C12 are only expressed in female liver whereas CYP2C11 and CYP2C13 are only expressed in male liver. CYP2C6, CYP2C22, and CYP2C23 do not exhibit sex-specific expression (Agraval and Shapiro, 1997; Mugford and Kedderis, 1998). Rat CYP2C enzymes are involved in the hydroxylation of endogenous steroids (Lewis, 1996; Martucci and Fishman, 1993; Nedelcheva and Gut, 1994). For example, CYP2C11 converts testosterone to 2α-hydroxy-testosterone and 16α- hydroxy-testosterone (Cheng et al., 1983; Chovan et al., 2007). The mechanisms that regulate expression of rat CYP2C enzymes are currently poorly understood. CYP2D6 is the only CYP enzyme expressed in the CYP2D subfamily in humans. CYP2D6 is expressed mainly in the liver but also in kidney, small intestine, and brain (Martignoni et al., 2006). CYP2D6 accounts for 4-5% of the total hepatic CYP content in human liver andexhibits inter-individual variability (Madani et al., 1999; Zuber et al., 2002). Hepatic microsomal CYP2D6 protein concentration and catalytic activity were found to vary by 13- and 14-fold, respectively, among individuals (Madani et al., 1999). CYP2D6 is a highly polymorphic enzyme wtih more than 80 polymorphic variants (Brockmoller et al., 2000; Guengerich, 2005). Depending on the number of copies of wild-type and polymorphic 49  alleles inherited, individuals have been classified as poor, intermediated, extensive and ultra rapid metabolizers of debrisoquine (Johansson et al., 1993). CYP2D6 is involved in the metabolism of approximately 30% of drugs presently on the market (Zuber et al., 2002), including bufuralol, debrisoquine, and propranolol (Hiroi et al., 2002). Therefore, CYP2D6 polymorphism has clinical relevance, especially for drugs with a narrow therapeutic index. The regulation of CYP2D6 expression is poorly understood. However, CYP2D6 protein content and catalytic activity were shown to increase developmentally (Stevens et al., 2008). The rat CYP2D subfamily consists of six enzymes, namely CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18. The six CYP2D enzymes are expressed in various tissues including liver, kidney, and small intestine (Hiroi et al., 1998) and exhibit partial overlap in substrate specificity. For example, formation of 1'-hydroxy-bufuralol is catalyzed by all the rat CYP2D enzymes. However, formation of bufuralol 1',2'- ethenylbufuralol is specifically catalyzed by CYP2D4. Debrisoquine 4-hydroxylation and propranolol 7-hydroxylation activities were specific to CYP2D2. These catalytic activities are useful as marker activities for individual rat CYP2D enzymes (Hiroi et al., 2002). The regulation of CYP2D enzyme expression is poorly understood. However, CYP2D mRNA and protein levels were increased in the brains of rats treated with testosterone or nicotine (Baum and Strobel, 1997; Yue et al., 2008). In rats treated with nicotine, the increase in CYP2D protein level did not involve alterations in mRNA levels, indicating a post transcriptional mechanism. CYP2E1 is the only member of the CYP2E subfamily in humans and in rats. CYP2E1 is constitutively expressed in liver, lungs, and nasal mucosa (Wang et al., 2002). CYP2E1 is the only CYP enzyme involved in the biotransformation of small hydrophilic 50  molecules. Substrates of CYP2E1 include organic solvents (i.e. acetaldehyde, acetone and methyl tert-butyl-ether), nitrosamines (i.e. N-nitrosodimethylamine), and drugs including acetaminophen, caffeine and paracetamol (Hong et al., 1997; Levin et al., 1986;  Terelius et al., 1991; Zuber et al., 2002). CYP2E1-mediated metabolism can produce reactive oxygen intermediates that can lead to oxidative stress and liver injury (Wu and Cederbaum, 2003). Bromozene, vinylidene chloride, p-nitrophenol and ethanol are examples of CYP2E1 substrates that are metabolizedto reactive oxygen species and result in liver injury (Dai et al., 1993; Hetu et al., 1983; Siegers et al., 1983). The regulation of CYP2E1 expression is complex (Novak and Woodcroft, 2000). Ethanol was shown to induce CYP2E1 protein expression via post-transcriptional mechanisms (Novak and Woodcroft, 2000).   1.5.7 CYP3 Family CYP3A is the only subfamily of the CYP3 family in humans and rats. CYP3A enzymes are the most abundant CYP enzymes in human liver and intestine, representing approximately 30% and 80% of the total CYP content, respectively (Guengerich, 2005; Martignoni et al., 2006; Paine et al., 2006). CYP3A enzymes are involved in the metabolism of many xenobiotics, including drugs and environmental pollutants, and of endogenous molecules, including bile acids and steroid hormones (Hrycay and Bandiera, 2008). The human CYP3A subfamily consists of CYP3A4, CYP3A5, CYP3A7, and CYP3A43 enzymes. CYP3A4 is the predominant CYP3A enzyme in human liver and small intestine (Shimada et al., 1994; Paine et al., 2006; Wrighton et al., 1990). CYP3A4 is involved in the metabolism of several environmental pollutants, including chlorpyrifos, 51  parathion and malathion (Buratti et al., 2003, 2006; Butler and Murray, 1997) and endosulfan and methoxychlor (Casabar et al., 2006; Hu and Kupfer, 2002a; Lee et al., 2006; Stresser and Kupfer, 1997). Many drugs are metabolized by CYP3A4, including erythromycin, midazolam, and quinidine (Christensen et al., 2009; Maekawa et al., 2009; Nielsen et al., 1999; Riley and Howbrook, 1998). Steroid hormones including progesterone and testosterone are also metabolized by CYP3A4 (Yamazaki and Shiamda, 1997). CYP3A5 is expressed in liver and intestine at levels up to 60 and 25% of those of CYP3A4, respectively (Paine et al., 2006; Yamaori et al., 2004). CYP3A5 shows overlapping catalytic selectivity with CYP3A4 (Wrighton et al., 1989) but has equal or lower activity than CYP3A4 for metabolism of many substrates (Williams et al., 2002). CYP3A7 is mainly expressed in fetal liver, where it accounts for 30 to 50% of the total hepatic CYP content (Daly, 2006; Gellner et al., 2001). The rat CYP3A subfamily is composed of five members, namely CYP3A1, CYP3A2, CYP3A9, CYP3A18 and CYP3A62. The CYP3A enzymes show sexually- dimorphic expression. For example, CYP3A2 and CYP3A18 are male-specific enzymes (Robertson et al., 1998; Yamazoe et al., 1988), whereas CYP3A9 is female dominant (Robertson et al., 1998). CYP3A1 is not detectable in the liver of untreated rats. In contrast, CYP3A2 is constitutively expressed in the liver of immature and adult male rats (Cooper et al., 1993). CYP3A62 is predominately expressed in the small intestine (Martignoni et al., 2006). CYP3A1 and CYP3A2 are involved in the metabolism of several drugs, including midazolam, triazolam, phenacetin, coumarin, and R-mephenytoin (Chovan et al., 2007; Kobayashi et al., 2002). 52  Levels of CYP3A enzymes are highly inducible. Xenobiotics that cause up- regulation of CYP3A enzymes include dexamethasone, phenobarbital and rifampin (Cooper et al., 1993). Dexamethasone causes up-regulation of CYP3A enzymes in rat liver slices and human hepatocytes (Lu and Li, 2001; Martignoni et al., 2004). In contrast, ripampin is a strong inducer of CYP3A in humans but not in rats (Lu and Li, 2001; Martignoni et al., 2006). Induction of CYP3A levels occurs via transcriptional activation, mainly through the activation of the pregnane X receptor. The constitutive androstane receptor and the glutocorticoid receptor are also thought to be involved in CYP3A induction (Burk and Wojnowski, 2004).   1.6 Oxidative Metabolism of PBDE Mixtures in Rodents CYP enzymes typically catalyze the first step in the biotransformation of lipophilic xenobiotics (Parkinson and Ogilvie, 2008). PBDEs are lipophilic xenobiotics (Table 1.3). Therefore, some of the CYP enzyme(s) discussed in the previous sections could be involved in the biotransformation of PBDEs in mammals. The formation of hydroxylated metabolites of PBDEs has been investigated in plasma and feces of rodents that had been treated with PBDE mixtures. For example, mice were treated with the PBDE mixture, DE-71, at 45 mg/kg per day for 5 days a week for 34 days by subcutaneous injection or oral gavage. Several hydroxylated PBDE metabolites, including 4ʹ-hydroxy-BDE-17 (4ʹ-OH-BDE-17), 2'-hydroxy-BDE-28 (2ʹ-OH-BDE-28), 4- hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), 3-hydroxy-BDE-47 (3-OH- BDE-47), and 6-hydroxy-BDE-47 (6-OH-BDE-47), 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl 53  ether (4ʹ-OH-BDE-49), 2,4-dibromophenol (2,4-DBP), 2,4,5-tribromophenol (2,4,5-TBP), and 2,4,6-tribromophenol were detected in plasma samples from mice 24h after the last treatment (Qiu et al., 2007). Sixteen hydroxylated PBDEs were detected in plasma samples of rats that had been treated with a single intraperitoneal injection of DE-71, along with BDE-183 and BDE-209, but only 4-OH-BDE-42, 3-OH-BDE-47, and 4ʹ-OH-BDE-49 were identified (Malberg et al., 2005). Five mono-OH-tetrabrominated-PBDEs and five mono- OH-pentabrominated-PBDEs were detected but were not structurally identified in plasma and fecal samples of mink fed DE-71 (Zhang et al., 2008). In these studies, it was not possible to determine which of the hydroxylated PBDEs detected were derived from parent PBDE congeners because rats, mice, and mink were treated with PBDE mixtures. In addition, the structures of most of the hydroxylated metabolites formed could not be elucidated because of the limited number of authentic standards. Moreover, the rates of formation of the hydroxylated PBDE metabolites were not calculated.   1.7 Metabolism of BDE-47 in Mammals A few studies have been conducted to investigate the metabolism of BDE-47 in rats and mice that were treated in vivo with this congener (Orn and Klasson-Weheler, 1998; Marsh et al., 2006; Sanders et al., 2006; Staskal et al., 2006). In addition, a limited number of studies investigated the in vitro metabolism of BDE-47 by rat or human hepatic preparations (Hamers et al., 2008; Lupton et al., 2009, 2010; Marteau et al., 2012) and biomonitoring studies revealed the presence of hydroxylated PBDEsin human plasma and 54  serum samples (Athanasiadou et al., 2008; Kawashiro et al., 2008; Qiu et al., 2009; Zota et al., 2011). Five hydroxylated metabolites of BDE-47 were detected in the feces of rats and mice that had been treated with 30 moles of 14C-BDE-47/kg per day for 5 days. Two hydroxylated metabolites of BDE-47 were detected in the plasma of rats and mice and only at trace levels (Orn and Klasson-Weheler, 1998). In another study, rats and mice were treated by oral gavage with 1 µmole of 14C-BDE-47/kg per day for ten days (Sanders et al., 2006). BDE-47 derived metabolites made up to 39% and 18% of 14C in fecal extracts of rats and mice, respectively, suggesting that the oxidative metabolism of BDE-47 proceeded at a larger rate in rats than in mice. In addition, a 2,4-DBP glucuronide and a 2,4-DBP sulfate conjugate were detected in rat urine. With the exception of these last two metabolites, the structures of the other metabolites of BDE-47 were not elucidated. In a third study, three hydroxylated metabolites were detected (but not structurally identified) in fecal extracts of mice that had been intravenously administered 1 mg BDE-47/kg (Staskal et al., 2006). Using authentic standards, the hydroxylated metabolites formed by rats treated with 30 µmoles of 14C-BDE-47/kg per day for 5 days were identified for the first time as 4'-OH- BDE-17, 2'-OH-BDE-28, 3'-OH-BDE-28, 4-OH-BDE-42, 3-OH-BDE-47, 5-hydroxy-BDE- 47 (5-OH-BDE-47), 6-OH-BDE-47, and 4'-OH-BDE-49 (Marsh et al., 2006). Collectively, these studies showed that BDE-47 undergoes oxidative metabolism in vivo in rodents. However, the structures of the hydroxylated metabolites of BDE-47 were characterized only in one study and their rates of formation were not determined. Only one study investigated the in vitro metabolism of BDE-47 in rats (Hamers et al., 2008). Liver microsomes (1mg/mL) obtained from rats tha had been pretreated with 55  phenobarbital were incubated with 25 µM BDE-47 for 90 min. Six metablites were produced; 3-OH-BDE-47 and 4'-OH-BDE-49 (major metabolites), 2'-OH-BDE-66 (intermediate metabolite), and 4-OH-BDE-42, 5-OH-BDE-47, and 6-OH-BDE-47 (minor metabolites). The hydroxylated metabolites of BDE-47 formed by rat liver microsomes largely overlap those formed by rats treated with BDE-47 in vivo and identified by Marsh et al. (2006) (Figure 1.7). However, rates of metabolite formation by rat liver microsomes were not determined and the CYP enzymes involved in the formation of hydroxylated metabolites of BDE-47 were not identified. There is much less information regarding the in vitro hepatic biotransformation of BDE-47 in humans. Two hydroxylated metabolites (2,4-DBP and an unidentified di-OH- tetrabrominated-PBDE) were detected following incubation of human liver microsomes with BDE-47 for 2 h (Lupton et al., 2009, 2010). In contrast, three different hydroxylated metabolites (3-OH-BDE-47, 5-OH-BDE-47, and 6-OH-BDE-47) were detected following incubation of human liver microsomes with BDE-47 for 1 h (Feo et al., 2013). In addition, 5-OH-BDE-47, 6-OH-BDE-47 and an unidentified di-OH-tetrabrominated-PBDE were detected when human primary hepatocytes were incubated with BDE-47 for 24 h (Marteau et al., 2012). In these studies, relatively few metabolites of BDE-47 were identified, rates of formation of hydroxylated metabolites of BDE-47 were not calculated, and the CYP enzymes responsible for their formation were identified only by Feo et al. (2013). Hydroxylated PBDEs have been detected in human plasma and serum samples, suggesting in vivo metabolism of BDE-47. In plasma and serum samples of women and children who were environmentally (Kawashiro et al., 2008; Qiu et al., 2009; Zota et al., 2011) or occupationally (Athanasiadou et al., 2008) exposed to PBDE mixtures, the 56    Figure 1.7 Hydroxylated metabolites of BDE-47 produced in vivo by rats treated with BDE- 47 (A) and in vitro by rat liver microsomes (B). References: Hamers et al., 2008; Marsh et al., 2006.  A B 57  presence of 2,4-DBP, mono-OH-tetrabrominated-PBDEs (i.e. 4-OH-BDE-42, 3-OH-BDE- 47, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49) and mono-OH-tribrominated- PBDEs (4'-OH-BDE-17 and 2'-OH-BDE-28) was reported. The hydroxylated PBDEs detected in human plasma and serum samples likely are in vivo metabolites of BDE-47. Many more metabolites were detected in human plasma and serum samples than were formed when BDE-47 was incubated with human liver microsomes or hepatocytes (Figure 1.8). This discrepancy could be due to the incubation conditions used to investigate the in vitro metabolism of BDE-47. More effort is needed to characterize the in vitro metabolism of BDE-47 by human liver microsomes with a different experimental design and analytic method.   1.8 Metabolism of BDE-99 in Mammals Two mono-OH-tetrabrominated-PBDEs, four mono-OH-pentabrominated-PBDEs and one di-OH-pentabrominated-PBDE were detected in the feces of mice that had been injected intravenously with BDE-99 at a single dose of 1 mg/kg body weight (Staskal et al., 2006). Two mono-OH-tetrabrominated-PBDEs, two mono-OH-pentabrominated-PBDEs, and three di-OH-pentabrominated-PBDEs were detected in feces and bile of rats that had been orally treated with BDE-99 at a single dose of 2.2 mg/rat (Hakk et al., 2002). 2,4,5- TBP, one mono-OH-tetrabrominated-PBDE, and two mono-OH-pentabrominated-PBDEs were detected in the feces of rats that had been administered a single dose of BDE-99 (0.1 to 1,000 µmol/kg) by oral gavage (Chen et al., 2006). Glucuronide and sulphate conjugates of 2,4,5-TBP, mono-OH-tetrabrominated-PBDEs, and mono-OH-pentabrominated-PBDEs 58    Figure 1.8 Hydroxylated metabolites of BDE-47 formed by human hepatic preparations (A) and hydroxylated PBDEs detected in human plasma and serum samples (B). References: Athanasiadou et al., 2008; Feo et al., 2013; Kawashiro et al., 2008; Lupton et al., 2009, 2010; Marteau et al., 2012; Qiu et al., 2009; Zota et al., 2012. A B 59  metabolites of BDE-99 were detected in rat urine and bile samples (Chen et al., 2006). Most of the hydroxylated metabolites, other than 2,4,5-TBP, were not identified in these studies because of the lack of authentic standards and rates of formation of the hydroxylated metabolites of BDE-99 were not calculated. One study investigated the in vitro oxidative metabolism of BDE-99 in rats. Rat hepatocytes were incubated with BDE-99 for 72 h and production of 2,4,5-TBP, 5-OH- BDE-47, and 5'-hydroxy-BDE-99 (5ʹ-OH-BDE-99) was observed but the rates of formation were not quantified (Dong et al., 2010). The number of hydroxylated metabolites of BDE-99 formed by rat hepatocyte incubations was smaller than those detected in rats that had been treated in vivo with BDE-99 (Figure1.9). The poor correspondence between results of in vitro and in vivo studies suggests that further studies are needed to characterize the in vitro metabolism of BDE-99 by rat hepatic preparations. The in vitro formation of 5-OH-BDE-47 and the in vivo formation of unidentified mono-OH-tetrabrominated metabolites of BDE-99 (Chen et al., 2006; Hakk et al., 2002) suggest that BDE-99 can be oxidatively debrominated in the rat. However, in both in vivo and in vitro studies, the stock solutions of BDE-99 used were not checked for the presence of lower brominated PBDE impurities (i.e. BDE-47). The unidentified mono-OH-tetrabrominated metabolites of BDE-99 formed in vivo and the formation of 5-OH-BDE-47 by rat hepatocytes incubated with BDE-99 could be products of oxidative debromination of BDE-99 or of oxidation of BDE-47, if BDE-47 was present as an impurity in the BDE-99 stock solutions used. The in vitro hepatic oxidative metabolism of BDE-99 in humans has received limited attention. Lupton and co-workers reported that 2,4,5-TBP and an unidentified di-OH- pentabrominated-PBDE metabolite were formed when BDE-99 (20 µM) was incubated with 60    Figure 1.9 Hydroxylated metabolites of BDE-99 produced by rats and mice treated with BDE-99 (A) and by rat hepatocytes (B). References: Chen et al., 2006; Dong et al., 2010; Hakk et al., 2002; Staskal et al., 2006.  A B 61  human liver microsomes (0.5 mg/mL) for 2 hours (Lupton et al., 2009, 2010). 2,4,5-TBP, 5'- OH-BDE-99, and an unidentified mono-OH-pentabrominated-PBDE metabolite were detected when BDE-99 (10 µM) was incubated with cultured human hepatocytes for 72 h (Stapleton et al., 2009). The presence of hydroxylated metabolites of BDE-99 in human plasma and serum samples has been reported. 2,4,5-TBP, 4-hydroxy-2,2',3,4',5-pentabromodiphenyl ether (4- OH-BDE-90), 5'-OH-BDE-99, and 6'-hydroxy-BDE-99 (6'-OH-BDE-99) have been detected in human serum samples (Athanasiadou et al., 2008; Qiu et al., 2009) and may represent oxidative metabolites of BDE-99 formed in vivo. The hydroxylated metabolites of BDE-99 produced by human hepatic preparations partially matched the hydroxylated PBDEs detected in human plasma and serum samples (Figure 1.10). A more thorough characterization of the in vitro oxidative metabolism of BDE-99 using human hepatic preparations, a larger number of authentic standards for the hydroxylated metabolites of BDE-99 is needed for comparison with the results of the biomonitoring studies. Collectively, the studies described above showed that BDE-47 and BDE-99 are oxidatively metabolized in rodents and humans, both in vitro and in vivo. The formation of hydroxylated metabolites strongly suggests the involvement of CYP enzymes in the metabolism of BDE-47 and BDE-99. Further studies are needed to structurally characterize the hydroxylated metabolites of BDE-47 and BDE-99 formed by rodents and humans, quantify their rates of formation in vivo and in vitro and identify the CYP enzymes involved.   62      Figure 1.10 Hydroxylated metabolites of BDE-99 formed by human hepatic preparations (A) and hydroxylated PBDEs detected in human plasma and serum samples (B). References: Athanasiadou et al., 2008; Lupton et al., 2009, 2010; Qiu et al., 2009; Stapleton et al., 2009.    A B 63  1.9 Analytical Methods for Analysis of Hydroxylated PBDEs In the majority of the studies mentioned above, gas chromatography-mass spectrometry or gas chromatography-electron capture detector methods were used to detect the hydroxylated PBDEs in biological fluids of rodents that had been treated with PBDEs (Malberg et al., 2005; Marsh et al., 2006; Qiu et al., 2007; Staskal et al., 2006; Zhang et al. 2008) and in human plasma and serum samples (Athanasiadou et al., 2008; Kawashiro et al., 2008; Qiu et al., 2009; Zota et al., 2011). Gas chromatography-mass spectrometry or gas chromatography-electron capture detector methods were also used to detect the hydroxylated metabolites of BDE-47 and BDE-99 produced by rat and human hepatic preparations (Feo et al., 2013; Hamers et al., 2008; Stapleton et al., 2009). Gas chromatography-mass spectrometry or gas chromatography-electron capture detector methods are highly sensitive methods for the detection of hydroxylated PBDEs. However, analysis of hydroxylated PBDEs by gas chromatography-based methods requires derivatization, which involves extensive sample preparation, use of harmful derivatizing agents such as diazomethane, and the possibility of introducing errors in the quantification of hydroxylated PBDEs due to incomplete derivatization of the analytes. Liquid chromatography-mass spectrometry (LC/MS) is a sensitive analytical technique that is widely used for the separation and quantification of hydroxylated metabolites of diverse xenobiotic compounds. Hydroxylated metabolites can be efficiently ionized by the ionization techniques associated with LC/MS systems and do not require derivatization. Recently, three LC/MS methods for the determination of hydroxylated PBDEs have been developed. The LC/MS method by Mas et al. (2007) was developed for the analysis of mono-OH-tribrominated-PBDEs and mono-OH-tetrabrominated-PBDEs in fish muscle, 64  sediment, and water samples but was not used for the analysis of mono-OH- pentabrominated-PBDEs.  The method was not validated and was partially characterized only in terms of sensitivity and precision. The LC/MS method by Lupton et al. (2010) was developed for the analysis of mono-OH-tribrominated-PBDEs to mono-OH- hexabrominated-PBDEs. However, this method detected two out of the thirteen possible mono-hydroxylated metabolites of BDE-99. Also, the performance of this method was only characterized in terms of working range using authentic standards. Therefore, no knowledge is available regarding the accuracy, precision, and sensitivity of this method to identify and quantify hydroxylated metabolites of BDE-99 produced by liver microsomes. The method by Marteau et al. (2012) was developed for the analysis of mono-OH-tetrabrominated- PBDEs to mono-OH-octabrominated-PBDEs. However, only one out of the thirteen possible mono-hydroxylated metabolites of BDE-99 was detected by this method. The performance of this method was only characterized in terms of sensitivity using authentic standards. In addition, this method was used to identify some of the hydroxylated metabolites of BDE-47 produced by human hepatocytes and not to quantify the amount of metabolites formed and their rates of formation. Collectively, the LC/MS methods mentioned above show that LC/MS is a promising technique for the analysis of hydroxylated PBDEs. A validated LC/MS-based assay able to detect multiple hydroxylated metabolites of BDE-99 and BDE-47 and designed for the quantitative investigation of the oxidative metabolism of BDE-99 and BDE-47 by liver microsomes is needed and was the first goal of my project.   65  1.10 Rationale BDE-47 and BDE-99 are the two major PBE congeners in commercial Penta-BDE mixtures that have been predominantly used in North America. BDE-47 and BDE-99 levels in the Penta-BDE mixture and in several abiotic matrices were similar (Butt et al., 2004; Hassanin et al., 2004; North, 2004; Stapleton et al., 2005; Wilford et al., 2005). In human plasma and serum samples, however, levels of BDE-47 have been consistently found to be 3 to 10 times larger than those of BDE-99. This was true for samples collected from the general population in the U.S. (Qiu et al., 2009; Stapleton et al., 2011; Zota et al., 2011), Europe (Guvenius et al., 2003; Meijer et al., 2008; Roosens et al., 2010), and Asian countries (Bi et al., 2006; Kawashiro et al., 2008). A similar difference between BDE-47 and BDE-99 levels was reported for adults and children occupationally exposed to PBDEs (Athanasiadou et al., 2008; Lee et al., 2007; Stapleton et al., 2008). Larger levels of BDE-47 than BDE-99 in human plasma and serum samples might be due to a higher intake of BDE- 47, a higher absorption of BDE-47, a lower rate of metabolism of BDE-47, a lower rate of excretion of BDE-47 or a combination of any of these factors.        66  1.11 Research Hypothesis and Specific Aims The research hypothesis of my PhD project was:  Hepatic biotransformation of BDE-47 and BDE-99 by CYP enzymes is a major determinant of the different levels of BDE-47 and BDE-99 measured in humans and rats.  The specific aims of my project were: 1. To develop an in vitro biotransformation assay using liver microsomal preparations to investigate the formation of hydroxylated metabolites of BDE- 99 by:  developing and optimizing sample preparation, liquid chromatography separation, and mass spectrometry detection and quantification of the hydroxylated metabolites;  validating the assay for selectivity, sensitivity, linearity, range, accuracy, precision, and recovery rates. 2. To use the biotransformation assay developed to characterize the in vitro oxidative metabolism of BDE-99 in rat by:  identifying the hydroxylated metabolites of BDE-99 formed by rat liver microsomes and quantify their rates of formation;  determining enzyme kinetic parameters associated with the formation of hydroxylated metabolites of BDE-99 by rat liver microsomes; 67   assessing the role of individual rat recombinant CYP enzymes in the in vitro oxidative metabolism of BDE-99 using a panel of rat recombinant CYP enzymes and liver microsomes obtained from rats treated with inducers of CYP1A, CYP2B, or CYP3A enzymes. 3. To characterize the in vitro oxidative metabolism of BDE-99 and BDE-47 in humans by:  identifying the hydroxylated metabolites of BDE-99 and BDE-47 formed by human liver microsomes and quantifying their rates of formation;  determining enzyme kinetic parameters associated with the formation of hydroxylated metabolites of BDE-99 and BDE-47 by human liver microsomes and human recombinant CYP enzymes;  assessing the role of individual human CYP enzymes in BDE-99 and BDE-47 in vitro oxidative metabolism using a panel of human recombinant CYP enzymes, CYP-specific antibodies, and single donor human liver microsomes.        68  2. Materials and Methods    2.1 Chemicals and Reagents BDE-47 (neat, 98.8% or greater purity) and BDE-99 (neat, 97.7% or greater purity) were purchased from Chiron AS (Trondheim, Norway) and from AccuStandard (New Haven, CT). 4'-OH-BDE-17, 2'-OH-BDE-28, 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE- 47, 6-OH-BDE-47, 4'-OH-BDE-49, 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'- hydroxy-2,2',4,5,5'-pentabromodiphenyl ether (4'-OH-BDE-101), 2,4-DBP, and 2,4,5-TBP (10 or 50 μg/mL in acetonitrile, 97.7% grade purity or larger) were obtained from AccuStandard. 2-Hydroxy-2',3,4,4ʹ,5-pentabromodiphenyl ether (2-OH-BDE-123; neat) was a generous gift from Dr. R.J. Letcher (Environment Canada, Ottawa, Canada). 4'-Hydroxy- 2,2',4,6'-tetrachlorobiphenyl (4'-OH-CB-50) (neat, 99.9% purity) and 4-hydroxy-2',3,4',5,6'- pentachlorobiphenyl (4-OH-CB-121) (neat, 100% purity), which served as the internal standards (ISs) for the BDE-47 and BDE-99 biotransformation assays, respectively, were also purchased from AccuStandard. Bupropion hydrochloride (>98% purity), triprolidine hydrochloride (>99% purity), and NADPH were purchased from Sigma-Aldrich. Ethoxyresorufin, benzyloxyresorufin, resorufin, 7-benzyloxyquinoline, and 7-hydroxy- quinoline were purchased from BD Biosciences (Oakville, Ontario, Canada). Sodium phenobarbital (PB), dexamethasone (DEX), and 3-methylcholanthrene (MC) were purchased from Sigma-Aldrich Canada (Oakville, Ontario, Canada). Hydrochloric acid, sodium hydroxide, and organic solvents (HPLC grade or better) were purchased from 69  Fisher Scientific (Ottawa, Ontario, Canada). Ultra-pure water was obtained using a Millipore Milli-Q system (Billerica, MA). Baculovirus-insect cell microsomes containing expressed rat CYP enzyme (CYP1A1, CYP1A2, CYP2A1, CYP2A2, CYP2B1, CYP2C6, CYP2C11, CYP2C12, CYP2C13, CYP2D1, CYP2D2, CYP2E1, CYP3A1, or CYP3A2) co-expressed with rat CYP-oxidoreductase, or with rat CYP-oxidoreductase and rat cytochrome b5, and baculovirus-insect cell microsomes containing expressed human CYP enzyme (CYP1A1, CYP1A2, CYP1B1, CYP2A6,  CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) co-expressed with human CYP-oxidoreductase, or with human CYP-oxidoreductase and human cytochrome b5 (BD SUPERSOMES™ Enzymes) were purchased from BD Biosciences. Pooled human liver microsomes (mixed gender, n=50) were purchased from Xenotech (Lenexa, Kansas). Single donor human liver microsomes were purchased from Xenotech (H0426, H0435, H0442, H0444, and H0455) and BD Biosciences (HG95, HH13, HH18, and HH837). Human CYP2B6 selective mouse monoclonal antibody (MAB-2B6) was obtained from BD Biosciences. Information about the reactivity of the MAB-2B6 antibody towards 12 human CYP enzymes was provided by BD Biosciences (Table A1.1; Figure A1.1). Rabbit anti-rat CYP2B1 IgG was prepared as described previously (Panesar et al., 1996) and was shown to react with CYP2B1, CYP2B2, and CYP2B3 enzymes in liver microsomes from untreated, MC-, and PB-treated rats by immunoblot analysis (Bandiera et al., 1995). A polyclonal antibody against rat epoxide hydrolase was raised in female New Zealand rabbits immunized with the electrophoretically-homogeneous protein, which was purified from 70  Long Evans rats using the method described by Ryan and Levin (1990) IgG was purified from a pool of heat-inactivated high-titer sera obtained from multiple bleedings from several rabbits. The specificity of the antibody relative to purified enzymes and hepatic microsomal proteins was assessed using enzyme-linked immunosorbent assays and immunoblot analysis. Rabbit anti-rat epoxide hydrolase reacted with rat, mouse and human epoxide hydrolase but not with rat, mouse, or human CYP enzymes or other microsomal proteins (Bandiera et al., 1995).   2.2 Rat Treatment with Prototypical CYP Inducers and Preparation of Hepatic Microsomes Male Long-Evans rats (7 to 8 weeks of age) were purchased from Charles River Canada (Saint-Constant, Quebec, Canada).  Rats were housed in pairs in polycarbonate cages on corncob bedding (The Anderson’s, Maumee, OH) with free access to water and food (Laboratory Rodent Diet, PMI Feeds Inc., Richmond, IN).  Animal quarters were maintained at a constant temperature (23C) with controlled light (14 h) and dark (10 h) cycles.  Rats were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. Rats (n=6/7) were treated with PB (dissolved in phosphate buffer saline, 80 mg/kg/day), MC (dissolved in corn oil, 25 mg/kg/day), DEX (dissolved in corn oil, 100 mg/kg/day), or vehicle (corn oil, 2 mL/kg/day). Compounds were administered by intraperitoneal injection for 3 consecutive days and rats were killed by decapitation 24 h after the last treatment.  Microsomes were prepared from pooled livers as described 71  previously (Thomas et al., 1993).  Microsomal pellets were suspended in 0.25 M sucrose and aliquots were stored at –80C, until needed.   2.3 Determination of Total Protein and Cytochrome P450 Content, Marker Activities for CYP1A, 2B, and 3A Enzymes in Rat Hepatic Microsomes Protein concentration was measured by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Total CYP concentration was determined according to the method described in Omura and Sato (1964) using an extinction coefficient value of 91 (cm * mM)-1. Ethoxyresorufin O-deethylase (EROD) and benzyloxyresorufin O-dealkylase (BROD) activities were measured using a fluorometric assay as previously described (Edwards et al., 2007). 7-Benzyloxyquinoline O-dealkylation (BOQD) activity was measured fluorometrically as described by Renwick et al. (2001), with minor modifications. Briefly, all measurements were made using a Varian Cary Eclipse Fluorescence spectrophotometer (Mississagua, Ontario, Canada) equipped with a Cary temperature controller and a multicell holder. Excitation and emission wavelength values were 420 and 530 nm, respectively. A 5 nm slit width was used. Incubations were performed in a Hellma (Concord, Ontario, Canada) 3 mL fluorescence cuvette. Reaction mixtures contained 1.92 mL of 0.2 M potassium phosphate buffer (pH 7.4), 0.05 mL of rat hepatic microsomes (final protein concentration of 0.05 mg/mL for liver microsomes from DEX-treated rats and 0.4 mg/mL for liver microsomes from corn oil-, MC-, and PB-treated rats), and 0.02 mL of 7- 72  benzyloxyquinoline stock solution in methanol (final concentration of 200 μM) in a total volume of 1.99 mL. After preincubation for 2 min at 37ºC, reactions were initiated by the addition of 0.01 mL of NADPH (final concentration 250 μM) and allowed to proceed for 2 min. Fluorescence was measured at 0 and 2 min. Net fluorescence at 2 min was obtained subtracting the fluorescence reading obtained at 0 min from that obtained at 2 min. Negative control samples were prepared as described above but omitting NADPH. The calibration curve prepared using 7-hydroxyqinoline was used to convert net fluorescence reading into amount (pmol) of 7-hydroxyquinoline formed. Calibration standards for product formation were prepared by mixing 1.93 mL of 0.2 M potassium phosphate buffer (pH 7.4), 0.05 mL of rat hepatic microsomes (final total protein concentration 0.05 mg/mL), and 0.02 mL of 7- hydroxyquinoline stock solutions prepared in methanol (final concentrations of 0.10, 0.25, 0.50, 1.0, 5.0 and 10.0 μM) in a final volume of 2.0 mL. Blank samples (n=3) were prepared using methanol instead of the 7-hydroxyquinoline solution. Mixtures were incubated for 2 min at 37ºC before taking the fluorescence reading. For each 7-hydroxyquinoline calibration standard, the mean blank fluorescence value (n=3) was subtracted from the raw value to obtain the net fluorescence value. Linear regression analysis was performed using net fluorescence values. Samples were prepared in duplicate and average values were calculated.   2.4 Preparation of Metabolite Standard Stock Solutions for Method Validation A stock solution of metabolite standards containing 4-OH-BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99 (at 1.25 μM), and 2,4,5-TBP (at 15.62 μM) was prepared in methanol 73  and stored in an amber vial (final volume was 5.0 mL). A second stock solution was prepared by making a 10-fold dilution of the first stock solution. After preparation, the vials were capped, vortex-mixed vigorously for 1 min, and stored at -20ºC. A separate stock solution of the internal standard (4'-OH-CB-121, 75.0 μM) was prepared in methanol and stored at -20ºC. Two extra solutions were prepared for system suitability tests (SST). The first solution (SST1) contained 2,4,5-TBP (0.250 μM), 4-OH-BDE-90, 5-OH-BDE-99, 6- OH-BDE-99 (0.025 μM) and 4'-OH-CB-121 (3.0 μM) standards in methanol. The second solution (SST2) contained 2,4,5-TBP (5.0 μM), 4-OH-BDE-90, 5-OH-BDE-99, 6-OH- BDE-99 (0.500 μM) and 4'-OH-CB-121 (3.0 μM) standards in methanol. Aliquots of 100 μL each of SST1 and SST2 were stored in HPLC vials at -20ºC. Each aliquot was removed from storage before use and was used once.   2.5 Preparation of Calibration Standard and Quality Control Samples Five calibration standard (CS) and two quality control (QC) samples were prepared by mixing 0.50 mg of rat hepatic microsomal protein, 50 mM phosphate buffer containing 3 mM MgCl2 (pH=7.4), and an appropriate volume of one of the two stock solutions in a final volume of 1.0 mL. Final concentration of the metabolite standards in the CS samples were 0.010, 0.050, 0.100, 0.250 and 1.0 μM for 4-OH-BDE-90, 5-OH-BDE-99 and 6-OH-BDE- 99, and 0.0625, 0.125, 0.500, 1.25, 2.50 and 12.50 μM for 2,4,5-TBP. Final concentrations of the metabolite standards in the QC-Low sample were 0.025 μM for 4-OH-BDE-90, 5- OH-BDE-99 and 6-OH-BDE-99, and 0.250 μM for 2,4,5-TBP (QC-Low). Final 74  concentrations of the metabolite standards in the QC-High sample were 0.500 μM for 4-OH- BDE-90, 5-OH-BDE-99 and 6-OH-BDE-99, and 5.0 μM for 2,4,5-TBP (QC-High). The CS and QC samples were prepared on ice. Blank samples contained only rat hepatic microsomes and phosphate buffer. The CS, QC, and blank samples were extracted using a liquid-to-liquid extraction technique. Tubes were incubated at 37 ºC for 15 min in a shaking water bath. After incubation, 1.0 mL of ice-cold 0.5 M sodium hydroxide was added to each tube, and tubes were capped and inverted twice to mix the contents. IS was added to each tube (10.0 μL of 75.0 μM stock solution, final concentration of 3.0 μM). The tubes were vortex-mixed vigorously for 30 s and then heated in a water bath at 70ºC for 10 min. After cooling to room temperature, 2.0 mL of 6.0 M HCl were added to each tube followed by 1.0 mL of isopropanol. The tubes were vortex-mixed vigorously for 1 min and 2.0 mL of a mixture of methyl-tert-butyl ether:hexane (1:1 v/v) was then added to each tube. Tubes were vortex- mixed vigorously for 1 min and spun in a centrifuge at 2,500 rpm for 5 min. The top organic layer was transferred to a clean set of tubes and the extraction procedure was repeated two more times. The organic phases from each extraction of the same sample were pooled and dried under a gentle flow of nitrogen. The residue was reconstituted in 250 μL of methanol, vortex-mixed vigorously for 10 s, and filtered through a 0.45 μm polytetrafluoroethylene membrane into a 300 μL HPLC vial.   75  2.6 Ultra Performance Liquid Chromatography/Mass Spectrometry Conditions An ultra performance liquid chromatography/mass spectrometry (UPLC/MS) system was used to detect and quantify hydroxylated metabolites of BDE-99. The UPLC/MS system consisted of a Waters Acquity UPLC Sample Manager and a Waters Acquity UPLC Binary Solvent Manger connected to a Waters Quattro Premier XE triple quadrupole mass spectrometer equipped with a combined electrospray and atmospheric pressure chemical ionization probe (Waters, Milford, MA, U.S.). Chromatographic separation was achieved with a Waters Acquity UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) column, which was maintained at 50 ºC. The autosampler tray temperature was 4ºC and the injection volume was 5.0 μL. The mobile phase was composed of solvent A (water containing 0.1% formic acid) and solvent B (methanol containing 0.1% formic acid). Solvents were filtered through 0.22 μm filters (Millipore Durapore Membrane Filters, 0.22 μm GV, Billerica, U.S.). A gradient was used to resolve the hydroxylated metabolites. Gradient elution was as follows; solvent A:solvent B (35:65, v/v) from 0 to 7 min at a flow rate of 0.2 mL/min, followed by a linear increase to solvent A:solvent B (15:85, v/v) from 7 to 27 min at a flow rate of 0.2 mL/min. At 27.1 min, solvent B was increased to 100% and flow rate was increased to 0.3 mL/min and maintained for 2 min. The column was then re-equilibrated with solvent A:solvent B (35:65, v/v) for 3 min at a flow rate of 0.3 mL/min. The total analysis time was 32 min. The mass spectrometer was operated in negative electrospray ionization mode using selected ion recording at a capillary voltage of 3 kV, cone voltage of 40 V, source temperature of 120ºC, desolvation temperature of 400 ºC, and desolvation gas flow of 1005 L/h. The analytes of interest were identified by comparison of their mass-to-charge ratio 76  (m/z) and retention time values with those of authentic standards: m/z 328.7 for 2,4,5-TBP, m/z 578.5 for 4-OH-BDE-90, 5-OH-BDE-99 and 6-OH-BDE-99, and m/z 340.8 for 4-OH- CB-121. Because authentic standards for di-OH-pentabrominated-PBDEs are not commercially available, the m/z value used to monitor the formation of di-OH- pentabrominated-PBDEs (m/z 594.5) was derived from exact mass value of di-OH- pentabrominated-PBDEs and the ionization technique used (electrospray in negative mode). It should be noted that the present assay was unable to detect non-hydroxylated metabolites of BDE-99 (i.e. tetrabrominated-PBDEs) or 2,4-DBP. 2,4-DBP could not be detected because a calibration curve for 2,4-DBP could not be obtained due to a background contamination of 2,4-DBP. MassLynx v. 4.1software was used to control the UPLC/MS system. The UPLC/MS method was modified, after validation (Section 2.7), because standards for 4'-OH-BDE-101 and 2-OH-BDE-123 became available. The modification involved a slight change of the elution gradient in which the composition of the mobile phase (water:methanol) was increased linearly from 35:65 (v/v) to 25.5:74.5 (v/v) between 0 and 38 min. At 38.1 min, methanol was increased to 100% and maintained at 100% for 2 min. The column was then re-equilibrated with water:methanol 35:65 (v/v) for 3 min. Flow rate was maintained at 0.2 mL/min.   2.7 Assay Validation Using the UPLC/MS Method The assay was validated for selectivity, limit of quantification (LOQ), accuracy, precision, linearity, range, and recovery rates. The performance of the UPLC/MS system 77  was monitored using SST1 and SST2 samples. Each day, an aliquot of SST1 and of SST2 were removed from storage, thawed, and injected as the first two samples. Chromatographic parameters such as relative retention time, capacity factor, and resolution were monitored for each analyte in both SST1 and SST2 aliquots. Values of these chromatographic parameters were calculated as reported by Rely (1996). Acceptance criteria were relative retention time of 0.26±0.050, 0.90±0.050 and 0.97±0.050 (mean±S.D.) for 2,4,5-TBP, 4- OH-BDE-90 and 5ʹ-OH-BDE-99, respectively, capacity factor values larger than 5 for all the analytes, and an resolution value larger than 2 between 5-OH-BDE-99 and 6-OH-BDE- 99 peaks. Selectivity was assessed by comparing the chromatograms obtained from blank samples and spiked rat liver microsome samples for the presence of interfering peaks with m/z and retention time values overlapping those of 4-OH-BDE-90, 5-OH-BDE-99, 6-OH- BDE-99, 2,4,5-TBP, and 4-OH-CB-121. Selectivity was determined at the LOQ values of 4- OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP, and at 3.0 µM for 4-OH-CB- 121. The LOQ of each analyte was assessed by preparing a calibration curve and five replicates of the three lowest CS samples. LOQ was determined as the lowest CS concentration that met the following acceptance criteria; signal/noise (S/N) ratio at least 5 times the blank response, accuracy (percent deviation, %Dev) within ± 20% of the nominal concentration, and precision (percent relative standard deviation, %RSD) not exceeding 20%. The S/N ratio value was determined using MassLynx v. 4.1software and the peak-to- peak method. 78  Accuracy was calculated as %Dev between the mean measured concentration (n=6) and the nominal concentration. Precision was expressed as %RSD. Accuracy and precision values were determined in QC-Low and QC-High samples. Six replicates of freshly prepared QC-Low and QC-High samples were analyzed on the same day to determine the intra-day accuracy and precision values, and on six consecutive days to determine the inter- day accuracy and precision values. Acceptance criteria were intra-day and inter-day %Dev values within ± 15% of the nominal concentration and %RSD values not exceeding 15%. Linearity of the calibration curves was assessed using the correlation coefficient (r) and the reproducibility (%RSD) of the mean slope values (n=6). Calibration curves were constructed by plotting the analyte to internal standard peak area ratios (Y-axis) against the corresponding analyte nominal concentrations (µM, X-axis). Calibration curves were constructed using linear regression analyses with the weighting factor of 1/X2 to improve accuracy in the lower concentration range of the curve. Acceptance criteria were mean r values larger than 0.95 and %RSD of the mean slope value lower than 15%. The range was defined as the linear section of the calibration curve where the CS samples were determined accurately and precisely. Acceptance criteria were r values larger than 0.95 for each calibration curve, %Dev values within ±15% of the nominal concentration, and %RSD values not exceeding 15% for each CS sample. Recovery values were determined in QC-Low and QC-High samples. Recovery rates were calculated by comparing peak area of spiked microsomal samples with that of unextracted standards (i.e. SST1 and SST2) at the same nominal concentration. For each analyte, recovery rates were determined at two concentrations per day, for six consecutive days. The acceptance criterion was a mean recovery value between 80% and 120%. 79  The dataset generated was tested for the presence of outliers using the Dixon’s Q test (Rorabacher, 1991) with a 95% confidence interval (α=0.05). Outliers were to be excluded from the dataset, but no outlier was found.   2.8 Biotransformation Assays for BDE-47 and BDE-99 The bioassay used to investigate BDE-47 and BDE-99 in vitro metabolism is described in general terms below. Specific incubation conditions and preliminary experiments conducted using rat and human liver microsomes and recombinant CYP enzymes are presented in separate paragraphs. Reaction mixtures contained BDE-47 or BDE-99, hepatic microsomal protein, and 50 mM potassium phosphate buffer containing 3 mM magnesium chloride (pH 7.4) in a final volume of 0.99 mL. Reaction mixtures were preincubated for 5 min in a shaking water bath at 37°C. Reactions were initiated by addition of 0.01 mL of NADPH solution in assay buffer (1 mM final concentration) and terminated by addition of 1 mL of ice-cold 0.5 M sodium hydroxide. A fixed amount (100 μL) of internal standard was then added to each tube (4'- OH-CB50 or 4-OH-CB-121 final concentration of 0.5 μM for BDE-47 or BDE-99 metabolism assay, respectively). Samples were then prepared as described in Section 2.5 and analyzed as described in Section 2.6 and 2.9 for rat and human liver microsomal incubations, respectively. Blank and negative control samples were included in each assay. Blank samples did not contain substrate and NADPH. Negative control samples were devoid of NADPH or substrate. To determine if metabolite formation was enzyme-mediated, experiments were 80  conducted using heat-denatured microsomes.  Hepatic microsomal samples were boiled for 5 min in assay buffer prior to use. To determine if metabolite formation was CYP-mediated, carbon monoxide, which is a classic nonspecific CYP inhibitor, was bubbled for 2 min into an incubation mixture containing assay buffer, microsomes, and NADPH prior to use. For incubations with rat liver microsomes, the BDE-99 concentration ranged between 2.5 and 200 M and the incubation time was 10 min. Preliminary experiments using liver microsomes from corn oil- and DEX-treated rats were conducted to determine the linearity of product formation with respect to incubation time and protein concentration. The hepatic microsomal protein concentration was 0.1 or 0.5 mg/mL for incubations using hepatic microsomes obtained from DEX- or corn oil-treated rats, respectively. Samples were prepared in duplicate and three independent experiments were conducted. A preliminary experiment was conducted to characterize the catalytic activity in the four rat liver microsomal preparations available. BDE-99 at 100 M was incubated with liver microsomes from corn oil-, DEX-, MC-, and PB-treated rats (0.5 mg/mL) for 10 min. Incubations with rat recombinant CYP enzymes were performed as described above except that reaction mixtures contained 30 pmol of rat recombinant CYP enzyme instead of rat hepatic microsomes. Insect cell control microsomes containing expressed rat CYP- oxidoreductase and cytochrome b5 but without CYP enzyme were used as CYP negative controls at an equivalent amount of protein (0.30 mg). To ensure that product formation was linear with respect to incubation time and CYP concentration, preliminary experiments were performed using rat recombinant CYP3A1 enzyme. Samples were prepared singly and three independent experiments were performed. Samples containing rat microsomal preparations 81  or rat recombinant CYP enzymes were processed as described in Section 2.5 and analyzed as described in Section 2.6. For incubations with pooled human liver microsomes, BDE-47 and BDE-99 concentrations ranged between 0.5 and 200 M, liver microsomal protein concentration was 0.1 mg/mL, and the incubation time was 5 min for BDE-47 and 10 min for BDE-99. Preliminary experiments were conducted using pooled human liver microsomes to determine the linearity of BDE-47 and BDE-99 product formation with respect to incubation time and hepatic microsomal protein concentration. Samples were prepared in duplicate and replicate experiments were conducted on three different days. Incubations with single donor human liver microsomal preparations and BDE-99 were performed as described above except that a BDE-99 concentration, which was determined to be saturating for the formation of the metabolites produced by pooled human liver microsomes, (i.e. 100 M for 5'-OH-BDE-99 and 4'-OH-BDE-101 or 10 M for the other metabolites) was used. Incubations with single donor human liver microsomal preparations and BDE-47 were performed as described above except that a BDE-47 concentration of 100 M, which was saturating for all metabolites of BDE-47 produced by pooled human liver microsomes, was used. Samples were prepared in duplicate and replicate experiments were conducted on two different days. For incubations with human recombinant CYP enzymes, reaction mixtures contained individual recombinant CYP enzymes instead of human hepatic microsomal protein. Experiments to compare the activity of individual human recombinant CYP enzymes were conducted with BDE-47 and BDE-99 at a 100 M (final concentration), which was 82  saturating, 10 pmol of recombinant CYP/mL, and an incubation time of 10 min. Insect cell control microsomes containing expressed human CYP-oxidoreductase without CYP enzyme was used as a negative control at an equivalent amount of protein (50 g). Additional experiments were performed with human recombinant CYP2B6 to ensure that product formation of BDE-47 and BDE-99 was linear with respect to incubation time and CYP2B6 concentration. On the basis of the results obtained, rates of formation of hydroxylated metabolites of BDE-47 and BDE-99 by CYP2B6 were determined using concentrations of BDE-47 and BDE-99 between 0.5 and 200 M, 5 pmol of recombinant CYP2B6/mL, and an incubation time of 5 for BDE-47 and 10 min for BDE-99. Samples were prepared singly and three independent experiments were conducted. To investigate whether 2,4,5-TBP and the di-OH-pentabrominated-PBDE metabolite produced when BDE-99 was incubated with pooled human liver microsomes were primary or secondary hydroxylated metabolite of BDE-99, pooled human liver microsomes were incubated with 5'-OH-BDE-99, 6'-OH-BDE-99, or 4'-OH-BDE-101, at 0.1 to 100 nM (final concentration), instead of BDE-99. The concentration range was selected to bracket the concentration range (0.2 to 30 nM) at which 5'-OH-BDE-99, 6'-OH-BDE-99, and 4'-OH- BDE-101 were formed following incubation of human liver microsomes with BDE-99 (0.5 to 200 M). Samples were prepared in duplicate and one experiment was conducted. To investigate whether 2,4-DBP, 4'-OH-BDE-17, 2'-OH-BDE-28 and  the di-OH- tetrabrominated-PBDE metabolites produced when BDE-47 was incubated with pooled human liver microsomes were primary or secondary hydroxylated metabolite of BDE-47, pooled human liver microsomes were incubated with 4-OH-BDE-42, 5-OH-BDE-47, 6-OH- BDE-47 or 4'-OH-BDE-49 at 5.0 to 100 nM (final concentration), instead of BDE-47. In an 83  additional experiment, pooled human liver microsomes were incubated with BDE-47 (25 µM) plus increasing concentrations of 4-OH-BDE-42 or 4'-OH-BDE-49 (0 to150 nM). Samples were prepared in duplicate and one experiment was conducted. All the samples containing human enzymatic preparations were processed as described in Section 2.5 and analyzed as described in Section 2.9.   2.9 Ultra High Performance (UHP)LC/MS/MS Methods for Analysis of the Hydroxylated Metabolites of BDE-47 and BDE-99 In February 2010 the Waters UPLC/MS instrument failed and a new UHPLC/MS/MS instrument was purchased by the Faculty of Pharmaceutical Sciences in April 2011. Therefore, the previously validated and subsequently modified UPLC/MS method for the determination of hydroxylated metabolites of BDE-99 (Section 2.6) and the method for determination of hydroxylated metabolites of BDE-47 previously validated in Dr. Bandiera’s laboratory (Moffatt et al., 2011) were adapted to the new UHPLC/MS/MS instrument. The original UPLC column (BEH C18 100 x 2.1 mm, 1.7 μm) was replaced with a longer column (BEH130 C18 column 150 x 2.1 mm, 1.7 µm) and the elution program was changed, allowing baseline separation of nearly all the hydroxylated metabolites of BDE-99 and all the hydroxylated metabolites of BDE-47, including the two internal standards. The multiple reaction monitoring technique was used instead of the single ion monitoring technique. Multiple reaction monitoring transitions of the brominated isotopes of the precursor and product ions were experimentally determined using authentic standards and the total ion current of these transitions was used during the analyses to increase the 84  sensitivity and selectivity of the analytical method. The background contamination problem that had previously prevented detection of 2,4-DBP was found to be due to the hydrochloric acid dispenser. Dispensing hydrochloric acid using a clean pipette eliminated the contamination problem and allowed the detection of 2,4-DBP as candidate metabolite of BDE-47. The UHPLC/MS/MS system consisted of an Agilent 1290 Infinity UHPLC coupled with an AB Sciex QTrap® 5500 hybrid linear ion-trap triple quadrupole mass spectrometer equipped with a Turbo Spray source (Concord, Ontario, Canada). Chromatographic separation of standards for hydroxylated metabolites of BDE-99 (2,4,5-TBP, 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101, 2-OH-BDE-123) and the internal standard (4-OH-CB-121), and for hydroxylated metabolites of BDE-47 (2,4-DBP, 4'-OH- BDE-17, 2'-OH-BDE-28, 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, 4'-OH-BDE-49) and the internal standard (4'-OH-CB-50) was achieved using a Waters Acquity UPLC BEH130 C18 column (150 x 2.1 mm, 1.7 µm) and the following mobile phase composition: water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B). The elution was isocratic with 75% B for 43 min for the analysis of the hydroxylated metabolites of BDE-99 and 70% B for 45 min for the analysis of the hydroxylated metabolites of BDE-47 at a flow rate of 0.2 mL/min. The injection volume was 15 µL. The mass spectrometer was operated in electrospray negative ionization mode with the ion spray voltage of -4500 V, curtain gas of 20, nebulizing gas of 18, desolvation gas of 30 units, and temperature of 300 ºC. The analytes were detected in multiple reaction monitoring mode and identified by comparison of their retention times and isotopic mass to charge transition (parent/daughter ions) values with those of authentic standards (Table A1.2 and Table A2.1). 85  Because authentic standards for di-OH-tetrabrominated-PBDEs, di-OH-pentabrominated- PBDEs and some mono-OH-tetrabrominated-PBDEs and mono-OH-pentabrominated- PBDEs are not commercially available at present, the identity of these metabolites could not be determined. Range, linearity, and limit of quantification for the improved methods were determined as described in Section 2.7. The method used for the analysis of the hydroxylated metabolites of BDE-99 also yielded baseline separation of 4-OH-BDE-42, 3- OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47 and 4'-OH-BDE-49, but not of 4'-OH-BDE-17 and 2'-OH-BDE-28. These five metabolites were detected using the isotopic mass to charge transition values reported in Table A2.1. UHPLC/MS/MS data were acquired and processed using Analyst 1.5.2 software (Concord, Ontario, Canada).   2.10 Quality Control Along with each set of unknown samples, a calibration curve and three quality control samples were prepared daily to assess the linearity, inter-day accuracy, and inter-day precision values of the assay as described in Section 2.7. In experiments characterizing the metabolism of BDE-99, rat hepatic microsomes diluted in 50 mM potassium phosphate buffer (0.1 mg/mL final protein concentration) were spiked with authentic metabolite standards for 2,4,5-TBP, 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101, and 2-OH-BDE-123 at 2.5, 5.0, 10, 25, 50, 100, 250, and 500 nM (final concentrations) to prepare CS samples. QC-Low, QC-Mid, and QC-High samples were prepared as CS samples but at final concentrations of the authentic standards of 7.5, 80, and 400 nM. In experiments characterizing the metabolism of BDE-47, rat hepatic microsomes diluted in 50 86  mM potassium phosphate buffer (0.1 mg/mL final protein concentration) were spiked with authentic metabolite standards for 2,4-DBP, 4'-OH-BDE-17, 2'-OH-BDE-28, 4-OH-BDE- 42, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49 at 2.5, 5.0, 10, 25, 50, 100, 250, and 500 nM (final concentrations) to prepare CS samples. QC-Low, QC-Mid, and QC-High samples were prepared as CS samples but at final concentrations of the authentic standards of 7.5, 80, and 400 nM. For 2,4-DBP, 5.0, 10, 25, 50, 100, 250, and 500 nM (final concentrations) were used to prepare CS samples and 15, 80, and 400 nM (final concentrations) were used to prepare QC samples. The acceptance criteria were assessed on an inter-day basis as described in Section 2.7.  More details about the determination of linearity, specificity, LOQ, and inter-day accuracy and precision values of the assays used to quantify hydroxylated metabolites of BDE-47 and BDE-99 are reported in Table A1.3-A1.4 and A2.2-A2.3, respectively.   2.11 Bupropion Hydroxylation Assay Hepatic microsomal bupropion 4-hydroxylase activity was measured using UPLC/MS/MS analysis as described by Lau and Chang (2009). Briefly, reaction mixtures contained human liver microsomes (0.5 mg/mL final concentration), a saturating concentration of bupropion (100 µM final concentration), and 50 mM phosphate buffer with 3 mM magnesium chloride (pH 7.4). Reaction mixtures were prepared in a total volume of 0.49 mL. Samples were pre-incubated for 5 min in a shaking water bath at 37°C. Reactions were initiated by addition of 0.01 mL of NADPH solution in assay buffer (1 mM final concentration), allowed to proceed for 20 min, and terminated by addition of 0.5 mL of ice- 87  cold acetonitrile containing triprolidine (1 µM final concentration, internal standard). Samples were mixed on a vortex mixer for 30 sec and spun in a table-top centrifuge at 8,000 g for 10 min. An aliquot of the supernatant was then transferred to an autosampler vial for UPLC/MS/MS analysis. Blank and negative control samples were included in each assay. Blank samples did not contain substrate and NADPH. Negative control samples were devoid of either NADPH or substrate. Preliminary experiments using pooled human liver microsomes were conducted to ensure the linearity of product formation with respect to incubation time and protein concentration. On the basis of the results obtained, pooled human liver microsomes (0.5 mg/mL final concentration) were incubated with bupropion (25 µM to 1.0 mM, final concentration) for 20 min. Apparent Km and Vmax values were determined by nonlinear regression analysis as described in Section 2.13. The apparent Km value was then selected as the bupropion concentration for the antibody inhibition experiments (Section 2.12). The 4-OH metabolite of bupropion was quantified using the validated UPLC/MS/MS method described by Lau and Chang (2009). Briefly, the UPLC/MS/MS system consisted of a Waters Acquity UPLC Sample Manager and a Waters Acquity UPLC Binary Solvent Manger connected to a Waters Quattro Premier XE triple quadrupole mass spectrometer equipped with a combined electrospray and atmospheric pressure chemical ionization probe (Waters, Milford, MA, U.S.). Chromatographic separation was achieved with a Waters Acquity UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) column. The mobile phase was composed of solvent A (water containing 0.1% formic acid) and solvent B (methanol containing 0.1% formic acid). The elution gradient was as follows: isocratic at 2% B (0.0 to 1.5 min), linear gradient from 2% to 98% B (1.5 to 1.6 min), isocratic at 98% B (1.6 to 4.0 min), linear 88  gradient from 98% to 2% B (4.0 to 4.1 min), and isocratic at 2% B (4.1to 6.0 min). The mass spectrometer was operated in positive electrospray ionization mode with an electrospray capillary voltage of 3.5 kV, a cone voltage of 20 V, source temperature of 100°C, and desolvation temperature of 300°C. Nitrogen gas was used as the desolvation gas. Data were acquired and processed using MassLynx version 4.1 software with QuanLynx application manager (Waters).   2.12 Antibody Inhibition Experiments To evaluate the effect of CYP2B antibodies on BDE-99 metabolite formation, reaction mixtures containing pooled human liver microsomes (0.1 mg/mL) in 50 mM potassium phosphate buffer with 3 mM magnesium chloride (pH 7.4) were preincubated for 10 min in a shaking water bath at 37°C with various amounts of mouse anti-human CYP2B6 ascites fluid, control mouse serum, rabbit anti-rat CYP2B1 IgG, or control rabbit IgG. BDE- 99 was then added at 10 μM (final concentration) and the mixtures were incubated for an additional 5 min at 37°C. The reaction was initiated by addition of 0.01 mL of NADPH solution in assay buffer (1 mM final concentration), allowed to proceed for 10 min, and stopped by addition of 1.0 mL of ice-cold 0.5 M sodium hydroxide. A BDE-99 concentration that was approximately equal to the mean apparent Km value associated with the formation of hydroxylated metabolites of BDE-99 by human liver microsomes was selected for these experiments so that the effect of the antibody would be more apparent. Samples were processed as described in Section 2.5 and analyzed as described in Section 2.9. 89  To evaluate the effect of a CYP2B6 antibody on BDE-47 metabolite formation, BDE-47 was incubated with mouse anti-human CYP2B6 or control mouse serum at 10 or 50 µM for 5 min as described above for BDE-99 experiments. Samples were prepared in duplicate and two independent experiments were conducted. The effect of rabbit anti-rat CYP2B2B1 antibody on bupropion 4-hydroxylase activity, a well-established marker of CYP2B6 activity in human liver microsomes (Faucette et al., 2000; Hesse et al., 2000) was also evaluated. Experiments were conducted using pooled human liver microsomes at 0.5 mg/mL, various amounts of rabbit anti-rat CYP2B1 IgG or control rabbit IgG, bupropion at 50 μM (final concentration, approximately equal to the apparent Km value associated with the formation of 4-hydroxy-bupropion by human liver microsomes), and an incubation time of 20 min. Samples were prepared and 4-hydroxy- bupropion was analyzed as described in Section 2.11. The effect of mouse anti-human CYP2B6 monoclonal antibody on bupropion 4-hydroxylase activity was not measured in this study. This information was provided by the vendor (Figure A1.1). To assess the contribution of microsomal epoxide hydrolase to the formation of the di-OH-tetrabrominated-PBDE and di-OH-pentabrominated-PBDE metabolite of BDE-47 and BDE-99, respectively, by human liver microsomes, experiments were conducted using pooled human liver microsomes (0.1 mg/mL), various amounts of rabbit anti-rat microsomal epoxide hydrolase IgG (0.1 to 2.0 mg IgG/mg human liver microsomal protein) or an equivalent amount of rabbit control IgG, an incubation time of 5 min for BDE-47 experiments and of 10 min for BDE-99 experiments, and a BDE-47 or BDE-99 final concentration of 10 μM. A BDE-47 and BDE-99 concentration of 10 µM was selected to provide the appropriate analytical sensitivity to measure changes in rates of formation for 90  the di-OH-tetrabrominated-PBDE and di-OH-pentabrominated-PBDE metabolite of BDE-47 and BDE-99, respectively. Samples were processed as described in Section 2.5 and analyzed as described in Section 2.9. Samples were prepared in duplicate and two separate experiments were conducted.   2.13 Data Analysis To determine the enzyme kinetic model that best fit the rates of BDE-47 and BDE- 99 metabolite formation versus substrate concentration, the enzyme activity data were fitted to different enzyme kinetic models by nonlinear regression analysis using the SigmaPlot Enzyme Kinetics Module (v.1.1, Systat Software Inc., Richmond, CA). The equations used were the Michaelis-Menten equation (Equation 1), the Hill equation (Equation 2), and the substrate-inhibition kinetic equation (Equation 3):  ][ ][max SK SVv m                                       (Equation 1)  n n SK SVv ][ ][max                                     (Equation 2)  im KSSK Vv /][]/[1 max                      (Equation 3)  91  where v is initial velocity of the reaction, Vmax is the maximal velocity, [S] is the substrate concentration, K is the Hill dissociation constant, n is the Hill coefficient representing cooperativity in the reaction, Km is the Michaelis-Menten constant, and Ki is the dissociation constant of substrate binding to the inhibitory site. Selection of the appropriate model was determined by visual inspection of the metabolite formation versus substrate concentration plot and by statistical criteria to evaluate the goodness of the fit. The two statistical criteria used were Akaike Information Criterion corrected for small sample size (AICc) and the size of the standard deviation of the residuals. The model with the lowest values for AICc and the standard deviation of the residuals was considered to be the model that best fit the data. The relationship between rates of formation of hydroxylated metabolites of BDE-47 (or BDE-99) and marker activities for CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2E1 or CYP3A4 was analyzed by simple linear regression analysis. A p value <0.05 was considered statistically significant.       92  3. Results    The Results section is organized into four parts. In the first part, results pertaining to the development of a UPLC/MS-based assay to characterize the oxidative metabolism of BDE-99 by hepatic microsomal preparations are presented. Application of the newly developed assay to investigate the oxidative metabolism of BDE-99 by rat liver microsomes and rat recombinant CYP enzymes is presented in the second part. The third and fourth parts focus on the characterization of the oxidative metabolism of BDE-47 and BDE-99 by human liver microsomes and human recombinant CYP enzymes.   Part 1: Development and Validation of a New UPLC/MS-based Assay to Characterize the Oxidative Metabolism of BDE-99 The objective of the first part of my thesis was to develop a sensitive UPLC/MS- based assay to identify the hydroxylated metabolites of BDE-99 formed by hepatic microsomal incubations and to quantify the amount of metabolites formed without the need for derivatization. This required optimizing the mass spectrometer parameters for the detection of hydroxylated metabolites of BDE-99, optimizing the liquid chromatography parameters to achieve baseline separation of the hydroxylated metabolites of BDE-99 in a short run time, and developing an efficient sample preparation protocol.  93  3.1 Optimization of UPLC/MS Parameters Two ionization techniques, atmospheric pressure chemical ionization and electrospray, were compared for sensitivity of detection of 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP with the MS set in single ion recording mode. Electrospray operated in negative mode produced a larger signal for the hydroxylated metabolite standards than atmospheric pressure chemical ionization. BDE-99 was not ionized by either ionization technique. Flow injection analysis with individual standards (0.50 μM in methanol) was used to determine the molecular ions [M-H]- and optimal cone voltage values. The most intense signal for 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP was obtained at 40V. Source temperature, desolvation temperature, and desolvation gas flow values were also optimized (values reported in Section 2.6). To determine if the sensitivity of the analytical method could be improved, multiple reaction monitoring was assessed starting with the single ion recording conditions. Product ion scans of the molecular ions of 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP were performed at different collision energy values. The main product ion was a bromine fragment (m/z 79 and 81) with its highest intensity at a collision energy value of 40 eV. The sensitivity of the multiple reaction monitoring method was lower than that of the single ion recording method. Thus, the single ion recording method was used for subsequent analyses. Separation of 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP was achieved using a Waters Acquity UPLC BEH C18 (100 x 2.1 mm, 1.7 μm) column. Mobile phase composition was optimized using mixtures of water and acetonitrile, or water and methanol, with or without formic acid. Among the mobile phase compositions assessed, a 94  mixture of water and methanol containing 0.1% formic acid yielded the best peak shapes and the highest peak area counts and was selected for chromatographic separation. Formic acid (0.1%) was added to enhance ionization of the compounds analyzed. Baseline resolution of 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP was achieved using isocratic elution with methanol:water (65:35, v/v) for the first 7 min followed by a linear gradient to 85% methanol:15% water (from 7 to 27 min) at a flow rate of 0.2 mL/min.   3.2 Choice of the Internal Standard A hydroxylated pentachlorobiphenyl (i.e. 4-OH-CB-121) was chosen as the internal standard for the BDE-99 assay because of the structural similarity of PCBs and PBDEs (Figure 1.1), because 4-OH-CB-121 is a penta-halogenated, mono-hydroxylated PCB with similar physicochemical properties as OH-pentabrominated-PBDEs, and because 4-OH-CB- 121 is not likely to be present in wildlife samples, or may be present only at trace levels (Metcalfe et al., 2004; Sandala et al., 2004). Lastly, we preferred not to use an OH- pentabrominated-PBDE or OH-tetrabrominated-PBDE to avoid potential interference with possible hydroxylated metabolites of BDE-99 that may be formed by rat or human liver microsomes.   3.3 Optimization of Sample Preparation To achieve efficient extraction of 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP from the biological matrix of interest (i.e. rat liver microsomes) and to 95  minimize the possibility of spurious peaks that could interfere with the peaks of interest, various steps of the sample preparation protocol were optimized. Of the various organic solvents (acetone, dichloromethane, hexane) and mixtures tested, three extractions with methyl-tert-butyl ether:hexane (2 mL, 1:1 v/v) yielded the best recovery rates for 4-OH- BDE-90, 5-OH-BDE-99, 6-OH-BDE-99, and 2,4,5-TBP (≥85%) and did not produce spurious interfering peak(s). Addition of extra centrifugation steps or use of methanol or acetone in place of sodium hydroxide to terminate the biotransformation reaction did not improve recovery of the analytes of interest.   3.4 Assay Validation An aliquot of SST1 and SST2 were analyzed at the beginning of every batch analysis. The system suitability samples met the acceptance criteria for relative retention time, capacity factor, and resolution values with every batch analyzed (Table 1.1) and no batch had to be discarded. Comparison of chromatograms obtained with blank and spiked rat liver microsomes showed no interfering peaks at m/z and retention time values corresponding to those of 4- OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99, and 2,4,5-TBP (Figure 3.1). The LOQ concentration was 0.010 μM for 4-OH-BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99, and was 0.0625 μM for 2,4,5-TBP (Table 3.2). Inter-day and intra-day accuracy (0.47 to 8.0 and -0.80 to 7.7, respectively) and precision (2.0 to 8.5 and 2.2 to 8.6, respectively) values (Table 3.3) matched the acceptance criteria previously selected (Section 2.7). 96  Table 3.1Relative Retention Time, Capacity Factor, and Resolution Values for The Validated UPLC/MS-based assaya Metabolite standard Relative retention time Capacity factor Resolution 2,4,5-TBP 0.26±0.0011 5.3±0.044 - 4-OH-BDE-90 0.90±0.0010 21±0.074 - 5'-OH-BDE-99           0.97±0.00082 22±0.074 2.2±0.055 6'-OH-BDE-99               1 23±0.069 4'-OH-CB-121 0.78±0.0013 18±0. 078 -  a data are expressed as mean ± SD (n=6). An aliquot of SST and of SST2 was analyzed every day for 6 consecutive days.            97    Figure 3.1Representative chromatograms of rat liver microsomes spiked with methanol or authentic standards at the LOQ values. Rat liver microsomes (0.5 mg/mL) were spiked with an aliquot of methanol (A,C,E) or standards for 4-OH-BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99 (B), 2,4,5-TBP (D), and IS (F) at the LOQ values; 0.010 μM for 4-OH-BDE-90, 5-OH-BDE-99 and 6-OH-BDE-99, and 0.0625 μM for 2,4,5-TBP. IS was added at 3.0 μM. 98  Table 3.2 LOQ Values of Hydroxylated Metabolites of BDE-99 Using the UPLC/MS-based Assaya Metabolite standards Concentration Retention time (min) Blank S/N S/Nb %RSD %Dev 2,4,5-TBP     0.0625 μM 6.4 ≤ 3.0 38 ± 3.7 7.7 2.5 4-OH-BDE-90 0.010 μM 22.1 ≤ 3.0 22 ± 3.8 14 4.8 5ʹ-OH-BDE-99 0.010 μM 23.6 ≤ 3.0 28 ± 4.3 8.0 7.8 6ʹ-OH-BDE-99 0.010 μM 24.4 ≤ 3.0 8.6 ± 2.1 11 1.8  a n=5, 5 replicates per sample on the same day b mean ± SD     99  Table 3.3 Inter-Day and Intra-Day Precision (%RSD) and Accuracy (%Dev) Values Metabolite standards QC samples Nominal concentration (μM) Measured concentration (μM)c %RSD %Dev Inter-day a 2,4,5-TBP QC-Low 0.25 0.25 ± 0.013 5.0 0.67  QC-High 5.0       4.9 ± 0.26 5.3 1.8 4-OH-BDE-90 QC-Low 0.025   0.027 ± 0.002 8.5 8.0  QC-High 0.50     0.50 ± 0.025 5.0 0.57 5ʹ-OH-BDE-99 QC-Low 0.025   0.025 ± 0.002 8.0 1.3  QC-High 0.50     0.50 ± 0.010 2.0 0.47 6ʹ-OH-BDE-99 QC-Low 0.025   0.025 ± 0.002 7.6 2.0  QC-High 0.50 0.52 ± 0.020 3.9 3.5 Intra-day b 2,4,5-TBP QC-Low 0.25 0.24 ± 0.014 5.7 3.3  QC-High 5.0       5.0 ± 0.19 3.8 -0.80 4-OH-BDE-90 QC-Low 0.025   0.027 ± 0.002 8.6 7.3  QC-High 0.50     0.46 ± 0.010 2.2 7.7 5ʹ-OH-BDE-99 QC-Low 0.025   0.027 ± 0.002 7.1 6.0  QC-High 0.50 0.48 ± 0.014 3.0 3.5 6ʹ-OH-BDE-99 QC-Low 0.025   0.025 ± 0.001 5.9 0.67  QC-High 0.50 0.50 ± 0.029 5.7 0.83  a n=6, 1 replicate per day for 6 days b n=6, 6 replicates on the same day c mean ± SD    100  The assay was linear over a concentration range of 0.010 to 1.0 μM for 4-OH-BDE- 90, 5-OH-BDE-99 and 6-OH-BDE-99, and a concentration range of 0.0625 to 12.5 μM for 2,4,5-TBP (Table 3.4). Accuracy and precision values for CS samples met the acceptance criteria with the exception of two CS samples for 2,4,5-TBP, which resulted in slightly larger %Dev values (i.e. 20% vs 15%). As a result, the assay range was 0.010 to 1.0 μM for 4-OH-BDE-90, 5-OH-BDE-99 and 6-OH-BDE-99, and 0.0625 to12.5 μM for 2,4,5-TBP (Table 3.4). Recovery values between 85% and 100% were obtained across the assay range for 4-OH-BDE-90, 5-OH-BDE-99, 6-OH-BDE-99 and 2,4,5-TBP (Table 3.5). 101  Table 3.4 Calibration Curve Parameter Values Determined Using CS Samples Metabolite standard Nominal concentrations (μM) Measured concentration (μM)c %RSD %Dev  Slope r 2,4,5-TBP 0.063     0.075 ± 0.012 16 20 Mean 0.31 0.99  0.125 0.15 ± 0.020 13 22 SD 0.021  0.50 0.60 ± 0.048 8.0 20 %RSD 6.5  1.25 1.4 ± 0.10 7.3 14  2.5   2.6 ± 0.090 3.3 4.6  12.5  11 ± 0.83 7.4 -11 4-OH-BDE-90 0.010     0.010 ± 0.002 16 1.7 Mean 0.98 0.99  0.050 0.050 ± 0.004 6.7 8.1 SD 0.13  0.10   0.11 ± 0.010 9.6 12 %RSD 13  0.25   0.27 ± 0.011 4.3 6.1  1.0   0.86 ± 0.092 11 -14 5ʹ-OH-BDE-99 0.010 0.010 ± 0.001 6.4 6.8 Mean 1.1 0.99  0.050      0.051 ± 0.002 4.6 2.7 SD 0.11  0.10 0.110 ± 0.007 6.5 6.7 %RSD 10  0.25   0.26 ± 0.013 5.0 4.2  1.0   0.90 ± 0.090 10 -10 102  Metabolite standard Nominal concentrations (μM) Measured concentration (μM)c %RSD %Dev  Slope r 6ʹ-OH-BDE-99 0.010 0.010 ± 0.001 11 2.2 Mean 0.59 0.99  0.050 0.050 ± 0.003 5.7 -1.2 SD 0.092  0.10   0.110 ± 0.0089 8.7 8.6 %RSD 15  0.25   0.27 ± 0.014 5.3 6.4  1.0   0.96 ± 0.092 9.6 -4.4  a n=6, 1 replicate per day for 6 days b Y-intercept values were between -2.00*10-4 and 1.17*10-2 c mean ± SD 103  Table 3.5 Recovery Values Determined Using QC-Low and QC-High Samples Metabolite standards QC-Low QC-High 2,4,5-TBP 97 ± 16% 100 ± 7.5% 4-OH-BDE-90  94 ± 6.9%  97 ± 12% 5ʹ-OH-BDE-99  97 ± 9.7%   100 ± 7.1% 6ʹ-OH-BDE-99  85 ± 9.7%   85 ± 5.2% Internal standard 4-OH-CB-121 118 ± 12%    a data are expressed as mean±SD (n=6), 1 replicate per day for 6 days           104  Part 2: Oxidative Metabolism of BDE-99 by Rat Liver Microsomes and Rat Recombinant CYP Enzymes The objective of the second part of the thesis was to investigate the in vitro metabolism of BDE-99 by rat liver microsomes. Using the validated UPLC/MS-based assay, I identified the hydroxylated metabolites of BDE-99 formed by rat liver microsomes, quantified their rates of formation, and calculated values for the kinetic parameters associated with their formation. The role of individual rat CYP enzymes in the metabolism of BDE-99 was assessed using liver microsomes from rats treated with prototypical CYP inducers and using a panel of rat recombinant CYP enzymes.   3.5 Characterization of Rat Hepatic Microsomes Liver microsomes were prepared from rats treated with prototypical inducers of the CYP1A, CYP2B, and CYP3A enzymes to obtain insights into the contribution of individual subfamilies of CYP enzymes to the hepatic metabolism of BDE-99 in the rat. The hepatic microsomal preparations were characterized with respect to total protein concentration, cytochrome P450 content, and marker activities for CYP1A, 2B, and 3A enzymes. As shown in Table 3.6, the total CYP content of hepatic microsomes was increased 1.5- to 2-fold by treatment with DEX, MC, or PB when compared to the total CYP content of hepatic microsomes from corn oil-treated rats. EROD activity, which is a catalytic marker for CYP1A enzymes (Kobayashi et al., 2002), was increased 29-fold by treatment with MC. BROD and BOQD activities, which are catalytic markers for CYP2B and CYP3A enzymes, respectively (Kobayashi et al., 2002; Renwick et al., 2001), 105  Table 3.6 Effect of Treatment with CYP Inducers on Total Protein Content, Total CYP Content, and CYP Marker Activities in Pooled Rat Hepatic Microsomes (n=6) Treatment Total Protein Concentration (mg/mL)a Total CYP content (nmol/mg protein) EROD activity (pmol/min/mg) BROD activity (pmol/min/mg) BOQD activity (pmol/min/mg) Corn oil 45.67 1.25 (1.0) 840 (1.0) 175 (1.0) 3,910 (1.0) DEX 39.46 2.51 (2.0) 1,070 (1.3) 1,850 (11) 57,100 (15) MC 32.37 1.90 (1.5) 24,700 (29) 1,350 (7.7) 3,810 (1.0) PB 42.74 2.47 (2.0) 1,300 (1.5) 17,500 (100) 12,210 (3.1)  Note. Values in parentheses are the fold-increase relative to the corn oil (control) group. a All data reported as the average of two separate determinations (n=2).      106  were increased 100- and 15-fold by treatment with PB and DEX, respectively. The four pooled hepatic microsomal preparations were then used to study the oxidative metabolism of BDE-99.   3.6 Identification of the Hydroxylated Metabolites of BDE-99 Formed by Rat Liver Microsomes An initial set of experiments was conducted involving BDE-99 (50 µM, final concentration) and the four pooled rat liver microsomal preparations. Incubation of BDE- 99 with liver microsomes from DEX- or PB-treated rats yielded seven hydroxylated metabolites, six of which were identified as 2,4,5-TBP, 4-OH-BDE-90, 5ʹ-OH-BDE-99, 6ʹ-OH-BDE-99, 4ʹ-OH-BDE-101 and 2-OH-BDE-123 (Figure 3.2) based on the m/z ratio and retention time values of the authentic standards available. A seventh metabolite peak (M1), corresponding to a mono-OH-pentabrominated-PBDE based on its m/z value, was detected but could not be identified because its retention time did not match that of any of the authentic standards used. Incubation of BDE-99 with liver microsomes from corn oil- or MC-treated rats yielded three hydroxylated metabolites identified as 4-OH-BDE-90, 5ʹ-OH-BDE-99 and 6ʹ-OH-BDE-99 (Figure 3.2). Formation of 2,4,5-TBP, 4'-OH-BDE- 101 and 2-OH-BDE-123 was not detected. Metabolite peaks corresponding to mono-OH- tetrabrominated-PBDEs or di-OH-pentabrominated-PBDEs were not detected with any of the microsomal preparations used.   107     Figure 3.2 Rates of BDE-99 metabolite formation in liver microsomes from corn oil-, DEX-, MC-, and PB-treated rats. Rat liver microsomes (0.5 mg/mL) were incubated with BDE-99 (100 µM, final concentration) for 10 min. Metabolites were extracted and analyzed as described in Section 2.5 and 2.6, respectively.     0 50 100 150 200 250 300 350 400 Corn-oil DEX MC PB Pr od uc t f or m at io n (p m ol /m in /m g pr ot ei n) 4-OH-BDE-90A 0 5 10 15 20 25 30 35 Corn-oil DEX MC PB Pr od uc t f or m at io n (p m ol /m in /m g pr ot ei n) 5'-OH-BDE-99 6'-OH-BDE-99 2,4,5-TBP B 108  Metabolite formation was not observed when BDE-99 was incubated with boiled microsomal preparations, indicating that metabolite formation was enzyme-mediated. In addition, metabolite formation was not observed when NADPH or BDE-99 was omitted from the reaction mixture, or when carbon monoxide was bubbled into an incubation mixture for 2 min prior to addition of substrate, suggesting that formation of hydroxylated metabolites of BDE-99 was CYP-mediated. Hepatic microsomal preparations from DEX- and corn oil-treated rats showed the highest and the lowest BDE-99 metabolism activity, respectively (Figure 3.2). Therefore, hepatic microsomal preparations from DEX- and corn oil-treated rats were used to determine the linearity of BDE-99 metabolite formation with respect to incubation time and protein concentration. Formation of all the BDE-99 metabolites detected was linear up to 10 min and a protein concentration of 0.1 mg/mL using hepatic microsomal preparations from DEX-treated rats (Figure 3.3 and 3.4). Formation of the two major metabolites of BDE-99 (namely 4-OH-BDE-90 and 6'-OH-BDE-99) was linear up to 10 min and a protein concentration of 0.5 mg/mL using hepatic microsomal preparations from corn oil-treated rats (Figure 3.5). These incubation time and protein concentration values were then used to conduct substrate-course experiments using hepatic microsomal preparations from DEX- and corn oil-treated rats.      109    Figure 3.3 Effect of incubation time on formation of hydroxylated metabolites of BDE- 99 (mean ± SD) by liver microsomes from DEX-treated rats. Liver microsomes from DEX-treated rats (0.1 mg/mL) were incubated with BDE-99 (100 µM final concentration) and NADPH for 0 to 30 min. Samples were prepared in duplicate and three separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.6, respectively.  0 250 500 750 1,000 1,250 0 5 10 15 20 25 30 Pr od uc t f or m at io n (p m ol /m g pr ot ei n) 4-OH-BDE-90A 0 40 80 120 160 200 0 5 10 15 20 25 30 Pr od uc t f or m at io n (p m ol /m g pr ot ei n) Incubation time (min) 5'-OH-BDE-99 6'-OH-BDE-99 2,4,5-TBP B 110    Figure 3.4 Effect of total liver microsomal protein concentration on rates of BDE-99 metabolite formation (mean ± SD) by liver microsomes from DEX-treated rats. Liver microsomes from DEX-treated rats (0.05 to 0.5 mg/mL) were incubated with BDE- 99 (50 µM final concentration) and NADPH for 10 min. Samples were prepared in duplicate and three separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.6, respectively.  0 10 20 30 40 50 60 0 0.1 0.2 0.3 0.4 0.5 Pr od uc t f or m at io n (p m ol /m in ) 4-OH-BDE-90A 0 2 4 6 0 0.1 0.2 0.3 0.4 0.5 Pr od uc t f or m at io n (p m ol /m in ) Protein concentration (mg/mL) 5'-OH-BDE-99 6'-OH-BDE-99 2,4,5-TBP B 111      Figure 3.5 Effect of incubation time (A) and total liver microsomal protein (B) on formation of hydroxylated metabolites of BDE-99 (mean ± SD) by liver microsomes from corn oil-treated rats. BDE-99 (50 µM final concentration) was incubated with (A) liver microsomes from corn oil-treated rats (0.5 mg/mL) for 0 to 30 min or with liver microsomes from corn oil- treated rats (0 to 1.0 mg/mL) for 10 min (B). Samples were prepared in duplicate and three separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.6, respectively.  0.0 0.4 0.8 1.2 1.6 2.0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Protein concentration (mg/mL) 5'-OH-BDE-99 2,4,5-TBP 6'-OH-BDE-99 4-OH-BDE-90 B Pr od uc t f or m at io n (p m ol /m g pr ot ei n) 0 7 14 21 28 35 0 5 10 15 20 25 30 Pr od uc t f or m at io n (p m ol /m g pr ot ei n) Incubation time (min) 4-OH-BDE-90 6'-OH-BDE-99 5'-OH-BDE-99 2,4,5-TBP A 112  3.7 Kinetic Analysis of the Hydroxylated Metabolites of BDE-99 Formation by Liver Microsomes from DEX- and Corn Oil-treated Rats Rates of BDE-99 metabolite formation were quantified over a range of substrate concentrations (2.5 to 200 μM) using hepatic microsomes from DEX-treated rats. Data were fitted to different enzyme kinetic models (Section 2.13). The model with the lowest AICc and standard deviation of the residual values was considered to be the model best fitting the data. As shown in Figure 3.6, formation of 4-OH-BDE-90, 5ʹ-OH-BDE-99, 6ʹ- OH-BDE-99, and M1was best fitted by the Michaelis-Menten model (Equation 1 in Section 2.13). Formation of 2,4,5-TBP was best fitted by the substrate inhibition model (Equation 3 in Section 2.13). Kinetic analysis of 4ʹ-OH-BDE-101 and 2-OH-BDE-123 formation was not conducted because 4ʹ-OH-BDE-101 and 2-OH-BDE-123 were formed in quantifiable amounts only at substrate concentrations of 50 μM or greater. Eadie- Hoftsee plots (velocity vs velocity/substrate concentration ratio) were prepared separately for comparison purposes. Linear Eadie-Hoftsee plots were obtained for 6ʹ-OH-BDE-99 and M1 formation and an Eadie-Hoftsee plot indicative of substrate inhibition was obtained for 2,4,5-TBP formation (Figure 3.6). Eadie-Hoftsee plots for 2,4,5-TBP, 6ʹ- OH-BDE-99 and M1formation supported the statistically-derived best-fit model for their formation. Non linear Eadie-Hoftsee plots, signifying sigmoidal and biphasic kinetic models, were obtained for 4-OH-BDE-90 and 5'-OH-BDE-99 formation, respectively (Figure 3.6), not supporting the model best fitting their formation. Apparent Km and Vmax values for hepatic microsomal formation of 4-OH-BDE-90, 5ʹ-OH-BDE-99, 6ʹ-OH-BDE-99, and M1 were calculated using the Michaelis-Menten model (Equation 1 in Section 2.13) and apparent Km, Ki, and Vmax values for 2,4,5-TBP 113  Figure 3.6 Enzyme kinetic profiles for the formation of hydroxylated metabolites of BDE-99 by liver microsomes from DEX-treated rats. Rate of metabolite formation was plotted as a function of substrate concentration following a 10 min incubation of BDE-99 with hepatic microsomes (0.1 mg protein/mL) from DEX-treated rats.  Data points are the mean  SD of three separate experiments.  Lines represent rates of metabolite formation modeled by nonlinear regression analysis.  The insets depict Eadie-Hofstee plots. Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values. Due to the lack of an authentic standard, the plot of M1 formation was prepared using relative peak area.              114  formation were calculated using substrate-inhibition equation (Equation 3 in Section 2.13). The apparent Vmax value for 4-OH-BDE-90 formation was approximately 20-times larger than that for 5ʹ-OH-BDE-99 or 6ʹ-OH-BDE-99 formation and 46-times greater than that for 2,4,5-TBP formation, indicating that 4-OH-BDE-90 was the major hydroxylated metabolite of BDE-99 produced by hepatic microsomes from DEX-treated rats (Figure 3.6).  Apparent Km values associated with the formation of 2,4,5-TBP and 6ʹ- OH-BDE-99 were lower than those for 4-OH-BDE-90 and 5ʹ-OH-BDE-99 (Figure 3.6), suggesting that 2,4,5-TBP and 6ʹ-OH-BDE-99 metabolites would be produced preferentially at low BDE-99 concentrations. Rates of BDE-99 metabolite formation were quantified over a range of substrate concentrations (2.5 to 200 μM) also using hepatic microsomes from corn oil-treated rats. 4-OH-BDE-90, 5ʹ-OH-BDE-99, and 6ʹ-OH-BDE-99 were formed in quantifiable amounts only at BDE-99 concentrations between 50 and 200 µM (Figure 3.7). Therefore, kinetic analysis of 4-OH-BDE-90, 5ʹ-OH-BDE-99, and 6ʹ-OH-BDE-99 formation could not be conducted.   3.8 Effects of CYP Inducers on BDE-99 Biotransformation To investigate the influence of CYP induction on oxidative biotransformation of BDE-99, rates of BDE-99 metabolite formation were determined using hepatic microsomes prepared from rats pretreated with either corn oil, DEX, MC, or PB. A saturating substrate concentration of 100 μM was used based on enzyme kinetic results 115    Figure 3.7 Enzyme kinetic profiles of 4-OH-BDE-90, 5'-OH-BDE-99 and 6'-OH-BDE- 99 formation by hepatic microsomes from corn oil-treated rats. Rates of metabolite formation was plotted as a function of substrate concentration following a 10-min incubation of BDE-99 with hepatic microsomes from corn oil-treated rats (0.5 mg/mL). Data points are the mean of two separate experiments.          0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 25 50 75 100 125 150 175 200 Pr od uc t f or m at io n (p m ol /m in /m g pr ot ei n) BDE-99 concentration (uM) 4-OH-BDE-90 5'-OH-BDE-99 6'-OH-BDE-99 116  (Section 3.7).  Distinct profiles of BDE-99 metabolite formation were obtained with each hepatic microsomal preparation (Table 3.7). Three metabolites, 4-OH-BDE-90, 5'-OH- BDE-99, and 6'-OH-BDE-99, were detected and quantified when BDE-99 was incubated with hepatic microsomes from corn oil- or MC-treated rats. The major metabolites produced by hepatic microsomes from corn oil- and MC-treated rats were 4-OH-BDE-90 and 6'-OH-BDE-99, respectively (Table 3.7). Seven metabolites, 2,4,5-TBP, 4-OH-BDE- 90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101, 2-OH-BDE-123, and M1, were detected using hepatic microsomes from DEX- or PB-treated rats. The major metabolite produced by both hepatic microsomal preparations was 4-OH-BDE-90. On the basis of relative peak areas, M1 formation was greater in hepatic microsomes from DEX- than PB-treated rats and it appears to be a quantitatively important metabolite in both microsomal preparations. The structure of the metabolites produced incubating BDE-99 with rat liver microsomal preparations is depicted in Figure 3.8.   3.9 Oxidative Biotransformation of BDE-99 by Rat Recombinant CYP Enzymes Liver microsomes from DEX-treated rats showed the highest activity for BDE-99 metabolism (Figure 3.2). Rat treatment with DEX preferentially induced CYP3A activity (Table 3.6). Therefore, recombinant CYP3A1 was thought to be the most active rat CYP enzyme for BDE-99 biotransformation. Rat recombinant CYP3A1 was then selected to conduct initial experiments to ensure linearity of product formation with respect to incubation time and recombinant CYP enzyme concentration using a saturating concentration of BDE-99 (100 μM). Rates of product formation were linear up to 10 min 117  Table 3.7 Effect of Treatment with CYP Inducers on Rates of Formation of Hydroxylated Metabolites of BDE-99 by Rat Hepatic Microsomes Treatment Rates of metabolite formation (pmol/min/mg protein)  2,4,5-TBP 4-OH-BDE-90 5'-OH-BDE-99 6'-OH-BDE-99 4'-OH-BDE-101 2-OH-BDE-123 Corn oil N.D.a 2.7 ± 1.6 0.31 ± 0.11 0.62 ± 0.41 N.D. N.D. DEXb 8.2 ± 2.4      320 ± 51.6 18 ± 3.8        12 ± 3.7 3.6 ± 1.1  3.7 ± 0.63 MCc N.D. 6.0 ± 3.6  1.9 ± 0.72        20 ± 4.0 N.D. N.D. PBd 21 ± 1.4       76 ± 16 27 ± 5.4  27 ± 3.7 9.0 ± 1.8 7.5 ± 1.0  Note. Rat hepatic microsomes were incubated with BDE-99 at saturating substrate concentrations (100M) for 10 min. A protein concentration of 0.5 mg/mL was used with hepatic microsomes from corn oil-treated rats and a protein concentration of 0.1 mg/mL was used with hepatic microsomes from DEX-, MC-, or PB-treated rats, as described in Section 2.8. Values represent the mean  SD of three independent experiments. M1 was detected in hepatic microsomes from DEX- or PB-treated rats only.  Rates of M1 formation could not be expressed as pmol/min/mg protein because of the lack of an authentic standard. Using peak area count values, the rate of M1 formation was estimated to be 8 times larger in hepatic microsomes from DEX-treated than PB-treated rats. a N.D., not detected. b dexamethasone. c 3-methylcholanthrene. d phenobarbital. 118    Figure 3.8 Scheme showing the chemical structures of metabolites produced incubating BDE-99 with the four rat liver microsomal preparations used.               119  and a recombinant CYP3A1 concentration of 30 pmol/mL (Figure 3.9 and 3.10). Hydroxylated metabolites were not detected when BDE-99 was incubated with control insect cell microsomes containing expressed rat CYP-oxidoreductase without CYP enzymes. A recombinant CYP enzyme concentration of 30 pmol/mL, incubation time of 10 min, and a BDE-99 concentration of 100 µM were then used in subsequent experiments. The contribution of individual CYP enzymes to BDE-99 biotransformation was evaluated using a panel of fourteen rat recombinant CYP enzymes. Several recombinant CYP enzymes were involved in the oxidative biotransformation of BDE-99 (Figure 3.11). For example, formation of 2,4,5-TBP was catalyzed by seven of the fourteen CYP enzymes tested.  Biotransformation of BDE-99 to 4-OH-BDE-90 was catalyzed at a relatively high rate by CYP3A1.  Formation of 5ʹ-OH-BDE-99 and 6ʹ-OH-BDE-99 was catalyzed by several enzymes including CYP1A1, CYP2A2, CYP2B1, CYP3A1, and CYP3A2.  Biotransformation of BDE-99 to 4ʹ-OH-BDE-101 was catalyzed at a low rate by CYP1A2 and CYP2A2, and 2-OH-BDE-123 formation was mediated predominantly by CYP1A1 and CYP2B1, also at a low rate.  CYP2C11 and CYP3A1 enzymes were the major enzymes involved in M1 formation. Under the experimental conditions used, CYP3A1 was the most active recombinant CYP enzyme in the biotransformation of BDE-99 and 4-OH-BDE-90 was the major metabolite produced. There was no evidence for the formation of mono-OH- tetrabrominated-PBDE or di-OH-pentabrominated-PBDE metabolites of BDE-99, which is consistent with the results obtained using rat liver microsomes. 120     Figure 3.9 Effect of incubation time on formation of hydroxylated metabolites of BDE- 99 by rat recombinant CYP3A1. Rat recombinant CYP3A1 (30 pmol/mL) was incubated with BDE-99 (100 µM final concentration) and NADPH for 5 to 20 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.6, respectively.   0.000 0.002 0.004 0.006 0.008 0 5 10 15 20 Pr od uc t f or m at io n (r es po ns e/ pm ol  r ec om bi na nt  C Y P3 A 1) Incubation time (min) 5'-OH-BDE-99 6'-OH-BDE-99 M1 B 0.000 0.003 0.006 0.009 0.012 0.015 0.018 0 5 10 15 20 Pr od uc t f or m at io n (r es po ns e/  pm ol  r ec om bi na nt  C Y P3 A 1) 4-OH-BDE-90A 121     Figure 3.10 Effect of recombinant CYP3A1 concentration on rates of formation of hydroxylated metabolites of BDE-99. Rat recombinant CYP3A1 (10-50 pmol/mL) was incubated with BDE-99 (100 µM final concentration) and NADPH for 10 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.6, respectively.  0.000 0.005 0.010 0.015 0.020 0.025 0 10 20 30 40 50 Pr od uc t f or m at io n (r es po ns e/ m in ) 4-OH-BDE-90A 0.0000 0.0010 0.0020 0.0030 0.0040 0 10 20 30 40 50 Pr od uc t f or m at io n (r es po ns e/ m in ) Concentration of recombinant CYP3A1 (pmol/mL) 5'-OH-BDE-99 6'-OH-BDE-99 M1 B 122   Figure 3.11 Rates of formation of hydroxylated metabolites of BDE-99 by a panel of rat recombinant CYP enzymes. Rates of 2,4,5-TBP, 4-OH-BDE-90, and 5'-OH-BDE-99 (A) and of 6'-OH-BDE-99, 4'- OH-BDE-101, 2-OH-BDE-123, and M1 (B) formation were measured following a 10 min incubation of BDE-99 (100 M) with baculovirus insect cell microsomes containing expressed rat CYP enzymes (30 pmol/mL) and OR or OR only.  Plots show the mean values  SD of triplicate determinations.     123  Part 3: Oxidative Metabolism of BDE-99 by Human Liver Microsomes and Recombinant CYP Enzymes The objective of the third part of my thesis was to characterize the in vitro metabolism of BDE-99 by human liver microsomes and recombinant CYP enzymes. Using an improved UHPLC/MS/MS-based method, the hydroxylated metabolites of BDE-99 formed by human liver microsomes were identified, their rates of formation were quantified, and the values of kinetic parameters associated with their formation were determined. A possible pathway of secondary metabolite formation was also investigated. In addition, the role of CYP2B6 was assessed using a combined approach including a panel of human recombinant CYP enzymes, CYP2B-specific inhibitory antibodies, single donor human liver microsomes, and correlation analysis.   3.10 Identification of the Hydroxylated Metabolites of BDE-99 Formed by Human Liver Microsomes Incubation of BDE-99 with pooled human liver microsomes yielded ten hydroxylated metabolites (Figure 3.12). Six hydroxylated metabolites were identified as 2,4,5-TBP, 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101 and 2-OH- BDE-123 by comparison of their retention time and isotopic mass to charge transition (parent/daughter ions) values with those of authentic standards. On the basis of response values (area of the metabolite peak/area of the internal standard), the major metabolites were 2,4,5-TBP, 5'-OH-BDE-99 and 4'-OH-BDE-101. Three additional mono-OH- pentabrominated-PBDE metabolites (M1-M3) and a di-OH-pentabrominated-PBDE 124     Figure 3.12 Scheme showing the chemical structures of hydroxylated metabolites formed following the incubation of human liver microsomes with BDE-99. General structures for M1-M4 are also shown.        125  metabolite (M4) were detected but not identified because the retention times of these metabolites did not match those of the authentic standards. Metabolite peaks corresponding to mono-OH-tetrabrominated-PBDEs and 2,4-DBP were not detected. Metabolite formation was not observed when BDE-99, hepatic microsomes, or NADPH was omitted from the reaction mixture. A representative chromatogram showing all the hydroxylated metabolites of BDE-47 detected is presented in Figure 3.13. Experiments with single donor human liver microsomes confirmed the formation of the same ten hydroxylated metabolites of BDE-99. On the basis of response values (area of the metabolite peak/area of the internal standard), 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 were major metabolites and 4-OH-BDE-90, 6'-OH-BDE-99 and 2- OH-BDE-123 were minor metabolites of BDE-99, as was observed by pooled human liver microsomes. In addition, a 5- to 200-fold difference in the rates of formation of each hydroxylated metabolite of BDE-99 was observed among the individual human donor samples assessed (Table 3.8). Pooled human liver microsomes were used to determine the linearity of BDE-99 metabolite formation with respect to incubation time and protein concentration. Formation of the major identified and unidentified BDE-99 metabolites detected was linear up to 10 min and a protein concentration of 0.1 mg/mL (Figure 3.14 and 3.15). These incubation time and protein concentration values were then used to conduct the subsequent substrate-course experiments using pooled human liver microsomes.  126    Figure 3.13 Representative UHPLC/MS/MS chromatograms of 2,4,5-TBP (A), mono- OH-pentabrominated-PBDE (B), and di-OH-pentabrominated-PBDE (M4) (C) metabolites produced by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-99 (100 µM) for 10 min. Hydroxylated metabolites were detected operating the mass spectrometer in electrospray negative mode and identified by comparison of their isotopic mass to charge transition values (reported on each panel above) and retention time values with those of authentic standards.  127  Table 3.8 Rates of Formation of Hydroxylated Metabolites of BDE-99 by Single Donor Human Liver Microsomea Metabolite Rates of metabolite formation (pmol/min/mg protein)  HG95 HH837 HH13 HH18 H0435 H0426 H0455 H0442 H0444 2,4,5-TBP 1.1 61 20.5 31 4.9 240 38 91 79 4-OH-BDE-90 1.5 4.9 2.2 1.8 BDL 12 2.5 5.0 7.4 5'-OH-BDE-99 1.3 31 18 12 1.2 54 8.6 17 49 6'-OH-BDE-99 BDLb 1.7 1.0 1.0 0.49 4.0 1.2 1.9 2.8 4'-OH-BDE-101 0.6 16 9.1 6.6 0.71 29 4.6 9.2 25 2-OH-BDE-123 BDL 7.5 3.4 1.5 0.023 16 4.5 7.5 12 4-OH-Bupropion 10 598 244 141 24 1,200 190 440 710   128  Metabolite Rates of metabolite formation (response/min/mg protein)  HG95 HH837 HH13 HH18 H0435 H0426 H0455 H0442 H0444 M1 0.021 0.44 0.24 0.092 0.017 0.64 0.12 0.15 0.72 M2 BDL 0.24 0.11 0.059 BDL 0.028 0.091 0.098 0.31 M3 BDL 0.12 0.069 0.029 BDL 0.23 0.047 0.062 0.27 M4 0.0021 0.14 0.066 0.099 0.023 0.92 0.13 0.41 0.31  a Single donor human liver microsomes (0.1 mg/mL) were incubated with BDE-99 (10 or 100 µM) for 10 min as described in Section 2.8. Single donor human liver microsomes (0.5 mg/mL) were incubated with bupropion (100 µM) for 20 min as described in Section 2.8. b BDL, below detection limit     129     Figure 3.14 Effect of incubation time on formation of the identified (A) and unidentified (B) hydroxylated metabolites of BDE-99 produced by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-99 (100 µM final concentration) and NADPH for 2.5 to 20 min. Samples were prepared in duplicate and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.   0 500 1,000 1,500 2,000 2,500 0 100 200 300 400 0 2 4 6 8 10 12 14 16 18 20 22 2, 4, 5- T B P  fo rm at io n (p m ol /m g pr ot ei n) Pr od uc t f or m at io n (p m ol /m g pr ot ei n) 4-OH-BDE-90 5'-OH-BDE-99 6'-OH-BDE-99 4'-OH-BDE-101 2-OH-BDE-123 2,4,5-TBP A 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 0.0 3.0 6.0 9.0 12.0 0 2 4 6 8 10 12 14 16 18 20 22 M 2 fo rm at io n (r es po ns e/ m g pr ot ei n) M 1, M 3,  a nd  M 4 fo rm at io n (r es po ns e/ m g pr ot ei n) Incubation time (min) M1 M3 M4 M2B 130     Figure 3.15 Effect of total protein concentration on formation of the identified (A) and unidentified (B) hydroxylated metabolites of BDE-99 produced by pooled human liver microsomes. Pooled human liver microsomes (0.05 to 0.4 mg/mL) were incubated with BDE-99 (100 µM final concentration) and NADPH for 10 min. Samples were prepared in duplicate and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively. Rates of 2,4,5-TBP formation are referred to the Y-axis on the right side of the plot. 0 5 10 15 20 25 30 35 40 0 2 4 6 8 10 12 14 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 2, 4, 5- T B P  fo rm at io n (p m ol /m in ) Pr od uc t f or m at io n (p m ol /m in ) 4-OH-BDE-90 5'-OH-BDE-99 6'-OH-BDE-99 4'-OH-BDE-101 2-OHBDE-123 2,4,5-TBP A 0.00 0.03 0.06 0.09 0.12 0.15 0.18 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 M 4 fo rm at io n (r es po ns e/ m in ) M 1,  M 2 an d M 3 fo rm at io n (r es po ns e/ m in ) Protein concentration (mg /mL) M1 M2 M3 M4B 131  3.11 Kinetic Analysis of the Hydroxylated Metabolites of BDE-99 Formation by Human Liver Microsomes Rates of BDE-99 metabolite formation were quantified over a range of substrate concentrations (0.5 to 200 μM) using pooled human liver microsomes. Data were fitted to different enzyme kinetic models as described earlier (Section 2.13). The model with the lowest values for AICc and standard deviation of the residuals was considered to be the best fitting model. Formation of 2,4,5-TBP, M1, M2, M3and M4 was best described by the substrate-inhibition model (Equation 3 in Section 2.13) (Figures 3.16 and 3.17). Formation of 5'-OH-BDE-99, 4'-OH-BDE-101, 4-OH-BDE-90 and 2-OH-BDE-123 was best fitted by the Michaelis-Menten model (Equation 1 in Section 2.13). Formation of 6'- OH-BDE-99 was best described by the Hill model (mean Hill coefficient value > 1; Equation 2 in Section 2.13). Eadie-Hoftsee plots (velocity vs velocity/substrate concentration) were prepared separately for comparative purposes. Eadie-Hoftsee plots that were indicative of substrate inhibition were obtained for the formation of 2,4,5-TBP, M1, M2, M3and M4 and a curved plot, typical of the Hill model, was obtained for 6'-OH- BDE-99 formation. The Eadie-Hoftsee plots of 2,4,5-TBP, M1 to M4, and  6'-OH-BDE- 99 formation were consistent with the statistically-derived best fit model for the formation of these metabolites. Eadie-Hoftsee plots of 4-OH-BDE-90 and 2-OH-BDE- 123 formation did not completely match a typical linear Eadie-Hoftsee plot indicative of the Michaelis-Menten model. The Eadie-Hoftsee plots obtained for 5'-OH-BDE-99 and 4'-OH-BDE-101 formation were indicative of biphasic kinetics (Kramer and Tracy, 2008) and did not support the Michaelis-Menten model as the model best fitting their formation. 132    Figure 3.16 Enzyme kinetic profiles for the formation of the major identified and unidentified hydroxylated metabolites of BDE-99 by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-99 (0.5 to 200 µM) for 10 min. Data points are the meanSD of three separate experiments. Lines represent rates of metabolite formation modeled by nonlinear regression analyses. The insets depict Eadie-Hoftsee plots. Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n=3).         133    Figure 3.17 Enzyme kinetic profiles for the formation of the minor identified and unidentified hydroxylated metabolites of BDE-99 by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-99 (0.5 to 200 µM) for 10 min. Data points are the meanSD of three separate experiments.  Lines represent rates of metabolite formation modeled by nonlinear regression analysis.  The insets depict Eadie-Hoftsee plots.  Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n=3).    134  Apparent Km and Vmax values of metabolite formation were calculated using the Michaelis-Menten, Hill, or substrate-inhibition equations, as appropriate (Table 3.9). Apparent Vmax values for 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 were 10 to 90 times greater than those of other metabolites, confirming that 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 were the major hydroxylated metabolites of BDE-99 under the experimental conditions used. On the basis of response values, M1 was determined to be the major unidentified hydroxylated metabolite formed by human liver microsomes (Figure 3.16 and 3.17). Apparent Km values suggest that formation of 2,4,5-TBP, 4-OH- BDE-90, 6'-OH-BDE-99, 2-OH-BDE-123, and M4 was favored at low BDE-99 concentrations.   3.12 Identification of CYP2B6 as the Major CYP Enzyme Involved in the Oxidative Biotransformation of BDE-99 The contribution of individual CYP enzymes to BDE-99 biotransformation was evaluated using a panel of twelve human recombinant CYP enzymes (Figure 3.18). BDE- 99 (100 µM) was incubated with individual human recombinant CYP enzymes (10 pmol/mL) for 10 min. Among the human recombinant CYP enzymes tested in the CYP2B6 was the most active and, in many cases, the only enzyme that produced hydroxylated metabolites of BDE-99 at detectable levels. Formation of 4-OH-BDE-90 and 2-OH-BDE-123 was also catalyzed by recombinant CYP3A4, but at a rate that was <5% that of CYP2B6. Recombinant CYP2B6 catalyzed the formation of all ten hydroxylated metabolites. 135  Table 3.9 Apparent Vmax, Km, and Ki Values for the Formation of the Hydroxylated Metabolites of BDE-99 by Pooled Human Liver Microsomes BDE-99 metabolite Apparent Vmax (pmol/min/mg protein) Apparent Vmax (response/min/ mg protein) Apparent Km (µM) Apparent Ki (µM) 2,4,5-TBP 45 ± 2.2  1.6 ± 0.21 69 ± 14 4-OH-BDE-90  2.4 ± 0.26  1.7 ± 0.23            – 5'-OH-BDE-99 47 ± 2.3        32 ± 12            – 6'-OH-BDE-99a    1.1 ± 0.017  2.2 ± 0.55            – 4'-OH-BDE-101 24 ± 3.5        20 ± 7.2            – 2-OH-BDE-123 0.61 ± 0.051       2.3 ± 0.058            – M1  1.1 ± 0.31 4.9 ± 1.8      140 ± 55 M2  0.65 ± 0.078 4.0 ± 1.3      280 ± 200 M3     0.74 ± 0.23 6.2 ± 3.8      110 ± 88 M4     0.51 ± 0.11 1.7 ± 0.71        67 ± 14  Note. Values represent the mean  SD of three independent experiments.  Rates of M1- M4 formation could not be expressed as pmol/min/mg protein because of the lack of authentic standards and are therefore reported as response/min/mg protein.  a Hill coefficient mean value 2.0 ± 0.2 (n=3)        136    Figure 3.18 Rates of formation of 2,4,5-TBP, 5'-OH-BDE-99, 4'-OH-BDE-101, and M1following incubation of BDE-99 with a panel of human recombinant CYP enzymes. Individual human recombinant CYP enzymes (10 pmol/mL) and oxidoreductase or oxidoreductase (OR, 50 µg) alone were incubated with BDE-99 (100 µM) for 10 min. Data points are the meanSD of three separate experiments.         137  To determine if CYP2B6 was the most active CYP enzyme in the oxidative biotransformation of BDE-99 by human liver microsomes, BDE-99 was incubated with pooled human liver microsomes in the presence of varying amounts of two CYP2B- selective antibodies. Rabbit anti-rat CYP2B1 IgG and mouse anti-human CYP2B6 ascites fluid inhibited formation of all the ten hydroxylated metabolites of BDE-99 in a concentration-dependent manner (Figures 3.19-3.21). Almost complete inhibition of metabolite formation (>95% inhibition relative to control activity) was observed in the presence of 2.5 mg anti-CYP2B1 IgG or 2.5 µL anti-CYP2B6 ascites/mg microsomal protein, whereas no inhibition was observed with control rabbit IgG or control mouse serum (Figure 3.19-21), suggesting that formation of all ten hydroxylated metabolites of BDE-99 produced by human liver microsomes was predominantly mediated by CYP2B6. To confirm the importance of hepatic microsomal CYP2B6 activity in the oxidative biotransformation of BDE-99, bupropion 4-hydroxylase activity, a catalytic marker of CYP2B6 activity, was determined and compared with rates of hydroxylated BDE-99 metabolite formation using nine single donor liver microsomal samples. The rates of formation of all 10 hydroxylated metabolites were highly correlated (r>89 and r2>0.81) with those of 4-hydroxy-bupropion formation (Table 3.10), suggesting that the same CYP enzyme, namely CYP2B6, catalyzed bupropion 4-hydroxylation and BDE-99 biotransformation. For comparison, correlation analysis was also performed with CYP1A2-, CYP2C9-, CYP2C19-, CYP2E1-, and CYP3A4-mediated enzyme activities of the same nine single donor liver microsomal preparations. CYP1A2-, CYP2C9-, CYP2C19-, CYP2E1-, and CYP3A4-marker activity values were provided by 138    Figure 3.19 Effect of rabbit anti-rat CYP2B1 IgG and mouse anti-human CYP2B6 ascites on the formation of the major identified and unidentified metabolites of BDE-99 by human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were preincubated for 10 min with various amounts of anti-CYP2B1 IgG (open symbols), control rabbit IgG (closed symbols), anti- CYP2B6 ascites (open symbols), or control mouse serum (closed symbols) prior to addition of BDE-99 (10 µM) and a further incubation for 5 min. Data are expressed as percent of the activity measured with no antibody, IgG, or serum. Data points represent the average of two experiments. 139    Figure 3.20 Effect of rabbit anti-rat CYP2B1 IgG on the formation of the minor identified and unidentified metabolites of BDE-99 by human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were preincubated with various amounts of anti-CYP2B1 IgG (open symbols) or control rabbit IgG (closed symbols) for 10 min, prior to addition of BDE-99 (10 µM), and further incubation for 5 min. Data are expressed as a percent of the activity measured with no IgG and represent the average of two experiments.  140     Figure 3.21 Effect of mouse anti-human CYP2B6 ascites on the formation of the minor identified and unidentified metabolites of BDE-99 by human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were preincubated with various amounts of anti-CYP2B6 ascites (open symbols) or control mouse serum (closed symbols) for 10 min, prior to addition of BDE-99 (10 µM), and further incubation for 5 min. Data are expressed as a percent of the activity measured with no anti-CYP2B6 ascites or control mouse serum.     141  Table 3.10 Correlation Analysis of BDE-99 Hydroxylated Metabolite Formation and CYP Marker Activities Using Single Donor Human Liver Microsomes BDE-99 metabolites Correlation coefficient, r (and coefficient of determination, r2) values  CYP1A2a CYP2B6b CYP2C9c CYP2C19d CYP2E1e CYP3A4f 2,4,5-TBP 0.48 (0.23)  0.93 (0.87) 0.33 (0.11) 0.89 (0.80) 0.43 (0.19) 0.53 (0.28) 4-OH-BDE-90 0.40 (0.16) 0.98 (0.97) 0.46 (0.21) 0.81 (0.66) 0.63 (0.40) 0.47 (0.22) 5'-OH-BDE-99 0.41 (0.17) 0.96 (0.92) 0.64 (0.41) 0.63 (0.40) 0.79 (0.63) 0.46 (0.21) 6'-OH-BDE-99 0.37 (0.14) 0.98 (0.95) 0.48 (0.23) 0.75 (0.56) 0.62 (0.39) 0.45 (0.20) 4'-OH-BDE-101 0.41 (0.17) 0.96 (0.93) 0.64 (0.41) 0.65 (0.42) 0.78 (0.61) 0.46 (0.21) 2-OH-BDE-123 0.32 (0.10) 0.98 (0.97) 0.45 (0.20) 0.75 (0.56) 0.65 (0.42) 0.39 (0.15) M1 0.32 (0.10) 0.90 (0.81) 0.67 (0.45) 0.52 (0.27) 0.85 (0.73) 0.37 (0.14) M2 0.36 (0.13) 0.91 (0.83) 0.65 (0.42) 0.57 (0.33) 0.75 (0.56) 0.43 (0.19) M3 0.22 (0.05) 0.89 (0.80) 0.62 (0.38) 0.49 (0.24) 0.83 (0.78) 0.28 (0.08) M4 0.39 (0.15) 0.90 (0.81) 0.24 (0.06) 0.85 (0.73) 0.41 (0.17) 0.44 (0.19) Note. Marker activity for CYP2B6 was experimentally determined as described in the Section 2.11, whereas marker activity values for the other CYP enzymes were provided by the vendor (Table A1.5). aCYP1A2 marker activity: phenacethin O-deethylation;bCYP2B6 marker activity: bupropion 4-hydroxylation;cCYP2C9 marker activity: diclofenac 4'-hydroxylation; dCYP2C19 marker activity:(S)-mephenytoin 4'-hydroxylation;eCYP2E1 marker activity: chlorzoxazone 6-hydroxylation;fCYP3A4 marker activity: testosterone 6β-hydroxylation. 142  Xenotech and BD Biosciences (see product information sheets, Table A1.5). The correlation values obtained for rates of hydroxylated BDE-99 metabolite formation and CYP1A2-, CYP2C9-, CYP2C19-, CYP2E1-, and CYP3A4-mediated enzyme activities were lower than those obtained for rates of hydroxylated BDE-99 metabolite formation and CYP2B6-mediated enzyme activity (Table 3.10). Rates of formation for a few hydroxylated metabolites of BDE-99 (i.e. 4-OH-BDE-90, 6'-OH-BDE-99, 2-OH-BDE- 123, and M4), however, were highly correlated (0.75<r<0.85) with CYP2C19- and CYP2E1-marker activity values (Table 3.10). When CYP2B6- and CYP2C19-marker activity values of the single donor human liver microsomes were compared to each other, a relatively large correlation coefficient (r=0.78) was found (Figure A1.2), which indicates that the catalytic activities of CYP2B6 and CYP2C19 co-vary in this samples set.   3.13 Kinetic Analysis of the Hydroxylated Metabolites of BDE-99 Formation by Recombinant CYP2B6 Rates of BDE-99 metabolite formation were quantified over a range of substrate concentrations (0.5 to 200 μM) using a recombinant CYP2B6 concentration of 5 pmol/mL and an incubation time of 10 min. In preliminary experiments, rates of BDE-99 metabolite formation were found to be linear up to a recombinant CYP2B6 concentration of 5 pmol/mL and 10 min (Figure 3.22-3.25). Data were fitted to different enzyme kinetic models as described earlier (Section 2.13). The model with the lowest AICc and standard deviation of the residual values was considered to be the best fitting model. Formation of 143     Figure 3.22 Effect of incubation time on formation of 2,4,5-TBP, 5'-OH-BDE-99, and 4'- OH-BDE-101 (A) and of 4-OH-BDE-90, 6'-OH-BDE-99, and 2-OH-BDE-123 (B) by recombinant CYP2B6. Recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-99 (100 µM final concentration) and NADPH for 5 to 20 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.   0 1,000 2,000 3,000 4,000 0 5 10 15 20 Pr od uc t f or m at io n (p m ol / nm ol  r ec om bi na nt  C Y P2 B 6) 2,4,5-TBP 5'-OH-BDE-99 4'-OH-BDE-101 A 0 50 100 150 200 250 300 0 5 10 15 20 Pr od uc t f or m at io n (p m ol /n m ol  r ec om bi na nt  C Y P2 B 6) Incubation time (min) 4-OH-BDE-90 2-OH-BDE-123 6'-OH-BDE-99 B 144      Figure 3.23 Effect of incubation time on formation of M1 and M2 (A) and of M3 and M4 (B) by recombinant CYP2B6. Recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-99 (100 µM final concentration) and NADPH for 5 to 20 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.10, respectively.   0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 Pr od uc t f or m at io n (r es po ns e/ nm ol  r ec om bi na nt  C Y P2 B 6) M1 M2 A 0 2 4 6 8 10 12 0 5 10 15 20 Pr od uc t f or m at io n (r es po ns e/  nm ol  r ec om bi na nt  C Y P2 B 6) Incubation time (min) M4 M3 B 145      Figure 3.24 Effect of recombinant CYP2B6 concentration on rates of formation of 2,4,5- TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 (A) and of 4-OH-BDE-90, 6'-OH-BDE-99, and 2-OH-BDE-123 (B). Recombinant CYP2B6 (2.5 to 12 pmol/mL) was incubated with BDE-99 (100 µM final concentration) and NADPH for 5 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.  0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 2 4 6 8 10 12 Pr od uc t f or m at io n (p m ol /m in ) 2,4,5-TBP 5'-OH-BDE-99 4'-OH-BDE-101 A 0.00 0.05 0.10 0.15 0.20 0.25 0 2 4 6 8 10 12 Pr od uc t f or m at io n (p m ol /m in ) Concentration of recombinant CYP2B6 (pmol/mL) 4-OH-BDE-90 2-OH-BDE-123 6'-OH-BDE-99 B 146       Figure 3.25 Effect of recombinant CYP2B6 concentration on rates of formation of M1 and M2 (A) and of M3 and M4 (B). Recombinant CYP2B6 (2.5 to 12 pmol/mL) was incubated with BDE-99 (100 µM final concentration) and NADPH for 5 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.  0.000 0.005 0.010 0.015 0.020 0.025 0.030 0 2 4 6 8 10 12P ro du ct  fo rm at io n (r es po ns e/ m in ) M1 M2 A 0.000 0.002 0.004 0.006 0.008 0.010 0.012 0 2 4 6 8 10 12P ro du ct  fo rm at io n (r es po ns e/ m in ) Concentration of recombinant CYP2B6 (pmol/mL) M4 M3 B 147  2,4,5-TBP, 4-OH-BDE-90, 6'-OH-BDE-99, M1, M2, M3, and M4 by recombinant CYP2B6 was best fitted by the substrate-inhibition model and formation of 5'-OH-BDE- 99, 4'-OH-BDE-101 and 2-OH-BDE-123 by recombinant CYP2B6 was best fitted by the Michaelis-Menten model (Figures 3.26 and 3.27). Eadie-Hoftsee plots (velocity vs velocity/substrate concentration ratio) were prepared separately for comparison purposes. Eadie-Hoftsee plots indicative of substrate inhibition were obtained for the formation of 2,4,5-TBP, 4-OH-BDE-90, 6'-OH-BDE-99, M1, M2 and M4, and linear Eadie-Hoftsee plots, typical of the Michaelis-Menten model, were obtained for the formation of 4'-OH- BDE-101 and 2-OH-BDE-123 (Figures 3.26 and 3.27). The Eadie-Hoftsee plots obtained for these BDE-99 metabolites were consistent with the statistically-derived best fit model previously chosen. Non-linear Eadie-Hoftsee plots, typical of sigmoidal and biphasic kinetic models, were obtained for M3 and 5'-OH-BDE-99 formation, respectively. The non-linear Eadie-Hoftsee plots were not consistent with the best fit model previously chosen to describe the formation of these two metabolites. Kinetic parameters for the formation of all 10 hydroxylated metabolites of BDE- 99 by recombinant CYP2B6 were calculated using the Michaelis-Menten or substrate- inhibition model, as appropriate (Table 3.11). Consistent with the results obtained with pooled human liver microsomes, 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101 were the major identified hydroxylated metabolites and M1 was the major unidentified hydroxylated metabolite of BDE-99 produced by recombinant CYP2B6, as indicated by the Vmax values. Trace amounts of three mono-OH-tetrabrominated-PBDEs were detected following incubation of BDE-99 with recombinant CYP2B6. The mono-OH- tetrabrominated-PBDEs detected were identified as 5-OH-BDE-47, 6-OH-BDE-47, and 148   Figure 3.26 Enzyme kinetic profiles for the formation of the major identified and unidentified hydroxylated metabolites of BDE-99 by human recombinant CYP2B6. Human recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-99 (0.5 to 200 µM) for 10 min. Data points are the meanSD of three separate experiments.  Lines represent rates of metabolite formation modeled by nonlinear regression analyses.  The insets depict Eadie-Hoftsee plots.  Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n=3).        149    Figure 3.27 Enzyme kinetic profiles for the formation of the minor identified and unidentified hydroxylated metabolites of BDE-99 by human recombinant CYP2B6. Human recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-99 (0.5–200 µM) for 10 min. Data points are the meanSD of three separate experiments. Lines represent rates of metabolite formation modeled by nonlinear regression analysis. The insets depict Eadie-Hoftsee plots. Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n=3).     150  Table 3.11 Values of Vmax, Km, and Ki for the Formation of the Hydroxylated Metabolites of BDE-99 by Human Recombinant CYP2B6 BDE-99 metabolite Vmax (pmol/min/nmol rCYP2B6) Vmax (response/min/ nmol rCYP2B6) Km (µM) Ki (µM) 2,4,5-TBP 210 ± 37  0.061 ± 0.018 140 ± 52 4-OH-BDE-90          28 ± 6.8  0.33 ± 0.14   55 ± 14 5'-OH-BDE-99        460 ± 99    2.4 ± 0.70  – 6'-OH-BDE-99          14 ± 2.1  0.28 ± 0.15   64 ± 33 4'-OH-BDE-101        250 ± 31  0.89 ± 0.13  – 2-OH-BDE-123 6.8 ± 0.76      0.27 ± 0.076  – M1       2.2 ± 0.38     0.35 ± 0.054   48 ± 10 M2     0.83 ± 0.10     0.39 ± 0.079    39 ± 4.8 M3     0.73 ± 0.14 0.56 ± 0.17    22 ± 4.4 M4  0.78 ± 0.0053 0.088 ± 0.019   72 ± 25  Note. Values represent the mean  SD of three independent experiments.  Rates of M1- M4 formation could not be expressed as pmol/min/ nmol recombinant CYP2B6 because of the lack of authentic standards and are therefore reported as response/min/nmol recombinant CYP2B6.       151  4'-OH-BDE-49 using authentic standards. The amounts of 5-OH-BDE-47, 6-OH-BDE- 47, and 4'-OH-BDE-49 formed were below the limit of quantification. Thus, kinetic analysis of their formation could not be undertaken.   3.14 Investigation of the Mechanism of Formation of Secondary Metabolites of BDE-99 by Human Liver Microsomes To investigate whether 2,4,5-TBP or the di-OH-pentabrominated-PBDE metabolite (M4) were formed as secondary metabolites of BDE-99, pooled human liver microsomes were incubated with 5'-OH-BDE-99, 6'-OH-BDE-99 or 4'-OH-BDE-101 instead of BDE-99. 2,4,5-TBP and M4 were not detected when 5'-OH-BDE-99, 6'-OH- BDE-99 or 4'-OH-BDE-101 were used as substrate at concentrations of 0.1 to 100 nM. However, 2,4,5-TBP, but not M4, was formed when human liver microsomes were incubated with 5'-OH-BDE-99, 6'-OH-BDE-99 or 4'-OH-BDE-101 at 350 nM. Overall, the results suggest that 2,4,5-TBP and M4 are formed directly from BDE-99 and are not produced as secondary hydroxylated metabolites by human liver microsomes unless the concentration of primary metabolites of BDE-99 is 200 nM or greater. There was no difference in M4 formation when pooled human liver microsomes were incubated with BDE-99 in the presence of varying amounts of antibody to epoxide hydrolase or rabbit IgG (antibody negative control; Figure 3.28), suggesting that microsomal epoxide hydrolase is not involved in the formation of di-OH- pentabrominated-PBDE metabolite. A scheme showing possible pathways for the formation of 2,4,5-TBP and M4 is presented in Figure 3.29. 152             Figure 3.28 Effect of anti-epoxide hydrolase IgG on the formation of the unidentified di- OH-pentabrominated-PBDE metabolite of BDE-99 by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with 0 to 2.0 mg of rabbit anti-rat epoxide hydrolase IgG/mg human liver microsomes (full symbols) or an equivalent amount of rabbit IgG (empty symbols) for 10 min. BDE-99 was then added (10 µM, final concentration) and the mixtures were incubated for an additional 5 min. The reaction was initiated by addition of 0.01 mL of NADPH (1mM, final concentration), allowed to proceed for 10 min, and stopped with 1 mL of ice-cold sodium hydroxide. Samples were then processed and analyzed as described in Section 2.5 and 2.9, respectively.        0 20 40 60 80 100 120 140 160 0.0 0.5 1.0 1.5 2.0 %  a ct iv ity  re m ai ni ng mg of anti-epoxide hydrolase IgG/mg protein Rabbit IgG Anti EH antibody 153    Figure 3.29 Scheme showing possible pathways for the formation of 2,4,5-TBP and M4 from the three major primary hydroxylated metabolites (namely 5'-OH-BDE-99, 6'-OH- BDE-99, and 4'-OH-BDE-101) of BDE-99. Incubating pooled human liver microsomes (0.1 mg/mL) with 5'-OH-BDE-99, 6'-OH- BDE-99, or 4'-OH-BDE-101(0 to 100 nM) instead of BDE-99 as substrate for 10 min indicate the lack of formation of secondary hydroxylated metabolites (namely 2,4,5-TBP and M4) from any of the three primary BDE-99 metabolites used as substrates.          154  Part 4: Oxidative Metabolism of BDE-47 by Human Liver Microsomes and Recombinant CYP Enzymes The objective of the fourth part of my thesis was to investigate the in vitro metabolism of BDE-47 by human liver microsomes and human recombinant CYP enzymes. Using an improved UHPLC/MS/MS-based method, the hydroxylated metabolites of BDE-47 formed by human liver microsomes were identified, their rates of formation were quantified, and the values of kinetic parameters associated with their formation were determined. A possible pathway of secondary metabolite formation was also investigated. In addition, the role of CYP2B6 was assessed using a combined approach including a panel of human recombinant CYP enzymes, CYP2B-specific inhibitory antibodies, single donor human liver microsomes, and correlation analysis.   3.15 Identification of the Hydroxylated Metabolites of BDE-47 Formed by Human Liver Microsomes Incubation of BDE-47 with pooled human liver microsomes yielded 9 hydroxylated metabolites (Figure 3.30). Seven hydroxylated metabolites were identified as 2,4-DBP, 4'-OH-BDE-17, 2'-OH-BDE-28, 4-OH-BDE-42, 5-OH-BDE-47, 6-OH- BDE-47, and 4'-OH-BDE-49 by comparison of their retention time and isotopic mass to charge transition (parent/daughter ions) values with those of authentic standards. On the basis of response values (area of the metabolite peak/area of the internal standard), 5-OH- BDE-47 and 6-OH-BDE-47 were major metabolites, 4-OH-BDE-42 and 4'-OH-BDE-49 were intermediate metabolites, and 4'-OH-BDE-17 and 2'-OH-BDE-28 were minor 155    Figure 3.30 Scheme showing the chemical structures of the hydroxylated metabolites formed following the incubation of pooled human liver microsomes with BDE-47. General structures for M1 and M2 are also shown.          156  metabolites of BDE-47. A peak corresponding to mono-OH-tetrabrominated-PBDE (M1) that did not match the retention time of any of the authentic standards was also detected. Because the only possible OH-tetrabrominated-PBDE metabolite of BDE-47 for which an authentic standard was not available is 2'-OH-2,3',4,4'-tetrabromodipheyl ether (2'- OH-BDE-66), we suggest that M1 is 2'-OH-BDE-66. Similarly, a peak with m/z transition values corresponding to a di-OH-tetrabrominated-PBDE (M2) was detected but not identified. On the basis of response values, M1 was determined to be a major metabolite of BDE-47. 3-OH-BDE-47 is a possible metabolite of BDE-47 (Feo et al., 2013). In our study, formation of 3-OH-BDE-47 was below limit of quantification. Formation of hydroxylated metabolites was not observed when BDE-47, hepatic microsomes, or NADPH was omitted from the reaction mixture. A representative chromatogram showing all the hydroxylated metabolites of BDE-47 detected is presented in Figure 3.31. Experiments with single donor human liver microsomes confirmed the formation of the same nine hydroxylated metabolites of BDE-47 produced by pooled human liver microsomal incubations. On the basis of response values (area of the metabolite peak/area of the internal standard), 5-OH-BDE-47 and 6-OH-BDE-47 were major metabolites, 4-OH-BDE-42 was an intermediate metabolite, and 4'-OH-BDE-17 and 2'- OH-BDE-28 were minor metabolites of BDE-47. Formation of 3-OH-BDE-47 was below limit of quantification in all samples, which is consistent with the results obtained using pooled human liver microsomes. In addition, a 5- to 60-fold difference in the rates of formation of each hydroxylated metabolite of BDE-47 was observed among the individual human donor samples assessed (Table 3.12). 157    Figure 3.31 Representative UHPLC/MS/MS chromatograms of mono-OH- tetrabrominated-PBDE (A), di-OH-tetrabrominated-PBDE (B), mono-OH-tribrominated- PBDE (C), and 2,4-DBP (D) metabolites of BDE-47 produced by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (100 µM) for 5 min. Hydroxylated metabolites were detected operating the mass spectrometer in electrospray negative mode and identified by comparison of their isotopic mass to charge transition values (reported on each panel above) and retention time values with those of authentic standards.  158  Table 3.12 Rates of Formation of Metabolites of BDE-47 by Single Donor Human Liver Microsomesa BDE-47 metabolites Rates of identified metabolite formation (pmol/min/mg protein)  HG95 HH837 HH13 HH18 H0435 H0426 H0455 H0442 H0444 2,4-DBP 1.8 36 14 14 2.3 60 11 26 38 2'-OH-BDE-17 BDLb 3.4 2.2 1.1 0.3 5.4 1.3 2.3 5.0 4-OH-BDE-28 BDL 4.6 2.3 1.2 BDL 6.9 1.5 2.9 6.4 4-OH-BDE-42 BDL 20 10 8.0 0.7 43 7.8 14 37 5-OH-BDE-47 2.4 68 32 24 3.6 140 20 52 94 6-OH-BDE-47 2.3 67 29 22 3.7 120 20 46 92 4'-OH-BDE-49 1.7 56 25 16 3.0 97 16 35 87   Rates of unidentified metabolite formation (response/min/mg protein) M1 1.3 71 31 21 3.7 120 22 45 105 M2 0.023 1.8 0.42 0.68 0.063 4.3 0.69 1.6 1.1  a Single donor human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (100 µM) for 5 min as described in the Section 2.8. b BDL, below detection limit  159  Pooled human liver microsomes were used to determine the linearity of BDE-47 metabolite formation with respect to incubation time and protein concentration. Formation of all the BDE-47 metabolites was linear up to 5 min and a protein concentration of 0.1 mg/mL (Figure 3.32 and 3.33). These incubation time and protein concentration values were then used to conduct the subsequent substrate-course experiments using pooled human liver microsomes.   3.16 Kinetic Analysis of Hydroxylated Metabolites of BDE-47 Formation by Human Liver Microsomes Rates of hydroxylated metabolite formation were measured over a range of BDE- 47 concentrations (2.5 to 200 μM) using pooled human liver microsomes. Data were fitted to different enzyme kinetic models as described earlier (Section 2.13). The model with the lowest AICc and standard deviation of the residual values was considered to be the best fitting model. Formation of 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, 4'- OH-BDE-49, and M1 was best described by the Michaelis-Menten model (Equation 1 in Section 2.13) and formation of M2 was best fitted by the substrate inhibition model (Equation 3 in Section 2.13) (Figure 3.34). Formation of 2,4-DBP was observed only at BDE-47 concentrations greater than 10 µM and was not adequately described by any of the kinetic models tested, suggesting that 2,4-DBP could be a secondary metabolite of BDE-47 (see Section 3.19). The amounts of 4'-OH-BDE-17 and 2'-OH-BDE-28 formed over the BDE-47 concentration range assessed were between the limit of detection and the limit of quantification, suggesting that 4'-OH-BDE-17 and 2'-OH-BDE-28 are minor 160     Figure 3.32 Effect of incubation time on formation of 4-OH-BDE-42, 5-OH-BDE-47, 6- OH-BDE-47, and 4'-OH-BDE-49 (A), of 2,4,-DBP, 4'-OH-BDE-17, and 2'-OH-BDE-28 (B), and of M1 and M2 (C) by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (100 µM final concentration) and NADPH for 2.5 to 20 min. Samples were prepared in duplicate and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively. 0 50 100 150 200 250 0 5 10 15 20 Pr od uc t f or m at io n (p m ol /m g pr ot ei n) 4'-OH-BDE-49 4-OH-BDE-42 5-OH-BDE-47 6-OH-BDE-47 A 0 50 100 150 200 250 0 9 18 27 36 45 0 5 10 15 20 25 2, 4- D B P fo rm at io n (p m ol /m g pr ot ei n) Pr od uc t fo rm at io n (p m ol /m g pr ot ei n) 2'-OH-BDE-28 4'-OH-BDE-17 2,4-DBP B 0.0 1.0 2.0 3.0 4.0 5.0 0 30 60 90 120 150 0 5 10 15 20 25 M 2 fo rm at io n (r es po ns e/ m g pr ot ei n) M 1 fo rm at io n (r es po ns e/ m g pr ot ei n) Incubation time (min) M1 M2C 161      Figure 3.33 Effect of total protein concentration on rates of formation of 2,4-DBP, 4- OH-BDE-42, and 6-OH-BDE-47 (A), of 4'-OH-BDE-17, 2'-OH-BDE-28, 5-OH-BDE-47, and 4'-OH-BDE-49 (B), and of M1 and M2 (C) by pooled human liver microsomes. Pooled human liver microsomes (0.05 to 0.5 mg/mL) were incubated with BDE-47 (100 µM final concentration) and NADPH for 5 min. Samples were prepared in duplicate and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively. 0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 0.0 1.0 2.0 3.0 4.0 5.0 0.0 0.1 0.2 0.3 0.4 0.5 2, 4- D B P fo rm at io n (p m ol /m in ) Pr od uc t f or m at io n (p m ol /m in ) 6-OH-BDE-47 4-OH-BDE-42 2,4-DBP A 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 0.0 0.4 0.8 1.2 1.6 0.0 0.1 0.2 0.3 0.4 0.5 0.6 5- O H -B D E -4 7 fo rm at io n (p m ol /m in ) Pr od uc t f or m at io n (p m ol /m in ) 4'-OH-BDE-49 4'-OH-BDE-17 2'-OH-BDE-28 5-OH-BDE-47 B 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.0 2.0 4.0 6.0 8.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 M 2 fo rm at io n (r es po ns e/ m in ) M 1  fo rm at io n (r es po ns e/ m in ) Protein concentration (mg/mL) M1 M2C 162    Figure 3.34 Enzyme kinetic profiles for the formation of the hydroxylated metabolites of BDE-47 by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with BDE-47 (0.5 to 200 µM) for 5 min. Data points are the meanSD of three separate experiments. Lines represent rates of metabolite formation modeled by nonlinear regression analyses. The insets depict Eadie-Hoftsee plots. Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n=3). Because 2,4-DBP, 4'-OH- BDE-17, and 2'-OH-BDE-28 were only quantifiable at the highest BDE-47 concentrations used, their enzyme kinetic profiles could not be determined.    163  metabolites of BDE-47. Due to the small amounts of 4'-OH-BDE-17 and 2'-OH-BDE-28 formed, kinetic analysis of 4'-OH-BDE-17 and 2'-OH-BDE-28 formation could not be conducted. Eadie-Hoftsee plots (velocity vs velocity/substrate concentration ratio) were prepared separately for comparison purposes. Eadie-Hoftsee plots, typical of the Michaelis-Menten model, were obtained for 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE- 47, 4'-OH-BDE-49 and M1formation (Figure3.34). An Eadie-Hoftsee plot that was indicative of substrate inhibition was obtained for M2 formation. Therefore, the Eadie- Hoftsee plots supported the statistically-derived best fit model for the formation of the BDE-47 metabolites. Apparent Vmax , Km , and Ki values of metabolite formation were calculated using the Michaelis-Menten or substrate-inhibition equations, as appropriate (Table 3.13). The largest apparent Vmax values were obtained for 5-OH-BDE-47 and 6-OH-BDE-47, confirming that 5-OH-BDE-47 and 6-OH-BDE-47 were the major hydroxylated microsomal metabolites of BDE-47 produced by human liver microsomes under the experimental conditions used. Apparent Km values suggest that M2 and 4'-OH-BDE-49 are preferentially formed at lower BDE-47 concentrations.   3.17 Identification of CYP2B6 as the Major CYP Enzyme Involved in the Oxidative Biotransformation of BDE-47 The contribution of individual CYP enzymes to BDE-47 biotransformation was evaluated using a panel of twelve human recombinant CYP enzymes (Figure 3.35). BDE- 47 (100 µM) was incubated with individual human recombinant CYP enzymes (10 164  Table 3.13 Apparent Vmax, Km, and Ki Values for the Formation of the Hydroxylated Metabolites of BDE-47 by Human Liver Microsomes BDE-47 metabolite Apparent Vmax (pmol/min/mg protein) Apparent Vmax (response/min/mg protein) Apparent Km (µM) Apparent Ki (µM) 4-OH-BDE-42 14 ± 4.1  44 ± 42         – 5-OH-BDE-47 27 ± 3.9  36 ± 15         – 6-OH-BDE-47 24 ± 3.4       25 ± 6.3         – 4'-OH-BDE-49 13 ± 3.0       10 ± 2.9         – M1  5.7 ± 1.9      23 ± 3.9         – M2  0.55 ± 0.13 6.6 ± 4.3 86 ± 42  Note. Values represent the mean  SD of three independent experiments.  Rates of M1 and M2 formation could not be expressed as pmol/min/mg protein because of the lack of authentic standards and are therefore reported as response/min/mg protein.            165    Figure 3.35 Rates of formation of 5-OH-BDE-47, 6-OH-BDE-47, 4-OH-BDE-42, 4'- OH-BDE-49, M1 and M2 following incubation of BDE-47 with a panel of human recombinant CYP enzymes. Individual human recombinant CYP enzymes (10 pmol/mL) or oxidoreductase (OR, 50 µg) were incubated with BDE-47 (100 µM) for 5 min. Rates of formation of identified or unidentified metabolites are referred to the left or the right Y axis, respectively. Data points are the meanSD of three separate experiments. 2,4-DBP, 4'-OH-BDE-17, and 2'- OH-BDE-28 were also formed by rCYP2B6 only but were not included in the figure for sake of clarity.           166  pmol/mL) for 5 min. Among the human recombinant CYP enzymes tested, CYP2B6 was the most active and, in many cases, the only enzyme that catalyzed conversion of BDE- 47 to all 9 hydroxylated metabolites (Figure 3.35). Formation of 5-OH-BDE-47 and 6- OH-BDE-47 was catalyzed by recombinant CYP3A4 also, but at a rate that was <1% that of CYP2B6. The other hydroxylated metabolites were not produced at a detectable level by any human recombinant CYP enzyme other than CYP2B6. To assess the catalytic role of CYP2B6 in the biotransformation of BDE-99 by human liver microsomes, BDE-47 was incubated with pooled human liver microsomes in the presence of varying amounts of a monoclonal antibody to CYP2B6.  Mouse anti- human CYP2B6 ascites inhibited formation of all nine metabolites in a concentration- dependent manner (Figure 3.36).  Almost complete inhibition (<5% control activity left) of metabolite formation was observed in the presence of 2.5 µL anti- CYP2B6 ascites/mg microsomal protein, whereas no inhibition was observed with control mouse serum (Figure 3.36), showing that formation of all nine hydroxylated metabolites by human liver microsomes was mediated almost exclusively by CYP2B6. To confirm the importance of hepatic microsomal CYP2B6 activity in the oxidative biotransformation of BDE-47, bupropion 4-hydroxylase activity, a catalytic marker of CYP2B6 activity, was determined and compared with rates of hydroxylated BDE-47 metabolite formation using nine single donor liver microsome samples. The rates of formation of all nine hydroxylated metabolites were highly correlated (r>93 and r2>0.86) with those of 4-hydroxy-bupropion formation (Table 3.14), suggesting that the same CYP enzyme, namely CYP2B6, catalyzed bupropion 4-hydroxylation and BDE-47 biotransformation. For comparison, correlation analysis was also performed with 167    Figure 3.36 Effect of mouse anti-human CYP2B6 ascites on the formation of 5-OH- BDE-47 (A), 6-OH-BDE-47 (B), 4-OH-BDE-42 (C), 4'-OH-BDE-49 (D), M1 (D), and M2 (F) metabolites of BDE-47 by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were preincubated for 10 min with various amounts of anti-CYP2B6 ascites (open symbols) or control mouse serum (closed symbols) prior to addition of BDE-47 (10 or 50 µM) and a further incubation for 5 min. Data are expressed as percent of the activity measured with no antibody. Data points represent the average of two experiments.     168  Table 3.14 Correlation Analysis of Formation of Hydroxylated Metabolites of BDE-47 and CYP Marker Activities Using Single Donor Human Liver Microsomes BDE-47 metabolite Correlation coefficient, r (and coefficient of determination, r2) values  CYP1A2a CYP2B6b CYP2C9c CYP2C19d CYP2E1e CYP3A4f 2,4-DBP 0.52 (0.27) 0.99 (0.99) 0.56 (0.31) 0.82 (0.67) 0.59 (0.35) 0.58 (0.34) 4'-OH-BDE-17 0.36 (0.13) 0.95 (0.91) 0.52 (0.27) 0.62 (0.39) 0.80 (0.64) 0.33 (0.11) 2'-OH-BDE-28 0.36 (0.13) 0.95 (0.90) 0.56 (0.31) 0.58 (0.34) 0.81 (0.65) 0.26 (0.07) 4-OH-BDE-42 0.36 (0.13) 0.96 (0.92) 0.56 (0.32) 0.64 (0.41) 0.82 (0.67) 0.36 (0.13) 5-OH-BDE-47 0.45 (0.20) 0.99 (0.99) 0.56 (0.31) 0.77 (0.59) 0.68 (0.46) 0.50 (0.25) 6-OH-BDE-47 0.43 (0.19) 0.99 (0.98) 0.59 (0.35) 0.74 (0.54) 0.71 (0.50) 0.49 (0.24) 4'-OH-BDE-49 0.37 (0.14) 0.97 (0.94) 0.62 (0.38) 0.66 (0.44) 0.77 (0.59) 0.43 (0.19) M1 0.39 (0.15) 0.98 (0.95) 0.62 (0.38) 0.68 (0.46) 0.75 (0.56) 0.45 (0.20) M2 0.61 (0.37) 0.93 (0.86) 0.37 (0.14) 0.96 (0.92) 0.33 (0.11) 0.66 (0.43)  Note. Marker activity value for CYP2B6 was experimentally determined as described in the Section 2.11, whereas marker activity values for the other CYP enzymes were provided by the vendor (Table A1.5). aCYP1A2 marker activity: phenacethin O-deethylation; bCYP2B6 marker activity: bupropion 4-hydroxylation; cCYP2C9 marker activity: diclofenac 4'-hydroxylation; dCYP2C19 marker activity: (S)-mephenytoin 4'-hydroxylation;eCYP2E1 marker activity: chlorzoxazone 6-hydroxylation;fCYP3A4 marker activity: testosterone 6β-hydroxylation. 169  CYP1A2-, CYP2C9-, CYP2C19-, CYP2E1-, and CYP3A4-mediated enzyme activities for the same single donor liver microsomal preparations. CYP1A2-, CYP2C9-, CYP2C19-, CYP2E1-, and CYP3A4-marker activity values were provided on the Xenotech and BD Biosciences product information sheets (Table A1.5). The correlation values obtained for rates of hydroxylated BDE-47 metabolite formation and CYP1A2-, CYP2C9-, CYP2C19-, CYP2E1-, and CYP3A4-mediated enzyme activities were lower, in most cases, than those obtained for rates of hydroxylated BDE-47 metabolite formation and CYP2B6-mediated enzyme activity (Table 3.14). Rates of formation for a few hydroxylated metabolites of BDE-47 (i.e. 2,4-DBP, 5-OH-BDE-47, and M2) were highly correlated (0.77<r<0.96) with CYP2C19- and CYP2E1-marker activity values (Table 3.14). When CYP2B6- and CYP2C19-marker activity values of the single donor human liver microsomes were compared to each other, a relatively large correlation coefficient value (r=0.78) was found (Figure A1.2), which indicates that the catalytic activity of the two enzymes co-varies in this sample set.   3.18 Kinetic Analysis of the Hydroxylated Metabolites of BDE-47 Formation by Recombinant CYP2B6 Rates of BDE-47 metabolite formation were quantified over a range of substrate concentrations (0.5 to 200 μM) using a recombinant CYP2B6 concentration of 5 pmol/mL and an incubation time of 5 min. In preliminary experiments, formation of all the BDE-47 metabolites was found to be linear up to 5 pmol/mL and 10 min (Figure 3.37-3.40). Data were fitted to different enzyme kinetic models as described earlier 170     Figure 3.37 Effect of incubation time on formation of 2'-OH-BDE-28 and 4'-OH-BDE- 49 (A) and of 4'-OH-BDE-17, 5-OH-BDE-47, and 6-OH-BDE-47 (B) by recombinant CYP2B6. Recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-47 (100 µM final concentration) and NADPH for 2.5 to 15 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.   0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 0 5 10 15 Pr od uc t f or m at io n  (r es po ns e/ nm ol  r ec om bi na nt  C Y P2 B 6) 4'-OH-BDE-49 2'-OH-BDE-28 A 0 10,000 20,000 30,000 40,000 0 5 10 15 Pr od uc t f or m at io n (r es po ns e/ nm ol  r ec om bi na nt  C Y P2 B 6) Incubation time (min) 5-OH-BDE-47 6-OH-BDE-47 4'-OH-BDE-17 B 171     Figure 3.38 Effect of incubation time on formation of M1 and M2 (A) and of 2,4-DBP and 4-OH-BDE-42 (B) by recombinant CYP2B6. Recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-47 (100 µM final concentration) and NADPH for 2.5 to 15 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.    0 100 200 300 400 500 0 5,000 10,000 15,000 20,000 0 5 10 15 20 M 2 fo rm at io n (r es po ns e/ nm ol  r ec om bi na nt  C Y P2 B 6) M 1f or m at io n (r es po ns e/  nm ol  r ec om bi na nt  C Y P2 B 6) M1 M2 A 0 2,000 4,000 6,000 8,000 10,000 0 5 10 15 Pr od uc t f or m at io n  (r es po ns e/  nm ol  r ec om bi na nt  C Y P2 B 6) Incubation time (min) 2,4-DBP 4-OH-BDE-42 B 172     Figure 3.39 Effect of recombinant CYP2B6 concentration on rates of formation of 6- OH-BDE-47 and M1 (A) and of 5-OH-BDE-47 and 4'-OH-BDE-49 (B). Recombinant CYP2B6 (2.5 to 10 pmol/mL) was incubated with BDE-47 (100 µM final concentration) and NADPH for 5 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.  0.0 1.0 2.0 3.0 4.0 5.0 0 2 4 6 8 10 Pr od uc t f or m at io n (r es po ns e/ m in ) 6-OH-BDE-47 M1 A 0.00 0.40 0.80 1.20 1.60 0 2 4 6 8 10 Pr od uc t f or m at io n (r es po ns e/ m in ) Concentration of recombinant CYP2B6 (pmol/mL) 4'-OH-BDE-49 5-OH-BDE-47 B 173    Figure 3.40 Effect of recombinant CYP2B6 concentration on rates of formation of 4'- OH-BDE-17, 2'-OH-BDE-28, and 4-OH-BDE-42 (A), and of 2,4-DBP and M2 (B). Recombinant CYP2B6 (2.5 to 10 pmol/mL) was incubated with BDE-47 (100 µM final concentration) and NADPH for 5 min. Samples were prepared in single and two separate experiments were conducted. Metabolites were extracted and analyzed as described in Section 2.5 and 2.9, respectively.  0.00 0.10 0.20 0.30 0.40 0 2 4 6 8 10 Pr od uc t f or m at io n (r es po ns e/ m in ) 4'-OH-BDE-17 2'-OH-BDE-28 4-OH-BDE-42 A 0.000 0.010 0.020 0.030 0.040 0.050 0.060 0 2 4 6 8 10 Pr od uc t f or m at io n (r es po ns e/ m in ) Concentration of recombinant CYP2B6 (pmol/mL) 2,4-DBP M2 B 174  (Section 2.13). The model with the lowest AICc and standard deviation of the residual values was considered to be the best fitting model. Formation of 6-OH-BDE-47 and M1 was best fitted by the Michaelis-Menten model and formation of 4-OH-BDE-42, 5-OH- BDE-47, and 4'-OH-BDE-49 was best fitted by the substrate inhibition model. Formation of M2 was not well described by any of the kinetic models tested (Figure 3.41). Eadie- Hoftsee plots (velocity vs velocity/substrate concentration) were prepared separately for comparative purposes. None of the Eadie-Hoftsee plots obtained were indicative of the Michaelis-Menten, Hill, or substrate-inhibition models (Kramer and Tracy, 2008). Consistent with the results obtained with pooled human liver microsomes, 5-OH- BDE-47 and 6-OH-BDE-47 (but also 4'-OH-BDE-49) were the major identified hydroxylated metabolites of BDE-47 and M1was the major unidentified hydroxylated metabolite of BDE-47 produced by recombinant CYP2B6, as indicated by the Vmax values (Table 3.15). In addition, Km values suggest that M2 and 4'-OH-BDE-49 are preferentially formed by recombinant CYP2B6 at lower BDE-47 concentrations. The amounts of 2'-OH-BDE-28 and 4'-OH-BDE-17 detected following incubation of BDE-47 with recombinant CYP2B6 were between the limit of detection and the limit of quantification, which was consistent with the results obtained with human liver microsomes (Section 3.15), confirming that 2'-OH-BDE-28 and 4'-OH-BDE-17 are minor metabolites of BDE-47. Thus, kinetic analysis of 2'-OH-BDE-28 and 4'-OH-BDE-17 formation could not be undertaken. In addition, formation of 3-OH-BDE-47 by recombinant CYP2B6 was not detected, which was consistent with the results obtained with human liver microsomes (Section 3.15). 175    Figure 3.41 Enzyme kinetic profiles for the formation of hydroxylated metabolites of BDE-47 by recombinant CYP2B6. Human recombinant CYP2B6 (5 pmol/mL) was incubated with BDE-47 (0.5–200 µM) for 5 min. Data points are the meanSD of three separate experiments. Lines represent rates of metabolite formation modeled by nonlinear regression analyses. The insets depict Eadie-Hoftsee plots. Error bars are not shown on the insets to avoid obscuring the data points, which represent mean values (n=3). Because 2,4-DBP, 4'-OH-BDE-17, and 2'- OH-BDE-28 were only quantifiable at the highest BDE-47 concentrations used, their enzyme kinetic profiles could not be determined.   176  Table 3.15 Values of Vmax, Km, and Ki for the Formation of the Hydroxylated Metabolites of BDE-47 by Human Recombinant CYP2B6 BDE-47 metabolite Vmax (pmol/min/nmol rCYP2B6) Vmax (response/min/nmol rCYP2B6) Km (µM) Ki (µM) 4-OH-BDE-42 150 ± 49   2.7 ± 1.9 390 ± 150 5-OH-BDE-47 300 ± 27   5.8 ± 1.7     220 ± 75 6-OH-BDE-47 260 ± 37   2.8 ± 0.61 – 4'-OH-BDE-49 270 ± 23  1.2 ± 0.10 540 ± 190 M1  570 ± 330  6.6 ± 6.6 –  Note. Values represent the mean  SD of three independent experiments.  Rates of M1 formation could not be expressed as pmol/min/ nmol rCYP2B6 because of the lack of authentic standards and are therefore reported as response/min/nmol rCYP2B6. Rates of M2 formation were not adequately described by any kinetic model testedand values for Vmax, Km, and Ki were not generated.        177  3.19 Investigation of the Mechanism of Formation of Secondary Metabolites of BDE-47 by Human Liver Microsomes To investigate if the di-OH-tetrabrominated-PBDE metabolite (M2) of BDE-47 was formed by two sequential hydroxylation steps mediated by CYP enzyme(s), 4-OH- BDE-42, 5-OH-BDE-47, 6-OH-BDE-47 or 4'-OH-BDE-49, instead of BDE-47, were incubated with pooled human liver microsomes. Formation of M2 was not detected when any of the BDE-47 monohydroxylated metabolites was used as substrate, indicating that M2 was not as a secondary metabolite of BDE-47 and suggesting that M2 is produced directly from BDE-47. The effect of antibody to epoxide hydrolase on formation of M2 from BDE-47 was also examined to determine if M2 was produced through a stable epoxide intermediate. As shown in figure 3.42, no difference in rates of M2 formation was observed when BDE-47 was incubated with pooled human liver microsomes and NADPH in the presence of varying amounts of antibody to epoxide hydrolase or rabbit IgG (antibody negative control). This result indicated that microsomal epoxide hydrolase was not involved in M2 formation and suggested that M2 was not formed via a stable epoxide intermediate. To investigate whether 2,4-DBP, 4'-OH-BDE-17 and 2'-OH-BDE-28 were primary or secondary metabolites of BDE-47, their formation was monitored during the experiments described above. Formation of 4'-OH-BDE-17 and 2'-OH-BDE-28 was not detected, suggesting that 4'-OH-BDE-17 and 2'-OH-BDE-28 were primary metabolites of BDE-47 (i.e. produced via oxidative debromination of BDE-47 and not by reductive debromination of a primary hydroxylated metabolite of BDE-47). However, 2,4-DBP was formed when human liver microsomes were incubated with 4-OH-BDE-42, 5-OH- 178           Figure 3.42 Effect of anti-epoxide hydrolase IgG on the formation of the unidentified di- OH-tetrabrominated-PBDE metabolite of BDE-47 produced by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with 0 to 2.0 mg of rabbit anti-rat epoxide hydrolase IgG/mg human liver microsomes (full symbols) or an equivalent amount of rabbit IgG (empty symbols) for 10 min. BDE-47 was then added (10 µM, final concentration) and the mixtures were incubated for an additional 5 min. The reaction was initiated by addition of 0.01 mL of NADPH (1 mM, final concentration), allowed to proceed for 5 min, and stopped with 1 mL of ice-cold sodium hydroxide. Samples were then processed and analyzed as described in Section 2.5 and 2.9.        0 20 40 60 80 100 120 0.0 0.5 1.0 1.5 2.0 %  a ct iv ity  r em ai ni ng mg of anti-epoxide hydrolase IgG/mg protein Rabbit IgG Rabbit anti EH antibody 179  BDE-47, 6-OH-BDE-47 or 4'-OH-BDE-49, or with BDE-47 plus an increasing concentration of 4-OH-BDE-42 and 4'-OH-BDE-49 (Figure 3.43). This result indicate that 2,4-DBP is a secondary metabolite of BDE-47 and may also be a primary metabolites of BDE-47.                    180    Figure 3.43 Formation of 2,4-DBP as possible secondary metabolite of BDE-47 by pooled human liver microsomes. Pooled human liver microsomes (0.1 mg/mL) were incubated with the four major BDE- 47 primary hydroxylated metabolites (5 to100 nM) individually (A) or with BDE-47 plus increasing concentrations (25 to 150 nM) of 4-OH-BDE-42 or 4'-OH-BDE-49 (B). Samples were processed and analyzed as described in Section 2.5 and 2.9, respectively.   181  4. Discussion    The present study investigated the oxidative biotransformation of BDE-99 by rat and human liver microsomes and the oxidative biotransformation of BDE-47 by human liver microsomes. A new liquid chromatography/mass spectrometry-based assay was developed, optimized, and validated to characterize the oxidative metabolism of BDE-99. The validated assay was used to identify the hydroxylated metabolites of BDE-99 and quantify their rates of formation by rat liver microsomes and to determine the CYP enzymes involved in BDE-99 oxidative metabolism using a panel of rat recombinant CYP enzymes. A similar assay developed in Dr. Bandiera’s laboratory by Sarah Moffatt was used to characterize the oxidative metabolism of BDE-47 (Moffatt et al., 2011). Both assays were improved and adapted to a newly available tandem mass spectrometry instrument. The two improved assays were then used to study the metabolism of BDE-47 and BDE-99 by human liver microsomes and human recombinant CYP enzymes.   4.1 Validation of a Novel Assay for Metabolism of BDE-99 by Liver Microsomes As a first step, the liquid chromatography/mass spectrometr-based assay was validated for selectivity, sensitivity, linearity, range, accuracy, precision, and recovery. Studies investigating the in vitro formation of hydroxylated metabolites of PBDEs by 182  liquid chromatography/mass spectrometry-based assays have been described but the assays were not validated (Lupton et al., 2010; Marteau et al., 2012; McKinney et al., 2006; Stapleton et al., 2009). To my knowledge, we were the first to report the validation of a liquid chromatography/mass spectrometry-based assay to characterize the oxidative metabolism of BDE-99 and BDE-47 (Moffatt et al., 2011). With regard to sensitivity, the LOQ value for the determination of 4-OH-BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99 in rat liver microsomes (50 fmol injected on column) is similar to the LOQ values reported in other studies that employed liquid chromatography/mass spectrometry-based assays to investigate the metabolism of PBDEs. The previously reported LOQ values for the analysis of mono-OH- tribrominated-PBDEs, mono-OH-tetrabrominated-PBDEs, and mono-OH- pentabrominated-PBDEs range between 12 and 50 pmol injected onto the LC column (Marteau et al., 2012; Mas et al., 2007; Moffatt et al., 2011). However, the LOQ values reported by Marteau et al. (2012) and Mas et al. (2007) were determined using only the signal-to-noise ratio criterion and stock solutions of authentic standards. My experimental design differed in that the LOQ value was determined in samples containing the matrix of interest (i.e. liver microsomes) and was determined based on values for signal-to-noise, accuracy, and precision (Section 2.7). The LOQ value of the present assay was only 3- fold larger than that determined by Feo et al. (2013) for derivatized mono-OH- tetrabrominated-PBDEs using a gas chromatography/mass spectrometry-based method and authentic standards (LOQ=14 fmol injected on column). This result suggests that analysis of hydroxylated PBDEs by liquid chromatography/mass spectrometry without 183  the need for derivatization offers comparable sensitivity as analysis by gas chromatography/mass spectrometry. Ranges of intra-day and inter-day precision values for the determination of 4-OH- BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99 (%RSD values 2.2 to 8.6% and 3.9 to 8.0%, respectively) are consistent with those obtained for the determination of mono-OH- tribrominated-PBDEs and mono-OH-tetrabrominated-PBDEs by Mas et al. (2007; %RSD values 4 to 9% and 6 to10%, respectively) and by Moffatt et al. (2011; %RSD values 5.9 to 17% and 7.1 to 14%, respectively). However, precision values reported by Mas et al. (2007) were determined at a single concentration and using stock solutions of the OH-BDEs of interest. My experimental design differed in that both precision and accuracy values were determined, values for accuracy and precision were determined at two concentrations, and the biological matrix of interest (i.e., liver microsomes) was included in the sample preparation. Ranges of intra-day and inter-day accuracy values for the determination of 4-OH- BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99 (%Dev values -0.80 to 7.7% and 0.47 to 8.0%, respectively) were smaller than those reported by Moffatt et al. (2011) for the determination of mono-OH-tribrominated-PBDEs and mono-OH-tetrabrominated-PBDEs (%Dev values -12 to 9.3% and -11 to 5.3%, respectively). The common sample preparation and liquid chromatography-based analysis used in my assay and in that of Moffatt et al. (2011) seems to provide a more accurate determination of mono-OH- pentabrominated-PBDEs than of mono-OH-tribrominated-PBDEs and mono-OH- tetrabrominated-PBDEs. The absence of accuracy values in other studies that characterized the in vitro metabolism of PBDEs (Browne et al., 2009; Feo et al., 2013; 184  Lupton et al., 2009, 2010; Marteau et al., 2012; McKinney et al., 2006; Stapleton et al., 2009) prevented further comparison. The recovery values for 4-OH-BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99 were greater than those reported in previous studies involving similar biological matrices and using gas chromatography coupled with mass spectrometry or electron capture detection analysis. For example, recovery values of 57% and 65% were obtained by spiking rat liver microsomes with 2-OH-BDE-28 and 6-OH-BDE-47 standards, respectively (Hamers et al., 2008; McKinney et al., 2006). A recovery value of 50±7% was obtained spiking human hepatocytes with 6-OH-BDE-47 standard (Stapleton et al., 2009). Furthermore, analysis of hydroxylated PBDEs using gas chromatography coupled with mass spectrometry methods in derivatized extracts of human milk samples resulted in highly variable recovery values (57 to 102% for mono-OH-tribrominated-PBDEs and mono-OH-tetrabrominated-PBDEs; Lacorte et al., 2009), which could be due to the extraction and clean-up steps involved. The sample preparation protocol used in my assay coupled with UPLC/MS analysis yielded high and reproducible recovery rates for 4-OH- BDE-90, 5-OH-BDE-99, and 6-OH-BDE-99, providing evidence for the reliability of the assay. The validated assay was an essential first step in my project and has several potential applications. The validated assay can be used to investigate the oxidative metabolism of BDE-99 using liver microsomal preparations from any of several species. For example, a preliminary study investigating the oxidative metabolism of BDE-99 by starling (Sturnus vulgaris) liver microsomes was recently undertaken (Erratico et al., 2013). The sample preparation and UPLC/MS-based analysis used in the validated assay 185  can be modified to investigate the oxidative metabolism of any PBDE congener, as already shown for BDE-47 (Moffatt et al., 2011). Liquid chromatography was shown to be a flexible and promising technique for the separation of hydroxylated metabolites of PBDEs. The elution gradient used in the validated assay can be easily modified to separate new standards for hydroxylated metabolites of BDE-99 (Section 2.6) and to resolve a large number of mono-OH-pentabrominated-PBDE congeners (Section 2.9). Therefore, the newly developed assay is sufficiently flexible and can be easily modified to detect hydroxylated PBDEs in biological and environmental matrices (i.e. plasma, serum, water).   4.2 Oxidative Metabolism of BDE-99 by Rat Liver Microsomes and Rat Recombinant CYP Enzymes The UPLC/MS-based assay provided insights into the mechanisms of BDE-99 oxidation by rat liver microsomes and rat recombinant CYP enzymes. Under the experimental conditions used, the major oxidative metabolite in most of the rat liver microsomal preparations used was 4-OH-BDE-90, which resulted from oxygen insertion at a (para) brominated carbon atom with a NIH-shift of the bromine atom. Oxygen insertion at an unsubstituted ortho or meta position of  BDE-99 (resulting in the formation of 6'-OH-BDE-99 and 5'-OH-BDE-99, respectively) was less favorable than para-hydroxylation involving the NIH-shift mechanism. In turn, oxygen insertion without NIH-shift mechanism was more efficient than hydroxylation at the 1'carbon atom of the ether bond, which lead to O-dealkylation (resulting in the formation of 2,4,5-TBP). 186  Formation of mono-OH-tetrabrominated-PBDE or di-OH-pentabrominated-PBDE metabolites of BDE-99 by either rat hepatic microsomes or recombinant CYP enzymes was not observed. Therefore, oxidative debromination and dihydroxylation of BDE-99 are less important mechanisms for the metabolism of BDE-99 by rat liver microsomes and rat recombinant CYP enzymes. Formation of individual hydroxylated metabolites of BDE-99 by rat liver microsomes is predominantly mediated by different but overlapping sets of rat CYP enzymes. In rats, hepatic levels of CYP1A, 2B, and 3A enzymes are induced by MC, PB, and DEX treatments, respectively (Edwards et al., 2007; Hrycay and Bandiera, 2003). The results suggest that CYP2B and CYP3A enzymes are involved in 4-OH-BDE-90 formation, whereas CYP1A enzymes are involved in 6ʹ-OH-BDE-99 formation. CYP2B and CYP3A enzymes are likely involved in the formation of 2,4,5-TBP, 4ʹ-OH-BDE-101, 2-OH-BDE-123, and M1 also, as these metabolites were detected only in incubations containing hepatic microsomes from PB- and DEX-treated rats. The contribution of individual CYP enzymes to the formation of hydroxylated metabolites of BDE-99 was confirmed by experiments with a panel of rat recombinant CYP enzymes. More specifically, among the fourteen CYP enzymes tested, CYP3A1 and CYP1A1 were the most efficient catalysts of 4-OH-BDE-90 and 6ʹ-OH-BDE-99 formation, respectively. CYP2B1, CYP3A1, and CYP3A2 were among the most active recombinant CYP enzymes in the formation of 2,4,5-TBP, and CYP2B1 and CYP3A1 showed the highest catalytic activity towards 2-OH-BDE-123 and M1 formation, respectively. Some rat recombinant CYP enzymes preferentially oxidize a carbon atom at a specific position of the BDE-99 molecule, whereas other rat recombinant CYP enzymes 187  catalyze the oxidation of carbon atoms at different positions of the BDE-99 molecule. For example, CYP3A1 preferentially catalyzed para-hydroxylation of BDE-99 (producing 4- OH-BDE-90) and CYP1A1 demonstrated a preference for ortho-hydroxylation of BDE- 99 (producing 6'-OH-BDE-99; Figure 3.11). In contrast, CYP2B1 catalyzed the formation of 4-OH-BDE-90, 5ʹ-OH-BDE-99, 6ʹ-OH-BDE-99, and 2,4,5-TBP demonstrating that a single CYP enzyme mediated ortho-, meta-, and para- hydroxylation, as well as oxidative cleavage, of BDE-99. Collectively, the results show that the position of the carbon atom of BDE-99 that is oxidized by rat CYP enzyme(s) depends on the CYP enzyme involved and suggests that some CYP enzymes are able to accommodate BDE-99 in the binding pocket of their active site with different orientations. Some, but not all, of the hydroxylated metabolites of BDE-99 identified in the present study were reported in previous in vitro and in vivo studies using rodents. Of the 7 hydroxylated metabolites of BDE-99 detected in our study, two (i.e. 2,4,5-TBP and 5ʹ- OH-BDE-99) were identified following incubations  of rat hepatocytes with BDE-99 at 10 µM for up to 72 h (Dong et al., 2010). Formation of 2,4,5-TBP, two to four unidentified mono-OH-pentabrominated-PBDE metabolites, and one to three unidentified di-OH-pentabrominated-PBDE metabolites of BDE-99 was reported in rodents treated with BDE-99 (Chen et al., 2006; Hakk et al., 2002; Staskal et al., 2006). The absence of metabolite identification in the in vivo studies mentioned above prevented comparison with the hydroxylated metabolites of BDE-99 identified in the present study.   188  4.3 Comparison of Oxidative Metabolism of BDE-47 and BDE-99 by Rat Liver Microsomes The mechanisms of in vitro metabolism of BDE-99 and BDE-47 by rat liver microsomes and recombinant CYP enzymes are similar (Erratico et al., 2011). In most of the rat liver microsomal preparations used oxygen insertion at a para substituted carbon atom accompanied by a NIH-shift of the bromine atom was favored over oxygen insertion at an unsubstituted meta or ortho position of BDE-47. In addition, the rat recombinant CYP1A1 catalyzed exclusively para-hydroxylation of BDE-47, whereas CYP2A2 mediated ortho-, meta-, and para-hydroxylation of BDE-47. Collectively, the results show that the position of the carbon atom of BDE-47 that was oxidized by CYP enzyme(s) depends on the CYP enzyme involved and that the same CYP enzyme can bind BDE-47 with different orientations. Rates of formation of BDE-99 metabolites by rat hepatic microsomes are 5- to 10- fold larger than those of BDE-47 metabolites (Erratico et al., 2011). The different rates of BDE-99 than BDE-47 metabolite formation by rat liver microsomes can be explained by the constitutive level of expression, the inducibility, and the intrinsic catalytic activity of the major rat CYP enzymes involved in the biotransformation of BDE-47 and BDE-99. BDE-99 was metabolized by rat CYP enzymes belonging to the CYP2B, CYP2C, and CYP3A subfamilies, which together represent 45 to 70% of the total CYP content of adult male rat liver microsomes (Bandiera, 2001; Ryan and Levin, 1990). In addition, CYP3A1 activity was highly induced by DEX-treatment (Table 3.6) and CYP3A1 exhibited 6-fold greater activity in the formation of hydroxylated metabolites of BDE-99 (Figure 3.11) than BDE-47 (Erratico et al., 2011). Conversely, BDE-47 was mainly 189  metabolized by rat CYP1A1 and CYP2A2 (Erratico et al., 2011), which together represent up to 5% of the total CYP content of adult male rat liver microsomes (Thummel et al., 1988). CYP1A1 and CYP1A2 are highly induced by MC treatment (Table 3.6; Nebert et al., 2004) but not by PB or DEX treatments (Matsunaga et al., 1988; Thummel et al., 1988). In addition, CYP1A1 and CYP2A2 showed high catalytic activity for the formation of 4'-OH-BDE-49 and 5-OH-BDE-47, respectively (Erratico et al., 2011).   4.4 Comparison of Oxidative Metabolism of BDE-47 and BDE-99 by Human Liver Microsomes Results obtained from the studies involving the biotransformation of BDE-99 and BDE-47 provide insights into the reactivity of the carbon atoms of BDE-99 and BDE-47 and into the mechanisms of oxidation by human liver microsomes. Formation of 2,4,5- TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101as the three major hydroxylated metabolites of BDE-99 (Table 4.1) indicates that human hepatic microsomal hydroxylation of the less brominated ring is preferred over hydroxylation of the more brominated ring of BDE-99. Moreover, comparison of apparent Vmax values for the identified hydroxylated metabolites of BDE-99 reveals that the order of reactivity of the carbon atoms of BDE-99 seems to be meta- ~ ortho-> para-hydroxylation. If the pattern holds true for the unidentified mono-OH-pentabrominated-PBDE metabolites detected in the present study, then we predict that the major unidentified mono-OH-pentabrominated-PBDE metabolite of BDE-99 (M1) is 3'-OH-BDE-99. Structural identification of the unidentified metabolites of BDE-99 awaits the availability of more authentic standards. 190  TABLE 4.1 Structure and Mechanisms For the Formation of Hydroxylated Metabolites of BDE-99 by Pooled Human Liver Microsomes Substrate     Hydroxylated metabolite Position of the C atom oxidized Mechanism of substrate oxidation Apparent Vmax (pmol/min/mg protein) Apparent Km (µM) Name Structure 2,4,5-TBP ortho O-dealkylation 45±2.2 1.6±0.2 5'-OH-BDE-99 meta Aromatic hydroxylation at an unsubstituted C atom 47±2.3 32±12 4'-OH-BDE-101 para Aromatic hydroxylation at a substituted C atom (plus NIH shift) 24±3.5 20±7.2  191  Substrate     Hydroxylated metabolite Position of the C atom oxidized Mechanism of substrate oxidation Apparent Vmax (pmol/min/mg protein) Apparent Km (µM) Name Structure 4-OH-BDE-90 para Aromatic hydroxylation at a substituted C atom (plus NIH shift) 2.4±0.3 1.7±0.2 6'-OH-BDE-99 ortho Aromatic hydroxylation at an unsubstituted C atom 1.1±0.2 2.2±0.6 2-OH-BDE-123 ortho Aromatic hydroxylation at a substituted C atom (plus NIH shift) 0.6±0.1 2.3±0.1 di-OH-penta-BDE ? Aromatic hydroxylation - - 192  Formation of 2,4,5-TBP as a major metabolite of BDE-99 indicates that O- dealkylation is an important biotransformation pathway of BDE-99 by human liver microsomes. Oxygen insertion at an unsubstituted meta position of BDE-99 (i.e. formation of 5'-OH-BDE-99), which does not involve a NIH shift mechanism, was also a highly favorable mechanism of BDE-99 hydroxylation. In addition, one major (namely 4'-OH- BDE-101) and two minor (namely 4-OH-BDE-90 and 2-OH-BDE-123) oxidative metabolites of BDE-99 resulted from oxygen insertion accompanied by a NIH-shift of a bromine atom, possibly in concert with formation of an arene oxide intermediate. Overall, comparison of apparent Vmax and Km values of the identified metabolites of BDE-99 suggests that O-dealkylation and oxygen insertion without a NIH-shift are slightly favoured over oxygen insertion with a NIH-shift in the oxidative metabolism of BDE-99 by human liver microsomes. A di-OH-pentabrominated-PBDE primary metabolite of BDE-99 was also formed. Although this metabolite remains unidentified because of the lack of the appropriate authentic standard, peak area ratio values suggest that the di-OH-pentabrominated-PBDE metabolite is a major hydroxylated metabolite of BDE-99. Therefore, dihydroxylation could be an important mechanism for the oxidative metabolism of BDE-99 by human liver microsomes. In addition, the lack of detection of mono-OH-tetrabrominated-PBDE metabolites of BDE-99 indicates that oxidative debromination is a negligible mechanism of biotransformation of BDE-99 by human liver microsomes. Formation of 5-OH-BDE-47 and 6-OH-BDE-47 as major metabolites of BDE-47 (Tale 4.2) suggests that the presence of two adjacent unsubstituted carbon atoms on the phenyl rings favors CYP-mediated oxidative metabolism. This result is consistent with  193  TABLE 4.2 Structure and Mechanisms of BDE-47 Hydroxylated Metabolites Formation by Pooled Human Liver Microsomes Substrate    Hydroxylated metabolite Position of the C atom oxidized Mechanism of substrate oxidation Apparent Vmax (pmol/min/mg protein) Apparent Km (µM) Name Structure 2,4-DBP ortho O-dealkylation - - 5-OH-BDE-47 meta Aromatic hydroxylation at an unsubstituted C atom 27±3.9 36±15 6-OH-BDE-47 ortho Aromatic hydroxylation at an unsubstituted C atom 24±3.4 25±6.3 4-OH-BDE-42 para Aromatic hydroxylation at a substituted C atom (plus NIH shift) 14±4.1 44±42 194  Substrate    Hydroxylated metabolite Position of the C atom oxidized Mechanism of substrate oxidation Apparent Vmax (pmol/min/mg protein) Apparent Km (µM) Name Structure 4'-OH-BDE-49 para Aromatic hydroxylation at a substituted C atom (plus NIH shift) 13±3.0 10±2.9 4'-OH-BDE-17 para Oxidative dehalogenation - - 2'-OH-BDE-28 ortho Oxidative dehalogenation - - di-OH-tetra-BDE ? Aromatic hydroxylation - -   195  the lack of detection of 3-OH-BDE-47 and with the finding that 5'-OH-BDE-99 was a major hydroxylated metabolite of BDE-99 produced by human liver microsomes (Table 3.8 and 3.9). Moreover, comparison of apparent Vmax values for the identified hydroxylated metabolites of BDE-47 reveals that the order of reactivity of the carbon atoms of BDE-47 seems to be meta ~ ortho > para, which is consistent with the metabolism of BDE-99. Formation of 5-OH-BDE-47 and 6-OH-BDE-47 shows that BDE-47 can be oxidatively metabolized at an unsubstituted carbon atom. Formation of 4-OH-BDE-42 and 4'-OH- BDE-49 shows that BDE-47 can be metabolized also at a substituted carbon atom accompanied by a NIH-shift mechanism. However, the number of identified metabolites formed and the apparent Vmax and Km values calculated (Table 3.13) suggest that neither of these two oxidation mechanisms (oxidation with or without a NIH-shift) is favored over the other, which is consistent with the metabolism of BDE-99 by human liver microsomes. O- Dealkylation of BDE-47 results in the formation of 2,4-DBP. Although 2,4-DBP is a primary and secondary metabolite of BDE-47 produced by human liver microsomes, it does not appear to be a major metabolite of BDE-47. This result is in contrast with the importance of O-dealkylation in the metabolism of BDE-99 by human liver microsomes, suggesting that small differences in the PBDE structure (i.e. bromine substituted or unsubstituted carbon 5 atom) can affect the relative importance of O-dealkylation. An unidentified di-OH- tetrabrominated-PBDE primary metabolite of BDE-47 was also formed by human liver microsomes. Peak area ratio values suggest that the di-OH-tetrabrominated-PBDE is not a major metabolite of BDE-47. Therefore, dihydroxylation does not seem as important mechanism for the metabolism of BDE-47 as for the metabolism of BDE-99. Formation of 4'-OH-BDE-17 and 2'-OH-BDE28 by human liver microsomes shows that BDE-47 is 196  oxidatively debrominated by CYP enzymes. However, 4'-OH-BDE-17 and 2'-OH-BDE28 are minor metabolites of BDE-47, suggesting that oxidative debromination is a minor pathway of BDE-47 metabolism by human liver microsomes, which is consistent with its minor importance for metabolism of BDE-99. Previous reports of the biotransformation of BDE-99 and BDE-47 by human hepatic preparations described the formation of a smaller number of hydroxylated metabolites than those reported here. 2,4,5-TBP, 5'-OH-BDE-99 and an unidentified mono-OH- pentabrominated-PBDE metabolite were detected when human liver microsomes or cultured human hepatocytes  were incubated with BDE-99 (Lupton et al., 2009, 2010; Stapleton et al., 2009). The present study is the first to report the formation of 4-OH-BDE-90, 6'-OH- BDE-99, 4'-OH-BDE-101, 2-OH-BDE-123, three unidentified mono-OH-pentabrominated- PBDEs, and one unidentified di-OH-pentabrominated-PBDE along with 2,4,5-TBP and 5'- OH-BDE-99 as human hepatic microsomal metabolites of BDE-99. 2,4-DBP, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, and an unidentified di- OH-tetrabrominated-PBDE metabolite were formed when human liver microsomes or human hepatocytes were incubated with BDE-47 (Feo et al., 2013; Lupton et al., 2009, 2010; Marteau et al., 2012). The present study identified the formation of 4-OH-BDE-42, 4'- OH-BDE49, 4'-OH-BDE-17, 2'-OH-BDE28, an unidentified mono-OH-tetrabrominated- PBDE (M1), and an unidentified di-OH-tetrabrominated-PBDE (M2) along with 2,4-DBP, 5-OH-BDE-47, and 6-OH-BDE-47 (but not 3-OH-BDE-47) as human hepatic microsomal metabolites of BDE-47. Moroever, 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH- BDE-47 and 4'-OH-BDE-49 were formed when human recombinant CYP2B6 was incubated with BDE-47 (Feo et al., 2013). The present study identified the formation of 2,4-DBP, 4'- 197  OH-BDE-17, 2'-OH-BDE28, an unidentified mono-OH-tetrabrominated-PBDE (M1), and an unidentified di-OH-tetrabrominated-PBDE (M2) along with 4-OH-BDE-42, 5-OH-BDE- 47, 6-OH-BDE-47 and 4'-OH-BDE-49 (but not 3-OH-BDE-47) as human CYP2B6 metabolites. Differences in incubation conditions and the analytical method used can partly account for differences in BDE-99 and BDE-47 metabolite profiles between the current and previous studies. The results of the present study characterizing the in vitro metabolism of BDE-47 and BDE-99 by human liver microsomes are in good agreement with the identity and relative importance of mono-OH-tetrabrominated-PBDEs and mono-OH-pentabrominated- PBDEs detected in human plasma and serum samples (Athanasiadou et al., 2008; Kawashiro et al., 2008; Qiu et al., 2009; Ryden et al., 2012; Zota et al., 2012). Five of the hydroxylated metabolites of BDE-99 formed by human liver microsomes in the present study (namely 2,4,5-TBP, 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, and 4'-OH-BDE-101) have been detected in human plasma and serum samples (Athanasiadou et al., 2008; Qiu et al., 2009; Ryden et al., 2012) and may represent oxidative metabolites of BDE-99 formed in vivo. At present, there are no reports of 2-OH-BDE-123 in human plasma and serum, but unidentified mono-OH-pentabrominated-PBDE peaks have been detected in human serum samples (Athanasiadou et al, 2008; Ryden et al., 2012; Wan et al., 2010). It is feasible that some of the unidentified mono-OH-pentabrominated-PBDE detected in human plasma and serum samples could correspond to 2-OH-BDE-123 or M1to M4. Identification of additional hydroxylated PBDEs in human plasma and serum samples is likely to increase as more hydroxylated PBDE standards become available and as the sensitivity of analytical methods increases. 198  Five of the hydroxylated metabolites of BDE-47 formed in the present study (namely 2,4-DBP, 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49) have been detected in several human serum and plasma samples, suggesting that 2,4-DBP, 4-OH-BDE- 42, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49 are in vivo oxidative metabolites of BDE-47. In human plasma and serum samples, concentrations of 5-OH-BDE-47 and 6-OH- BDE-47 are larger than those of 4-OH-BDE-42 and 4'-OH-BDE-49, which is consistent with 5-OH-BDE-47 and 6-OH-BDE-47 being the major in vitro hydroxylated metabolites of BDE-47 produced by human liver microsomes in the present study. Moreover, human plasma and serum concentrations of 4'-OH-BDE-17 and 2'-OH-BDE-28 are usually equal to, or smaller than, those of 4-OH-BDE-42 and 4'-OH-BDE-49, which is consistent with 4'-OH- BDE-17 and 2'-OH-BDE-28 being minor metabolites of BDE-47 produced by human liver microsomes in the present study. Human plasma and serum concentrations of 3-OH-BDE-47 are 10 times lower than those of 5-OH-BDE-47 (Qiu et al., 2009) or lower than those of 4'- OH-BDE-17 (Athanasiadou et al., 2008), which is consistent with lack of 3-OH-BDE-47 detection in human liver microsomal and CYP2B6 incubations. It can be problematic to extrapolate in vitro metabolism findings to the in vivo situation, especially as human plasma and serum concentrations of BDE-99 and BDE-47 are in the 50 to 300 pM range (Daniels et al., 2010; Gómara et al., 2007a,b; Sjödin et al., 2008) and are much lower than the BDE-47 and BDE-99 concentrations used in the present in vitro biotransformation study (0.5 to 200 µM). However, we expect that human hepatic biotransformation of BDE-99 and BDE-47 will occur in vivo, albeit at much lower rates than those obtained with human liver microsomes, and will produce picomolar concentrations of hydroxylated metabolites in the blood. In support to this conjecture, Qiu et al., (2009) 199  showed that the total concentrations of the  hydroxylated metabolites of BDE-99 (i.e. 2,4,5- TBP, 5'-OH-BDE-99, and 6'-OH-BDE-99: 265 pM) and BDE-47 (2,4-DBP, 4-OH-BDE-42, 3-OH-BDE-47, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49: 650 pM) are similar to the concentration of the parent compound (170 and 620 pM for BDE-99 and BDE-47, respectively) in the combined human plasma samples. Experiments involving a panel of 12 human recombinant CYP enzymes, CYP2B- specific inhibitory antibodies, and correlation analysis using single donor human liver microsome samples with different CYP2B6 activity provided convincing evidence that CYP2B6 was the CYP enzyme predominantly responsible for the oxidative metabolism of BDE-99 and BDE-47 by human liver microsomes. This finding is consistent with a recently published study (Feo et al., 2013). Therefore, ortho-, meta-, and para-hydroxylation, oxidative cleavage, and dihydroxylation of BDE-99 and BDE-47 were catalyzed by CYP2B6. In addition, trace amounts of mono-OH-tetrabrominated-PBDEs or mono-OH- tribrominated-PBDEs were detected when recombinant CYP2B6 was incubated with BDE- 99 or BDE-47, respectively, suggesting that recombinant CYP2B6 can oxidatively debrominate BDE-99 and BDE-47. This versatility suggests that the active site of CYP2B6 can bind BDE-99 and BDE-47 in various orientations resulting in the oxidation of different carbon atoms of BDE-99 and BDE-47. The flexibility of CYP2B6 active site is confirmed by the diverse chemical structures of CYP2B6 substrates (i.e. α-endosulfan, methoxychlor, chlorpyrifos, and several organophosphate pesticides) and by the number of hydroxylated metabolites produced by CYP2B6 from each of these xenobiotics (Buratti et al., 2005; Foxemberg et al., 2007; Hu and Kupfer, 2002; Lee et al., 2006; Sams et al., 2004; Tang et al., 2001). For example, CYP2B6 was shown to catalyze O-dealkylation of methoxychlor, 200  producing a mono-hydroxylated metabolite, and to further oxidize the mono-hydroxylated metabolite into a di-hydroxylated metabolite (again by O-dealkylation) and a catechol metabolite (by oxidation of an unsubstituted carbon atom) (Hu and Kupfer, 2002). In addition, CYP2B6 was shown to biotransform chlorpyrifos into a paraoxon metabolite via oxidative desulfuration and into p-nitrophenol via O-dealkylation (Foxemberg et al., 2007). The role of CYP2B6 in the in vitro metabolism of BDE-47 and BDE-99 helps explain the large inter-individual variability observed in the rates of formation of the hydroxylated metabolites of BDE-47 and BDE-99 by single donor human liver microsomes (Table 3.8 and 3.12). CYP2B6 protein content was reported to range between 0.5 and 70 pmol/mg protein (Code et al., 1997; Ekins et al., 1998). CYP2B6 activity varied from 10- to 25-fold using S-mephenytoin N-demehtylation as CYP2B6 marker activity (Ekins et al., 1998; Lamba et al., 2003) and up to 80-fold using bupropion hydroxylation as CYP2B6 marker activity (Faucette et al., 2000) in single donor human liver microsomes. Gender and ethnic differences in CYP2B6 protein levels and catalytic activity has also been reported. Women had 1.7-fold more CYP2B6 protein and 1.6-fold more activity (measured as S- mephenytoin N-demehtylation) than men (Lamba et al., 2003). CYP2B6 activity (measured as S-mephenytoin N-demethylation) was greater by 3.6- and 5.0-fold in Hispanic women than in Caucasin or African-American women, respectively, but no difference was noted among men (Lamba et al., 2003). In addition, CYP2B6 is a highly polymorphic enzyme with 37 alleles and more than 100 single nucleotype polymorphic variants (http://www.cypalleles.ki.se/cyp2b6.htm). Some CYP2B6 variants have been shown to have greater or lesser catalytic activity and sometimes different binding affinity (expressed as Km) than the wild type enzyme, depending on the substrate used (Honda et al., 2011; Jinno et al., 201  2003; Xu et al., 2012). Collectively, these factors can explain the large inter-individual differences in rates of in vitro formation of hydroxylated metabolites of BDE-47 and BDE- 99 measured in single donor human liver microsomes (Table 3.8 and 3.12). CYP2B6 inducibility, drug therapy, and personal habits can also contribute to in vivo inter-individual differences in plasma and serum levels of BDE-99, BDE-47, and their hydroxylated metabolites.CYP2B6 mRNA, protein, and catalytic activity (measured as 4- hydroxy-bupropion formation) were induced by treatment with efavirenz, phenobarbital, dexamethasone, and rifampin treatment (Faucette et al., 2007; Wang et al., 2003) and by cigarette smoke extract (Washio et al., 2011) in human hepatocytes. In vivo, CYP2B6 protein level was induced in different regions of human brain by smoking (2.2- to 3.3-fold) and alcoholism (3.5- to 5.3-fold) (Mikyss et al., 2003). As a consequence, the inter- individual variability in the extent of BDE-99 and BDE-47 metabolism in vitro could account, together with CYP2B6 induction in vivo and differences in BDE-47 and BDE-99 exposure, for inter-individual differences in plasma and serum levels of BDE-47, BDE-99, and their hydroxylated metabolites (Kawashiro et al., 2008; Qiu et al., 2009; Zota et al., 2011). The role of CYP2B6 in the metabolism of BDE-47 and BDE-99 can have implications in determining human fetal exposure to PBDEs (Kawashiro et al., 2008; Qiu et al., 2009). CYP2B6 activity (measured as pentoxyresorufin O-dealkylation) was low (approximately 20 pmol/min/mg protein) in hematopoietic stem cells but CYP2B6 mRNA was induced 30-fold by rifampin treatment (Singh et al., 2012). However, CYP2B6 mRNA or protein levels could not be detected in fetal liver samples at 11 or 24 weeks of gestation, respectively (Maenpaa et al., 1993: Hakkola et al., 1994). In addition, CYP2B6 protein was 202  detected only in 2 out of 10 liver samples from infants (younger than 1 year of age) and was expressed at a level that was 10% of the mean CYP2B6 protein level measured in liver samples from children at 2 years of age and older (Tateishi et al., 1997). Collectively, the results suggest that CYP2B6-mediated metabolism of BDE-47 and BDE-99 would proceed at lower rate in the fetus than in adult humans and, therefore, that the hydroxylated PBDEs measured in cord blood are more likely to be produced by the mother than the fetus (Kawashiro et al., 2008; Qiu et al., 2009). The predominant role of CYP2B6 in the in vitro metabolism of BDE-47 and BDE-99 could have implications for the metabolism of other xenobiotics biotransformed by CYP2B6, such as organophosphate insecticides (i.e., chlorpyrifos, malathion, and parathion), organohalogen insecticides (i.e., endosulfan and methoxychlor), and polycyclic aromatic hydrocarbons (i.e., naphthalene) (Buratti et al., 2005; Cho et al., 2006; Foxemberg et al., 2007; Hu and Kupfer 2000a,b; Lee et al., 2006; Sams et al., 2004; Tang et al., 2001). Apparent Km values for the formation of CYP2B6-generated metabolites of the organophosphate and organohalogen xenobiotics mentioned above ranged between 10 and 70 µM and between 6 and 7 µM, respectively, except for the formation of chlorpyrifos oxon metabolite (apparent Km of 0.81 µM; Foxenberg et al., 2007). Apparent Km value for the formation of naphthalene metabolites ranged between 40 and 100 µM. In comparison, apparent Km values for the formation of metabolites of BDE-47 ranged between 10 and 44 µM (Table 3.13) and for the formation of metabolites of BDE-99 between 1.6 and 6.2 µM (Table 3.9). Therefore, BDE-99 is more likely than BDE-47 to affect the biotransformation of chlorpyrifos, malathion, parathion and naphthalene when these xenobiotics are present in 203  humans. Decreased metabolism of cholpyrifos, malathion, parathion and naphthalene can result in increased circulating levels of these compounds and toxic effects.   4.5 Comparison of Oxidative Metabolism of BDE-47 and BDE-99 by Rat and Human Liver Microsomes A comparison of the results of the present study with those of a previous study of metabolism of BDE-47 by rat liver microsomes (Erratico et al., 2011) reveals that the oxidative metabolism of BDE-99 and BDE-47 produces a different number and different patterns of hydroxylated metabolites, proceeds at different rates and is catalyzed by different CYP enzymes in rat and human liver microsomal incubations (Table 4.3 and 4.4). A larger number of hydroxylated metabolites of BDE-99 (ten) and BDE-47 (nine) were produced by human liver microsomes compared to liver microsomes obtained from rats treated with corn oil (3 and none, respectively), DEX (6 and 3, respectively), MC (3 and 2, respectively), or PB (6 and 4, respectively). The major hydroxylated metabolites of BDE-99 formed by human liver microsomes (namely 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101) were either intermediate or minor metabolites of BDE-99 produced by the rat liver microsomal preparations used. The major metabolite of BDE-99 produced by most of the rat liver microsomal preparations used (namely 4-OH-BDE-90) was a minor metabolite produced by human liver microsomes. In addition, one of the two major (namely 6-OH-BDE-47) hydroxylated metabolites of BDE- 47 produced by human liver microsomes was below the limit of detection in incubations containing all of the rat liver microsomal preparations assessed (Erratico et al., 2011). 204  TABLE 4.3 Comparison of BDE-99 Hydroxylated Metabolite Formation by Human and Rat Liver Microsomes Human Liver Microsomes Liver Microsomes from C.O.-treated rats Liver Microsomes from DEX-treated rats Liver Microsomes from MC-treated rats Liver Microsomes from PB-treated rats Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) 5'-OH-BDE-99 (47±2.3 pmol/min/mg) 4-OH-BDE-90  (2.7±1.6 pmol/min/mg) 4-OH-BDE-90  (320±52 pmol/min/mg) 6'-OH-BDE-99 (20±4.0 pmol/min/mg) 4-OH-BDE-90 (76±16 pmol/min/mg) 2,4,5-TBP  (45±2.2 pmol/min/mg) 6'-OH-BDE-99  (0.6±0.4 pmol/min/mg) 5'-OH-BDE-99 (18±3.8 pmol/min/mg) 4-OH-BDE-90 (6.0±3.6 pmol/min/mg) 5'-OH-BDE-99 (27±5.4 pmol/min/mg) 4'-OH-BDE-101 (24±3.5 pmol/min/mg) 5'-OH-BDE-99  (0.3±0.1 pmol/min/mg) 6'-OH-BDE-99 (1.2±3.7 pmol/min/mg) 5'-OH-BDE-99  (1.9±0.7 pmol/min/mg) 6'-OH-BDE-99 (27±3.7 pmol/min/mg) 205  TABLE 4.4 Comparison of BDE-47 Hydroxylated Metabolite Formation by Human and Rat Liver Microsomesa Human Liver Microsomes Liver Microsomes from C.O.-treated rats Liver Microsomes from DEX-treated rats Liver Microsomes from MC-treated rats Liver Microsomes from PB-treated rats Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) 5-OH-BDE-47   (27±3.9 pmol/min/mg) Not detected 3-OH-BDE-47  (3.6±1.3 pmol/min/mg) 4'-OH-BDE-49  (11±2.2 pmol/min/mg) 4'-OH-BDE-49  (17±0.6 pmol/min/mg) 6-OH-BDE-47   (24±3.4 pmol/min/mg) Not detected 5-OH-BDE-47   (1.6±0.1 pmol/min/mg) Not detected 3-OH-BDE-47   (6.8±0.1 pmol/min/mg) 4-OH-BDE-42    (14±4.1 pmol/min/mg) Not detected 4'-OH-BDE-49 (1.5±0.2 pmol/min/mg) Not detected 4-OH-BDE-42   (3.6±0.2 pmol/min/mg) 206  Human Liver Microsomes Liver Microsomes from C.O.-treated rats Liver Microsomes from DEX-treated rats Liver Microsomes from MC-treated rats Liver Microsomes from PB-treated rats Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) Hydroxylated metabolite (app. Vmax) 4'-OH-BDE-49  (13±3.0 pmol/min/mg) Not detected Not detected Not detected 5-OH-BDE-47   (1.3±0.04 pmol/min/mg)  a Data about metabolism of BDE-47 by rat liver microsomes from Moffatt et al., 2011. C.O. = corn oil. 207  A second major hydroxylated metabolite of BDE-47 produced by human liver microsomes (namely 5-OH-BDE-47) was below the limit of detection in liver microsomes from corn oil- or MC-treated rats and was a minor and an intermediate metabolite produced by liver microsomes from PB- and DEX-treated rats, respectively (Erratico et al., 2011). In contrast, 3-OH-BDE-47, the major hydroxylated metabolite of BDE-47 produced by liver microsomes obtained from DEX- and PB-treated rats, respectively (Erratico et al., 2011), was below the limit of quantification in incubations containing human liver microsomes. Finally, the number of unidentified mono- and di-hydroxylated metabolites of BDE-99 and BDE-47 produced by human liver microsomes were different than those produced by rat liver microsomes (Erratico et al., 2011). Rates of formation of hydroxylated metabolites of BDE-99 by rat and human liver microsomal incubations (Table 3.7 and 3.9, respectively) suggest that hydroxylation of the more brominated ring is preferred over the less brominated ring by rat liver microsomes, whereas the opposite is true for human liver microsomes. With rat liver microsomes, para-hydroxylation of BDE-99 accompanied by a NIH- shift mechanism (formation of 4-OH-BDE-90) was more favorable than hydroxylation at an unsubstituted ortho or meta position (formation of 6'-OH-BDE-99 and 5'-OH-BDE-99, respectively) and was favored over oxidation of the 1' carbon atom resulting in O- dealkylation (formation of 2,4,5-TBP). In contrast, neither hydroxylation at an unsubstituted ortho or meta carbon position (formation of 6'-OH-BDE-99 and 5'-OH-BDE-99, respectively) nor O-dealkylation (formation of 2,4,5-TBP) was favored over the other by human liver microsomes and both mechanisms were slightly favored over para- 208  hydroxylation of a substituted carbon atom accompanied by a NIH-shift mechanism (formation of 4-OH-BDE90, 4'-OH-BDE-101, and 2-OH-BDE-123). The same pattern was observed for the metabolism of BDE-47 by rat and human liver microsomes (Erratico et al., 2011). Para-hydroxylation of a brominated carbon atom accompanied by a NIH-shift mechanism (formation of 4-OH-BDE-42 and 4'-OH-BDE-49) was favored over oxygen insertion at unsubstituted meta or ortho positions (formation of 3- OH-BDE-47, 5-OH-BDE-47, or 6-OH-BDE-47, respectively). Conversely, results obtained with human liver microsomes (Table 3.13) suggest that neither oxygen insertion at an unsubstituted ortho or meta carbon atom (formation of 5-OH-BDE-47 and 6-OH-BDE-47, respectively) nor at a brominated para carbon atom involving a NIH-shift mechanism (formation of 4-OH-BDE-42 or 4'-OH-BDE-49) is favored over the other mechanism. Rates of BDE-99 and BDE-47 metabolite formation by rat and human liver microsomes differed greatly. For example, rates of formation of the three major identified hydroxylated metabolites of BDE-99 produced by human liver microsomes (namely 2,4,5- TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101) were 150-, 90-, and 50-times larger than those of the same metabolites produced by liver microsomes from corn oil-treated rats. Rates of formation of the two major identified hydroxylated metabolites of BDE-47 produced by human liver microsomes (namely 5-OH-BDE-47 and 6-OH-BDE-47) were at least 90- and 45-times larger than those of the same metabolites produced by liver microsomes from corn oil-treated rats. Several rat recombinant CYP enzymes including CYP1A1, 2A2, 2B1, 2C6, and 3A1 catalyzed the oxidative metabolism of BDE-99. A previous study showed that CYP1A1, 2A2, and 3A1 catalyzed the metabolism of BDE-47 (Erratico et al., 2011). In contrast, 209  CYP2B6 was the predominant CYP enzyme involved in the oxidative metabolism of both BDE-99 and BDE-47 using human liver microsomes and a panel of twelve human recombinant CYP enzymes. The different number, patterns, and rates of hydroxylated metabolite formed together with the different main mechanisms of oxidation and number of CYP enzymes involved in BDE-99 and BDE-47 metabolism by rat and human liver microsomes strongly suggest that hepatic metabolism of PBDEs (and possibly of other organohalogen xenobiotics) should be assessed in a species-specific manner and results should not be extrapolated across mammalian species. Differences in in vitro metabolism among species was previously shown for 2,2',5,5'-tetrachloro biphenyl using rat and hamster liver microsomal incubations (Koga et al., 1996; Preston et al., 1983) and for benzo(a)pyrene using liver microsomal preparations from several mammalian species (Harris et al., 2009). In humans, oxidative metabolism of BDE-47 and BDE-99 can be of toxicological concern for thyroid hormone homeostasis. Transthyretin is a major transport protein for 3,3',5,5'-tetraiodothyronine (T4) in blood in humans and rats (Visser et al., 1996). Several hydroxylated metabolites of BDE-47 produced by human liver microsomes (namely 4-OH- BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49) were shown to be 250- to 5,000-fold and 160- to 1,600-fold more potent than BDE-47 as inhibitors of T4 binding to human and rat transthyretin, respectively. The concentrations that produced a 50% decrease (IC50) of T4 binding to human and rat transthyretin were between 4 and 900 nM and between 20 and 200 nM, respectively, for 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, and 4'-OH-BDE-49, compared to 25 µM for BDE-47 (Cao et al., 2010; Hamers et al., 2006; Hamers et al., 2008; Marchesini et al., 2008; Ren and Guo, 2012). In addition, the combined 210  potency of hydroxylated metabolites of BDE-47 as inhibitors of T4 binding to rat transthyretin was shown to be well predicted according to the concentration-addition model (Hamers et al., 2008). Some hydroxylated metabolites of BDE-99 and BDE-47 are more potent than the parent compounds as inhibitors of the biotransformation of T4 into 3,3',5-triiodothyronine (T3) by human liver microsomes. For example, Butt et al. (2011) showed that 5'-OH-BDE- 99 (IC50 value of 0.4 µM) but not 6'-OH-BDE-99, 4'-OH-BDE-101, BDE-99 or 5-OH-BDE- 47 inhibited the biotransformation of T4 into T3 by human liver microsomes. Further studies are needed to investigate the ability of more hydroxylated metabolites of BDE-99 and BDE- 47 to inhibit the biotransformation of T4 to T3 by human liver microsomes. Some hydroxylated metabolites of BDE-99 and BDE-47 are also in vitro agonists or antagonists of the human thyroid receptor. In Chinese hamster ovary cells transfected with human thyroid receptor, 4-OH-BDE-90 at a concentration of 8.1 µM inhibited 20% of the human thyroid receptor-mediated transcriptional activity yielded by 10 nM T3. In comparison, BDE-99 at a concentration up to 10 µM did not elicit an antagonistic effect in the same assay (Kojima et al., 2009). These data suggest that 4-OH-BDE-90 is a slightly more potent antagonist than BDE-99 for human thyroid receptor-mediated transcriptional activity induced by T3 in vitro. The agonistic activity of hydroxylated PBDEs for human thyroid receptor was investigated in a T3-mediated transcription assay. Yeast cells were transfected with human thyroid receptor regulating the expression of the gene coding for β- galactosidase activity. Concentrations of 4-OH-BDE-42, 5-OH-BDE-47, 6-OH-BDE-47, 4'- OH-BDE-49, 5'-OH-BDE-99, and 6'-OH-BDE-99 between 37 pM and 24 nM induced 20% of the β-galactosidase activity yielded by 1 µM T3 (Li et al.,  2010). In comparison, in the 211  same assay no agonistic activity was found for BDE-47 and BDE-99 up to 1 µM concentration (Kojima et al., 2009). These data suggest that the hydroxylated metabolites of BDE-99 and BDE-47 tested are 40- to 25,000-fold more potent agonists of human thyroid receptor-mediated in vitro transcriptional activity than BDE-47 and BDE-99. The in vivo thyroid toxicity of PBDE and hydroxylated PBDEs in humans is currently unknown. The concentration of total hydroxylated metabolites of BDE-47 and BDE-99 detected in human plasma and serum samples is only 1 to 20 times lower than those of BDE-47 and BDE-99, respectively (Athanasiadou et al., 2008; Kawashiro et al., 2008; Qiu et al., 2009; Zota et al., 2012). Therefore, if the greater potency of hydroxylated metabolites of BDE-47 and BDE-99 than BDE-47 and BDE-99 as disruptors of thyroid homeostasis observed in vitro occurs also in vivo and if potency of total hydroxylated metabolites of BDE-47 and BDE-99 for binding to transthyretin is exerted according to the concentration addition model also in vivo, the hydroxylated metabolites of BDE-47 and BDE-99 rather than BDE-47 and BDE-99 might be able to disrupt thyroid hormones homeostasis in humans. Therefore, the in vivo oxidative metabolism of BDE-47 and BDE- 99 could be an important determinant of PBDEs toxicity in humans. The parent compounds (PBDEs) are not the major toxicants but are pro-toxicants activated by CYP enzymes to form hydroxylated metabolites that are able to alter thyroid hormone transport, peripheral metabolism, and nuclear receptor-mediated gene transcription more potently than their parent compounds. Decreased blood levels of thyroid hormones are a concern, particularly during pregnancy because thyroid hormones insufficiency is associated with detrimental neurodevelopmental effects in children (Zoeller et al., 2007).  212  In addition to affecting thyroid hormone homeostasis, BDE-47 and BDE-99 hydroxylated metabolites can contribute to neurodevelopmental disorders in rodents. Neurodevelopmental disorders caused by BDE-47 or BDE-99 exposure include increased locomotor activity (Suvorov et al., 2009), hyperactivity and delayed development of neuromotor function (Gee et al., 2008; Gee and Moser, 2008), altered learning and memory functions (Cheng et al., 2009) and altered spontaneous motor behavior (Eriksson et al., 2002; Viberg et al., 2002). The contribution of hydroxylated PBDEs to these neurodevelopmental disorders was shown in in vitro studies. In rat PC12 cell lines, 5-OH- BDE-47, 6-OH-BDE-47 and 4'-OH-BDE-49 metabolites of BDE-47 were shown to be more potent than BDE-47 in altering Ca2+ intracellular homeostasis (Dingemans et al., 2008, 2010, 2011) via modulation of ryanodine receptor activity (Kim et al., 2011). As a consequence, 5-OH-BDE-47, 6-OH-BDE-47 and 4'-OH-BDE-49 were shown to alter Ca- controlled release of catecholamines (Dingemans et al., 2008, 2010a, b). In addition, in Xenopus levis oocytes, partial agonistic and antagonistic effect on GABA and nicotinic acethylcohline receptors was demonstrated for 6-OH-BDE-47 but not for BDE-47 (Hendriks et al., 2010). Because cathecolamines are important neurotransmitters and because GABA and nicotinic acethylcohline receptors are critical for neurodevelopment (Represa and Ben- Ari, 2007), the in vitro effects of hydroxylated metabolites of BDE-47 suggest an important role for OH-BDEs in neurotoxicity. 213  5. Concluding Remarks    5.1 Conclusions The present study was the first to thoroughly assess the in vitro oxidative metabolism of BDE-99 using rat and human liver microsomes and recombinant enzymes and the in vitro oxidative metabolism of BDE-47 using human liver microsomes and human recombinant enzymes. Three to six hydroxylated metabolites of BDE-99 were produced by rat liver microsomal preparations or rat recombinant CYP enzymes. 4-OH-BDE-90 was the major metabolite, 5'-OH-BDE-99 and 6'-OH-BDE-99 were the intermediate metabolites, and 2,4,5-TBP was the minor metabolite of BDE-99, although differences in rates of metabolite formation exist among the four liver microsomal preparations assessed. Among the rat recombinant CYP enzymes tested, CYP3A1, CYP2A2, and CYP2B1 were the most active CYP enzymes in metabolizing BDE-99. Collectively, the present data show that oxidative metabolism of BDE-99 by rat liver microsomes proceeds at relatively slow pace, which is consistent with the ability of PBDEs to bioaccumulate in rats. The major hydroxylated metabolites of BDE-47 produced by human liver microsomes were 5-OH-BDE-47 and 6-OH-BDE-47 and the major hydroxylated metabolites of BDE-99 were 2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101. CYP2B6 was the CYP enzyme predominantly responsible for the in vitro oxidative metabolism of BDE-47 and BDE-99 and was responsible for producing all the mono- and di- 214  hydroxylated metabolites of BDE-47 and BDE-99 detected in the present study. The rate of in vitro oxidative metabolism of BDE-47 and BDE-99 is relatively low compared to that of other xenobiotics, which suggests that in vivo oxidative metabolism of BDE47 and BDE-99 is relatively slow and could be a key determinant of the bioaccumulation of BDE-47 and BDE-99 in humans. However, a similar number of identified hydroxylated metabolites of BDE-47 and BDE-99 are produced by human liver microsomes at similar rates of formation, suggesting that oxidative metabolism of BDE-47 and BDE-99 in vivo is not likely a major determinant of their different levels in human plasma and serum. Striking inter-species differences have been noted in the in vitro oxidative metabolism of BDE-99 by rat and human liver microsomes. The oxidative metabolism of BDE-99 produces a different number and different patterns of hydroxylated metabolites, proceeds via different main oxidative mechanisms and at different rates, and is catalyzed by a different number of CYP enzymes in rat and human liver microsomal incubations. Therefore, the present study suggests that hepatic metabolism of PBDEs should be assessed in a species-specific manner and that results should not be extrapolated across mammalian species. In addition, the number of hydroxylated metabolites of BDE-99 formed by rat (3) and human (10) liver microsomes and their rates of formation being 10 times larger in humans than in rat liver microsomes suggest that in vivo BDE-99 undergoes more extensive oxidative metabolism in humans than in rats.    215  5.2 Strengths and Weaknesses of the Present Study The major strengths of the present study are as follows: 1. The quality and reliability of the data generated. Validating the in vitro assay used to assess the oxidative metabolism of BDE-99 by liver microsomal preparation, extending the assay to analyze newly available standards for possible hydroxylated metabolites of BDE-99, improving the specificity of the LC/MS using a LC/MS/MS method, and preparing fresh calibration standard and quality control samples for every batch of unknown samples provided a solid approach to generate precise and accurate data. 2. The reliability of the experimental design used to determine kinetic parameters associated to formation of BDE-47 and BDE-99 metabolites.  To my knowledge, this is the first study in which a comprehensive approach has been taken to determine kinetic parameters associated with the in vitro formation of PBDE metabolites by liver microsomes and recombinant CYP enzymes. 3. The combined experimental approach used to identify the major CYP enzyme responsible for BDE-47 and BDE-99 metabolism by human liver microsomes. Comparing results from different experiments with  human recombinant CYP enzymes, pooled human liver microsomes, CYP-specific antibodies, and single donor human liver microsomes provided solid and supporting evidence of the role of individual CYP enzymes in the in vitro metabolism of BDE-47 and BDE-99.   216  The major weaknesses of the present study are as follows: 1. The limited number of mono- and di-OH-BDE standards presently available. Several mono- and di-hydroxylated metabolites were not identified due to the lack of authentic standards. Lack of complete structure elucidation limited interpretation of the mechanisms of oxidation of BDE-47 and BDE-99 by human liver microsomes and recombinant CYP2B6 and prevented quantification of rates of metabolite formation. It also prevented the comparison of all the hydroxylated metabolites formed by human liver microsomes with the OH-BDEs identified in biomonitoring studies in human plasma and serum samples. 2. The low throughput of the assay. Although effort was directed to optimize sample preparation and chromatographic separation of the hydroxylated metabolites of BDE-99, the throughput of the assay was low (i.e. 20 to 30 samples per day). Liquid chromatography columns with greater selectivity for hydroxylated metabolites of PBDEs (and of organohalogen compounds in general) can produce improvement for higher throughput assays. 217  References   Abdallah, M. 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Med. 6:189-198.   274  Appendix 1- Metabolism of BDE-99 by Human Liver Microsomes   Characterization of the Modified UHPLC/MS/MS Method for Determination of the Hydroxylated Metabolites of BDE-99: Selectivity, Linearity, Sensitivity, and Quality Control Values Representative retention time and mass spectrometry parameter values used for determination of hydroxylated metabolites of BDE-99 using the modified UPLC/MS/MS- based assay are reported in Table A1.2. Spurious peaks with the same mass-to-charge transition values and eluting at the same retention times as the authentic standards used were not detected in the blank samples. The assay was linear over the 2.5 to 500 nM concentration range for all the analytes of interest (r>0.95). For each analyte, the limit of quantitation was 2.5 nM with  accuracy and precision values of less than 20% bias and less than 20% percent relative standard deviation, respectively, and a signal-to-noise value larger than 10 (Table A1.3). For every experiment conducted to characterize the oxidative metabolism of BDE-99 by human liver microsomes and recombinant CYP enzymes, accuracy and precision values were also monitored using freshly prepared QC–Low, QC–Mid, and QC–High samples (n=1 per experiment) as described in Section 2.7. Inter-day accuracy and precision values for QC– Low, QC–Mid, and QC–High samples were less than 20% bias and less than 20% percent relative standard deviation, respectively (n=8; Table A1.4). 275  One di-OH-pentabrominated-PBDE metabolite of BDE-99 (M4) with a retention time of 8.01 min was detected. A peak eluting at 8.01 min with multiple reaction monitoring transitions corresponding to those of mono-OH-pentabrominated-PBDEs was detected. The other unidentified mono-OH-pentabrominated-PBDE metabolites of BDE-99 (M1-M3) eluted later (i.e., retention time values of 13.05 min or larger). Thus, it is likely that the peak eluting at 8.01 min and corresponding to a mono-OH-pentabrominated-PBDE is a product of in-source fragmentation, whereby M4 undergoes a loss of -OH (17 AMU) in the source of the mass spectrometer and generates a signal with multiple reaction monitoring transitions corresponding to a mono-OH-pentabrominated-PBDE metabolite of BDE-99. Therefore, the peak eluting at 8.01 min was not considered a mono-OH-pentabrominated-PBDE metabolite of BDE-99.             276  Table A1. 1 Cross Reactivity of Mouse Anti-Human CYP2B6 Antibodya  aThe table was provided by the Antibody Vendor (BD Biosciences)           277  Table A1. 2 Retention Time (RT), Precursor and Product ion (Q1/Q3) Transitions, Declustering Potential (DP), and Collision Energy (CE) Values of the Hydroxylated BDE-99 Metabolites and Internal Standard (IS) Using the Modified UPLC/MS/MS- based Assay Analyte RT (min) Q1/Q3 transitions (m/z)a DP (V) CE (eV) 2,4,5-TBP 6.54 328.8/78.9 -100 -68 328.8/81.0 -100 -68  4-OH-BDE-90 27.80 5'-OH-BDE-99 33.81 576.4/78.8 -80 -98 6'-OH-BDE-99 36.15 578.6/78.8 -80 -98 4'-OH-BDE-101 37.49 580.6/78.8 -80 -80 2-OH-BDE-123 29.10 580.6/81.0 -80 -80 M1 13.05 582.6/81.0 -80 -80 M2 24.82 576.4/78.8 -80 -98 M3 39.00 M4 8.01 592.5/78.8 -80 -80 594.5/78.8 -80 -80 596.5/78.8 -80 -80 596.5/81.0 -80 -80 598.5/81.0 -80 -80 4-OH-CB-121 (IS) 19.89 340.9/304.9 -100 -40  a Total ion current (TIC) of the Q1/Q3 transitions were used for analysis.    278  Table A1. 3 % Relative Standard Deviation (%RSD), Percent Deviation (%Dev), and Signal-to-Noise (S/N) Values of the Limit of Quantification for the Analytes of Interesta Analyte Nominal concentration (nM) Measured concentration (nM) %RSD %Dev S/N  2,4,5-TBP 2.5 2.2 ± 0.39 17 -10 230 4-OH-BDE-90 2.5 2.8 ± 0.16 6.0 11 57 5ʹ-OH-BDE-99 2.5 2.6 ± 0.29 11 3.5 91 6ʹ-OH-BDE-99 2.5 2.3 ± 0.46 18 -9.4 58 4'-OH-BDE-101 2.5 2.2 ± 0.44 20 -11 75 2-OH-BDE-123 2.5 2.7 ± 0.25 9.4 6.5 99  a n=6, 6 replicates per sample on the same day.               279  Table A1. 4 Inter-day Precision (%RSD) and Accuracy (%Dev) Values Determined With QC Samples at Low, Mid, and High Concentrations (n=8) Using the Modified UPLC/MS/MS-based Assay Analyte QC sample Nominal concentration (nM) Measured concentration (nM) %RSD %Dev 2,4,5-TBP QC-Low 7.5 7.9 ± 1.6 20 5.7  QC-Mid 80      77 ± 10 13 -3.6  QC-High 400    350 ± 63 18 -11 4-OH-BDE-90 QC-Low 7.5 8.0 ± 1.3 16 6.1  QC-Mid 80 78 ± 6.0 7.7 -2.2  QC-High 400    360 ± 39 11 -10 5'-OH-BDE-99 QC-Low 7.5 7.0 ± 0.72 10 -6.9  QC-Mid 80 76 ± 7.3 10 -5.5  QC-High 400    340 ± 37 11 -14 6'-OH-BDE-99 QC-Low 7.5     7.7 ± 0.87 11 2.7  QC-Mid 80 76 ± 4.1 5.5 -5.5  QC-High 400    340 ± 39 11 -14 4'-OH-BDE-101 QC-Low 7.5     7.4 ± 0.86 11 -0.65  QC-Mid 80 75 ± 4.8 6.3 -5.9  QC-High 400    350 ± 42 12 -12 2-OH-BDE-123 QC-Low 7.5     7.6 ± 0.77 10 1.1  QC-Mid 80 73 ± 6.6 9.0 -8.2  QC-High 400    340 ± 31 9.1 -16   280  Table A1. 5 Marker Activity Values for Major CYP and UGT Enzymes in the Single Donor Human Liver Microsomesa  aTable provided by Xenotech 281    Figure A1.1 The specificity of inhibition by mouse anti-CYP2B6 monoclonal antibody. Data provided by BD Biosciences.              282                    Figure A1.2 Correlation between CYP2B6 and CYP2C19 marker activities for single donor human liver microsomal samples. Data provided by BD Biosciences.               Y = 17.7 X + 94.4 (r=0.78; r² = 0.61) 0 500 1,000 1,500 2,000 2,500 3,000 3,500 4,000 4,500 0 50 100 150 200 C Y P2 B 6 m ar ke r ac tiv ity  (b up ro pi on  4 -h yd ro xy la se ) CYP2C19 marker activity (S-mephenytoin 4'-hydroxylase) 283  Appendix 2 - Metabolism of BDE-47 by Human Liver Microsomes   Characterization of the Modified UHPLC/MS/MS Method for Determination of the Hydroxylated Metabolites of BDE-47: Selectivity, Linearity, Sensitivity, and Quality Control Values  Representative retention time and mass spectrometry parameter values used for determination of hydroxylated metabolites of BDE-47 using the modified UPLC/MS/MS- based assay are reported in Table A2.1. Spurious peaks with the same mass-to-charge transition values and eluting at the same retention times as the authentic standards used were not detected in the blank samples (data not shown). With the exception of 2,4-DBP and 2'-OH-BDE-17, the method was linear over the 1.0 to 500 nM concentration range for all the analytes of interest (r>0.95). However, the accuracy and precision values obtained at 1.0 nM did not match the acceptance criteria previously established for LOQ determination (Section 2.7). Therefore, 1.0 nM was considered the limit of detection of the present method for all the analytes except 2,4-DBP and 2'-OH-BDE-17. For all of the analytes, the LOQ value was 2.5 nM (5.0 nM for 2,4-DBP) with accuracy and precision values of less than 20% bias and less than 20% relative standard deviation, respectively. The signal-to-noise value was larger than 10 for all the analytes (Table A2.2).  Therefore, method was linear over 2.5 to 500 nM concentration range for all the analytes of interest (5.0 to 500 nM for 2,4-DBP; r>0.95). 284  For every experiment conducted to characterize the oxidative metabolism of BDE-47 by human liver microsomes and recombinant CYP enzymes, accuracy and precision values were monitored using freshly prepared QC–Low, QC–Mid, and QC–High samples (n=1 per experiment) as described in Section 2.7. Inter-day accuracy and precision values for QC– Low, QC–Mid, and QC –High samples were less 20% bias and less than 20% relative standard deviation, respectively (n=8; Table A2.3). In theory, only six mono-OH-tetrabrominated-PBDE metabolites can be formed from BDE-47. The five authentic standards used in the present study allowed the identification of four OH-tetra-BDE metabolites eluting between 25.2 and 42.0 min. 3-OH- BDE-47 was below limit of detection in all samples. However, two peaks, eluting at 8.48 and 27.8 min, with multiple reaction monitoring transitions corresponding to those of mono- OH-tetrabrominated-PBDEs were consistently detected in the samples analyzed. The unknown mono-OH-tetrabrominated-PBDE peak with a retention time of 8.48 min elutes much earlier than all the identified mono-OH-tetrabrominated-PBDE metabolites of BDE- 47. In addition, a di-OH-tetrabrominated-PBDE metabolite of BDE-47 (M2) with the same retention time of 8.48 min was detected. Thus, it is likely that the mono-OH- tetrabrominated-PBDE peak eluting at 8.48 min is a product of in-source fragmentation, whereby M2 undergoes a loss of -OH (17 AMU) in the source of the mass spectrometer and generates a signal with multiple reaction monitoring transitions corresponding to a mono- OH-tetrabrominated-PBDE metabolite of BDE-47, as noted for the di-OH-pentabrominated- PBDE metabolite of BDE-99 (Appendix no. 1). Conversely, an elution time of 27.8 min of the other unidentified mono-OH-tetrabrominated-PBDE peak is more consistent with those of identified mono-OH-tetrabrominated-PBDE metabolites of BDE-47 (25.2-42 min). In 285  addition, no peak corresponding to a di-OH-tetrabrominated-PBDE could be detected at 27.8 min (data not shown). Therefore, the peak eluting at 27.8 min is considered the unidentified mono-OH-tetrabrominated-PBDE metabolite of BDE-47, M1.                     286  Table A2.1 Retention Time (RT), Precursor and Product ion (Q1/Q3) Transitions, Declustering Potential (DP), and Collision Energy (CE) Values of the Hydroxylated BDE-47 Metabolites and Internal Standard (IS) Using the Modified UPLC/MS/MS- based Assay Analyte RT (min) Q1/Q3 transitions (m/z)a DP (V) CE (eV) 2,4-DBP 5.50 251.0/78.9 -100 -68 253.0/78.9 -100 -68   419.1/78.9 -80 -98 4'-OH-BDE-17 20.10 421.1/78.9 -80 -98 2'-OH-BDE-28 21.60 421.1/81.0 -80 -80   423.1/81.0 -80 -80   425.1/81.0 -80 -80 4-OH-BDE-42 29.60 496.6/78.9 -80 -98 3-OH-BDE-47 25.50 498.6/78.9 -80 -98 5-OH-BDE-47 35.80 500.67/8.9 -80 -80 6-OH-BDE-47 42.00 500.6/81.0 -80 -80 4'-OH-BDE-49 40.60 502.6/81.0 -80 -80 M1 27.80 M2 8.5 512.6/78.9 -80 -80 514.6/78.9 -80 -80 516.6/78.9 -80 -80 516.6/81.0 -80 -80 518.6/81.0 -80 -80 4-OH-CB-50 (IS) 11.90 340.9/304.9 -100 -40  a Total ion current (TIC) of the Q1/Q3 transitions were used for analysis.  287  Table A2.2 % Relative Standard Deviation (%RSD), Percent Deviation (%Dev), and Signal-to-Noise (S/N) Values of the Limit of Quantification for the Analytes of Interesta Analyte Nominal concentration (nM) Measured concentration (nM) %RSD %Dev S/N 2,4-DBP 5.0 4.2 ± 0.74 17 -16 37 4'-OH-BDE-17 2.5 2.3 ± 0.12 5.1 -9.4 25 2'-OH-BDE-28 2.5 2.1 ± 0.17 8.0 15 21 4-OH-BDE-42 2.5     2.4 ± 0.048 2.0 -4.3 21 3-OH-BDE-47 2.5 2.3 ± 0.24 10 -8.3 14 5-OH-BDE-47 2.5 2.5 ± 0.12 4.8 0.9 21 6-OH-BDE-47 2.5       2.4 ± 0.091 3.7 -2.5 43 4'-OH-BDE-49 2.5 2.4 ± 0.22 9.5 -5.0 30  a n=6, 6 replicates per sample on the same day.              288  Table A2.3 Inter-day %RSD and %Dev Values Determined With QC Samples at Low, Mid, and High Concentrations (n=8) Analyte QC sample Nominal concentration (nM) Measured concentration (nM) %RSD %Dev 2,4-DBP QC-Low 15 13 ± 2.0 15 -16  QC-Mid 80      77 ± 19 20 -3.2  QC-High 400    300 ± 84 27 -23 4'-OH-BDE-17 QC-Low 7.5     7.8 ± 0.68 8.8 3.5  QC-Mid 80 79 ± 4.0 5.0 -0.6  QC-High 400    370 ± 15 4.2 -8.3 2'-OH-BDE-28 QC-Low 7.5     7.5 ± 0.75 10 0.10  QC-Mid 80      80 ± 4.3 5.4 0.27  QC-High 400    370 ± 21 5.7 -8.3 4-OH-BDE-42 QC-Low 7.5     7.6 ± 0.94 12 1.6  QC-Mid 80 79 ± 6.2 7.9 -1.4  QC-High 400    380 ± 36 9.5 -5.2 3-OH-BDE-47 QC-Low 7.5     7.5 ± 0.76 10 -0.33  QC-Mid 80 78 ± 4.8 6.2 -2.9  QC-High 400    370 ± 27 7.3 -8.2 5-OH-BDE-47 QC-Low 7.5     7.6 ± 0.71 9.3 1.4  QC-Mid 80      76 ± 7.3 9.6 -4.5  QC-High 400    360 ± 34 9.5 -9.8 6-OH-BDE-47 QC-Low 7.5     7.8 ± 0.61 7.8 4.2  QC-Mid 80 77 ± 7.2 9.4 -3.5  QC-High 400    350 ± 29 8.3 -12 4'-OH-BDE-49 QC-Low 7.5     7.4 ± 0.52 7.0 -1.7  QC-Mid 80 76 ± 5.7 7.4 -4.6  QC-High 400    380 ± 28 7.3 -5.4 

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