Human metabolism of polybrominated diphenyl ethers: Identification of metabolites using gas chromatography and supercritical fluid chromatography with mass spectrometric detection
Gross, Michael S.
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Human exposure to polybrominated diphenyl ethers (PBDEs) results in deleterious health effects. Hydroxylated analogs of PBDEs (OH-BDEs) are products of oxidative metabolism via cytochrome P450s (CYPs), atmospheric reactions between anthropogenic PBDEs and hydroxyl radicals, and biogenic sources in marine environments. Recent mechanistic studies have suggested that OH-BDEs have significantly greater endocrine disrupting and neurotoxic potential than their PBDE counterparts. Consequently, it is critical to develop a better understanding of PBDE metabolism in humans. The research conducted herein aimed to characterize the enzyme-specific and congener-specific metabolism of PBDEs using gas chromatography-mass spectrometry (GC-MS). Unknown OH-BDE biotransformation products were later identified through a correlation between calculated boiling points and experimental GC-MS retention times. Lastly, an alternative supercritical fluid chromatography-mass spectrometry (SFC-MS) instrumental method was developed for the trace analysis of OH-BDEs in in vitro metabolism and biological samples. The focus of Chapter 2 was to qualitatively and quantitatively characterize the in vitro metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), an environmentally and biologically abundant PBDE congener. Samples were incubated with either individual recombinant CYPs, or with pooled human liver microsomes (HLMs), and analyzed using GC-MS. Results revealed that CYP2B6 was the predominant enzyme responsible for the biotransformation of BDE-47 into eight OH-BDE oxidative metabolites. Five metabolites, including 3-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (3-OH-BDE-47), 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47), 4-hydroxy-2,2',3,4'-tetrabromodiphenyl ether (4-OH-BDE-42), and 4'-hydroxy-2,2',4,5'-tetrabromodiphenyl ether (4'-OH-BDE-49), were identified by use of authentic reference standards. The three remaining metabolites were hypothesized to be 2'-hydroxy-2,3',4,4'-tetrabromodiphenyl ether (2'-OH-BDE-66), a di-OH-tetraBDE, and a di-OH-tetrabrominated dioxin. The rates of formation (Vmax) for 3-OH-BDE-47, 5-OH-BDE-47, and 6-OH-BDE-47, the three major metabolites of BDE-47, were determined using Michaelis-Menten kinetics. The Vmax values ranged from 1.93 to 230 pmol/min/nmol P450 (0.91 to 107 pmol/min/mg protein) for HLMs and from 10.6 to 950 pmol/min/nmol P450 for recombinant human CYP2B6. Chapter 3 characterized the in vitro metabolism of 2,2',4,4',6-pentabromodiphenyl ether (BDE-100), one of the most abundant PBDE congeners found in humans, by recombinant CYPs and pooled HLMs. CYP2B6 was again found to be the predominant enzyme responsible for nearly all formation of six mono-OH-pentaBDE and two di-OH-pentaBDE metabolites during the metabolism of BDE-100. Four metabolites were identified as 3-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (3-OH-BDE-100), 5'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (5'-OH-BDE-100), 6'-hydroxy-2,2',4,4',6-pentabromodiphenyl ether (6'-OH-BDE-100), and 4'-hydroxy-2,2',4,5',6-pentabromodiphenyl ether (4'-OH-BDE-103) through use of reference standards. The two remaining mono-OH-pentaBDE metabolites were hypothesized based on characteristic mass spectral fragmentation patterns of derivatized OH-BDEs, which allowed prediction of an ortho-OH-pentaBDE and a para-OH-pentaBDE positional isomers. Additional information based on theoretical boiling point calculations using COnductor-like Screening MOdel for Realistic Solvents (COSMO-RS) and experimental chromatographic retention times were used to identify the hypothesized metabolites as 2'-hydroxy-2,3',4,4',6-pentabromodiphenyl ether (2'-OH-BDE-119) and 4-hydroxy-2,2',4',5,6-pentabromodiphenyl ether (4-OH-BDE-91). Kinetic studies of BDE-100 metabolism using CYP2B6 and HLMs revealed Km values ranging from 4.9 to 7.0 μM and 6 to 10 μM, respectively, suggesting a high affinity towards OH-BDE formation. In Chapter 4, COSMO-RS was used to predict the boiling points of several PBDEs and methylated derivatives (MeO-BDEs) of OH-BDE metabolites. A linear correlation was achieved by plotting COSMO-RS calculated theoretical boiling points against experimentally determined GC-MS retention times. The model was applied towards the identification of “unknown” PBDEs and previously unidentified BDE-47 and BDE-100 oxidative metabolites. Characteristic fragmentation patterns of MeO-BDE positional isomers facilitated identification of metabolites where reference standards are not available. The relationship between calculated boiling points and experimental retention times allowed prediction of retention times to within 5%. Using calculated boiling points, retention times, and mass spectral fragmentation patterns of the MeO-BDE positional isomers, the identities of the unknown mono-hydroxylated metabolites were proposed to be 2'-OH-BDE-66 from BDE-47, and 2'-OH-BDE-119 and 4-OH-BDE-91 from BDE-100. Issues with resolution have limited the analysis of OH-BDEs via conventional methods such as liquid chromatography-mass spectrometry (LC-MS), where published methods have only been able to analyze for 13 congeners. Likewise, derivatization of OH-BDEs to convert them to GC amenable compounds adds to sample preparation time and limits the column lifetime due to the introduction of trace residues of highly reactive derivatization agents. Chapter 5 reported the development of a SFC-MS/MS method for the analysis of 22 OH-BDEs. The optimized method had four pairs of co-eluting isobaric congeners, but selective MS/MS transitions allowed for all but one pair to be distinguishable from each other. Instrumental limits of detection and limits of quantitation ranged from 2 to 106 fg and 8 to 353 fg injected on column, respectively, which are lower than conventional LC-MS and GC-MS methods. Furthermore, the developed SFC-MS/MS method was successfully applied towards the trace analysis of OH-BDEs produced from in vitro metabolism studies and in human serum samples, demonstrating the applicability of the method for analyzing OH-BDEs different biological matrices.