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Binding and metabolism of tetrachlorobiphenyls by Cytochrome P450 enzymes : structure-activity relationships Edwards, Patrick Robert 2006

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BINDING AND METABOLISM OF TETRACHLOROBIPHENYLS BY CYTOCHROME P450 ENZYMES: STRUCTURE-ACTIVITY RELATIONSHIPS by Patrick Robert Edwards B . S c , University of Guelph, 2004 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in The Faculty of Graduate Studies (Pharmaceutical Sciences) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A October 2006 © Patrick R. Edwards, 2006 Abstract Polychlorinated biphenyls (PCBs) are a class o f persistent organic pollutants that bioaccumulate in humans and wildlife. Cytochrome P450 ( C Y P ) enzymes play a critical role in the biotransformation of PCBs . The objective of this study was to explore structure-activity relationships between the chlorine substitution pattern of P C B s and their interaction with C Y P enzymes. Benzyloxy-, ethoxy- and methoxyresorufin (2-dealkylase ( B R O D , E R O D and M R O D , respectively) activities were determined in the presence of four symmetrically substituted PCBs : 2,2',4,4'-tetrachlorobiphenyl (PCB 47), 2,2',5,5'-tetrachlorobiphenyl (PCB 52), 2,2',6,6'-tetrachlorobiphenyl (PCB 54) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77). The most potent inhibitor of M R O D and E R O D activities was P C B 77 with approximate K i values of 10 n M and 46 n M , respectively. P C B 47 and P C B 52 inhibited M R O D activity with Kj values of 15 u M and 9 u M , respectively, and inhibited E R O D activity with K j values of 5.5 u M and 6.5 u M , respectively. P C B 54 was unable to inhibit E R O D or M R O D activity. P C B 47, 52 and 54 inhibited B R O D activity with K j values of 440 n M , 60 n M and 100 n M , respectively. B R O D activity was unaffected by P C B 77. A validated and optimized method to analyze P C B 54 biotransformation was developed using liquid chromatography-electrospray mass spectrometry. The major metabolite produced was a monohydroxyl metabolite. The hydroxylation position on the biphenyl ring could not be determined due to a lack of P C B 54 metabolite standards. The rate of formation of the monohydroxyl metabolite was compared in hepatic microsomes from corn oi l - , 3-methylcholanthrene (MC)- , phenobarbital (PB)- and dexamethasone (DEX)-treated rats. N o metabolite was detectable when P C B 54 was incubated with i i hepatic microsomes from M C - or corn oil-treated rats. Metabolite formation was greater using hepatic microsomes from PB-treated rats than DEX-treated rats. The involvement of C Y P enzymes was investigated further using a panel of recombinant rat C Y P enzymes. The rate of monohydroxyl metabolite formation was 5.9 pmol/min/pmol C Y P in S U P E R S O M E S expressing C Y P 2 B 1 . The only other recombinant C Y P enzyme that metabolized P C B 54 was C Y P 3 A 1 , which had a rate of formation of 0.03 pmol/min/pmol C Y P . In summary, the results demonstrate the involvement of the C Y P 2 B enzymes and C Y P 3 A enzymes in the biotransformation of P C B 54. i i i Table of Contents Page Title Page i Abstract ii Table of Contents iv List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xii 1. Introduction 1 1.1. Poly chlorinated biphenyls 1 1.1.1. Physical properties of P C B s 1 1.1.2. History of P C B use 3 1.1.3. Toxicological effects : 4 1.1.4. P C B detection in the environment and humans 6 1.1.5. Toxicokinetics of P C B s 10 1.2. Cytochrome P450 enzymes.. 14 1.2.1. C Y P reaction cycle 15 1.2.2. C Y P classification 18 1.2.3. C Y P 1A subfamily 19 1.2.4. C Y P 2 B subfamily 21 1.2.5. C Y P 3 A subfamily 23 1.2.6. C Y P probe substrates 25 1.3. CYP-mediated metabolism of P C B s 27 1.4. Rationale 32 1.5. Hypotheses 34 1.6. Specific aims 34 2. Materials and Methodology 35 2.1. Materials , 35 2.2. Methodology 37 2.2.1. Animal treatments 37 2.2.2. Hepatic microsome preparation 37 2.2.3. Total protein determination 38 2.2.4. Total cytochrome P450 determination 38 2.2.5. Alkoxyresorufin O-dealkylase assays 39 2.2.5.1. Experimental procedure 39 i v - 2.2.5.2. Data analysis 41 2.2.6. P C B 54 biotransformation assay 42 2.2.6.1. Experimental procedure 42 2.2.6.2. Extraction procedure.. 44 2.2.6.3. Sample analysis 45 2.2.6.4. Assay validation 46 2.2.6.4.1. 4 -OH-CB54 standard curve variability 46 2.2.6.4.2. Limits of detection and quantitation ( L O D and L O Q ) 46 2.2.6.4.3. Extraction efficiencies of the monohydroxyl P C B s 47 2.2.6.5. Assay optimization.. 47 2.2.6.5.1. Length of incubation 48 2.2.6.5.2. Microsomal protein concentration 48 2.2.6.5.3. Saturating substrate concentration 49 3. Results 50 3.1. Alkoxyresorufin (9-dealkylase inhibition 50 3.1.1. Inhibition of alkoxyresorufin (9-dealkylase activities by tetrachlorobiphenyls 50 3.1.2. Fitting of data to competitive and mixed models of inhibition 51 3.1.3. Effect of duration of preincubation of microsomes with P C B s on alkoxyresorufin (9-dealkylase activities 64 3.2. P C B 54 biotransformation assay 69 3.2.1. Identification of metabolites 69 3.2.2. Assay validation 75 3.3.2.1. Standard curve validation 75 3.3.2.2. Limits of detection and quantitation ( L O D and L O Q ) 78 3.3.2.3. Extraction efficiencies of the monohydroxyl P C B s 78 3.2.3. Assay optimization 79 3.2.3.1. Length of incubation 79 3.2.3.2. Microsomal protein concentration 80 3.2.3.3. Saturating substrate concentration 85 3.2.4. P C B 54 biotransformation with CYP-induced microsomes 88 3.2.5. Antibody Inhibition Studies '. 90 3.2.6. P C B 54 biotransformation with recombinant C Y P enzymes 93 4. Discussion 95 4.1. Inhibition of alkoxyresorufin (9-dealkylase activities 95 4.2. P C B 54 biotransformation assay 103 4.3. Conclusions '. 108 4.4. Future experiments 110 5. References I l l v Appendices 120 Appendix I: Inhibition of E R O D and M R O D activity 120 Appendix II: Immunoblot analysis of C Y P enzymes expression in hepatic microsomes 122 1. Materials and Methodology 122 1.1. Materials '. '. , 122 1.2. Methodology 124 2. Results 125 3. References 128 Appendix III: Measurement of N A D P H - C Y P reductase activity in hepatic microsomes 129 1. Materials and Methodology 129 1.1. Materials '. 129 1.2. Methodology 130 2. Results '. 130 3. Reference 131 v i List of Tables Page Table 1. Physical-chemical properties of P C B 47, 52, 54 and 77 ........3 Table 2. Total P C B concentration of 103 congeners in Lake Michigan biota 8 Table 3. Comparison of C Y P enzyme levels in rat and human liver microsomes ..19 Table 4. C Y P enzymes responsible for B R O D , E R O D and M R O D activities in microsomes prepared from untreated, M C and PB-treated rats 27 Table 5. Hepatic microsomal metabolites of P C B 52, 77 and 80 30 Table 6. Values for the goodness of fit tests and Kj values 62 Table 7. Inhibitory constants (Kj) of PCBs for inhibition of M R O D , E R O D and B R O D activities .63 Table 8. V and K m values for M R O D , E R O D and B R O D activities 63 Table 9. P C B concentrations used to inhibit alkoxyresorufin O-dealkylase activities 64 Table 10. Masses and relative abundance of hydroxylated P C B s 70 Table 11. Inter-assay variability of 4-OH-CB54 78 Table 12. Intra-assay variability of 4-OH-CB54 78 Table 13. Percent recovery of 4-OH-CB54 and 4-OH-CB104 from the microsomal matrix 79 Table 14. Summary of optimal assay conditions for the formation of the mono- and dihydroxyl metabolites 85 Table 15. Protein Levels of Rat Hepatic Microsomal Cytochrome P450 Enzymes 127 Table 16. N A D P H - C Y P reductase activity in the presence of P C B s 131 v i i List of Figures Figure 1. Generalized P C B structure 2 Figure 2. Tissue distribution of total P C B s in Aroclor 1254-treated rats 11 Figure 3. Potential pathways of biotransformation of P C B s 13 Figure 4. Cytochrome P450 catalytic cycle 17 Figure 5. (9-dealkylation of an alkoxyresorufin compound where x represents the alkyl group 26 Figure 6. Inhibition of M R O D activity by P C B 47 53 Figure 7. Inhibition of M R O D activity by P C B 52 54 Figure 8. Inhibition of M R O D activity by P C B 77 55 Figure 9. Inhibition of E R O D activity by P C B 47 : 56 Figure 10. Inhibition of E R O D activity by P C B 52 ...57 Figure 11. Inhibition of E R O D activity by P C B 77 58 Figure 12. Inhibition of B R O D activity by P C B 47 59 Figure 13. Inhibition of B R O D activity by P C B 52 60 Figure 14. Inhibition of B R O D activity by P C B 54. 61 Figure 15. Effect of preincubation of rat liver microsomes with P C B 77 on M R O D activities 66 Figure 16. Effect of preincubation of rat liver microsomes with P C B 77 on E R O D activities 67 Figure 17. Effect of preincubation of rat liver microsomes with P C B 54 on B R O D activities 68 Figure 18. Negative E S I - M S m/z spectrum of 4 -OH-CB54 70 Figure 19. Negative E S I - M S m/z spectrum of the dihydroxyl-CB54 metabolite 71 Figure 20. Negative E S I - M S m/z spectrum of 4-OH-CB104 71 v i i i Figure 21. Separation of P C B 54 metabolism products and internal standard 73 Figure 22. Time dependent formation of the monohydroxyl P C B 54 metabolite.......74 Figure 23. Time dependent formation of the dihydroxyl P C B 54 metabolite 74 Figure 24. Peak area response of 4-OH-CB54 and 4-OH-CB104 76 Figure 25. Standard curve for 4-OH-CB54 77 Figure 26. Time dependent formation of the monohydroxyl P C B 54 metabolite 81 Figure 27. Time dependent formation of the dihydroxyl P C B 54 metabolite 82 Figure 28. Protein dependent formation of the monohydroxyl P C B 54 metabolite....83 Figure 29. Protein dependent formation of the dihydroxyl P C B 54 metabolite 84 Figure 30. Effect of substrate concentration on the rate of formation of the monohydroxyl P C B 54 metabolite 86 Figure 31. Effect of substrate concentration on the formation of the dihydroxyl P C B 54 metabolite 87 Figure 32. Effect of C Y P inducers on formation of the monohydroxyl P C B 54 metabolite 89 Figure 33. Effect of ant i -CYP2B IgG on monohydroxyl P C B 54 metabolite formation 91 Figure 34. Effect of ant i -CYP2B IgG on monohydroxyl P C B 54 metabolite formation in the presence of sodium cholate 92 Figure 35. Comparison of monohydroxyl P C B 54 metabolite formation by a panel of recombinant C Y P enzymes 94 Figure 36. Inhibition of E R O D activity by P C B 77 120 Figure 37. Inhibition of M R O D activity by P C B 77 121 i x List of Abbreviations (-)-ESI electrospray negative ionization 4-OH-CB54 2,2',6,6'-pentachloro-4-biphenylol 4-OH-CB72 2,3 ',5,5 '-tetrachloro-4-biphenylol 4-OH-CB104 2,2',4',6,6'-pentachloro-4-biphenylol A h R aryl hydrocarbon receptor A I C c Akaike information criterion A U C area under the curve B R O D benzyloxyresorufin (9-dealkylase C A R constitutive androstane receptor C . V . coefficient of variation C Y P cytochrome P450 D E X dexamethasone E R O D ethoxyresorufin (9-dealkylase G C - E C D gas chromatography- electron capture detector G C / L R M S gas chromatography-low resolution mass spectrometry G C / H R M S gas chromatography-high resolution mass spectrometry IgG immunoglobulin-G ip intraperitoneal IS internal standard L C / M S liquid chromatography/mass spectrometry L O D limit of detection L O Q limit of quantitation M C 3 -methy lcho 1 anthrene M D R multidrug resistance transporter M R O D methoxyresorufin (9-dealkylase M S mass spectrometer m/z mass to charge ratio P A R peak area ratio P B phenobarbital P C B polychlorinated biphenyl P C B 47 2,2',4,4'-tetrachlorobiphenyl P C B 52 2,2',5,5'-tetrachlorobiphenyl P C B 54 2,2',6,6'-tetrachlorobiphenyl P C B 77 3,3',4,4'-tetrachlorobiphenyl P C B 80 3,3', 5,5'-tetrachlorobiphenyl P C B 101 2,2',4,5,5'-pentachlorobiphenyl P C B 153 2,2',4,4',5,5'-hexachlorobiphenyl P C D D polychlorinated dibenzodioxin P C D F polychlorinated dibenzofuran P C N pregnenolone 16a-carbonitrile P X R pregnane X receptor R X R retinoid X receptor SD standard deviation X S E M standard error of the mean Sy.x standard deviation of the residuals T C D D 2',3',7',8'-tertrachorobibenzo-/?-dioxin TOF time-of-flight U P L C ultra-performance liquid chromatography x i Acknowledgements Thank you to my supervisor, Dr. Stelvio Bandiera, for his support and guidance. I would also like to thank my committee members, Dr. H . Burt, Dr. A . Frankel, Dr. M . Levine and Dr. W . Riggs for their advice. I would also like to acknowledge Dr. E . Hrycay for quantifying the C Y P enzyme levels and N A D P H - C Y P reductase activities in the hepatic microsomes used in this study and M r . R. Burton for his help operating the L C / M S . I am also thankful to the University of British Columbia and the Natural Sciences and Engineering Research Council of Canada ( N S E R C ) for providing me with funding during my studies. x i i 1. Introduction 1.1. Polychlorinated biphenyls 1.1.1. Physical properties of PCBs Polychlorinated biphenyls (PCBs) are a class of polyhalogenated aromatic hydrocarbons. The basic structure of P C B s is a biphenyl molecule with a variable number of chlorine atoms on the phenyl rings. The general chemical formula of P C B s is C^Hio-nClp, where n represents the number of chlorine atoms on the molecule (Figure 1). There are a total of 209 possible congeners, which differ in the number and position of chlorine atoms on the phenyl rings. P C B s are numbered by a system introduced by Ballschmiter and Ze l l that was subsequently recognized by the International Union of Pure and Applied Chemistry ( IUPAC) . Congeners are numbered according to the number (1-10) and positions (ortho, meta and para) of chlorine atoms on the phenyl rings (Maervoet et al., 2004a). The 2, 6, 2' and 6' positions on the phenyl rings are called the orr/zo-positions. The 3, 5, 3' and 5' positions are called the meta-positions and the 4 and 4' positions are called the /?ara-positions. The presence of chlorine atoms in the ortho-positions reduces the relative planarity of the molecule through steric hindrance (McKinney and Singh, 1981). P C B s that lack chlorine atoms in the orr/zo-positions such as 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 3,3',5,5'-tetrachlorobiphenyl (PCB 80) are called coplanar P C B s because the two phenyl rings are nearly parallel to one another. PCBs containing two or more or/yw-chlorine atoms such as 2,2',4,4'-tetrachlorobiphenyl (PCB 47), 2,2',5,5'-tetrachlorobiphenyl (PCB 52) and 2,2',6,6'-tetrachlorobiphenyl (PCB 1 54) are called noncoplanar P C B s because the two phenyl rings are nearly perpendicular to each other. The coplanarity of a P C B can be measured by the dihedral angle between the two phenyl rings (McKinney and Singh, 1981). A s the number of chorine atoms in the ortho positions increases, the coplanarity is reduced, which is seen by an increase in the dihedral angle between the phenyl rings. Table 1 summarizes the dihedral angle for P C B 47, 52, 54 and 77. 5 6 6' 5 ' Figure 1. Generalized P C B structure General P C B structure with carbon atoms numbered and arene substitutions indicated. Meta is abbreviated as m, ortho as o and para as p. 2 Table 1. Physical-chemical properties of P C B 47, 52, 54 and 77 Congener Dihedral Angle 3-dimensional structure P C B 47 (2,2',4,4'-tetrachlorobiphenyl) 73.0° P C B 52 (2,2',5,5'-tetrachlorobiphenyl) 73.0° P C B 54 (2,2',6,6'-tetrachlorobiphenyl) 89.9° P C B 77 (3,3',4,4'-tetrachlorobiphenyl) 37.8° ^ j 1 3-Dimensional structures were generated with ACD/ChemSketch (v. 8.0, Advanced Chemistry Development Inc., Toronto, ON). Dihedral angles were taken from Hrycay and Bandiera (2003). 1.1.2. History of PCB use P C B s were first synthesized in 1864; however, their production for commercial use did not begin until the 1930s (Noren and Meironyte, 2000; W H O , 1993). The physical-chemical properties of PCBs , such as their chemical inertness, low vapour pressure, high electrical impedance and resistance to thermal degradation made them suitable for a variety of uses. They were predominantly used as dielectric fluids in electrical transformers and capacitors and were also used in carbonless copy paper, adhesives, plasticizers and inks ( W H O , 1993). 3 P C B s were commercially manufactured by the progressive chlorination of biphenyl in the presence of a catalyst, such as iron chloride. Due to the production process, commercial products were a complex mixture o f P C B congeners with varying degrees of chlorination (Hardy, 2002). The major North American manufacturer was Monsanto Company, which marketed a number of P C B mixtures under the name Aroclor. The most common Aroclor mixtures were Aroclor 1242, 1248, 1254 and 1260 (Norstrom, 1988). A l l Aroclor mixtures contained four numbers, in which the first two digits indicated that the molecule was a biphenyl molecule and the last two indicated the chlorine content by weight (Hardy, 2002; Norstrom, 1988). PCBs were first recognized as environmental contaminants in the late 1960s (Jensen et al., 1969). Due to evidence of adverse health effects and their widespread presence in the environment, the production of P C B s was banned in the United States at the end of the 1970s ( A T S D R , 2001). B y this time, an estimated 501 600 000 kg of PCBs had been sold in the United States (Hardy, 2002). In Canada, the import, manufacture and sale of P C B s was made illegal in 1977 and the release of P C B s into the environment was made illegal in 1985 (Environment Canada, 2003). 1.1.3. T o x i c o l o g i c a l effects P C B s are known to produce a wide spectrum of toxic effects. Administration of commercial P C B mixtures to animals produced many adverse health effects including thymic atrophy, neurotoxicity, hepatotoxicity, reproductive and developmental toxicity, immunotoxicity and endocrine disrupting effects (Safe, 1994). The effects o f P C B exposure in humans have been investigated through epidemiological studies of exposed populations and the effects are less clear than in animal studies. One group that has been 4 extensively studied is a group of Taiwanese individuals who consumed P C B contaminated rice-bran oi l between 1978 and 1979. In addition to being contaminated with P C B s , the rice-bran oil was also contaminated with polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzodioxins (PCDDs). Victims of this incident displayed a number of adverse effects including, chloracne, hepatic damage and alterations in hepatic enzyme and blood hormone levels. Children of affected mothers experienced developmental delays, lower birth weight and neuropsychological abnormalities (Hsu et al., 2005). Safe (1993) states that the health effects seen in these individuals were more likely attributable to the high levels of P C D F s than the P C B s in the rice-bran o i l v Epidemiological studies have also been conducted in workers occupationally exposed to high levels of PCBs . The results of these studies led to contradictory conclusions with regard to P C B exposure and cancer risk. In a retrospective cohort study of 3569 workers at an electrical capacitor manufacturing plant in Indiana that were exposed to PCBs , workers were found to have reduced overall cancer mortality but increased melanoma and brain cancer mortality compared to the general population in Indiana (Ruder et al., 2006). Other studies have reported increased incidence of pancreatic cancer, liver cancer and lymphoma in workers exposed to P C B s (Ruder et al., 2006). In a review of six previous retrospective cohort studies, Bosetti et al. (2003) reported no increased cancer mortality due to occupational exposure to PCBs . Furthermore, no increase in mortality from liver, breast, lymphatic or haematopoietic cancers was found. The International Agency for Research on Cancer lists P C B s as probable human carcinogens due to sufficient evidence of carcinogenicity in animals, but lack of evidence in humans (Ruder et al., 2006). 5 Studies using individual congeners of differing substitution patterns have shown that coplanar and noncoplanar P C B s exhibit distinct toxicological effects in rats. Noncoplanar P C B s have carcinogenic, neurotoxic and endocrine effects. They also interfere with second messenger signaling mechanisms and cellular calcium ion homeostasis (Fischer et al., 1998; Gillette et al., 2001). Exposure to coplanar P C B s has been shown to cause weight loss, chloracne, thymic atrophy, tumor promoter activity and immune suppression (Safe, 1994). 1.1.4. PCB detection in the environment and humans Despite the ban on production and restrictions on their use, large amounts of PCBs continue to be found in the environment due to their resistance to microbial, photochemical and chemical degradation (Letcher et al., 1996; W H O , 1993). P C B s have been detected worldwide, including in isolated regions such as the Arctic, and they have been identified in nearly every organism and matrix examined (Connor et al., 1997; Letcher et al., 1996). P C B s are able to reach remote regions (such as the Arctic) from Europe, As ia and North America through atmospheric transport and ocean currents (Borga et a l , 2005; Sandala et al., 2004). The hydrophobicity of P C B s and their resistance to biological degradation contributes to the accumulation of these compounds in the fatty tissues of animals and their biomagnification in food webs (Letcher et al., 1996; Sandala et al., 2004). Due to bioaccumulation, animals at higher trophic levels have much greater P C B levels than animals in the lower trophic levels. The magnitude of bioaccumulation between different trophic levels is influenced by a number of factors. In aquatic food webs, P C B concentrations in water, sediment and (food all affect trophic transfer. Trophic transfer is 6 J also affected by feeding behaviour, food chain length, uptake kinetics and metabolism of PCBs (Kwon et al., 2006). Congeners that are easily metabolized show a lower bioaccumulation potential than congeners that are resistant to metabolism. A s a result, the congener pattern changes between trophic levels. The congener pattern varies between animals of the same species due to differences in diet, age and sex (Boon et al., 1997). Table 2 presents the results of a study examining P C B bioaccumulation in a food web in Lake Michigan. From this table it is evident that animals in the higher trophic levels such as lake trout and whitefish have higher levels of P C B s than animals in the lower tropic levels, such as shrimp and zooplankton. In a study o f a Lake Ontario food web, the length of the food chain was shown to significantly affect P C B concentrations. Each trophic level was found to contribute to a 3.5 fold biomagnification in P C B tissue concentrations (Rasmussen et al., 1990). Similar findings were shown by K w o n et al. (2006), who found that P C B concentrations increased three- to five-fold per trophic level in a Lake Erie food web. A n important implication of these findings was that in areas of low contamination, P C B concentrations can be biomagnified through the food chain resulting in levels in fish that exceed the Canadian guidelines for human consumption (Rasmussen et al., 1990). 7 Table 2. Total PCB concentration of 103 congeners in Lake Michigan biota Sample n I P C B (ng/g ww) Lake trout 5 1200 ± 460 Whitefish 6 280 ± 2 3 0 Deepwater sculpin 6 120 ± 5 5 Bloater chub 9 3 1 0 ± 97 Alewife 8 230 ± 7 0 Rainbow smelt 4 80 ± 2 4 Benthic amphipods 10 25 ± 8 Mys id shrimp 5 12 ± 4 Bulk zooplankton 5 20 ± 11 Data are presented as the mean ± the standard deviation (SD). Whole body P C B concentrations were the sum of 103 congeners. Data presented as ng/g wet weight (ww). Data from Stapleton and Baker (2003). Human exposure to P C B s mainly occurs through food. M i l k , meat and fish are the most important sources of dietary exposure (Maervoet et al., 2004b). Intake of PCBs varies considerably depending upon national and regional diets. In Sweden, based upon estimated intakes of various food groups, fish were the main source of P C B s in the diet, contributing to 57% of the total intake. This was followed by dairy products and meats, which contributed to 14 and 12% of the total intake, respectively (Darnerud et al., 2006). In an analysis of foods in the Canadian diet, fish and butter were found to contain the highest concentration of P C B s . Based upon the estimated consumption of various foods, the total daily intake of P C B s was estimated to be 5.7 ng/kg/day (Newsome et al., 1998). Dairy products were the greatest contributor to the daily intake, representing 40% of the daily intake. The second greatest contributor was meat products (26%), which included beef, pork, veal, lamb, eggs and processed meats (Newsome et a l , 1998). In contrast to Sweden where fish was the source of 57% of P C B s in the diet, the consumption of fish by 8 Canadians only represented 16% of the total daily P C B intake (Darnerud et al., 2006; Newsome et al., 1998). Inuit populations are particularly susceptible to accumulating high levels of PCBs and other organohalogens as a result of their traditional diet that contains sea mammal fat (Ayotte et al., 1997). In a study of plasma P C B concentrations, Inuit residing in Nunavik had a mean total P C B concentration of 4.1 mg/kg lipid, while residents of southern Quebec had levels that were 30-fold lower (Ayotte et al., 1997). A comparison of the levels of P C B 138 (2,2',3,4,4',5-hexachlorobiphenyl) and P C B 153 (2,2',4,4',5,5'-hexachlorobiphenyl) in umbilical cord plasma between Nunavik and southern Quebec groups found that plasma concentrations were on average four-fold higher in the Nunavik group (Pereg et al., 2002). Similarly, the concentration of P C B s in breast milk was shown to be five times higher in women from Nunavik compared to women from southern Quebec (Ayotte et al., 1997). Levels of organohalogens in breast milk are an indicator o f the total levels in adipose tissue. The concentrations o f organohalogens in the breast milk of Swedish women have been studied to provide an indication of the environmental contamination and background levels in the Swedish population since 1967. Beginning in the 1970s, when the use of P C B s became restricted, P C B levels in breast milk of Swedish women have decreased exponentially (Noren and Meironyte, 2000). A decrease in P C B levels in humans has also been shown in other countries including Canada (Noren and Meironyte, 2000; Craan and Haines, 1998). In 1997, PCBs represented 67% of the total 483 ng of organohalogens/g l ipid in Swedish human breast milk (Noren and Meironyte, 2000). P C B s are also often the most predominant 9 organohalogen contaminant in the environment (Berger et al., 2004). In a study of organochlorine contamination of bald eagle eggs and nestlings from the Canadian great lakes, P C B s were the greatest contributor to the organohalogen burden (Donaldson et al., 1999). In an analysis of diets of harbor seals inhabiting Puget Sound ( W A , U S A ) and the Straight of Georgia ( B C , Canada), the major contaminant was P C B s (Cullon et al., 2005). 1.1.5. Toxicokinetics of PCBs The toxicokinetics of P C B s are complicated by the fact that 209 congeners exist. In general terms, the lipophilic nature of P C B s allows for high absorption following oral and dermal exposure (James, 2001). In studies examining dermal absorption of PCBs , the degree of chlorine substitution was found to affect the rate at which P C B s were absorbed. Lower chlorinated P C B s had the highest rate of absorption and increasing chlorination reduced the rate of absorption (Garner et al., 2006). Once absorbed into the systemic circulation, P C B s are rapidly distributed by the blood throughout the body. In the blood, P C B s bind nonspecifically to blood cells and plasma proteins (Matthews and Dedrick, 1984). The initial distribution of P C B s is determined by tissue volume, binding to proteins and perfusion rate. Highly perfused tissues such as the liver and large tissues such as muscles, serve as the initial sites of P C B accumulation. During the initial distribution phase, P C B s are also redistributed to lipid-rich tissues, which eventually leads to accumulation o f P C B s in adipose tissue (Matthews and Dedrick,. 1984). Figure 2 shows the tissue distribution of total P C B s in rats one week after a single treatment of 0.05 mmol/kg Aroclor 1254 administered intraperitoneally (ip). Adipose and skin tissue contained the greatest concentrations of PCBs but they were also detectable in the spleen, serum, lung, liver, kidney, heart and brain (Kania-Korwel et al., 2005). P C B s can be 10 excreted unchanged in the urine and feces; the extent of excretion is congener dependent and can vary between species. In some female animals, l ipid sinks such as milk or eggs provide an alternate route for P C B excretion (Hardy, 2002). Clearance of P C B s is minimal without metabolism to more water soluble metabolites (Hardy, 2002). Figure 2. Tissue distr ibution of total P C B s in Aroc lor 1254-treated rats Levels of total PCBs per wet weight of tissue in rats one week following a single ip treatment with 0.05 mmol/kg Aroclor 1254. Figure reprinted from Kania-Korwel et al. In rats administered a single ip injection of 2,2',4,5, 5'-pentachlorobiphenyl (PCB 101), analysis of liver and serum detected mono- and di-hydroxylated metabolites as well as methylsulfonyl metabolites (Haraguchi et al., 2004). Hydroxylated and methylsulfonyl P C B metabolites have also been detected in the blood of wildlife and humans (Letcher et al., 2005; Sandala et al. 2004). A generalized overview of the possible routes of P C B metabolism is shown in Figure 3. The first step in the Blood (2005). 1 1 biotransformation of P C B s is oxidation by cytochrome P450 ( C Y P ) , producing an arene oxide or hydroxylated metabolite. Unstable arene oxide intermediates spontaneously rearrange to form hydroxylated metabolites, while more stable arene oxide metabolites can undergo further metabolism by epoxide hydrolase and glutathione-S-transferase. Glutathione conjugates can then be converted via a series of enzymatic reactions to methylsulfonyl metabolites, which have been shown to reduce thyroid hormone levels in rats and produce antiestrogenic effects (Safe, 1994; McGraw and Waller, 2006; Haraguchi et al., 2005). Hydroxylated metabolites can be excreted unchanged, undergo further hydroxylation or be conjugated with glucuronide or sulfate (Sandau et al., 2000; James, 2001; Daidoji et al., 2005). The involvement of dihydrodiol dehydrogenase in the formation of dihydrodiol metabolites has been suggested, but not proven (James, 2001). The Phase III enzymes are important in the efflux of a variety of lipophilic xenobiotics. The multidrug resistance ( M D R ) transporter is located on the plasma membrane of endothelial cells that make up the blood brain barrier, as well as the plasma membrane of lung, testes and intestinal cells. In studies examining the role of the human M D R 1 transporter as an efflux transporter of PCBs , the P C B congeners tested were found to bind to M D R 1 but were not transported by it, suggesting that P C B s are not substrates of M D R 1 (Tampal et al., 2003). Due to the fact that the biotransformation of P C B s is dependent upon initial C Y P mediated oxidation, C Y P enzymes are important in determining the bioaccumulation and elimination of P C B s (Bandiera, 2001). 12 4-monochlorobiphenyl Figure 3. Potential pathways of biotransformation of P C B s Enzymatic pathways by which PCBs can be biotransformed, using 4-monochlorobiphenyl as an example. E H : epoxide hydrolase; D H D : dihydrodiol dehydrogenase; U G T : UDP-glucuronosyltransferase; S U L T : sulfotransferase; GST: glutathione S-transferase (Adapted from James, 2001). 13 1.2. Cytochrome P450 enzymes The biotransformation of lipophilic xenobiotics is a two phase process. Phase I reactions are typically oxidative, reductive or hydrolytic reactions that expose or add a functional group on to the xenobiotic. Phase I biotransformation reactions serve to slightly increase the polarity of the xenobiotic and to provide a functional group for Phase II biotransformation. Phase II biotransformation is a conjugation reaction in which an endogenous moiety is added on to the xenobiotic producing a more water soluble product (Williams, 2002). The C Y P superfamily of hemoproteins are quantitatively and qualitatively the most important Phase I enzymes (Bandiera, 2001). In addition to catalyzing the biotransformation of xenobiotics, C Y P enzymes are also involved in the biotransformation of endogenous compounds and the biosynthesis of signaling molecules used to control homeostasis and development (Denisov et al., 2005). C Y P enzymes are present in all organisms including mammals, birds, amphibians, insects, plants, fungi and bacteria (Lewis, 1996; Guengerich et al., 2005). In eukaryotes, C Y P enzymes are located in the membrane of the endoplasmic reticulum (Josephy et al., 1997). In mammals, C Y P enzymes are found in almost all tissues including the mucosa of the small intestine, the lung, kidney, brain, olfactory mucosa and the skin (Paine et al., 2006). Concentrations and expression of individual C Y P enzymes differ between tissues; the liver contains the highest concentration of C Y P enzymes involved in xenobiotic biotransformation (Parkinson, 2001). 14 1.2.1. C Y P reaction cycle The C Y P reaction cycle is a series of steps that involves binding of substrate, the cleavage of the dioxygen bond of molecular oxygen producing an oxygen atom and insertion of the oxygen atom into the substrate. The overall stoichiometry of a C Y P -mediated hydroxylation reaction is represented as follows: P v H + N A D P H + H + + 0 2 - » R O H + N A D P + + H 2 0 where R H is the substrate and R O H is the hydroxylated product. Mammalian C Y P enzymes use N A D P H as a cofactor (Josephy et al., 1997). The catalytic cycle of C Y P enzymes occurs in several discrete steps (Figure 4). Substrates bind in a cavity that is in close proximity to the heme moiety. In the absence of substrate, the heme iron of C Y P is hexacoordianted, with the four nitrogen atoms of the porphyrin molecule, a cysteine residue of the C Y P enzyme and the sixth coordinate site is occupied by a molecule of water. Substrate binding displaces the water molecule that occupies the sixth coordinate site of the heme iron and results in a change in the iron spin state from a low to high-spin state (Josephy et al., 1997). The low-spin state is characterized by an absorbance maximum between 416 and 419 nm and the high-spin state is characterized by an absorbance maximum between 390 and 416 nm. Binding of a substrate typically results in a type I difference spectrum, which contains a peak at approximately 390 nm and a trough at 419 nm, depending upon the C Y P enzyme and substrate (Josephy et al., 1997). The high-spin Fe has a more positive redox potential and this facilitates the transfer of an electron from N A D P H via C Y P reductase to the heme iron. Transfer of an electron reduces the heme iron to its ferrous form, which then allows molecular oxygen to bind forming an iron dioxygen complex (Fe -O2). The 15 reduction of the iron dioxygen complex by a second electron produces a peroxo-iron intermediate (Fe 3 +-C>2 2"), which is followed by protonation at the distal oxygen atom forming a hydroperoxo-iron intermediate ( F e 3 + - 0 - O F f ) . The addition of a second proton leads to cleavage of the bond linking the two oxygen molecules, forming a molecule of water and a reactive iron-oxo complex ((Fe 5 + =0) 3 + ) that transfers the oxygen atom to the substrate (Denisov et al., 2005; Josephy et al., 1997; Lewis, 1996; Yasui et al., 2005). 16 Figure 4. Cytochrome P450 catalytic cycle Enzymatic steps by which C Y P enzymes catalyze the hydroxylation of a substrate (RH) to a product (ROH). (Adapted from Yasui et al., 2005) 17 1.2.2. C Y P classification A wide variety of structurally diverse compounds are substrates for C Y P enzymes, due to the presence of multiple C Y P isoforms, which have broad and overlapping substrate specificity (Bandiera, 2001). To date, nearly 4000 C Y P genes have been identified (Denisov et al., 2005). C Y P enzymes are classified based upon their amino acid sequence homology; those in the same family have at least 40% sequence similarity, while C Y P enzymes in the same subfamily have greater than 55% sequence similarity. Individual C Y P enzymes are identified by a number indicating the family, a letter that identifies the subfamily and a final number that specifies the individual protein (e.g. C Y P 1 A 2 ) (Nebert and Gonzalez, 1987). In humans, 57 C Y P genes and 17 families of C Y P enzymes have been identified thus far (Guengerich et al., 2005; Honkakoski and Negishi, 2000). The major substrate classes for C Y P enzymes in humans are sterols, xenobiotics, fatty acids, eicosanoids and vitamins. C Y P 5 and C Y P 8 A families are involved in the production of thromboxane and prostacyclin. The C Y P 1 1 , C Y P 17, C Y P 19 and CYP21 families play an important role in the synthesis of steroids and C Y P enzymes in the C Y P 7 , C Y P 8 B , C Y P 2 4 , C Y P 2 7 , C Y P 4 6 and C Y P 5 1 gene families are involved in the synthesis of bile acid, vitamin D 3 and cholesterol (Honkakoski and Negishi, 2000). Although many C Y P enzymes have been identified, the roles of some C Y P enzymes such as C Y P 2 A 7 , C Y P 2 S 1 , C Y P 2 U 1 and C Y P 2 W 1 remain unknown, as substrates for these enzymes have not been identified (Guengerich et al., 2005). The levels of the different C Y P enzymes vary among species. Table 3 compares the C Y P enzyme levels in human and rat liver microsomes. In humans, the C Y P 3 A enzymes are the predominant C Y P enzymes expressed in the liver 18 and the intestinal tract, while in rats, the C Y P 2 C enzymes are the major hepatic form (Bandiera, 2001; Kliewer et al., 2002). The first four C Y P families are responsible for the biotransformation of a wide variety of anthropogenic and endogenous compounds (Honkakoski and Negishi, 2000). In the following three sections, the expression, regulation and substrate specificities of the C Y P 1 A , C Y P 2 B and C Y P 3 A subfamilies wi l l be briefly discussed. Table 3. Comparison of CYP enzyme levels in rat and human liver microsomes Rat Liver Microsomes Human Liver Microsomes C Y P Enzyme Specific Percent of Specific Percent of Content Total C Y P Content Total C Y P (pmol/mg) (pmol/mg) C Y P 1 A 5-20 . 1-3 1-65 7-18 C Y P 2 A 20-40 3-5 1-27 1-7 C Y P 2 B 5-20 1-3 0-3 0-0.5 C Y P 2 C 380-650 40-65 30-90 12-24 C Y P 2 D Not Known ? 1-9 0.2-4 C Y P 2 E 60-80 8-10 10-34 4-10 C Y P 3 A 40-100 5-12 45-147 18-40 (adapted from Bandiera, 2001) 1.2.3. CYP1A subfamily Two C Y P enzymes are present in the 1A subfamily, C Y P 1 A l and C Y P 1 A 2 . These enzymes are highly conserved among species (Lewis, 1996). Both C Y P 1 A 1 and C Y P 1 A 2 metabolize planar molecules; however, they exhibit overlapping but different substrate specificity. Substrates for C Y P 1 A 2 include some drugs such as caffeine, olanzapine and phenacetin (Williams, 2002). C Y P 1 A 2 is also responsible for the metabolism of many environmental aromatic amines including 4-aminobiphenyl, 2-napthylamine and 2-acetylaminofluorene (Nebert et al., 2004; Nerurkar et al., 1993). 19 C Y P 1 A 1 preferentially metabolizes neutral polycyclic aromatic hydrocarbons and nitroarenes such as benzo(a)pyrene and nitropyrene (Lewis, 1996). C Y P 1 A 2 is constitutively expressed in the liver but is not detectable in other tissues and C Y P 1 A 1 expression is not detectable in the liver of uninduced animals (Nebert et al., 2004; Sesardic et al., 1990). Both C Y P 1 A 1 and C Y P 1 A 2 are inducible enzymes, which are up-regulated by the activation of the aryl hydrocarbon receptor (AhR) (Fujii-Kuriyama and Mimura, 2005; Nebert et al., 2004). Following treatment with the C Y P 1 A inducer 3-methylcholanthrene ( M C ) , C Y P 1 A l levels in rats increase from undetectable levels to account for 40% of the total C Y P content in the liver (Sesardic et al., 1990). C Y P 1 A 1 levels are also induced in the lung, small intestine and kidneys after treatment with C Y P 1 A inducers. C Y P 1 A 2 levels in the liver also increase but C Y P 1 A 2 remains undetectable in extrahepatic tissues following administration of inducers such as M C (Sesardic etal. , 1990). The A h R is a ligand-activated transcription factor that is conserved across many species including mammals and invertebrates (Fujii-Kuriyama and Mimura, 2005). Once activated, the A h R translocates to the nucleus where it forms a heterodimer with the aryl hydrocarbon nuclear translocator. This heterodimer complex then binds to a xenobiotic response element in the promoter region upstream of the C Y P 1 A 1 and C Y P 1 A 2 genes. Binding of the heterodimer complex to the xenobiotic response element results in recruitment of coregulatory proteins and relaxation of chromatin structure, which promotes transcription (Fujii-Kuriyama and Mimura, 2005). One o f the most potent inducers of C Y P 1 A expression is the exogenous compound, 2',3',7',8'-tetrachorodibenzo-/7-dioxin (TCDD) ; other exogenous A h R ligands include M C , indolo[2,3-6]carbazole and 20 coplanar P C B s (Fujii-Kuriyama and Mimura, 2005; Safe, 1997). Endogenous A h R ligands include tryptamine, bilirubin and indirubin (Fujii-Kuriyama and Mimura, 2005). Upregulation of C Y P 1 A 1 m R N A expression in hepatic and endothelial cells has been seen upon administration of the glucocorticoid, dexamethasone ( D E X ) . This is thought to be due to protein-protein interactions between the glucocorticoid receptor and other transcription factors (Honkakoski and Negishi, 2000). However, upregulation following treatment with D E X is much less compared to treatment with A h R agonists such as M C (2 to 4-fold versus 50-fold induction, respectively) (Honkakoski and Negishi, 2000; Lewis, 1996). 1.2.4. C Y P 2 B s u b f a m i l y The C Y P 2 B subfamily contains C Y P s that are responsible for steroid hydroxylation and the biotransformation of a variety of xenobiotics that are typically hydrophobic and noncoplanar. Some exogenous substrates include phenobarbital, related barbiturates and organochlorine pesticides including ;?,/?'-dichlorodiphenyltrichloroethane (DDT), chlordane, heptachlor and aldrin (Lewis, 1996). C Y P 2 B enzymes display distinct sex and tissue-specific regulation (Honkakoski and Negishi, 2000). Members of the C Y P 2 B subfamily in rats include, C Y P 2 B 1 , C Y P 2 B 2 and C Y P 2 B 3 (Nelson, 2005). C Y P 2 B 2 and C Y P 2 B 3 are constitutively expressed in the liver while C Y P 2 B 1 is constitutively expressed in the lung and testis. Both C Y P 2 B 1 and C Y P 2 B 2 are induced in the liver following exposure to phenobarbital (PB) while C Y P 2 B 3 is not inducible (Lewis 1996; Soucek and Gut, 1992). C Y P 2 B 1 is highly inducible and levels of C Y P 2 B 1 were observed, in our study, to increase from undetectable levels to 32% of total C Y P content in hepatic microsomes following PB-treatment (Appendix II). 21 C Y P 2 B 2 is only moderately inducible, with levels increasing 12-fold following P B -treatment (Lewis 1996). Two C Y P 2 B genes have been identified in humans. Transcripts of both C Y P 2 B 6 and C Y P 2 B 7 are detectable in human liver samples but only C Y P 2 B 6 is believed to be translated into a protein (Hanna et al., 2000). C Y P 2 B 6 shows sex and ethnic differences in expression and activity. Levels of C Y P 2 B 6 protein and activity are approximately 1.6 fold higher in females than males and Hispanic females have been shown to have higher C Y P 2 B 6 activity than Caucasian or African-American females (Lamba et a l , 2003). In cultured human hepatocytes, C Y P 2 B 6 expression increases following treatment with rifampicin, dexamethasone and phenobarbital (Ekins et al., 1998). Induction of the C Y P 2 B enzymes by PB-type inducers occurs mainly through the activation of the constitutive androstane receptor ( C A R ) . Following exposure to PB-type inducers, C A R dissociates from a protein complex that sequesters it in the cytoplasm. C A R then translocates to the nucleus and dimerizes with R X R . The heterodimer binds to and activates a PB-responsive element upstream of the C Y P 2 B genes (Qatanani and Moore, 2005; Wang and Negishi, 2003). In addition to P B , the C Y P 2 B enzymes can be induced by a variety of antimicrobials, barbiturates and pesticides. Some examples include clotrimazole, rifampicin, phenytoin, methoxychlor and D D T ( L i et al., 1995; Wang and Negishi, 2003). Exposure to noncoplanar P C B s has also been shown to induce the C Y P 2 B enzymes (Denomme et a l , 1983). Transcriptional activation of the C Y P 2 B genes appears to be mainly regulated by C A R ; however, the pregnane X receptor (PXR) and the glucocorticoids receptor may be required for optimal activation (Wang and Negishi, 2003). 22 1.2.5. C Y P 3 A s u b f a m i l y In rats, the C Y P 3 A subfamily includes, C Y P 3 A 1 , C Y P 3 A 2 , C Y P 3 A 9 , C Y P 3 A 1 8 and C Y P 3 A 6 2 (Matsubara et al., 2004; Nelson, 2005). A sixth rat C Y P 3 A enzyme, C Y P 3 A 2 3 , has also been reported in the literature, but this is now believed to be the product of an allelic variant of C Y P 3 A 1 (Matsubara et al., 2004; Rekka et al., 2002). The C Y P 3 A enzymes catalyze the biotransformation of a wide variety of compounds, such as bile acids, steroids and numerous drugs. C Y P 3 A 1 and C Y P 3 A 2 have similar substrate specificities and both are responsible for 6P-hydroxylation of testosterone (Lewis et al., 1996). C Y P 3 A 2 , C Y P 3 A 9 and C Y P 3 A 1 8 display sex specific regulation (Cooper et al., 1993; Mahnke et al., 1997). In male rat livers, C Y P 3 A 2 is the predominant C Y P 3 A form (Rekka et al., 2002). C Y P 3 A 2 is constitutively expressed in hepatic microsomes of immature rats and adult male but not adult female rats (Cooper et al., 1993). C Y P 3 A 1 8 is also a male specific form and its expression has been reported to be independent of age (Mahnke et al., 1997; Matsubara et al., 2004). Expression of C Y P 3 A 9 is developmentally controlled in both sexes and is undetectable in rats less than 5 weeks of age (Mahnke et al., 1997). In adult rats, C Y P 3 A 9 is a female-dominant form (Matsubara et al., 2004). Along with C Y P 3 A 9 , C Y P 3 A 6 2 is a major intestinal C Y P 3 A form in male and female rats but C Y P 3 A 6 2 protein is only detectable in the liver of female rats (Matsubara et al., 2004). C Y P 3 A 1 is non-detectable in hepatic microsomes from untreated mature rats of either sex (Cooper et al., 1993). Following administration of D E X , C Y P 3 A 1 is induced and accounts for 30-37% of total C Y P content in hepatic microsomes of immature and mature rats of both sexes (Cooper et al., 1993). Levels of C Y P 3 A 2 increase in hepatic microsomes of immature rats of both sexes and mature male 23 rats following D E X treatment but no increase is seen in hepatic C Y P 3 A 2 levels of adult female rats (Cooper et a l , 1993). C Y P 3 A 9 , C Y P 3 A 1 8 and C Y P 3 A 6 2 expression are also induced in livers of adult male and female rats following administration of D E X (Cooper et a l , 1993; Matsubara et al., 2004). Other inducers of rat C Y P 3 A enzymes include P B , rifampicin, isosafrole and pregnenolone 16a-carbonitrile (PCN) (Cooper et al., 1993). C Y P 3 A 4 is the predominant C Y P enzyme in human liver (Kliewer et al., 2002). The C Y P 3 A enzymes are also the most abundant C Y P enzymes in the small intestine (Paine et al., 2006). The C Y P 3 A subfamily is responsible for the biotransformation of greater than 50% of all prescription medications (Kliewer et al., 2002). The human C Y P 3 A subfamily also contains C Y P 3 A 5 , C Y P 3 A 7 and C Y P 3 A 4 3 . C Y P 3 A 5 is expressed in only 20% of human livers. Absence of C Y P 3 A 5 is due to a splice site mutation (Daly, 2006). C Y P 3 A 7 accounts for 30-50% of total C Y P content in the fetal liver.. C Y P 3 A 7 is now recognized to be expressed in adult livers as well and is estimated to account for approximately 20% of C Y P 3 A content in the liver (Daly, 2006). C Y P 3 A 4 3 m R N A is expressed at low levels in the liver and at relatively high levels in the testis and prostate. In earlier studies, a fifth C Y P 3 A enzyme ( C Y P 3 A 3 ) was identified. This enzyme showed strong homology to C Y P 3 A 4 ; however, it is now believed that C Y P 3 A 3 in fact arises from an allelic variant of C Y P 3 A 4 (Daly, 2006). Both C Y P 3 A 5 and C Y P 3 A 7 have similar substrate specificities to C Y P 3 A 4 . The substrate specificity of C Y P 3 A 4 3 is unknown since, only low levels of expression has been achieved in recombinant systems (Daly, 2006). 24 The C Y P 3 A 4 and C Y P 3 A 5 enzymes are inducible following administration of rifampicin, although C Y P 3 A 5 is induced to a lesser extent than C Y P 3 A 4 (Daly, 2006). Induction of the C Y P 3 A subfamily occurs mainly through activation of P X R . P X R binds as a heterodimer with the retinoid X receptor ( R X R ) to xenobiotic response elements in the proximal promoter regions of the C Y P 3 A genes (Kliewer et al., 2002). Induction of the C Y P 3 A subfamily show distinct species-specific differences. For example, rifampicin is an efficacious inducer of human C Y P 3 A 4 but rat C Y P 3 A 2 is only weakly induced (Kliewer et al., 2002). Studies have also suggested that C A R may be involved in transcriptional activation of the C Y P 3 A genes since C A R can bind to xenobiotic response elements in the promoter region of the C Y P 3 A genes (Kliewer et al., 2002). Experimental evidence has also suggested that the glucocorticoid receptor is involved in the induction of C Y P 3 A 4 ; however, the molecular mechanism is not clear (Burk and Wonjnowski, 2004). 1.2.6. CYP probe substrates The (9-dealkylase activities of alkoxyresorufin compounds are commonly used probes to characterize and quantify the induction of C Y P enzymes in experimentally induced animals (Burke et al., 1994). A number of alkoxyresorufin compounds are available that differ in the alkyl group attached to the resorufin molecule by an ether linkage. C Y P enzymes catalyze the oxidation at the aliphatic carbon a to the ether linkage producing an unstable hemiacetal that dissociates producing the fluorescent compound, resorufin (Chen et al., 2003). A generalized dealkylase reaction is shown in Figure 5. C Y P enzymes show differences in catalytic activity towards alkoxyresorufin compounds with varying alkyl groups. Furthermore, the contribution of an individual 25 C Y P enzyme catalyzing an alkoxyresorufin (9-dealkylase reaction is dependent upon the levels of that C Y P enzyme in the liver. In hepatic microsomes prepared from MC-treated rats, ethoxyresorufin (9-dealkylation (EROD) activity is mainly catalyzed by C Y P 1 A 1 , while in hepatic microsomes prepared from untreated rats, E R O D activity is predominately catalyzed by C Y P 2 C 6 (Burke et al., 1994). This demonstrates that alkoxyresorufin (9-dealkylase activities are not specific probes for an individual C Y P enzyme in all cases. The C Y P enzymes involved in benzyloxy-, ethoxy- and methoxyresorufin (9-dealkylase ( B R O D , E R O D and M R O D , respectively) activities in microsomes from untreated, MC-treated and PB-treated rats are shown in Table 4. C Y P N A D P H , 0 2 7-alkoxyresorufin resorufin Figure 5. 0-dealkylation of an alkoxyresorufin compound where x represents the alkyl group 26 Table 4. CYP enzymes responsible for BROD, EROD and MROD activities in microsomes prepared from untreated, MC and PB-treated rats Activity Microsomes Untreated MC-treated PB-treated B R O D C Y P 2 B 1 , C Y P 2 C 1 1 C Y P 1 A 1 , C Y P 1 A 2 CYP2B1, C Y P 2 B 2 E R O D CYP2C6, C Y P 2 B 1 . C Y P 2 C 1 1 , C Y P 3 A 1 , C Y P 3 A 2 C Y P l A l CYP2B1, C Y P 2 B 2 C Y P 2 C 6 M R O D CYP1A2 CYP1A2, C Y P l A l C Y P enzymes in bold indicate the predominant enzyme catalyzing the reaction. Data from Burke etal . (1994). 1.3. CYP-mediated metabolism of PCBs Early research into the C Y P mediated oxidation of P C B s found that the rate of hydroxylation was negatively correlated with the degree of chlorination, while the presence of unsubstituted meta-para positions was found to facilitate metabolism (Borlakoglu and Wilkins , 1993a). The importance of unsubstituted meta-para positions to the metabolism of P C B s was further illustrated by the fact that P C B s lacking unsubstituted meta-para carbon atoms on both rings contributed to 70% of the total PCBs in human tissues (Borlakoglu and Wilkins, 1993b). The presence of adjacent unsubstituted carbon atoms is thought to facilitate the formation of arene oxide intermediates, which can rearrange to form hydroxylated metabolites (Matthews and Dedrick, 1984). Several in vitro studies have shown that hydroxylation more frequently occurs through an arene oxide intermediate than direct insertion of a hydroxyl group. This has been demonstrated by performing P C B metabolism assays in the presence of glutathione or serum. The reactive arene oxide intermediates react with the sulfhydryl-groups of glutathione and the cysteine residues of serum proteins, thereby reducing the 27 formation of hydroxylated metabolites (Koga et a l , 1998; Schnellmann et al., 1983). Previous research has also shown that P C B s chlorinated on only one ring are readily metabolized, while chlorination of both rings greatly impedes metabolism (Matthews and Dedrick, 1984). The C Y P enzyme that catalyzes the hydroxylation of a particular P C B congener is dependent upon the substitution of chlorine atoms on the biphenyl rings (James, 2001). Spectral binding studies using antibody inhibition have shown that P C B 47, 52 and 54 bind preferentially to C Y P 2 B enzymes and also bind to a lesser extent to the C Y P 3 A enzymes in hepatic microsomes from PB-treated male rats. P C B 52 bound primarily to C Y P 3 A enzymes in hepatic microsomes from DEX-treated rats while P C B 52 bound to the C Y P 3 A and C Y P 2 C enzymes in microsomes from control male rats (Hrycay and Bandiera, 2003). The usefulness of spectral interaction studies is limited because substrate binding is only one part of the reaction cycle and does not necessarily indicate whether a compound is a substrate for a particular C Y P enzyme (Hrycay and Bandiera, 2003). Some studies reported that the shift in spin state that occurs upon substrate binding affected the rate of substrate hydroxylation, while other studies have reported no correlation between spectral changes and substrate turnover. It is also known that C Y P 2 E 1 and C Y P 1 A 2 do not undergo a spectral change when a substrate binds (Hrycay and Bandiera, 2003). In vitro metabolism studies have shown that tetrachlorobiphenyls are selectively metabolized to hydroxylated metabolites by specific C Y P enzymes. In rats, numerous studies have shown the importance of the C Y P 1 A and C Y P 2 B enzymes to the hydroxylation of P C B s (Ishida et al., 1991; Koga et al., 1994; Koga et al., 1995a; Koga et 28 al., 1995b; Koga et al., 1998). Incubation of P C B 77, a coplanar P C B , with hepatic microsomes from MC-treated rats produced 5-hydroxyl and 4-hydroxyl metabolites while incubation with hepatic microsomes from untreated and PB-treated rats produced no detectable metabolites. Incubation of the coplanar congener P C B 80 with hepatic microsomes from MC-treated rats produced a 4-hydroxyl metabolite while no metabolites were detected when P C B 80 was incubated with hepatic microsomes from corn oi l or P B -treated rats. A 3-hydroxyl metabolite was produced when the noncoplanar congener P C B 52 was incubated with hepatic microsomes from PB-treated rats; however, no metabolites were detected when P C B 52 was incubated with hepatic microsomes from corn oil or MC-treated rats (Koga et al., 1995a). These results suggest that coplanar P C B s are metabolized preferentially by the C Y P 1 A enzymes and noncoplanar P C B s are metabolized preferentially by the C Y P 2 B enzymes. Table 5 summarizes the metabolites of P C B 52, 77 and 80 when incubated with untreated and induced C Y P hepatic microsomes. 29 Table 5. Hepatic microsomal metabolites of PCB 52, 77 and 80 Treatment Corn o i l / Untreated M C P B P C B 52 (2,2',5,5'-tetrachlorobiphenyl) N . D . N . D . 3-hydroxy-2,2',5,5'-tetrachlorobiphenyl P C B 77 (3,3',4,4'-tetrachlorobiphenyl) N . D . 4- hydroxy-3,3',4',5-tetrachlorobiphenyl 5- hydroxy-3,3',4,4'-tetrachlorobiphenyl N . D . P C B 80 (3,3',5,5'-tetrachlorobiphenyl) N . D . 4-hydroxy-3,3',5,5'-tetrachlorobiphenyl N . D . N . D . , not detected. Data taken from Koga et al. (1995a). Studies have also been conducted using purified C Y P enzymes. Ishida et al. (1991) found that P C B 77 was metabolized by purified C Y P 1 A 1 while no detectable metabolites were produced when P C B 77 was incubated with purified C Y P 2 B 1 , C Y P 2 B 2 or C Y P 1 A 2 . Purified C Y P 2 B 1 and C Y P 2 B 2 were found to metabolize P C B 52 while no metabolites were detected when P C B 52 was incubated with purified C Y P 1 A 1 and C Y P 1 A 2 (Ishida et al., 1991). The results of these studies suggest that the substrate specificity of C Y P enzymes for P C B s is affected by the coplanarity of the P C B molecule. Coplanar P C B s were preferentially metabolized by C Y P 1 A enzymes while noncoplanar P C B s were preferentially metabolized by the C Y P 2 B enzymes. The role of C Y P 2 C enzymes in the metabolism of P C B s has also been explored using C Y P 2 C enzymes purified from dog liver microsomes. The results of this study suggested that C Y P 2 C enzymes are not involved in P C B metabolism in dogs (Ariyoshi et al., 1995a). 30 There is a limited amount of data regarding the in vitro metabolism of P C B congeners by C Y P enzymes in other species including hamsters, birds, aquatic mammals and humans (Koga et al., 1995b; Borlakoglu and Wilkins, 1993b; McKinney et al., 2005; McGraw and Waller, 2006). Using cDNA-expressed human C Y P 2 B 6 , 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153) was metabolized to a 3-hydroxyl metabolite at a low rate (Ariyoshi et al., 1995b). N o metabolites were detectable when P C B 153 was incubated with human liver microsomes (Schnellmann et al., 1983). In contrast, liver microsomes prepared from dogs metabolized P C B 153 to a 3-hydroxyl metabolite at a rate of 0.56 nmol/h/mg protein (Ariyoshi et al., 1992). Interestingly, when a panel of recombinant human C Y P enzymes was screened for hydroxylation activity towards 2,2',4,5,5'-pentachlorobiphenyl ( P C B 101), C Y P 2 A 6 displayed the highest activity. Human C Y P 3 A 4 and C Y P 2 C 1 8 also displayed activity, but to a much lesser extent. Although P C B 101 is noncoplanar, recombinant C Y P 2 B 6 displayed no activity toward P C B 101 (McGraw and Waller, 2006). These findings highlight species differences in the metabolism of PCBs . 31 1.4. Rationale In contrast to previous studies that thoroughly characterized the structure-function relationships relating P C B structure and induction of C Y P enzymes (Denomme et al., 1983; Safe et al., 1985); the structure-activity relationships regarding P C B structure and metabolism by C Y P enzymes has not been completed. Furthermore, the ability of the C Y P 3 A enzymes, which are the second most abundant C Y P subfamily in rat liver, to metabolize P C B s is not known. Evidence from spectral interaction studies suggests that noncoplanar P C B s bind to rat C Y P 3 A enzymes (Hrycay and Bandiera, 2003). In my thesis research, the interaction of four symmetrically substituted tetrachlorobiphenyls with C Y P enzymes was investigated by their ability to inhibit alkoxyresorufin O-dealkylase reactions that are catalyzed by specific C Y P enzymes. Because it is not feasible to test the C Y P specificity for all 209 congeners, four tetrachlorobiphenyls with varying degrees of coplanarity were chosen. Tetrachlorobiphenyls were chosen because they are more soluble and easily metabolized than the more highly chlorinated P C B s and are better suited for developing structure-activity relationships than less chlorinated P C B s (Hrycay and Bandiera, 2003). The C YP-mediated metabolism of P C B 54 was also examined using a liquid chromatography/ mass spectrometry ( L C / M S ) method to detect its hydroxylated metabolites. P C B 54 is a tetra-ortho substituted tetrachlorobiphenyl. The biotransformation of P C B 54 was further examined because little is known about the metabolism of highly orr/zo-substituted PCBs . Hepatic microsomes prepared from rats treated with C Y P inducers, ant i -CYP antibodies and recombinant C Y P enzymes were used to assess the contribution of individual C Y P enzymes in the hydroxylation of P C B 54. The results of this study w i l l be used to 32 develop a structure-activity relationship that can be used to predict the predominant C Y P enzymes involved in the metabolism of a P C B congener. The development of an in vitro assay to detect CYP-mediated P C B 54 metabolism with rat liver microsomes is an important first step for later studies that w i l l use liver microsomes derived from other species. The ability to measure P C B metabolism in vitro w i l l allow for the identification of species with low biotransformation capacity toward P C B s and thus, at greatest risk of toxicity. 33 1.5. Hypotheses 1. The degree of inhibition of alkoxyresorufin O-dealkylase activities by P C B s is dependent upon the chlorine substitution pattern. 2. 2,2',6,6'-Tetrachlorpbiphenyl (PCB 54), a noncoplanar P C B , is preferentially metabolized by C Y P 2 B and C Y P 3 A enzymes. 1.6. Specific aims 1. Determine inhibition of hepatic microsomal E R O D , M R O D and B R O D activities by P C B 47, P C B 52, P C B 54 and P C B 77. 2. Develop and validate an in vitro assay to analyze hepatic biotransformation of P C B 54. 3. Identify the C Y P enzymes involved in the hepatic biotransformation of P C B 54 through the use of C Y P inducers, ant i-CYP antibodies and recombinant C Y P enzymes. 34 2. Materials and Methodology 2.1. Materials Reagents were obtained from the following sources: AccustandardInc. (New Haven, Connecticut, U.S.A.): 2,2',4,4'-Tetrachlorobiphenyl (PCB 47); 2,2',5,5'- tetrachlorobiphenyl (PCB 52); 2,2',6,6'-tetrachlorobiphenyl (PCB 54); 3,3',4,4'- tetrachlorobiphenyl (PCB 77). BDH chemicals (Toronto, Ontario, Canada): Ethylenediaminetetracetic acid (EDTA), disodium salt; magnesium chloride; phenobarbital; potassium hydroxide. Eastman Kodak Co. (Rochester, New York, U.S.A.): 3-Methylcholanthrene Fischer Scientific Ltd. (Vancouver, British Columbia, Canada): Dimethyl sulfoxide (DMSO); hexanes, HPLC-grade; hydrochloric acid, optima-grade; isopropanol, HPLC-grade; methanol, HPLC-grade; methyl-r-butyl ether, HPLC-grade; potassium phosphate dibasic; potassium phosphate monobasic; sodium carbonate; sodium hydroxide. GENTEST (Woburn, Massachusetts, U.S.A.): Insect cell control microsomes; rat CYP1A2 and CYP2D1 SUPERSOMES™, which also expressed CYP-reductase; rat CYP2A2, CYP2B1, CYP2C6, CYP2C11, CYP2C13, CYP3A1 and CYP3A2 SUPERSOMES™ which also expressed C Y P -reductase and cytochrome b$. A l l products were produced in insect cells (BTI-TN-5B1-4) using a baculovirus (Autographa californica) expression system. 35 Gibco BRL (Gaithersburg, Maryland, U.S.A.): Glycerol ICNBiomedicals Inc. (Cleveland, Ohio, U.S.A): Sodium eholate J.T. Baker Chemical Co. (Phillipsburg, New Jersey, U.S.A.): Sodium dithionite Molecular Probes Inc. (Eugene, Oregon, U.S.A.): Benzyloxyresorufin; ethoxyresorufin; methoxyresorufin; resorufin. PMI Feeds Inc. (Richmond, Indiana, U.S.A.): Rodent laboratory diet™, No . 5001 Praxair (Vancouver, British Columbia, Canada): Carbon monoxide; nitrogen. Sigma Chemical Co. (St. Louis, Missouri, U.S.A.): Bovine serum albumin; corn o i l ; cupric sulfate pentahydrate; dexamethasone; N -2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) ; nicotinamide adenine dinucleotide phosphate tetra-sodium salt ( N A D P H , reduced form); potassium chloride; sucrose; tris(hydroxymethyl)aminomethane hydrochloride (Trizma HCI). Wellington Laboratories (Guelph, Ontario, Canada): 2,2',6,6'-Tetrachloro-4-biphenylol; 2,2',4',6,6'-pentachloro-4-biphenylol. Dr. S. M. Bandiera (Faculty of Pharmaceutical Sciences, University of British Columiba, Vancouver, British Columbia, Canada): Control rabbit IgG; polyclonal rabbit anti-rat C Y P 2 B 1 IgG. 36 2.2. Methodology 2.2.1. Animal treatments Twenty-four male Long-Evans rats, eight to ten weeks old, were obtained from Charles River Laboratories (Montreal, Quebec, Canada). Rats were housed in polycarbonate cages with corn cob bedding at a temperature of 20-23 °C and a 14 h light and 10 h dark photoperiod. Rats were provided with water and food ad libitum and were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. The rats were allocated equally to four treatment groups, corn oi l - , M C - , P B - and DEX-treated. Rats were allowed to acclimatize to their new housing and handlers for the first week after their arrival. Treatments began during the second week. Animals in the M C group were injected ip with M C dissolved in corn oi l 25 mg/kg/day, D E X dissolved in corn oi l was injected ip at 100 mg/kg/day and P B dissolved in saline was administered ip at 80 mg/kg/day. A l l treatments lasted three days and animals were killed by decapitation 24 h following the last treatment. 2.2.2. Hepatic microsome preparation Immediately following decapitation, livers were excised and then homogenized in 20 m L of ice-cold 0.05 M Tr i s -HCl buffer, p H 7.5, containing 1.15% KC1 in a Potter Elvehjem tissue grinder. Microsomes were prepared from pooled livers homogenates by differential centrifugation according to previously published protocols (Wong and Bandiera, 1996). The liver homogenate was spun at 9,000 x g for 20 min at 5 °C. Following centrifugation, the supernatant was filtered through cheese cloth and then spun at 105,000 x g for 60 min at 5 °C. The microsomal pellet was resuspended in ice-cold 10 37 mM E D T A buffer, pH 7.4, containing 1.15% KC1 and then centrifuged again at 105,000 x g for 60 min at 5 °C. The microsomal pellet was resuspended in ice-cold 0.25 M sucrose solution and stored at -75 °C in 1 mL aliquots. 2.2.3. Total protein determination Total protein content of the prepared hepatic microsomes was determined by the Lowry protein assay using bovine serum albumin as a standard (Lowry et al., 1951). Samples were measured in duplicate using a Shimadzu UV-160 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). 2.2.4. Total cytochrome P450 determination Total CYP content was determined by the reduced carbon monoxide difference spectrum originally described by Omura and Sato (1964). Hepatic microsomes prepared from MC- , DEX- and corn oil-treated rats were diluted 20-fold while hepatic microsomes from PB treated rats were diluted 40-fold with 0.1 M NaP0 4 buffer, pH 7.4, containing 20% glycerol and 0.1 mM EDTA. Diluted samples were added to a sample and reference cuvette. Carbon monoxide was bubbled into the sample cuvette for one minute and sodium dithionite was then added to both cuvettes. The difference absorption spectrum was obtained using a SLM-Aminco DW-2 spectrophotometer (SLM Instruments Inc., Urbana, IL, USA). Total CYP content was calculated using an extinction coefficient of 91 cnf'mM"1. 38 2.2.5. Alkoxyresorufin 0-dealkylase assays 2.2.5.1. Experimental procedure Hepatic microsomal B R O D , E R O D and M R O D activities were determined using a fluorescence based assay, originally described by Burke and Mayer (1974), with some modifications. The increase in fluorescence, associated with resorufin formation, was measured using a Shimadzu RF-540 spectrofluorometer (Shimadzu Corporation, Kyoto, Japan) with excitation and emission wavelengths set at 530 nm (5 nm slit width) and 582 nm (5 nm slit width), respectively. A resorufin standard curve was used to convert fluorescence readings to product formation. Enzymatic reactions were optimized such that product formation was linear with respect to both incubation time and protein concentration. Fluorescence was recorded at 1.5 min for B R O D and E R O D activities and 3 min for M R O D activity. For both E R O D and M R O D assays, a final protein concentration of 0.025 mg/mL of hepatic microsomes from MC-treated rats was used. A final concentration of 0.025 mg protein/mL of hepatic microsomes from PB-treated rats was used for measurement of B R O D activity. To study the effect of the tetrachlorobiphenyls on alkoxyresorufin 0-dealkylase activities, P C B s were preincubated with hepatic microsomes (50 pL) in 0.1 M H E P E S buffer with 5 m M M g C l 2 , pH 7.8 (1.92 mL) for 5 min at 37 °C before addition of the alkoxyresorufin substrate (10 pL) . Reactions were initiated by the addition of 10 p L of 50 m M N A D P H . Enzyme activity curves were generated by using five concentrations of substrate. Final ethoxyresorufin concentrations were 0.05, 0.1, 0.2, 0.3 and 0.75 p M and final methoxyresorufin concentrations were 0.1, 0.13, 0.2, 0.3, and 0.75 p M . Enzyme 39 activity was measured in the presence of four concentrations of P C B s , except where noted. A similar procedure was used to determine the effect of the tetrachlorobiphenyls on B R O D activity, except that hepatic microsomes from PB-treated rats were used instead of hepatic microsomes from MC-treated rats and benzyloxyresorufin was used as the substrate. Substrate concentrations at which B R O D activity was measured were 0.05, 0.1, 0.2, 0.3 and 0.75 p M . Enzyme activity curves were generated in the absence of PCBs and in the presence of four different concentrations of P C B s , except where noted. To determine the effect of preincubation of hepatic microsomes from MC-treated rats with P C B 77 and N A D P H on E R O D and M R O D activities, a similar procedure as that described above was used. Hepatic microsomes from MC-treated rats (0.025 mg/mL) were incubated at 37 °C with buffer and N A D P H , buffer with D M S O and N A D P H , buffer with P C B 77 or buffer with P C B 77 and N A D P H . The control group consisted of hepatic microsomes incubated in buffer with 0.25 m M N A D P H . The vehicle control group contained hepatic microsomes, 0.5% D M S O and 0.25 m M N A D P H incubated in buffer. In the third group, hepatic microsomes were incubated in buffer in the presence of 20 u M P C B 77 and the final group consisted of 20 u M P C B 77, 0.25 m M N A D P H and hepatic microsomes. Reactions were initiated by the addition of 0.1 u M ethoxyresorufin or methoxyresorufin. For the third group, in addition to the alkoxyresorufin substrate, 0.25 m M N A D P H was also added. E R O D activity was measured at 1.5 min while M R O D activity was measured at 3 min. A similar procedure was performed to determine the effect of duration of preincubation of hepatic microsomes from PB-treated rats with P C B 54 and N A D P H on B R O D activities except 40 that hepatic microsomes from PB-treated rats (0.025 mg/mL) were used instead of hepatic microsomes from MC-treated rats, P C B 54 (20 p M ) was used instead of P C B 77 and benzyloxyresorufin was used as the substrate. B R O D activity was determined at 1.5 min. 2.2.5.2. Data analysis For experiments in which alkoxyresorufin (9-dealkylase activities were determined in the presence of varying concentrations of inhibitor, data were analyzed using the Enzyme Kinetics module (v. 1.10, Systat Software Inc., Richmond, California, U.S .A. ) of SigmaPlot 200l(v. 7.0, Systat Software Inc., Richmond, California, U.S .A. ) . A single substrate-single inhibitor model was used and the data were fitted to the equations for competitive inhibition (Eq. 1) and mixed inhibition (Eq. 2). V[S] Eq. 1 v = [S] + tfm(l+ [/]/*,.) V[S] E q 2 / [5](i + [/]/^,) + / : m ( i + [/]//:,) v is the initial rate o f product formation, V is the limiting rate o f product formation, K m is the Michaelis constant, [S] is the substrate concentration, [I] is the inhibitor concentration, Kj is the equilibrium dissociation constant for the enzyme-inhibitor complex and K j ' is the equilibrium dissociation constant of the inhibitor from the enzyme-substrate-inhibitor complex. The term limiting rate was used instead of maximal velocity ( V m a x ) as recommended by the Nomenclature Committee of the International Union of Biochemistry (NC- IUB) ( N C - I U B 1983). Maximal velocity is discouraged because it does not define a maximum in the mathematical sense, but a limit. 41 The appropriate model of inhibition was chosen based upon statistical goodness of fit tests including the Akaike information criterion (AICc) (Eq. 3) and the size of the standard deviation of the residuals (Sy.x) (Eq. 4). Visual inspection of the Lineweaver-Burk plots was also used to choose the appropriate model. E q . 3 AICc = n*H^-) + 2K+2K(K + l ) n n- K - 1 Eq. 4 Sy.x= V n - 2 RSS is the sum of squares of residuals, n is the number of data points and K is the total number of estimated parameters plus one for a 2 . For both A I C c and Sy.x, the model of inhibition with the lowest value was considered the model that best fit the data. A l l data are expressed as mean ± standard error of the mean ( S E M ) except for graphs of inhibition of alkoxyresorufin (9-dealkylase activities by tetrachlorobiphenyls, which are plotted as mean ± standard deviation (SD). Standard deviation was used instead of standard error of the mean because plots were generated automatically with SigmaPlot software. 2.2.6. PCB 54 biotransformation assay 2.2.6.1. Experimental procedure The in vitro assay used to assess the C Y P mediated metabolism of P C B 54 was based upon previously published methods that examined the metabolism of P C B 52 and P C B 77 by rat liver microsomes (Ishida et al., 1991; Koga et al., 1995a). In a 1 mL final reaction volume, hepatic microsomes (50 uL) were added to a 50 m M KPO4 buffer, p H 42 7.4 containing 3 m M MgCl2. P C B 54 dissolved in methanol (20 uL) was added and the reaction was initiated by the addition of 10 p L of 100 m M N A D P H . Reactions were terminated by the addition of 1 m L of 0.5 M N a O H and stored on ice until the extraction procedure. Once the incubation was complete, 1460 pmol (20 p L of 25 pg/mL) of the internal standard (IS), 2,2',4',6,6'-pentachloro-4-biphenylol (4-OH-CB104), was added and the hydroxy-PCBs were then extracted. To determine the contribution of the C Y P 2 B enzymes to the metabolism of P C B 54 in hepatic microsomes from PB-treated rats, a polyclonal rabbit anti-rat C Y P 2 B 1 IgG was used. Hepatic microsomes were preincubated for 15 min at room temperature, in the presence of ant i -CYP2B or control IgG (0.5, 1, 2.5, or 5 mg IgG/ mg of microsomal protein). In an attempt to increase antibody binding to the C Y P enzyme, hepatic microsomes were solubilized by preincubation with sodium cholate in a subsequent experiment. Hepatic microsomes were preincubated with antibody and 0.06% sodium cholate for 15 min at room temperature. In experiments using sodium cholate, an additional concentration of IgG was used, 10 mg IgG/mg of microsomal protein. Following the preincubation 153 p M (10 p L of 2.24 mg/mL) P C B 54 was added and the reaction was then initiated with N A D P H . The rest of the procedure was carried out in the same manner as experiments performed in the absence of antibody. Biotransformation assays using recombinant rat C Y P enzymes, expressed in insect cell microsomes (SUPERSOMES™), were conducted in a similar approach as the biotransformation assays using rat hepatic microsomes. Insect cell microsomes expressing C Y P 1 A 2 , C Y P 2 A 2 , C Y P 2 B 1 , C Y P 2 C 6 , C Y P 2 C 1 1 , C Y P 2 C 1 3 , C Y P 3 A 1 , C Y P 3 A 2 and C Y P 2 D 1 were screened for their catalytic activity toward P C B 54. Insect 43 cell microsomes not expressing any C Y P enzymes were used as a control. In a 1 m L final reaction volume, insect cell microsomes (50 uL) were added to a 50 m M KPO4 buffer, p H 7.4 containing 3 m M M g C l 2 . 153 u M P C B 54 dissolved in methanol (20 uL) was added and the reaction was initiated by the addition of 10 u L of 100 m M N A D P H . Reactions were terminated after 20 min by 1 m L of 0.5 M N a O H . The rest of the procedure was carried out in the same manner as the experiments conducted with hepatic microsomes. 2.2.6.2. Extraction procedure A number of extraction procedures were tested for their ability to extract the hydroxyl metabolites from the aqueous matrix. The procedure that yielded the highest percent recovery was used in future experiments. The procedure used was as follows: tubes were heated at 70 °C for 10 min, 2 m L of 6 M HC1 and 1 m L of isopropanol was then added to each tube and vortex mixed for 1 min. A 1:1 mixture of hexane and methyl /-butyl ether was added (2 mL) and each tube was vortex mixed for 1 minute. To facilitate separation of the aqueous and organic phases, tubes were spun at 2,000 x g for 5 min. The organic layer was transferred to a clean tube and the procedure, starting from the addition of 2 m L of a 1:1 mixture of hexane and methyl /-butyl ether, was repeated two additional times. Once the extraction was complete, the organic phase was evaporated under a gentle stream of nitrogen and reconstituted with 1 m L of methanol. Prior to transferring the reconstituted samples to H P L C sample vials, the samples were filtered through syringe filters containing a 0.45 um polytetrafluoroethylene membrane to remove particulates. 44 2.2.6.3. Sample analysis To identify and quantify the metabolites, a Micromass Quattro Premier X E mass spectrometer (MS) (Waters Corporation, Milford, M A , U S A ) interfaced with a Waters A C Q U I T Y ultra-performance liquid chromatography ( U P L C ) system (Waters Corporation, Mil ford , M A , U S A ) was used. MassLynx (v. 4.1, Micromass Ltd., Altrincham, United Kingdom) controlled the instrumentation and data acquisition. Samples (5 pL) were injected by an autoinjector and separation of the hydroxylated P C B s was achieved using a Waters A C Q U I T Y U P L C bridged ethylsiloxane/silica hybrid (BEH) Ci8 column (1.7 pm x 2.1 mm x 100 mm) that was heated to 35 °C. The mobile phase consisted of methanol and water containing 10 m M ammonium acetate. Linear gradient conditions were as follows: 75-100% methanol from 0 to 5 min, 100% methanol from 5 to 7 min and a return to 75% methanol from 7 to 10 min. F low rate was 0.2 mL/min and the total run time was 10 min per sample. P C B 54 metabolites were detected by the M S operating in electrospray negative ionization ((-)-ESI) mode. Conditions were optimized for maximum ion current of the [M-H]" ion by directly infusing 2,2',6,6'-pentachloro-4-biphenylol (4-OH-CB54) into the M S . Nitrogen was used as the desolvation gas at a flow rate of 400 L/h . Source and desolvation temperatures were 100 and 200 °C, respectively while the capillary and cone voltages were 3 k V and 50 V , respectively. For the detection of hydroxylated P C B 54 metabolites, a mass to charge ratio (m/z) from 300 to 400 was scanned with a scan time of 0.5 s. 45 2.2.6.4. Assay validation Due to the lack of an authentic standard for the monohydroxyl P C B 54 metabolite, 4 -OH-CB54 was used instead for the assay validation. The assay could not be validated with respect to the dihydroxyl P C B 54 metabolite because dihydroxyl tetrachlorobiphenyls were not commercially available. 2.2.6.4.1. 4-OH-CB54 standard curve variability The 4-OH-CB54 standard curve was assessed for inter- and intra-day variability by comparing the peak area ratio (PAR) o f the area under the curve ( A U C ) of the 4 -OH-CB54 peak to the A U C of the internal standard peak at each concentration tested. Inter-day variability was determined by injecting the standards once per day for six separate days and calculating the coefficient of variation (C.V.) of the P A R . A coefficient of variation below 15% was considered acceptable. Intra-day variability was determined by injecting each standard six times in one day and calculating the coefficient of variation of the P A R . A coefficient of variation below 15% was considered acceptable. 2.2.6.4.2. Limits of detection and quantitation (LOD and LOQ) The limits of detection and quantitation for the monohydroxyl metabolite were determined using 4-OH-CB54. Both limits were determined using a calibration curve containing standards in the range of the detection and quantitation limits. Standard solutions of 4 -OH-CB54 at concentrations of 10, 20, 41 and 81 pmol/mL were prepared. 3 3cr The detection limit was calculated by LOD = ' and the quantitation limit was calculated by LOQ = where a is the standard deviation of the response and S is the 46 slope of the calibration curve. The residual standard deviation of the regression line was used as an estimate of a. The L O D and L O Q values were determined from experiments performed in duplicate on three separate days. 2.2.6.4.3. Extraction efficiencies of the monohydroxyl PCBs Extraction efficiency of recovering the monohydroxyl metabolite from the assay mixture was determined by using the 4-OH-CB54 standard as a surrogate. Two sets of samples, the non-extracted and extracted were used. In the extracted set, 4 -OH-CB54 and the internal standard were extracted from the assay mixture using the extraction procedure described in Materials and Methodology, section 2.2.6.2. The non-extracted sample containing 4 -OH-CB54 and the internal standard was prepared in methanol and injected directly into the L C / M S . Peak areas of the extracted samples were determined as a percentage of the non-extracted samples to calculate the percent recovery. Three concentrations of 4 -OH-CB54 were used to assess the recovery at different points on the standard curve. Recovery of 4-OH-CB104 was determined at the concentration in which it was used as the internal standard in the metabolism assay (1461 pmol/mL). Extraction recovery experiments were performed in duplicate on three separate days. 2.2.6.5. Assay optimization The P C B 54 biotransformation assay was optimized with respect to the length of incubation, microsomal protein concentration and a saturating substrate concentration using liver microsomes of PB-treated rats. 47 2.2.6.5.1. Length of incubation For the monohydroxyl metabolite, P C B 54 was incubated in a reaction mixture containing 0.1 mg/mL microsomal protein for varying lengths of time (0.5, 1, 2, 3, 5 and 7 min). Data were also plotted as monohydroxyl metabolite formed per mg of protein versus time and an optimal incubation time was chosen from the linear portion of the curve. For the dihydroxyl metabolite, P C B 54 was incubated in a reaction mixture containing 0.5 mg/mL microsomal protein for 1,5, 10, 15, 20, 30, 45 and 60 min. The longest incubation time in which product formation was linear with respect to time was chosen as the optimal incubation time for the formation of the dihydroxyl metabolite. This time point was chosen to maximize dihydroxyl metabolite formation as the rate of product formation was slow. 2.2.6.5.2. Microsomal protein concentration To determine the optimal microsomal protein concentration for monohydroxyl metabolite formation, P C B 54 was incubated for 1 min with varying microsomal protein concentrations (0.025, 0.05, 0.1, 0.25 and 0.5 mg/mL). Monohydroxyl metabolite formed per minute versus protein concentration was plotted as a function of microsomal protein concentration and an optimal microsomal protein concentration was chosen from the linear portion of the curve. The highest protein concentration in which product formation was linear with respect to protein concentration was chosen as the optimal protein concentration. This allowed for a maximal amount of product to be formed. For the dihydroxyl metabolite, P C B 54 was incubated in a reaction mixture for 20 min and the microsomal protein concentrations used were 0.25, 0.5, 0.75, 1 and 1.25 48 mg/mL. The optimal protein concentration for the formation of the dihydroxyl metabolite was performed in a similar manner as the monohydroxyl metabolite. 2.2.6.5.3. Saturating substrate concentration Assay conditions to determine a saturating substrate concentration for monohydroxyl metabolite formation were, 0.1 mg/mL microsomal protein and an incubation time of 1 min. For dihydroxyl metabolite formation, a microsomal protein concentration of 0.5 mg/mL and an incubation time of 20 min were used. In both cases P C B 54 substrate concentration varied from 34 to 171' u M . A saturating concentration for formation of the monohydroxyl metabolite was determined from a plot of P C B 54 hydroxylase activity versus substrate concentration. The saturating substrate concentration for dihydroxyl metabolite formation was determined from a plot of P A R of the metabolite relative to the internal standard versus substrate concentration. 49 3. Results 3.1. Alkoxyresorufin O-dealkylase inhibition 3.1.1. Inhibition of alkoxyresorufin O-dealkylase activities by tetrachlorobiphenyls Hepatic microsomal M R O D , E R O D and B R O D activities were measured at five substrate concentrations and increasing concentrations of P C B 47, P C B 52, P C B 54 and P C B 77. Figure 6 shows M R O D activity in the presence o f P C B 47. Increasing concentrations of P C B 47 led to increasing inhibition of M R O D activity. Incubation of hepatic microsomes from MC-treated rats with P C B 52 also inhibited M R O D activity with increasing concentrations of P C B 52 resulting in increasing inhibition of M R O D activity (Figure 7). P C B 54 at the highest concentration tested, 15 p M , did not inhibit M R O D activity. In Figure 8, increasing concentrations of P C B 77 led to a progressive decline in M R O D activity. M R O D activity was inhibited by more than 70% at the highest concentration of P C B 77 tested. Incubation of hepatic microsomes from MC-treated rats with P C B 47 resulted in a decrease in E R O D activity (Figure 9). In Figure 10, increasing concentrations of P C B 52 produced a decline in E R O D activity. A t the highest concentration of P C B 54 tested, 15 p M , E R O D activity was not affected. P C B 77 caused the greatest reduction in E R O D activity. Increasing concentrations of P C B 77 caused a progressive decline in E R O D activity (Figure 11). 50 P C B 47 produced a concentration-dependent reduction in B R O D activity (Figure 12). Incubation of hepatic microsomes from PB-treated rats with P C B 52 or P C B 54 also inhibited B R O D activity (Figures 13 and 14, respectively). B R O D activity was inhibited by more than 90% at the highest concentration of P C B 52 or P C B 54 tested. In contrast, B R O D activity was not inhibited by P C B 77 at the highest concentration tested (20 pM) . 3.1.2. Fitting of data to competitive and mixed models of inhibition Data for inhibition of alkoxyresorufin (9-dealkylase activities by P C B s were fitted to mixed and competitive models of inhibition by nonlinear regression. Data were also fit to an uncompetitive model of inhibition but these results were not included because the data poorly fit this model. Data were not fit to a noncompetitive model of inhibition since this type of inhibition has been suggested to be a theoretical concept and an unlikely mechanism of inhibition (Cornish-Bowden and Wharton, 1988). Selection of the appropriate mode of inhibition was determined by visual inspection of the Lineweaver-Burk plots and statistical criteria to evaluate the goodness of fit. The two statistical criteria used were A I C c values and the size of the standard deviation of the residuals (Sy.x). Table 6 summarizes the Kj values and the values of the goodness of fit tests for mixed and competitive modes of inhibition. In most cases, the A I C c and Sy.x values were similar when the data were modeled to mixed and competitive modes of inhibition. A similar trend was noted for Kj values. In all but one case, the difference in the Kj values determined using the two modes of inhibition was not greater than 2.1 fold. The mode of inhibition with the lowest A I C c and Sy.x values was considered to the best fit for the data. A mixed mode of inhibition was found to best fit the inhibition of M R O D and B R O D activities by P C B s , whereas a competitive mode of inhibition was found to 51 best fit the inhibition of E R O D activity. M R O D activity measured in the presence of P C B 47, 52 and 77, modeled to a mixed mode of inhibition, is presented in Figures 6, 7 and 8, respectively. Figures 9, 10 and 11 show E R O D activity in the presence of P C B 47, 52 and 77, modeled to a competitive mode of inhibition and Figures 12, 13 and 14 display B R O D activity in the presence of P C B 47, 52 and 54, respectively, modeled to a mixed mode of inhibition. 52 A P C B 47 B 1200 1000 E c E o E Q. > < Q O a: 800 600 400 200 H [Methoxyresorufin] (uM) P C B 47 0.007 g> 0.006 c J 0.005 o • I = 0 nM o I = 7500 nM T I = 15000 nM -2 0 2 4 6 1/[Methoxyresorufin] (uM) 0.8 12 Figure 6. Inhibi t ion of M R O D activity by P C B 47 Michaelis-Menten (A) and Lineweaver-Burk (B) plots o f rat liver microsomal M R O D activity in the presence of P C B 47. Varying concentrations of methoxyresorufin were incubated with hepatic microsomes from MC-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 47 dissolved in D M S O . Concentration of P C B 47 used in reaction mixture indicated by '1= '. Reactions were initiated with N A D P H and M R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 53 PCB 52 1200 1000 | o E CL o < O O or 800 600 400 200 0.0 0.1 B 0.2 0.3 0.4 0.5 0.6 [Methoxyresorufin] (uM) PCB 52 0.8 0.008 -i -2 0 2 4 6 1/[Methoxyresorufin] (uM) Figure 7. Inhibi t ion of M R O D activity by P C B 52 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal MROD activity in the presence of PCB 52. Varying concentrations of methoxyresorufin were incubated with microsomes of MC-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either DMSO or PCB 52 dissolved in DMSO. Concentration of PCB 52 used in reaction mixture indicated by '1= '. Reactions were initiated with NADPH and MROD activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 54 P C B 77 1200 1000 ro E o E Q . < Q O or 800 H 600 400 200 0.0 B 0.1 0.2 0.3 0.4 0.5 0.6 [Methoxyresorufin] (pM) P C B 77 0.018 -i • o I = 0 nM I = 5 nM 0.016 - • I = 25 nM in/mg) I = 50 nM in/mg) 0.014 - • I = 75 nM in/mg) E 0.012 -(pmol 0.010 -Activity 0.008 -'MROD 0.006 -0.004 -0.002 -1 1 i i i i 0 2 4 6 1/[Methoxyresorufin] (|jM) 10 12 Figure 8. Inhibition of MROD activity by PCB 77 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal M R O D activity in the presence of P C B 77. Varying concentrations of methoxyresorufin were incubated with microsomes from MC-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 77 dissolved in D M S O . Concentration of P C B 77 used in reaction mixture indicated by T= '. Reactions were initiated with N A D P H and M R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 55 A P C B 47 [Ethoxyresorufin] (pM) B P C B 47 -5 0 5 10 15 20 25 1/[Ethoxyresorufin] (uM) Figure 9. Inhibit ion of E R O D activity by P C B 47 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal E R O D activity in the presence of P C B 47. Varying concentrations of ethoxyresorufin were incubated with microsomes of MC-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 47 dissolved in D M S O . Concentration of P C B 47 used in reaction mixture indicated by T= ' . Reactions were initiated with N A D P H and E R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a competitive mode of inhibition by nonlinear regression analysis. 56 PCB 52 B 14000 12000 I = 0 nM I = 3000 nM I = 10000 nM CD E 10000 o E CL O < Q O or LU 8000 6000 4000 H 2000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 [Ethoxyresorufin] (uM) PCB 52 0.0012 i • l = 0nM o I = 3000 nM T I = 10000 nM 0.0010 CD E 0.0008 A -5 5 10 15 1/[Ethoxyresorufin] (uM) Figure 10. Inhibi t ion of E R O D activity by P C B 52 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal E R O D activity in the presence of P C B 52. Varying concentrations of ethoxyresorufin were incubated with microsomes from MC-treated rats (0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 52 dissolved in D M S O . Concentration of P C B 52 used in reaction mixture indicated by '1= ' . Reactions were initiated with N A D P H and E R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a competitive mode of inhibition by nonlinear regression analysis. 57 A P C B 77 14000 12000 o < Q O CC LU B 10000 A CO E | | 8000 Q . > 6000 4000 2000 • I = 0 nM o I = 25 nM T I = 50 nM V I = 100 nM • I = 200 nM 0.0 0.004 -, 0.1 0.2 0.3 0.4 0.5 [Ethoxyresorufin] (uM) P C B 77 CD E 0.003 • l = 0 n M o I = 25 nM T I = 50 nM V l = 100nM • I = 200 nM -5 0 5 10 15 20 25 1/[Ethoxyresorufin] (uM) Figure 11. Inhibition of EROD activity by PCB 77 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal E R O D activity in the presence of P C B 77. Varying concentrations of ethoxyresorufin were incubated with microsomes of MC-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 77 dissolved in D M S O . Concentration of P C B 77 used in reaction mixture indicated by T= '. Reactions were initiated with N A D P H and E R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a competitive mode of inhibition by nonlinear regression analysis. 58 A PCB 47 [Benzyloxyresorufin] (uM) B PCB 47 -5 0 5 10 15 20 25 1 / [Benzyloxyresoruf in] (uM) Figure 12. Inhibition of BROD activity by PCB 47 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal BROD activity in the presence of PCB 47. Varying concentrations of benzyloxyresorufin were incubated with microsomes of PB-treated rats (at 0.025 mg protein/mL). Reactions also contained either DMSO or PCB 47 dissolved in DMSO. Concentration of PCB 47 used in reaction mixture indicated by T= '. Reactions were initiated with NADPH and BROD activity was measured at 1.5 min. Control incubations contained DMSO. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 59 A PCB 52 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 [Benzyloxyresorufin] (uM) •5 0 5 10 15 20 25 1/[Benzyloxyresorufin] (uM) Figure 13. Inhibi t ion of B R O D activity by P C B 52 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal B R O D activity in the presence of P C B 52. Varying concentrations of benzyloxyresorufin were incubated with microsomes from PB-treated rats (at 0.025 mg protein/mL). Reactions mixtures also contained either D M S O or P C B 52 dissolved in D M S O . Concentration of P C B 52 used in reaction mixture indicated by '1= ' . Reactions were initiated with N A D P H and B R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 60 PCB 54 12000 10000 E > ts < Q O or CO B 8000 6000 4000 2000 0.0 0.1 0.014 0.2 0.3 0.4 0.5 0.6 [Benzyloxyresorufin] (uM) PCB 54 5 10 15 1/[Benzyloxyresorufin] (uM) 25 Figure 14. Inhibi t ion of B R O D activity by P C B 54 Michaelis-Menten (A) and Lineweaver-Burk (B) plots of rat liver microsomal B R O D activity in the presence of P C B 54. Varying concentrations of benzyloxyresorufin were incubated with microsomes from PB-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 54 dissolved in D M S O . Concentration of P C B 47 used in reaction mixture indicated by T= '. Reactions were initiated with N A D P H and B R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 61 Table 6. Values for the goodness of fit tests and K, values Activity P C B 47 P C B 52 P C B 54 P C B 77 C M C M C M C M M R O D A I C c 787 691 702 676 N o inhibition 1362 1349 Sy.x 73 43 66 57 44 43 Kj (nM) 3000 15000 4300 9000 8 10 E R O D A I C c 1141 1143 1145 1147 No inhibition 1754 1756 Sy.x 548 551 562 565 340 341 Kj (nM) 5500 5700 6500 6700 46 46 B R O D A I C c 2140 2122 2229 2224 2154 2118 N o inhibition Sy.x 364 346 481 474 379 342 Kj (nM) 230 440 40 60 50 100 Goodness of fit tests and Kj values were generated when inhibition of alkoxyresorufin O-dealkylase activities by P C B s were fitted to competitive and mixed modes of inhibition using the Enzyme Kinetics module (v. 1.10, Systat Software Inc., Richmond, California, U.S .A. ) of Sigmaplot 2001 (v. 7.0, Systat Software Inc., Richmond, California, U .S .A. ) . Competitive mode of inhibition is abbreviated as ' C and mixed mode of inhibition is abbreviated as ' M ' . M R O D and E R O D activities were determined with hepatic microsomes from MC-treated rats and B R O D activity was determined with hepatic microsomes from PB-treated rats. The Kj values for the inhibition of M R O D , E R O D and B R O D activities by the four tetrachlorobiphenyls tested are summarized in Table 7. P C B 77, the most coplanar P C B tested, was the most potent inhibitor of E R O D and M R O D activity with Kj values of 46 and 10 n M , respectively. The two di-ortho substituted congeners, P C B 47 and P C B 52, also inhibited E R O D and M R O D activity, but to a lesser extent. The Kj values for inhibition of E R O D activity by P C B 47 and P C B 52 were 5.5 and 6.5 p M , respectively and the Kj values for inhibition of M R O D activity by P C B 47 and P C B 52 were 15 and 9 p M , respectively. The noncoplanar PCBs , especially P C B 52 and P C B 54, were more potent inhibitors of B R O D activity with Kj values of 60 and 100 n M , respectively; whereas P C B 77 did not inhibit B R O D activity. 62 V and K m values for M R O D , E R O D and B R O D activities are summarized in Table 8. Table 9 summarizes the concentrations of P C B s that were used to determine the K i values. Initial estimates of the Kj values were determined by measuring alkoxyresorufin O-dealkylase activity at a single substrate concentration in the presence of varying concentrations of inhibitor. The range of inhibitor concentrations used for subsequent experiments, as shown in Table 9, were chosen so that they were 1/7 to 7 times the estimated Kj value. Table 7. Inhibitory constants (Kj) of PCBs for inhibition of MROD, EROD and BROD activities Activity Kj ± Standarc Error (nM) P C B 47 P C B 52 P C B 54 P C B 77 M R O D 15000 ± 3 3 0 0 9 0 0 0 ± 1 7 0 0 N o inhibition 1 0 ± 1 E R O D 5500 ± 400 6500 ± 5 0 0 N o inhibition 46 ± 2 B R O D 440 ± 50 60 ± 10 100 ± 10 N o inhibition Kj values for each P C B were calculated from 3 sets of data performed in duplicate. M R O D and E R O D activities were determined with hepatic microsomes from MC-treated rats and B R O D activity was determined with hepatic microsomes from PB-treated rats. Table 8. V and K m values for MROD, EROD and BROD activities Activity K m ± Standard Error V ± Standard Error (nM) (pmol/min/mg) M R O D 230 ± 2 0 1250 ± 4 0 E R O D 490 ± 40 19,380 ± 8 8 0 B R O D 770 ± 40 20,830 ± 7 3 0 Data were fitted to the Michaelis-Menten equation by nonlinear regression analysis. Values were calculated from 9 sets of data performed in duplicate. V and K m values for M R O D activity were determined from 0 inhibitor concentration of experiments that determined M R O D activity in the presence of P C B 47, 52 and 17. Similarly, V and K m values for E R O D activity were determined from 0 inhibitor concentration of experiments that determined E R O D activity in the presence of P C B 47, 52 and 77. V and K m values for B R O D activity were determined from 0 inhibitor concentration of experiments that determined B R O D activity in the presence of P C B 47, 52 and 54. 63 Table 9. PCB concentrations used to inhibit alkoxyresorufin O-dealkylase activities Activity Inhibitor Concentrations (nM) P C B 47 P C B 52 P C B 54 P C B 77 M R O D 7500,15000 7500, 15000 20000 5, 25, 50, 75 E R O D 3000, 10000 3000, 10000 20000 25, 50, 100, 200 B R O D 100, 400, 800, 10, 50, 400, 20, 100, 400, 15000 1600 800 800 3.1.3. Effect of duration of preincubation of microsomes with PCBs on alkoxyresorufin O-dealkylase activities Figures 15 to 17 show the effect of the length of preincubation of hepatic microsomes with N A D P H and a P C B on alkoxyresorufin O-dealkylase activities. The effect of preincubation of hepatic microsomes with N A D P H and P C B 77 on M R O D activity is shown in Figure 15. M R O D activity was similar when hepatic microsomes were preincubated with N A D P H compared to preincubations with N A D P H and D M S O . Compared to preincubations with N A D P H or N A D P H and D M S O , M R O D activity was reduced when hepatic microsomes were preincubated with P C B 77 and N A D P H . Preincubation with P C B 77 resulted in lower M R O D activity than when hepatic microsomes were preincubated with P C B 77 and N A D P H . When hepatic microsomes were preincubated with P C B 77, MROD,act ivi ty decreased with increasing preincubation time after 5 min. M R O D activity increased with preincubation time when hepatic microsomes were preincubated with P C B 77 and N A D P H . For other preincubation mixtures, M R O D activity increased with preincubation time up to 10 min. 64 The effect of preincubation of hepatic microsomes with N A D P H and P C B 77 on E R O D activity is shown in Figure 16. No difference in activity was seen when hepatic microsomes were preincubated with N A D P H compared to preincubations with N A D P H and D M S O . E R O D activity was reduced when hepatic microsomes were preincubated with P C B 77 and N A D P H in comparison to preincubation with N A D P H or N A D P H and D M S O . Incubation with N A D P H and P C B 77 resulted in higher E R O D activity than when hepatic microsomes were preincubated with P C B 77 alone. For all incubation mixtures, E R O D activity increased with preincubation time up to 10 min. In Figure 17, the effect of preincubation of hepatic microsomes with N A D P H and P C B 54 on B R O D activity is shown. Incubation with both N A D P H and P C B 54 resulted in higher B R O D activities than incubation of microsomes with P C B 54 but no N A D P H . N o difference in activity was seen when hepatic microsomes were preincubated with N A D P H compared to preincubations with N A D P H and D M S O . A t 15 and 20 minutes, B R O D activity was approximately 30% lower when hepatic microsomes were preincubated with P C B 54 and N A D P H than when hepatic microsomes were preincubated with N A D P H or N A D P H and D M S O . B R O D activities increased with preincubation length and reached a plateau at 10 min for all reaction mixtures. 65 c o o < 180 160 140 120 100 $ 80 60 O D 1 4 0 Y * 20 0 V • V T • NADPH o DMSO + NADPH T P C B 77 V P C B 77 + NADPH 10 15 Preincubation Time (min) 20 25 Figure 15. Effect of preincubation of rat liver microsomes with P C B 77 on M R O D activities Hepatic microsomes from MC-treated rats (0.025 mg/mL) were incubated in buffer at 37 °C with or without D M S O , P C B 77 and N A D P H , as indicated in the figure. Final concentrations were: 0.5 % D M S O , 0.25 m M N A D P H and 20 p M P C B 77 dissolved in D M S O . Reactions were initiated by the addition of 0.1 p M methoxyresorufin and M R O D activity was measured at 3 min. Activities are presented as a percentage of the control activity (1180 pmol/min/mg), which is the 0 min preincubation time for the no D M S O group. Each data point is the average of two replicates determined in a single experiment. 66 140 120 c 100 f o O o 80 > j> 60 o < Q o or LU o • NADPH o DMSO + NADPH • PCB 77 V PCB 77 + NADPH 40 <7 20 10 15 Preincubation Time (min) 20 25 Figure 16. Effect of preincubation of rat liver microsomes with PCB 77 on EROD activities Hepatic microsomes from MC-treated rats (0.025 mg/mL) were incubated in buffer at 37 °C with or without D M S O , P C B 77 and N A D P H , as indicated on the figure. Final concentrations were: 0.5 % D M S O , 0.25 m M N A D P H and 20 u M P C B 77 dissolved in D M S O . Reactions were initiated by the addition of 0.1 u M ethoxyresorufin and E R O D activity was measured at 1.5 min. Activities are presented as a percentage of the control activity (6100 pmol/min/mg), which is the 0 min preincubation time for the no D M S O group. Each data point is the average of two replicates determined in a single experiment. 67 140 120 A c 100 + o CJ 80A o .£ 60 o < Q o or CD 40 <? 20 o • NADPH o DMSO + NADPH T P C B 54 V PCB 54 + NADPH 10 15 Preincubation Time (min) 20 25 Figure 17. Effect of preincubation of rat liver microsomes with PCB 54 on BROD activities Hepatic microsomes from PB-treated rats (0.025 mg/mL) were incubated in buffer at 37 °C with or without D M S O , P C B 54, and N A D P H , as indicated in the figure. Final concentrations were: 0.5 % D M S O , 0.25 m M N A D P H and 20 u M P C B 54 dissolved in D M S O . Reactions were initiated by the addition of 0.1 u M benzyloxyresorufin and B R O D activity was measured at 1.5 min. Activities are presented as a percentage of the control activity (13610 pmol/min/mg), which is the 0 min preincubation time for the no D M S O group. Each data point is the average of two replicates determined in a single experiment. 68 3.2. PCB 54 biotransformation assay The interaction of P C B 54 with C Y P enzymes was further studied by investigating the CYP-mediated biotransformation of P C B 54. C Y P enzymes involved in the biotransformation of P C B 54 were identified through the use of hepatic microsomes from rats treated with C Y P inducers and recombinant rat C Y P enzymes. 3.2.1. Identification of metabolites Hydroxylated P C B s were identified by L C / M S operated in (-)-ESI mode. The hydroxylated P C B s were identified by both their expected molecular mass and their chlorine ion spectra. Chlorine exists naturally as two isotopes, CI, which accounts for 75.77% of total chlorine and 3 7 C 1 , which accounts for the remaining 24.23% (Pretsch et al., 1983). When hydroxylated P C B s are analyzed by M S , the presence of these two chlorine isotopes results in the production of a characteristic mass spectrum that makes hydroxylated P C B s easily detectable. Table 10 summarizes the relative abundance of each mass for tetra- and pentachlorinated hydroxylated PCBs . Figures 18, 19 and 20 are representative m/z spectra of mono- and dihydroxylated tetrachlorobiphenyls and monohydroxylated pentachlorobiphenyls. From these mass spectra, it is evident that the ratio of the different masses matches the expected relative abundances in Table 10. The most abundant m/z for 4-OH-CB54 was 307. Thus, a m/z of 307 was used to quantify production of the monohydroxyl P C B 54 metabolite in future experiments. For the dihydroxyl P C B 54 metabolite, a m/z of 323 was monitored and a m/z of 341 was used to quantify 4-OH-CB104. 69 c Table 10. Masses and relative abundance of hydroxylated P C B s Monohydroxyl Tetrachlorobiphenyl Dihydroxyl Tetrachlorobiphenyl Monohydroxyl Pentachlorobiphenyl [M-H] ' Relative Abundance [M-H]" Relative Abundance [M-H] ' Relative Abundance 304.9 78 320.9 78 338.9 63 306.9 100 322.9 100 340.9 100 308.9 48 324.9 48 342.9 64 310.9 10 326.9 10 344.9 20 Relative abundances of isotopes were determined by values stated in Pretsch et al. (1983). 100n 306.9 04 305.1 308.9 / 311.0 / MVT"fTt I I I I I I i i i i I • ' f ^ l - f l . " | - ' l ' T ' l ' ! " | T I V , 300 305 310' 315 320' 325 " 330 335 340 345 350 355 360 365 370 375. 380 385 390 395 400 r1'r1''l-T"'r^"TT fW|' f)M'T'i' Figure 18. Negative E S I - M S m/z spectrum of 4 - O H - C B 5 4 70 B27.0 AJ\hhl\hJ 310 315 320 325 330 335 350 355 360 365 370 375 380 385 390 395 400 Figure 19. Negative E S I - M S m/z spectrum of the d ihydroxyl -CB54 metabolite 100-341.0 338.8 \ 315 342.9 TV i m/z 300 305 310 320 325 >330 335 340 345 350 355 360 365 370 375 380 385 390 395 400 Figure 20. Negative E S I - M S m/z spectrum of 4 - O H - C B 1 0 4 71 Initial experiments identified a monohydroxyl and a dihydroxyl metabolite as products of P C B 54 metabolism by hepatic microsomes. A representative chromatogram is shown in Figure 21. Metabolite formation was not observed with reactions conducted in the absence of N A D P H and in reaction mixtures containing carbon monoxide-treated microsomes or boiled microsomes. The only synthetic monohydroxyl P C B 54 compound commercially available is 4-OH-CB54. Comparison of retention times indicated that the metabolite produced by hepatic microsomes was not 4-OH-CB54. The identity of the dihydroxyl metabolite could not be confirmed due to the lack of dihydroxyl metabolite standards. Formation of the mono- and dihydroxyl metabolites conducted with low substrate concentrations under non-optimized conditions are shown in Figures 22 and 23. Formation of the monohydroxyl metabolite, expressed as peak area ratio, quickly reached a maximum and then declined with increasing incubation time. This decrease in peak area of the monohydroxyl metabolite coincided with an increase in the formation of the dihydroxyl metabolite. In incubations lasting two hours, no other metabolites were detectable. Based on the assumption that all monohydroxyl P C B 54 metabolites have a similar ionization efficiency, 4 -OH-CB54 was used to quantify production of the monohydroxyl metabolite. Quantification of the substrate was attempted using electrospray and atmospheric pressure chemical ionization (APCI) , but neither method was able to detect P C B 54. 72 100-4.42 3695067 A internal standard m/z 341 B.27 49058 i i I i i i i | i i i i | i i i i | i i i i | i i i i | i i i i | i i i i | . . . . . . . . . 1.00 2 . 0 0 3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0 m/z 323 100-| 523033 ~\ ct'*^H m e t a b o l ' t e I \ 2.91 3.31 3:70 4.95 1 0 Q 6 2 I V.. 5 1 8 6 2 , 8 2 1 5 6990 7522 ' 8 3 5 8 5 6.09 B 42 8.74 39968 0-1-TTTTTTTTT-n i i i | M I 1 1 1 1 I I I I 1.00 2 . 0 0 3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0 m/Z 307 100 0-1 2.49 7691573 A m°no-OH metabolrte 2.90 232475 I I | I I I I | I I I I | I I lT| I I I' I | I I I I | I I I I | I I I I | | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | I I I I | M 1 ' | I I I I | I I I I | 1.00 2 . 0 0 3 . 0 0 4 . 0 0 5 . 0 0 6 . 0 0 7 . 0 0 8 . 0 0 9 . 0 0 1 0 . 0 0 TIC 100-, „ , / f u 4 4 2 7.33 5.80 6.80 A r r ,A n i \ 8 14 8.758.91 ~M~rrT~M i i I"I i i i i i i i i i ' i i i i . i i i i i i i i i i i i i i i i i Time 1.00 2 . 0 0 3 . 0 0 4 . 0 0 . 5 . 0 0 6 . 0 0 7 .00 8 . 0 0 9 . 0 0 1 0 . 0 0 Figure 21. Separation of PCB 54 metabolism products and internal standard Representative chromatograph showing separation of P C B 54 metabolites and the internal standard from an extracted reaction mixture (154 u M P C B 54, incubation time 20 min and 0.5 mg/mL protein). The top three graphs show the ion abundance of m/z of 307, 323 and 341 with respect to time. The total ion current (TIC), m/z of 300-400, with respect to time is shown in the bottom graph. 73 10000 Time (min) Figure 22. Time dependent formation of the monohydroxyl PCB 54 metabolite P C B 54 was incubated with hepatic microsomes of PB-treated rats, 0.5 mg/mL microsomal protein, for varying lengths of time. Reactions were initiated with 1 m M N A D P H . Each data point represents the average of 2 determinations. CO 0.5 H o < CO o CL Q o < 0.1 10 20 30 Time (min) Figure 23. Time dependent formation of the dihydroxyl PCB 54 metabolite P C B 54 was incubated with hepatic microsomes of PB-treated rats, 0.5 mg/mL microsomal protein, for varying lengths of time. Reactions were initiated with 1 m M N A D P H . Each data point represents the average of 2 determinations. 74 3.2.2. Assay validation 3.3.2.1. Standard curve validation The peak areas determined for 4-OH-CB54 and the internal standard (4-OH-CB104) were tested over a range of concentrations (Figure 24). Peak area response was linear throughout the concentrations of 4-OH-CB54 and internal standard tested. The amounts of monohydroxyl metabolite and internal standard injected into the L C / M S from the metabolism assays were determined to be within the linear range. A standard curve (Figure 25) generated using various concentrations of 4 -OH-CB54 and spanning the range at which the monohydroxyl metabolite was formed, was validated for inter-and intra-assay variability. Both the inter- and intra-assay variabilities were below 10% at the concentrations of 4 -OH-CB54 tested. Tables 11 and 12 summarize the coefficients of variation for the individual concentrations tested. 75 4 - O H - C B 5 4 3 e + 7 - i 3 e + 7 A 3 0 0 0 0 Amount Injected (pg) 1.6e+7 -1.4e+7 -1.2e+7 -1 .0e+7 -o ID 8 . 0 e + 6 -< 6 . 0 e + 6 -4 . 0 e + 6 -2 . 0 e + 6 -0 . 0 -4 -OH-CB104 2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0 Amount Injected (pg) 1 0 0 0 0 1 2 0 0 0 Figure 24. Peak area response of 4-OH-CB54 and 4-OH-CB104 Peak area responses of 4 -OH-CB54 was tested over injection amounts ranging from 25 to 25000 pg. Linearity of response for 4-OH-CB104 was tested over injection amounts ranging from 25 to 10000 pg. Detector response was linear over the range of concentrations tested for 4 -OH-CB54 and 4-OHCB104. Each data point represents a single determination. 76 0.6 4-OH-CB54 (pmol/mL) Figure 25. Standard curve for 4 - O H - G B 5 4 Standard curve for 4 -OH-CB54 over the range of 41-1136 pmol/mL. Data presented as the mean value ± SD of 6 determinations. Each determination was performed on a separate day. 77 Table 11. Inter-assay variability of 4-C )H-CB54 4-OH-CB54 (pmol/mL) 41 325 649 974 1136 Amount Injected (pg) 62.5 500 1000 1500 1750 C . V . (%) 6.9 7.0 7.5 4.9 8.1 The inter-assay variability of 4 -OH-CB54 was assessed at 5 concentrations by calculating the C . V . of the P A R from 6 determinations performed on 6 different days. Table 12. Intra-assay variability of 4-OH-CB54 4-OH-CB54 (pmol/mL) 41 325 649 974 1136 Amount Injected (pg) 62.5 500 1000 1500 1750 C . V . (%) 5.8 5.1 3.5 2.4 3.1 Intra-assay variability of 4-OH-CB54 was determined at the same 5 concentrations used to determine inter-assay variability. Intra-assay variability was evaluated by calculating the C . V . of the P A R response of 6 determinations all performed on the same day. 3.3.2.2. Limits of detection and quantitation (LOD and LOQ) The L O D and L O Q were determined to be 8 pmol/mL and 26 pmol/mL, respectively, according to the criteria described in the Materials and Methodology section. 3.3.2.3. Extraction efficiencies of the monohydroxyl PCBs The extraction of 4 -OH-CB54 and 4-OH-CB104 from the microsomal assay mixture was evaluated as described in the Materials and Methodology section. Both 4-OH-CB54 and 4-OH-CB104 were found to have approximate percent recoveries of 100%. Table 13 summarizes the extraction efficiencies of 4 -OH-CB54, evaluated at three concentrations. The extraction efficiency for 4-OH-CB104, which was used as the internal standard, is also presented in Table 13. 78 Table 13. Percent recovery of 4-OH-CB54 and 4-OH-CB104 from the microsomal matrix Mean % Recovery ± S E M 4-OH-CB54 (pmol/mL) 162 107 ± 7 487 103 ± 2 974 97 ± 3 4-OH-CB104 (pmol/mL) 1461 99 ± 1 Extraction efficiencies of 4-OH-CB54 and 4-OH-CB104 were determined by comparing the peak area of the monohydroxyl P C B s extracted from the microsomal matrix to the peak area of the monohydroxyl P C B s from a non-extracted sample. 4 -OH-CB54 percent recoveries are presented as the mean value ± S E M of 6 replicates and 4-OH-CB104 percent recoveries are presented as the mean value ± S E M of 18 replicates. 3.2.3. A s s a y o p t i m i z a t i o n Assay conditions were optimized with microsomes from PB-treated rats to ensure that substrate concentration was saturating and product formation was linear with respect to incubation time and protein concentration. The optimized assay conditions for the formation of the mono- and dihydroxyl metabolites are summarized in Table 14, at the end of section 3.2.3. 3.2.3.1. Length of incubation To optimize monohydroxyl metabolite formation with respect to time, P C B 54 (153 pM) was incubated with 0.1 mg/mL microsomal protein for 0.5, 1, 2, 3, 5 and 7 min. Dihydroxyl metabolite formation was optimized using the same substrate concentration but 0.5 mg/mL microsomal protein was used and reactions were terminated after 1,5, 10, 15, 20, 30, 45 and 60 min. Time dependent formation of the monohydroxyl metabolite is shown in Figure 26. Metabolite formation was linear up to 2 min and 1 min 79 was chosen as the incubation time for subsequent experiments. Formation of the dihydroxyl metabolite with respect to time is shown in Figure 27. Dihydroxyl metabolite formation was only detectable after 5 min and it then increased linearly up to 40 min. For later experiments, 20 min was chosen as the optimal incubation time. 3.2.3.2. Microsomal protein concentration To optimize monohydroxyl metabolite formation with respect to microsomal protein concentration, P C B 54 (153 p M ) was incubated for 1 min with varying concentrations of protein (0.025, 0.05, 0.1, 0.25 and 0.5 mg protein/mL). Dihydroxyl metabolite formation was optimized using the same substrate concentration but the incubation length was 20 min and the protein concentrations used were, 0.25, 0.5, 0.75, 1 and 1.25 mg protein/mL. Monohydroxyl metabolite formation (Figure 28) was linear to 0.1 mg protein/mL and this microsomal protein concentration was chosen as the optimal protein concentration for further experiments. Figure 29 shows dihydroxyl metabolite formation with respect to protein concentration. Dihydroxyl metabolite formation was linear up to 0.75 mg protein/mL and in subsequent experiments, 0.5 mg protein/mL was used to measure dihydroxyl metabolite formation. 80 18000 16000 ~ 14000 c: O Q. |> 12000 o E Q-"O E T3 O 10000 H 8000 -\ i i 6000 4000 2000 I T i m e (min) Figure 26. Time dependent formation of the monohydroxyl PCB 54 metabolite P C B 54 (153 uM) was incubated with hepatic microsomes from PB-treated rats, 0.1 mg/mL microsomal protein, for 0.5, 1, 2, 3, 5 and 7 min. Reactions were initiated with 1 m M N A D P H . Monohydroxyl metabolite formation was linear up to 2 min. Mean values ± S E M from 3 sets of data performed in duplicate are presented. The optimal incubation time for monohydroxyl metabolite formation used in future experiments was 1 min. 81 0.6 CO 0.5 4 O < in o • £ 0.2 b o 3 5 < 0.1 H I o.o «» o Jfc 10 20 30 40 50 60 70 T i m e (min) Figure 27. Time dependent formation of the dihydroxyl PCB 54 metabolite P C B 54 (153 uM) was incubated with hepatic microsomes from PB-treated rats, 0.5 mg/mL microsomal protein, for 1,5, 10, 15, 20, 30, 45 and 60 min. Reactions were initiated with 1 m M N A D P H . Mean values ± S E M from 2 sets of data performed in duplicate are presented. Dihydroxyl metabolite formation was linear for 30 min. For future experiments, 20 min was used as the optimal incubation time. 82 o E CL T3 E o 2500 2000 1500 1000 500 0 4-0.0 0.1 0.2 0.3 0.4 Protein Concent ra t ion (mg/mL) 0.5 0.6 Figure 28. Protein dependent formation of the monohydroxyl PCB 54 metabolite P C B 54 (153 uM) was incubated with varying concentrations of hepatic microsomes from PB-treated rats (0.025, 0.05, 0.1, 0.25 and 0.5 mg protein/mL). Reactions were initiated with 1 m M N A D P H and terminated after 1 min. Monohydroxyl metabolite formation was linear at 0.1 mg protein/mL. Mean values ± S E M from 3 sets of data performed in duplicate are presented. For further experiments, a protein concentration of 0.1 mg/mL was used to measure monohydroxyl metabolite formation. 83 0.35 CO O 3 < m CD O CL o u_ T3 5^ . C b o < 0.30 0.25 H 0.20 0.15 0.10 H 0.05 H 0.00 0.0 f 0.2 0.4 0.6 0.8 1.0 Protein Concent ra t ion (mg/mL) 1.2 1.4 Figure 29. Protein dependent formation of the d ihydroxyl P C B 54 metabolite P C B 54 (153 uM) was incubated with 0.25, 0.5, 0.75, 1 or 1.25 mg protein/mL of hepatic microsomes from PB-treated rats. Reactions were initiated with 1 m M N A D P H and terminated after 20 min. Dihydroxyl metabolite formation was linear throughout the range of protein concentrations tested. Mean values ± S E M from 3 sets of data performed in duplicate are presented. For further experiments, a protein concentration of 0.5 mg/mL was used to measure dihydroxyl metabolite formation. 84 3.2.3.3. Saturating substrate concentration A saturating substrate concentration for monohydroxyl metabolite formation was determined by incubating 0.1 mg protein/mL of microsomes with varying substrate concentrations (34-171 p M ) for 1 min. For the dihydroxyl metabolite, varying substrate concentrations (34-171 p M ) were incubated with hepatic microsomes (0.5 mg protein/mL) for 20 min. Formation of the monohydroxyl metabolite (Figure 30) was saturated at substrate concentrations exceeding 68 p M . Formation of the dihydroxyl metabolite (Figure 31) was only performed in duplicate. From the results obtained, the saturating substrate concentration was not evident. Table 14. Summary of optimal assay conditions for the formation of the mono- and dihydroxyl metabolites Parameter Monohydroxyl Metabolite Dihydroxyl Metabolite Optimal Incubation Length 1 min 20 min Optimal Amount of Microsomal Protein 0.1 mg/mL 0.5 mg/mL Saturating Substrate Concentration >68 p M 85 •- 8000 0 O i— CL O) E . • i 6000 .5 4000 o < CD to ro >> o 2000 -\ T3 X m 20 40 60 — i — 80 100 120 140 160 180 [PCB 54] uM Figure 30. Effect of substrate concentration on the rate of formation of the monohydroxyl PCB 54 metabolite Varying concentrations o f P C B 54 were incubated with hepatic microsomes from P B -treated rats at 0.1 mg/mL microsomal protein, for 1 min. Monohydroxyl formation did not increase at P C B 54 concentrations exceeding 68 p M . Mean values ± S E M from 3 sets of data performed in duplicate are presented. 86 0.12 CO 0.10 O 3 < -5 0.08 IT) DO. O - 0.06 S? o TJ >. b o < 0.02 0.04 0.00 20 40 60 80 100 120 [PCB 54] (u.M) 140 160 180 Figure 31. Effect of substrate concentration on the formation of the dihydroxyl PCB 54 metabolite V a r y i n g concentrations o f P C B 54 were incubated w i t h hepatic microsomes f rom P B -treated rats at 0.5 m g / m L mic rosoma l protein, for 20 m i n . Average values f rom 1 set o f data performed i n duplicate are presented. 87 3.2.4. PCB 54 biotransformation with CYP-induced microsomes The biotransformation of P C B 54 to the monohydroxyl metabolite was examined in hepatic microsomes prepared from corn oi l - , M C - , P B - and DEX-treated rats to identify the C Y P enzymes involved. A l l experiments were conducted under the optimal assay conditions determined in the previous section. Formation of the monohydroxyl metabolite was detectable in incubations with hepatic microsomes prepared from P B - and DEX-treated rats while no metabolites were detected when P C B 54 was incubated with hepatic microsomes from corn o i l - and MC-treated rats. Figure 32 compares P C B 54 hydroxylase activity of the four microsomal preparations. P C B 54 hydroxylase activity was highest in hepatic microsomes from PB-treated rats, 7200 pmol/min/mg protein. P C B 54 hydroxylase activity in hepatic microsomes from DEX-treated rats was 550 pmol/min/mg protein. 88 8000 - i DEX Corn oil MC Hepa t ic M i c r o s o m a l Prepara t ion Figure 32. Effect of C Y P inducers on formation of the monohydroxyl P C B 54 metabolite P C B 54 hydroxylase activity was compared using a saturating concentration of P C B 54 (153 uM) incubated with 0.1 mg/mL microsomal protein for 1 min. Mean values ± S E M from 3 sets of data performed in duplicate are presented. 89 3.2.5. Antibody Inhibition Studies The C Y P 2 B enzymes are the predominant C Y P enzymes expressed in hepatic microsomes of PB-treated rats. The involvement of the C Y P 2 B enzymes in P C B 54 hydroxylase activity was investigated using polyclonal rabbit anti-rat C Y P 2 B 1 IgG. As shown in Figure 33, at the highest concentration of a n t i - C Y P 2 B l IgG tested (5 mg IgG/ mg protein), P C B 54 hydroxylase activity was inhibited by approximately 40% in comparison to the control. Based upon the previous experiments using CYP-induced microsomes it was expected that P C B 54 hydroxylase activity should be inhibited by at least 70% because C Y P 2 B is the major C Y P enzyme in microsomes from PB-treated rats. C Y P 2 B enzymes were the predominant enzymes involved in P C B 54 biotransformation. In an attempt to allow greater access of the antibody to the C Y P 2 B enzymes, the antibody inhibition experiment was repeated in the presence of a detergent, sodium cholate. The presence of 0.06% sodium cholate did not increase the inhibition of P C B 54 hydroxylase activity (Figure 34). Due to relatively low levels of inhibition observed, further studies using microsomes of PB-treated rats were not performed and antibody inhibition studies were not conducted with microsomes of DEX-treated rats. 90 120 i 20 -0 -I 1 1 1 1 1 1 0 1 2 3 4 5 6 m g IgG/ mg M i c r o s o m a l Pro te in Figure 33. Effect of anti-CYP2B IgG on monohydroxyl PCB 54 metabolite formation Hepatic microsomes from PB-treated rats (0.1 mg) were incubated in the presence of varying concentration of polyclonal rabbit anti-rat C Y P 2 B 1 IgG (-o-) or control IgG (-•-) for 15 min at 37 °C prior to the addition of P C B 54 (153 uM) . Results are expressed as a percent o f the control activity, which is activity in the presence of 0 mg IgG. Mean values ± S E M from 2 sets of data performed in duplicate are presented. 1 2 0 - | 2 0 -0 -I 1 1 1 1 1 1 0 2 4 6 8 1 0 1 2 mg IgG/ mg Microsomal Protein Figure 34. Effect of a n t i - C Y P 2 B IgG on monohydroxyl P C B 54 metabolite formation in the presence of sodium cholate Hepatic microsomes from PB-treated rats (0.1 mg) were incubated in the presence of 0.06% sodium cholate and varying concentration of polyclonal rabbit anti-rat C Y P 2 B 1 IgG (-o-) or control IgG (-•-) for 15 min at 37 °C prior to the addition of P C B 54 (153 pM). Results are expressed as a percent of the control activity, which is activity in the presence of 0 mg IgG. Values represent the average of 1 determination performed in duplicate. 92 3.2.6. P C B 54 biotransformation with recombinant C Y P enzymes A panel of nine recombinant rat C Y P enzymes was screened for formation of the monohydroxyl P C B 54 metabolite. As shown in Figure 35, C Y P 2 B 1 was the most active recombinant C Y P enzyme. The rate of formation of the monohydroxyl metabolite was 5.9 pmol/min/pmol C Y P with S U P E R S O M E S ™ expressing C Y P 2 B 1 . The only other C Y P enzyme that showed catalytic activity was C Y P 3 A 1 , which had a rate of formation for the monohydroxyl P C B 54 metabolite of 0.03 pmol/min/pmol C Y P . In the experiments using recombinant C Y P enzymes, incubations were terminated after 20 min. A longer incubation time was used to explore whether the monohydroxyl metabolite was metabolized to the dihydroxyl metabolite; however, no dihydroxyl metabolite was detectable. 93 D_ o a. o E C L O < CD en 7 -, 6H 5 ^ J3 S? 2 T3 >> X 10 m o Q. 1 H o O A2 CD c o CM T— < A2 CQ O *— < < Q CN CM CN o o CO CO CM K 0 . 0 . CM CM D . CL CL >• >- > > D_ >- >- >-o o o o AO CY O o o Figure 35. Comparison of monohydroxyl PCB 54 metabolite formation by a panel of recombinant C Y P enzymes P C B 54 hydroxylase activity was compared using 153 p M of P C B 54 incubated with baculovirus-insect cell microsomes containing expressed rat C Y P enzymes (50 pmol) for 20 min. Control reaction mixtures contained insect cell control microsomes that expressed CYP-reductase. Control reaction mixtures contained an equivalent amount of protein as other reaction mixtures (0.25 mg). P C B 54 hydroxylase activity is presented as mean values ± S E M of 3 determinations for microsomes expressing recombinant C Y P 2 B 1 and C Y P 3 A 1 . P C B 54 hydroxylase activity in control microsomes and microsomes expressing other recombinant C Y P s was from a single determination. 94 4. Discussion The lipophilic nature of P C B s allows them to be easily absorbed and bioaccumulate in organisms (Letcher et al., 1996; Sandala et al., 2004). C Y P enzymes are responsible for the initial oxidation of PCBs and are important in the elimination of P C B s from the body (Bandiera, 2001). Previous studies suggested that C Y P specificity for P C B binding and metabolism is affected by the coplanarity of the P C B molecule (Hrycay and Bandiera, 2003; Ishida et al., 1991; Koga et al., 1995a). The present study investigated the structure activity relationship between P C B chlorine substitution patterns and C Y P specificity. This was accomplished by studying the inhibition of various alkoxyresorufin (9-dealkylase activities by four tetrachlorobiphenyls and also through the development of an in vitro metabolism assay to study the CYP-mediated metabolism of P C B 54. 4.1. Inhibition of alkoxyresorufin 0-dealkylase activities Alkoxyresorufin (9-dealkylase activities were differentially inhibited by the four PCBs tested. P C B 54 did not inhibit E R O D or M R O D activity at the highest concentration tested (20 uM) and P C B 77 did not inhibit B R O D activity at the highest concentration tested (15 pM) . For the other P C B and alkoxyresorufin combinations, a concentration-dependent inhibition, by the PCBs , of alkoxyresorufin (9-dealkylase activities was observed. The <9-dealkylation of alkoxyresorufin compounds is commonly used to measure that activity of specific C Y P enzymes under defined conditions of inducer treatment (Burke et al., 1994). It was demonstrated previously that in hepatic microsomes from 95 MC-treated rats, E R O D and M R O D activities are predominantly catalyzed by C Y P l A l and C Y P 1 A 2 , respectively, and that B R O D activity in hepatic microsomes from P B -treated rats is mainly catalyzed by C Y P 2 B 1 and C Y P 2 B 2 (Burke et al., 1994). Based upon previous studies, we expected that the tetrachlorobiphenyls tested could be metabolized by different C Y P enzymes, depending upon their chlorine substitution pattern. Hence, we hypothesized that the tetrachlorobiphenyls would competitively inhibit the various CYP-mediated activities i f they were substrates for that enzyme. The results of the inhibition of the alkoxyresorufin (9-dealkylase assays showed that the substitution pattern of chlorine atoms on the biphenyl molecule was an important determinant in the ability of P C B s to inhibit the various alkoxyresorufin (9-dealkylase activities. The most coplanar P C B tested, P C B 77, with a dihedral angle o f 37.8°, was the most potent inhibitor of E R O D and M R O D activities while it showed no inhibition of B R O D activity. These findings suggest that P C B 77 is a substrate for the C Y P l A l and C Y P 1 A 2 enzymes. The three noncoplanar P C B s tested, P C B 47, P C B 52 and P C B 54 that had dihedral angles of 73.0°, 73.0° and 89.9°, respectively, all inhibited B R O D activity. P C B 47 and P C B 52 were also able to inhibit E R O D and M R O D activities but P C B 54, the most noncoplanar P C B tested, did not inhibit either of these activities. P C B 47 was 12 times more potent at inhibiting B R O D activity than E R O D activity and more than 30 times more potent at inhibiting B R O D activity than M R O D activities. P C B 52 was approximately 100 times more potent at inhibiting B R O D activity than E R O D activity and 150 times more potent at inhibiting B R O D activity than M R O D activity. These results suggest that P C B 47, P C B 52 and P C B 54 are likely to be substrates for the C Y P 2 B 1 and C Y P 2 B 2 enzymes. Both P C B 52 and P C B 54 had similar Kj values for 96 inhibition of B R O D activity (60 and 100 n M , respectively) that were less than the Kj value for inhibition of B R O D activity by P C B 47 (440 nM) . P C B 47 has chlorine atoms substituted in two ortho- (2 and 2') and the two para-positions, P C B 52 is chlorinated in two ortho- (2 and 2') and two meta-positions (5 and 5') and P C B 54 has chlorine atoms in all four orr/zo-positions. The differences in Kj values indicate that coplanarity alone cannot explain the inhibition of B R O D activity and suggest that chlorine substitution of the meta- and para-positions is also an important determinant of the potency of inhibition. In order to analyze the data and compare the potency of inhibition, data were modeled to competitive and mixed modes of inhibition and a Kj value was generated for each. Kj is an equilibrium constant between free inhibitor and inhibitor bound to an enzyme. The reciprocal of Kj is the enzyme-inhibitor affinity (Dixon and Webb, 1964). Competitive inhibition occurs when the substrate and inhibitor compete for the enzyme and when one binds, the other can not. Competitive inhibitors increase the K m of a reaction but the limiting rate (V) is unaffected. Mixed inhibitors contain both competitive and uncompetitive components (Cornish-Bowden and Wharton, 1988). Similar to competitive inhibitors, mixed inhibitors can bind to the free enzyme, which prevents substrate from binding. M i x e d inhibitors can also bind to the enzyme-substrate complex thereby preventing product formation. Mixed inhibitors reduce the limiting rate of a reaction while the K m may increase or decrease depending upon the affinity of the inhibitor for the enzyme and enzyme-substrate complexes (Cornish-Bowden and Wharton, 1988). 97 The mode of inhibition that was chosen to best fit the data was based upon A I C c and Sy.x values and visual inspection of the Lineweaver-Burk plots. For most alkoxyresorufin and P C B combinations, the A I C c and Sy.x values were very similar. For comparative purposes, the Michaelis-Menten and Lineweaver-Burk graphs for inhibition of E R O D activity by P C B 77 fit to a mixed mode of inhibition are shown in Figure 36 (Appendix I). Also in Appendix I, are the Michaelis-Menten and Lineweaver-Burk plots for inhibition of M R O D activity by P C B 77 fit to a competitive mode of inhibition (Figure 37). It is not apparent by visual inspection whether the competitive or mixed modes of inhibition better fit the data for inhibition of E R O D activity by P C B 77 (Figures 11 and 36). A comparison of A I C c and Sy.x values for two modes of modes of inhibition showed that when the data were modeled to a competitive mode of inhibition, the values were slightly lower (1754 vs. 1756 and 340 vs. 341, respectively). When the Lineweaver-Burk plots for inhibition of M R O D activity by P C B 77 modeled to competitive and mixed modes of inhibition are examined, it can be seen that a mixed mode of inhibition better fits the data (Figures 8 and 37). This is also apparent by a comparison of the A I C c and Sy.x values for the mixed and competitive modes of inhibition (1349 vs. 1362 and 43 vs. 44, respectively). The small differences in A I C c and Sy.x values when data are modeled to mixed and competitive modes of inhibition prevents definitive conclusions regarding the mode of inhibition by which P C B s are acting. However, the similarity in Kj values when data were modeled to mixed and competitive modes of inhibition allows for comparisons of the inhibitory effects of PCBs on the three alkoxyresorufin O-dealkylase activities tested. 98 Initial estimates of the K i values were determined by measuring alkoxyresorufin 0-dealkylase activity at a single substrate concentration in the presence o f varying concentrations of inhibitor. The range of inhibitor concentrations used for subsequent experiments were chosen so that they were 1/7 to 7 times the estimated Kj value. In the case of B R O D activity with P C B 47, P C B 52 and P C B 54, the concentrations of inhibitor chosen were not optimal. A s a result, the Kj values determined may not reflect the true K i values. Two previous studies examined the inhibition of M R O D activity by a number of P C B s with different substitution patterns. The first study by Chen et al. (2003) tested the inhibition of M R O D activity in hepatic microsomes from MC-treated rats by seven P C B s (terra-, penta- and hexachlorobiphenyls). The second study by Staskal et al. (2005) tested the ability of six P C B s (penta- and hexachlorobiphenyls) to inhibit M R O D activity in S U P E R S O M E S ™ expressing C Y P 1 A 2 . The results of these two studies showed the same general trend, coplanar P C B s were the most potent inhibitors of M R O D activity and noncoplanar P C B s inhibited M R O D activity to a lesser extent; however, the magnitude of differences in K , values between noncoplanar and coplanar P C B s reported by Chen et al. (2003) was quite different. The coplanar PCBs , P C B 77 and P C B 126 (3,3',4,4',5-pentachlorobiphenyl) were reported to be the most potent inhibitors with Kj values of 31 and 26 n M , respectively. The K i values of the noncoplanar P C B s , P C B 52 and P C B 153 (2,2',4,4',5,5'-hexachlorobiphenyl) were very similar, both with a Kj value of 80 n M . In the present study, the approximate Kj values of P C B 52 and P C B 77 were 9000 and 10 n M , respectively. This large difference in the estimated potency of inhibition is likely due to differences in experimental design. Chen et al. (2003) determined the K i value by 99 measuring M R O D activity in the presence of only one concentration of inhibitor. Kj values were calculated by plotting the data as a Lineweaver-Burk plot and using the slope of the regression line to determine the Kj . The current study determined inhibition of M R O D activity by P C B 52 using two concentrations of inhibitor and inhibition by P C B 77 with four concentrations of inhibitor. Kj values were determined by nonlinear regression. The present results, which indicate that the chlorine substitution pattern is an important determinant in the ability of a P C B to inhibit B R O D activity, are also consistent with the results of spectral interaction studies of P C B s with hepatic microsomes. Hrycay and Bandiera (2003) showed that the magnitude of the absorbance change, an indication of substrate binding, for P C B 47 was less than that of P C B 52 and P C B 54. To the best of our knowledge, the present study is the first to investigate PCBs as reversible inhibitors of E R O D and B R O D activities. In hepatic microsomes from Aroclor 1254-treated rats, it was previously shown that E R O D activity decreased by 15% after a 30 minute preincubation of microsomes with P C B 77 and N A D P H in comparison to preincubation with P C B 77 alone (Schlezinger et al., 1999). Based upon the results of this prior study, the possibility that PCBs were inhibiting alkoxyresorufin O-dealkylase activities through mechanism based inactivation in the present study was investigated. Liver microsomes were preincubated with P C B s and N A D P H for up to 20 min before the alkoxyresorufin substrate was added. The measured E R O D and B R O D activities were higher in comparison to preincubation with P C B s in the absence of N A D P H . N o reduction in M R O D activity was seen when hepatic microsomes from MC-treated rats were preincubated with P C B 77 and N A D P H compared to preincubation with P C B 77 in the absence of N A D P H . These results 100 demonstrate that the alkoxyresorufin O-dealkylase activities were not inhibited through mechanism-based inactivation. Previously it had been shown with purified rat C Y P enzymes that P C B 77 is metabolized by C Y P l A l but not C Y P 1 A 2 (Ishida et al., 1991). In this study it was shown that S U P E R S O M E S ™ expressing C Y P 2 B 1 metabolized P C B 54. When hepatic microsomes were preincubated in the presence of P C B 54 and N A D P H , P C B 54 was metabolized to a hydroxylated metabolite. Based upon the results of the P C B 54 biotransformation assay, it is expected that the amount of P C B 54 available to bind to C Y P 2 B 1 decreased with increasing preincubation time. A s a result, B R O D activity was increased in comparison to preincubations with P C B 54 in the absence of N A D P H . A similar explanation is plausible for P C B 77 and C Y P l A l -mediated E R O D activity. In this study, P C B 77 was found to be a potent inhibitor of C Y P 1 A2-mediated M R O D activity indicating that P C B 77 binds to C Y P 1 A 2 . However, Ishida et al. (1991) showed that P C B 77 is not metabolized by C Y P 1 A 2 . When P C B 77 binds to C Y P 1 A 2 it is not readily released because it is not metabolized, regardless whether N A D P H is present or not. This prevents methoxyresorufin from binding, resulting in a reduction in M R O D activity. A general trend that was noted for all three alkoxyresorufin O-dealkylase activities was that activity increased with longer preincubation time. The reason for the increase in alkoxyresorufin O-dealkylase activities with preincubation time is unclear. It was not a temperature effect because buffer was warmed to 37 °C prior to the experiments. N A D P H - C Y P reductase is responsible for the transfer of electrons from N A D P H to C Y P enzymes (Gutierrez et al., 2003). The effect of P C B s on N A D P H - C Y P reductase activity was investigated to ensure that the inhibition seen in the alkoxyresorufin O-101 dealkylase assays was due to the P C B s acting directly on the C Y P enzymes. N A D P H -cytochrome P450 reductase activity in rat liver microsomes was determined using cytochrome c as an artificial electron acceptor and measuring its rate of reduction at 550 nm. The effect of various P C B s on activity was determined by preincubation of hepatic microsomes with P C B s in the phosphate buffer mixture before addition of cytochrome c and N A D P H . Incubation of hepatic microsomes of MC-treated rats with 15 u M of P C B 47, 52 or 77 resulted in no significant reduction in NADPH-cytochrome c reductase activity. Similarly, incubation of hepatic microsomes of PB-treated rats with 1.6 u M of P C B 47'or 0.8 p M of P C B 52 and P C B 54 led to no significant reduction in activity (appendix III). The results confirm that P C B s did not affect N A D P H - C Y P reductase activity and suggest that P C B s are exerting their inhibitory effect on the C Y P enzymes directly. Preliminary studies were also conducted to investigate the inhibition of C Y P 2 C 6 activities in hepatic microsomes of corn-oil treated rats by P C B s using ethoxyresorufin as a probe substrate. Burke et al., (1994) previously showed that C Y P 2 C 6 was the predominant enzyme catalyzing E R O D activity in hepatic microsomes of corn-oil treated rats using antibody inhibition. The C Y P 2 B 1 , C Y P 2 C 1 1 , C Y P 3 A 1 and C Y P 3 A 2 enzymes were also shown to be involved in E R O D activity, but to a lesser extent.. The results of the preliminary experiments involving inhibition of E R O D activity by P C B 47 and 52 demonstrated that multiple enzymes were catalyzing the O-dealkylase reaction and the effects the P C B s on C Y P 2 C 6 could not be discerned. Due to this reason, further experiments were not conducted. 102 The results of the inhibition of alkoxyresorufin O-dealkylase activities by the four tetrachlorobiphenyls tested support the notion that noncoplanar P C B s are metabolized by the C Y P 2 B enzymes while coplanar P C B s are metabolized by the C Y P 1 A enzymes. However, like spectral interaction studies, these inhibition studies only provide evidence that the P C B s are binding to the active site of the C Y P enzymes, which does not necessarily mean that they undergo biotransformation. One advantage of inhibiting the metabolism of methoxyresorufin is that it provides an indication of C Y P 1 A 2 substrate specificity. Spectral interaction studies cannot do this because C Y P 1 A 2 does not undergo a shift in spin state when a substrate binds. 4.2. PCB 54 biotransformation assay Induced C Y P microsomes and recombinant C Y P S U P E R S O M E S ™ were used to identify the C Y P enzymes responsible for the biotransformation of P C B 54. P C B 54 metabolites were analyzed using L C / M S . Previous in vitro biotransformation studies of PCBs have identified and quantitated metabolites by gas chromatography equipped with an electron capture detector ( G C - E C D ) . A n advantage of using L C / M S is that the hydroxylated metabolites do not need to be derivatized in order to be analyzed. G C -based approaches require derivitization of the hydroxylated metabolites. Derivitization has been reported to result in sample loss (Letcher et al., 2005) and it is also lengthens the time required before samples can be analyzed. The L C / M S method was validated with respect to inter- and intra-assay variability and limits of detection and quantitation. The response of the L C / M S showed low variability when 4-OH-CB54 standards were analyzed on the same day and on different days. The L O D and L O Q were determined to be 8 and 26 pmol/mL, respectively. 5 p L of sample was injected into the L C / M S for 103 analysis; thus the L O D and L O Q expressed as a mass were 15 and 40 pg, respectively. A previous study that compared L C / M S (equipped with a time-of-flight (TOF) detector) against G C low resolution mass spectrometry ( G C / L R M S ) and high resolution mass spectrometry ( G C / H R M S ) to analyze hydroxylated P C B s determined the instrument L O D s to be 2, 0.1 and 0.05 pg, respectively (Berger et al., 2004). The sensitivity of L C / T O F - M S towards P C B s is known to increase with the degree of chlorination (Berger et al., 2004). However, in this study it was unclear which hydroxylated P C B s were used to determine the L O D s and also how the L O D s were determined. In another study that analyzed hydroxylated P C B s by LC- ( - ) -ESI -MS-MS the instrument L O D for 4 -OH-CB72 (2,3',5,5'-tetrachloro-4-biphenylol) was 10 pg (Letcher et al., 2005). The L O D for 4-OH-CB54 determined in the present study is comparable to the L O D for 4-OH-CB72 determined by L C - ( - ) - E S I - M S - M S . Based upon L O D values, G C / L R M S and G C / H R M S are able to detect lower concentrations of hydroxylated PCBs . Analysis of the P C B 54 metabolism products by L C / M S operated in (-)-ESI mode revealed the production of a mono- and dihydroxyl metabolite. When analyzed by (-)-ESI -MS the hydroxy group of the metabolites is deprotonated producing a charged molecule that can be detected. P C B 54, the substrate, lacks any reactive groups that can be deprotonated by (-)-ESI and thus can not be detected by the M S . Due to a lack of metabolite standards, the mono and dihydroxyl metabolites could not be identified. Based upon a comparison of retention times, the monohydroxyl metabolite was not 4-OH-CB54 . There are two possible structures for the monohydroxyl metabolite, a hydroxylation at a meta- position yielding, 2,2',6,6'-tetrachloro-3-biphenylol (3-OH-CB54) or the formation of an arene oxide intermediate which undergoes a N I H shift to 104 form 2',3,6,6'-tetrachloro-2-biphenylol. Several lines of evidence suggest that 3-OH-CB54 is the most likely structure. In previous in vitro metabolism studies involving P C B 52, the sole monohydroxyl metabolite produced (2,2',5,5'-tetrachloro-3-biphenylol) was hydroxylated at a me/a-position (Ishida et al., 1991; Koga et al., 1995a; Koga et al;, 1995b; Preston et al., 1983). Hydroxylation at the meto-position is also thought to occur because with other in vitro P C B metabolism studies to date, no metabolites were found to be hydroxylated at the orzTzo-positions (Koga etal., 1994; Koga et al., 1995a; Goto et al., 1974). Furthermore, analyses of hydroxylated PCBs in human blood, liver and adipose tissue and polar bear blood and adipose tissue have only detected monohydroxyl metabolites that are hydroxylated in the meta- or para-positions (Guvenius et al., 2002; Sandala et al., 2004; Sandau et al., 2000). Based upon the results of preliminary experiments, which showed a decline in monohydroxyl metabolite levels as dihydroxyl metabolite increased (Figure 22 and 23), it is hypothesized that the monohydroxyl metabolite is a precursor to the dihydroxyl metabolite. In comparison to the monohydroxyl metabolite, formation of the dihydroxyl metabolite showed much greater variability. A s the optimization experiments progressed, the formation of the dihydroxyl metabolite decreased with each experiment and eventually became undetectable. Due to this, only four determinations of optimization of dihydroxyl metabolite formation with respect to time and two determinations of a saturating substrate concentration were conducted. A number of experiments were performed to determine the cause of the disappearance of the dihydroxyl metabolite. The m/z range was broadened to scan from 20 to 400 in order to detect any degradation products; however no degradation was detectable. Fresh assay buffer and sucrose 105 solution were prepared but this did not solve the problem either. The cause of the variability associated with the production of the dihydroxyl metabolite could not be determined. P C B 54 hydroxylase activity was highest in liver microsomes from PB-treated rats, converting substrate to the monohydroxyl metabolite at a rate of 7200 pmol/min/mg protein. Hepatic microsomes from DEX-treated rats catalyzed the formation of the monohydroxyl metabolite at a rate of 550 pmol/min/mg protein. N o metabolites were detectable when P C B 54 was incubated with hepatic microsomes from corn oi l and M C -treated rats. A number of studies have looked at the rates of metabolism of P C B 52, P C B 77 and P C B 80. The reported rates of metabolism of P C B 52 in hepatic microsomes from PB-treated rats have varied from 324 to 1450 pmol/min/mg protein (Ishida et al., 1991; Koga et al., 1995a). In hepatic microsomes from MC-treated rats, the reported rate of metabolism of P C B 77 has varied from 5 to 34.5 pmol/min/mg protein and the rate of P C B 80 metabolism in hepatic microsomes from MC-treated rats has been reported at 45.1 pmol/min/mg protein (Ishida et al., 1991; Koga et al., 1995a). The rates of P C B 54 metabolism determined in this study are much higher than the rates of metabolism of other P C B s reported in other studies. The reason for this may be due to differences in the extraction procedures between this and the previous studies, rather than P C B 54 being a better substrate. Ishida et al. (1991) and Koga et al. (1995a) used the same extraction procedure. In both studies, the reaction was terminated by the addition o f 7 m L of a 2:1 mixture of chloroform-methanol and the metabolites were extracted from the aqueous phase using 14 m L of n-hexane (Ishida et al., 1991; Koga et al., 1995a). The aqueous phase was then extracted twice more with 20 m L each of rc-hexane. In the extraction 106 procedure the aqueous phase was not acidified, which may reduce the extraction of the hydroxyl metabolites because some of the hydroxyl metabolite w i l l be deprotonated. Neither study used an internal standard to account for differences in extraction efficiency nor was the extraction efficiency stated. Therefore, it is not known i f the activities reported were accurate or i f they were underestimated due to low extraction efficiencies of the metabolites. Administration of P B to rats resulted in a large induction of the hepatic C Y P 2 B enzymes. Treatment of rats with D E X predominately induced hepatic C Y P 3 A enzymes. The C Y P 2 B enzymes were also slightly induced by D E X in comparison to the corn o i l -treatment as seen in the immunoblot results (Appendix II). Comparison of the P C B 54 hydroxylase activities determined using the four microsomal preparations, suggested that the C Y P 2 B enzymes were involved in the metabolism of P C B 54. To further investigate this result, P C B 54 metabolism assays with hepatic microsomes of PB-treated rats were conducted in the presence of ant i -CYP2B IgG. It was anticipated that formation of the monohydroxyl metabolite would be reduced by at least 70%; however, at the highest concentration of antibody tested, 5 mg IgG/mg microsomal protein, formation of the monohydroxyl metabolite was only inhibited by approximately 40%. A possible explanation for this result was that the lipid bilayer of the microsomes was preventing the antibody from binding to the C Y P 2 B enzymes. Because o f this, the antibody inhibition studies were repeated in the presence of an anionic detergent, sodium cholate. Sodium cholate was used to help solubilize the membrane proteins and increase the interaction of the antibody with the C Y P 2 B enzymes. The use of sodium cholate did not improve the inhibition of P C B 54 hydroxylase activities by C Y P 2 B antibodies. The results of the 107 immunoblots, shown in Table 15 (Appendix II), indicate that the a n t i - C Y P 2 B l IgG used reacted with the C Y P 2 B enzymes. It is difficult to provide an explanation for the observed lack of inhibition without further experiments. Further antibody inhibition studies were not conducted and studies using recombinant C Y P enzymes were performed instead to identify the C Y P enzymes that can metabolize P C B 54. O f the recombinant C Y P s examined only C Y P 2 B 1 and C Y P 3 A 1 hydroxylated P C B 54 to the monohydroxyl metabolite. C Y P 2 B 1 had the highest activity, 5.9 pmol/min/pmol C Y P , which was approximately 200 fold greater than that of C Y P 3 A 1 . No metabolites were detectable when P C B 54 was incubated with control S U P E R S O M E S ™ or S U P E R S O M E S ™ expressing recombinant rat C Y P 1 A 2 , C Y P 2 A 2 , C Y P 2 C 6 , C Y P 2 C 1 1 , C Y P 2 C 1 3 , C Y P 3 A 2 a n d C Y P 2 D l . In summary, the data from the experiments using hepatic microsomes from rats treated with C Y P inducers and recombinant C Y P enzymes suggest that C Y P 2 B enzymes are the predominant C Y P enzymes involved in the biotransformation of P C B 54. These results also, to our knowledge, show for the first time the ability of C Y P 3 A enzymes to hydroxylate P C B s . 4.3. Conclusions The inhibition of alkoxyresorufin O-dealkylase activities by P C B s demonstrated that the chlorine substitution pattern is an important determinant in the inhibition of these activities. Noncoplanar P C B s , P C B 47, P C B 52 and P C B 54 were the most potent inhibitors of CYP2B-mediated B R O D activity in hepatic microsomes from PB-treated rats. In addition to chlorine atoms substituted in the or/Tzo-positions that affect the planarity of a molecule, substitution at the meta- and para-positions affects the potency 108 of inhibition. P C B 77, a coplanar P C B , was the most potent inhibitor of C Y P 1 A l -mediated E R O D activity and CYPlA2-media ted M R O D activity in hepatic microsomes from MC-treated rats. A n in vitro assay was developed and validated to analyze the hepatic biotransformation o f P C B 54. The major metabolite was a monohydroxyl metabolite. Due to a lack of authentic standards, the position of hydroxylation on the biphenyl ring could not be identified. Biotransformation assays using induced C Y P microsomes and S U P E R S O M E S ™ expressing recombinant rat C Y P enzymes demonstrated that the biotransformation of P C B 54 to the monohydroxyl metabolite was predominately catalyzed by the C Y P 2 B enzymes. These results also confirm the conclusions reached by the alkoxyresorufin O-dealkylase inhibition experiments, which suggested that the C Y P 2 B enzymes were involved in the biotransformation of P C B 54. 109 4.4. Future experiments 1. Addi t ional evidence for a structure-activity relationship In order to provide additional evidence that C Y P specificity for PCBs is determined by the chlorine substitution pattern, additional experiments would need to be conducted with P C B s of varying degrees of chlorination (tri-, tetra-, penta- and hexachlorobiphenyls) that have different coplanarities. 2. Identify the monohydroxyl P C B 5 4 metabolite The monohydroxyl P C B 54 metabolite can be identified by dechlorination of the molecule using a previously published method (Goto et al., 1974). The dechlorinated metabolite, a hydroxylated biphenyl, can then be identified using L C / M S by comparing the retention time to that o f known hydroxylated biphenyl standards. 3. Extension of the assay to use liver microsomes from humans and wildlife The method developed in this study can be extended to include microsomes derived from other species. 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Pereg D , Dewailly E , Poirier G G , and Ayotte P (2002) Environmental exposure to polychlorinated biphenyls and placental C Y P l A l activity in Inuit women from northern Quebec. Environ Health Perspect 110: 607-612. Preston B D , Mi l le r J A , and Mi l le r E C (1983) Non-arene oxide aromatic ring hydroxylation of 2,2',5,5'-tetrachlorobiphenyl as the major metabolic pathway catalyzed by phenobarbital-induced rat liver microsomes. J Biol Chem 258: 8304-8311. Pretsch E, Clerc T, Seibl J, and Simon W (1983) Mass spectrometry. Tables of spectral data for structure determination of organic compounds (Boschke F L , Fresenius W, Huber J F K , Pungor E , Rechnitz G A , Simon W and West ThS, eds.). Springer-Verlag, New York, N Y . M 5 - M 1 7 0 . 117 Qatanani M and Moore D D (2005) C A R , the continuously advancing receptor, in drug metabolism and disease. Curr Drug Metab 6: 329-339. Rasmussen JB , Rowan D J , Lean D R S , and Carey J H (1990) Food chain structure in Ontario lakes determines P C B levels in lake trout (Salvelinus namaycush) and other pelagic fish. Can J Fish Aquat Sci 47: 2030-2038. Rekka E , Evdokimova E , Eeckhoudt S, Labar G , and Calderon P B (2002) Role of temperature on protein and m R N A cytochrome P450 3 A ( C Y P 3 A ) isozymes expression and midazolam oxidation by cultured rat precision-cut liver slices. Biochem Pharmacol 64:633-643. Ruder A M , Hein M J , Nilsen N , Waters M A , Laber P, Davis-King K , Prince M M , and Whelan E (2006) Mortality among workers exposed to polychlorinated biphenyls (PCBs) in an electrical capacitor manufacturing plant in Indiana: an update. Environ Health Perspect 114: \8-23. Safe S (1993) Toxicology, structure-function relationship, and human and environmental health impacts of polychlorinated biphenyls: progress and problems. Environ Health Perspect 100: 259-268. Safe S (1994) Polychlorinated biphenyls (PCBs): environmental impact, biochemical and toxic responses, and implications for risk assessment. Crit Rev Toxicol 24: 87-149. Safe S'(1997) Limitations of the toxic equivalency factor approach for risk assessment of T C D D and related compounds. Teratog Carcinog Mutagen 17: 285-304. Safe S, Bandiera S, Sawyer T, Robertson L , Safe L , Parkinson A , Thomas PE , Ryan D E , Reik L M , Levin W , Denomme M A , and Fujita T (1985) P C B s : structure-function relationships and mechanism o f action. Environ Health Perspect 60: 47-56. Sandala G M , Sonne-Hansen C, Dietz R, Mui r D C G , Valters K , Bennett E R , Born E W , and Letcher R J (2004) Hydroxylated and methyl sulfone P C B metabolites in adipose and whole blood of polar bear (Ursus maritimus) from East Greenland. Sci Total Environ 331: 125-141. Sandau C D , Ayotte P, Dewailly E , Duffe J, and Norstrom R J (2000) Analysis of hydroxylated metabolites of P C B s (OH-PCBs) and other chlorinated phenolic compounds in whole blood from Canadian Inuit. Environ Health Perspect 108: 611-616. Schlezinger JJ, White R D , and Stegeman JJ (1999) Oxidative inactivation of cytochrome P-450 1A (CYP1 A ) stimulated by 3,3',4,4'-tetrachlorobiphenyl: production of reactive oxygen by vertebrate C Y P 1 As . Mol Pharmacol 56: 588-597. 118 Schnellmann R G , Putnam C W , and Sipes IG (1983) Metabolism of 2,2',3,3',6,6'-hexachlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl by human hepatic microsomes. Biochem Pharmacol 32: 3233-3239. Sesardic D , Cole K J , Edwards R J , Davies DS, Thomas P E , Levin W , and Boobis A R (1990) The inducibility and catalytic activity of cytochromes P450c (P450IA1) and P450d (P450IA2) in rat tissues. Biochem Pharmacol 39: 499-506. Soucek P and Gut I (1992) Cytochrome P-450 in rats: structures, functions, properties and relevant human forms. Xenobiotica 22: 83-103. Stapleton H M and Baker JE (2003) Comparing polybrominated diphenyl ether and polychlorinated biphenyl bioaccumulation in a food web in Grand Traverse Bay, Lake Michigan. Arch Environ Contam Toxicol 45: 227-234. Staskal D F , Diliberto JJ, Devito M J , and Birnbaum L S (2005) Inhibition of human and rat C Y P 1 A 2 by T C D D and dioxin-like chemicals. Toxicol Sci 84: 225-231. Tampal N M , Robertson L W , Srinivasan C, and Ludewig G (2003) Polychlorinated biphenyls are not substrates for the multidrug resistance transporter-1. Toxicol Appl Pharmacol187: 168-177. Wang H and Negishi M (2003) Transcriptional regulation of cytochrome P450 2B genes by nuclear receptors. Curr Drug Metab 4:515-525. Williams D A (2002) Drug metabolism. Foye's Principles of Medicinal Chemistry, 5th edition (Williams D A and Lemke T L , eds.). Lippincott Will iams & Will iams, Baltimore, M D . pp. 174-233. Wong A and Bandiera S M (1996) Inductive effect of Telazol® on hepatic expression of cytochrome P450 2B in the rat. Biochem Pharmacol 52: 735-742. World Health Organization (WHO) (1993) Environmental Health Criteria 140: Polychlorinated Biphenyls and terphenyls second edition. October 8, 2005 <http://www.inchem.org/documents/ehc/ehc/ehcl40.htm>. Yasui H , Hayashi S, and Sakurai H (2005) Possible involvement of singlet oxygen species as multiple oxidants in P450 catalytic reactions. Drug Metab Pharmacokinet 20: 1-13. 119 Appendix I: Inhibition of EROD and MROD activities PCB 77 14000 12000 E 10000 H o E a. & '> o < Q O CC LU B 8000 6000 4000 2000 0.0 0.004 0.1 0.3 0.4 0.5 [Ethoxyresorufin] (pM) PCB 77 •5 0 5 10 15 1/[Ethoxyresorufin] (pM) Figure 36. Inhibi t ion of E R O D activity by P C B 77 Michaelis-Menten (A) and Lineweaver Burk (B) plots of rat liver microsomal E R O D activity in the presence o f P C B 77. Varying concentrations of ethoxyresorufin were incubated with hepatic microsomes (0.025 mg protein/mL) from MC-treated rats. Reaction mixtures also contained either D M S O or P C B 77 dissolved in D M S O . Concentration of P C B 77 used in reaction mixture indicated by '1= '. Reactions were initiated with N A D P H and E R O D activity was measured at 1.5 min. Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a mixed mode of inhibition by nonlinear regression analysis. 120 A PCB 77 •6 -4 -2 0 2 4 6 8 10 12 1 / [ M e t h o x y r e s o r u f i n ] ( u M ) Figure 37. Inhibit ion of M R O D activity by P C B 77 Michaelis-Menten (A) and Lineweaver Burk (B) plots of rat liver microsomal M R O D activity in the presence of P C B 77. Varying concentrations of methoxyresorufin were incubated with microsomes from MC-treated rats (at 0.025 mg protein/mL). Reaction mixtures also contained either D M S O or P C B 77 dissolved in D M S O . Concentration of P C B 77 used in reaction mixture indicated by T= '. Reactions were initiated with N A D P H and M R O D activity was measured at 1.5 min. Control incubations contained D M S O . Mean values ± SD from 3 sets of data performed in duplicate are presented. Data were fitted to a competitive mode of inhibition by nonlinear regression analysis. 121 Appendix II: Immunoblot analysis of CYP enzymes expression in hepatic microsomes Immunoblot analysis was performed to quantify the levels of C Y P 1 A , C Y P 2 B , CYP2C11 and C Y P 3 A enzymes in hepatic microsomes that were used in the alkoxyresorufin O-dealkylase assays and P C B 54 biotransformation assays. Immunoblot experiments were performed by Dr. Eugene Hrycay. 1. Materials and Methodology 1.1. Materials Anachemia (Montreal, Quebec, Canada): Hydrogen peroxide, 30% solution BDH chemicals (Toronto, Ontario, Canada): Ethylenediaminetetracetic acid ( E D T A ) , disodium salt Bio-Rad Laboratories (Mississauga, Ontario, Canada): Acrylamide; N,N'-methylene-bis-acrylamide (BIS); P-mercaptoethanol; N , N , N ' , N'-tetramethylethylenediamine ( T E M E D ) . Biosource International (Camarillo, California, U.S.A.): Alkaline phosphatase-conjugated goat anti-mouse; goat anti-rabbit F[ab']2 immunoglobulin G (IgG). 122 Fischer Scientific (Vancouver, British Columbia, Canada): Dimethylformamide ( D M F ) ; glycine; methanol, reagent grade; potassium phosphate monobasic; sodium chloride; sodium dodecyl sulphate (SDS); sodium phosphate dibasic. GENTEST (Woburn, Massachusetts, U.S.A.): Recombinant rat C Y P 3 A 1 ; recombinant rat C Y P 3 A 2 ICN Biomedicals Canada Ltd. (St-Laurent, Quebec, Canada): Bovine serum albumin (BSA) ; tris(hydroxymethyl)aminomethane (Trizma base). Mqndel Scientific Company Ltd. (Edomnton, Alberta, Canada): Blotting paper; nitrocellulose membrane. Pacific Milk Division (Vancouver, British Columbia, Canada): Skim milk powder Pierce (Rockford, Illinois, U.S.A.): 4- Nitro-blue tetrazolium chloride Schwarz/Mann Biotech (Cleveland, Ohio, U.S.A.): Ammonium persulphate Sigma Chemical Co. (St. Louis, Missouri, U.S.A.): Bromphenol blue; potassium chloride; tris(hydroxymethyl)aminomethane hydrochloride (Trizma HCI); poloxyethylene sorbitan monolaurate (Tween 20). Xymotech Biosystems (Mt. Royal, Quebec, Canada): 5- Bromo-4-chloro-3-indolyl phosphate disodium salt 123 Dr. S.M. Bandiera (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, British Columbia, Canada): Polyclonal rabbit anti-rat C Y P 1 A 2 IgG; polyclonal rabbit anti-rat C Y P 2 B 1 IgG; purified rat C Y P 1 A l ; purified rat C Y P 2 B 1 ; purified rat C Y P 2 C 1 1 . Dr. Wayne Levine (Hoffmann-La Roche Inc., Nutley, New Jersey, U.S.A.): Purified rat C Y P 1A2 Dr. P. Thomas (Rutgers University, Piscataway, New Jersey, U.S.A.): Monoclonal mouse anti-rat C Y P 1 A 1 IgG 1.2. Methodology Sodium dodecyl sulfate-polyacrylamide gel electrophoresis on a 7.5% gel followed by immunoblot analysis was used to identify and measure rat C Y P 1 A 1 , C Y P 1 A 2 , C Y P 2 B 1 , C Y P 2 B 2 , C Y P 2 C 1 1 , C Y P 3 A 1 , C Y P 3 A 2 , and C Y P 3 A 1 8 proteins. Varying concentrations of the appropriate internal standards consisting of rat C Y P 1 A l (0.05-0.375 pmol), C Y P 1 A 2 (0.025-0.25 pmol), C Y P 2 B 1 (0.03125-0.25 pmol), CYP2C11 (0.1-0.4 pmol), and recombinant C Y P 3 A 1 and C Y P 3 A 2 (0.125-0.375) were also included on the gels. Electrophoresis was carried out for 3.5 h as described previously (Wong and Bandiera, 1996; Hrycay and Bandiera, 2003). The resolved proteins were transferred onto nitrocellulose membranes and incubated for 2 h at 37° C with the following primary antibodies: monoclonal a n t i - C Y P l A l (6 pg/ml), polyspecific a n t i - C Y P l A 2 (1:860 dilution), polyspecific a n t i - C Y P 2 B l (2 ug/ml), monospecific anti-CYP2C11 (10 pg/ml), and polyspecific an t i -CYP3A (5 pg/ml) IgG (Hrycay and Bandiera, 2003; Wong and Bandiera, 1996). Mouse monoclonal anti-rat C Y P 1 A 1 IgG provided by Dr. Thomas reacts specifically with rat C Y P 1 A 1 and does not recognize • 124 C Y P 1 A 2 . Rabbit polyclonal a n t i - C Y P l A 2 IgG reacts strongly with C Y P 1 A 2 and weakly with rat C Y P l A l . Rabbit polyclonal a n t i - C Y P 2 B l IgG reacts equally well with C Y P 2 B 1 and C Y P 2 B 2 . After a three-cycle wash step, bound primary antibody was located by incubating immunoblots with secondary antibody (alkaline phosphatase-conjugated goat anti-mouse or goat anti-rabbit F[ab']2 IgG) at a 1:1500 or 1:3000 dilution, respectively. After a further three-cycle wash step, C Y P protein bands were detected by addition of substrate solution (Wong and Bandiera, 1996; Hrycay and Bandiera, 2003) and were quantified by densitometric analysis. The amount of immunoreactive C Y P protein was determined by means o f calibration curves prepared with purified rat C Y P standards as described previously (Wong and Bandiera, 1996; Hrycay and Bandiera, 2003). Because rabbit ant i -CYP2B polyclonal IgG reacts equally with C Y P 2 B 1 and C Y P 2 B 2 , purified rat C Y P 2 B 1 was used as the calibration standard for measurement of microsomal C Y P 2 B 1 and C Y P 2 B 2 enzymes. 2. Results Table 15 summarizes the protein levels of the various C Y P enzymes in hepatic microsomes from the four treatment groups. Treatment of rats with M C resulted in a marked elevation of the C Y P l A l and C Y P 1 A 2 enzymes. Administration of P B resulted in an increase of C Y P 2 B 1 and C Y P 2 B 2 enzyme levels (318 and 102 pmol/nmol total C Y P , respectively). D E X treatment also increased C Y P 2 B 1 and C Y P 2 B 2 enzyme levels (20 and 15 pmol/nmol total C Y P , respectively), but to a lesser extent. Expression of CYP2C11 was lower in hepatic microsomes from M C - , P B - and DEX-treated rats in comparison to hepatic microsomes from corn oil-treated rats. C Y P 3 A 2 has a slightly greater electrophoretic mobility than C Y P 3 A 1 , but C Y P 3 A 1 does not separate from 125 C Y P 3 A 2 and are visualized as one band. Because of this, the protein band was quantified as the sum of C Y P 3 A 1 and C Y P 3 A 2 . Administration of D E X resulted in an increase in C Y P 3 A 1 / 2 expression in hepatic microsomes. C Y P 3 A 1 8 protein levels increased following treatment with D E X . Treatment of rats with P B also increased the hepatic microsomal expression of C Y P 3 A 1 / 2 (71 pmol/nmol total C Y P ) but to a lesser extent than treatment with D E X . In hepatic microsomes prepared PB-treated rats, C Y P 3 A 1 8 was not detectable. 126 Table 15. Protein Levels of Rat Hepatic Microsomal Cytochrome P450 Enzymes Treatment Cytochrome P450 enzyme a (pmol/nmol total C Y P ) C Y P 3 A 1 8 C Y P 3 A 1 (R.O.D units/ + nmol total C Y P l A l C Y P 1 A 2 C Y P 2 B 1 C Y P 2 B 2 CYP2C11 C Y P 3 A 2 c C Y P ) b Corn oil . <1 (6 ) 7(6) <1(6) 2(6) 122 (2) 19(2) 15(2) M C 371 (9) 219(12) <1(8) <1(8) 63 (2) 17(2) 7(2) P B <1(8 ) 3 (8) 318(8) 102 (8) 46 (2) 71 (2) n.d. (2) D E X <1(3) 2(3) 20(3) 15(3) 45 (2) 127 (2) 89 (2) a Values shown are the mean of the number of determinations indicated in parentheses. A l l C Y P protein levels are expressed in terms of pmoles CYP/nmole total microsomal C Y P , except for C Y P 3 A 1 8 levels which are expressed as R .O .D . units/nmole total C Y P because a rat C Y P 3 A 1 8 standard was not available. b Abbreviations: n.d., non-detectable; R .O.D. , relative optical density. C Y P 3 A 1 levels are non-detectable (n.d.) in hepatic microsomes of corn o i l - or MC-treated rats but both C Y P 3 A 1 and C Y P 3 A 2 are induced in hepatic microsomes after P B or D E X treatment of rats (Cooper et al., 1993). C Y P 3 A 2 has a slightly greater electrophoretic mobility than C Y P 3 A 1 , but C Y P 3 A 1 does not separate from C Y P 3 A 2 and are visualized as one band. Consequently, the protein band has been quantified as the sum of C Y P 3 A 1 and C Y P 3 A 2 . — i 3. References Cooper KO, Reik L M , Jayyosi Z, Bandiera S, Kelley M , Ryan DE, Daniel R, McCluskey SA, Levin W, and Thomas PE (1993) Regulation of two members of the steroid-inducible cytochrome P450 subfamily (3A) in rats. Arch Biochem Biophys 301: 345-354. Hrycay E G and Bandiera SM (2003) Spectral interactions of tetrachlorobiphenyls with hepatic microsomal cytochrome P450 enzymes. Chem Biol Interact 146: 285-296. Wong A and Bandiera SM (1996) Inductive effect of Telazol on hepatic expression of cytochrome P450 2B in rats. Biochem Pharmacol 52: 735-742. 128 Appendix III: Measurement of NADPH-CYP reductase activity in hepatic microsomes NADPH-CYP reductase activity in hepatic microsomes from M C and PB-treated rats was determined by Dr. Eugene Hrycay. NADPH-CYP reductase activity was determined in the absence and presence of PCBs to determine if PCBs affected CYP reductase activity. 1. Materials and Methodology 1.1. Materials AccustandardInc. (New Haven, Connecticut, U.S.A.): 2,2',4,4'-Tetrachlorobiphenyl (PCB 47); 2,2',5,5'- tetrachlorobiphenyl (PCB 52); 2,2',6,6'-tetrachlorobiphenyl (PCB 54); 3,3',4,4'- tetrachlorobiphenyl (PCB 77). BDH chemicals (Toronto, Ontario, Canada): Ethylenediaminetetracetic acid (EDTA), disodium salt; magnesium chloride Fischer Scientific Ltd. (Vancouver, British Columbia, Canada): Dimethyl sulfoxide (DMSO); potassium phosphate dibasic; potassium phosphate monobasic. Sigma Chemical Co. (St. Louis, Missouri, U.S.A.): Bovine heart cytochrome c; nicotinamide adenine dinucleotide phosphate tetra-sodium salt (NADPH, reduced form); sucrose. 129 1.2. M e t h o d o l o g y NADPH-CYP reductase activity in rat liver microsomes was determined using cytochrome c as an artificial electron acceptor and measuring its rate of reduction at 550 nm in a final volume of 2.5 ml using the method of Strobel and Dignam (1978). Reaction rates were calculated using an extinction coefficient of 21 mM"'cm"' for reduced cytochrome c. The effect of various PCBs on CYP reductase activity was determined by preincubation of hepatic microsomes with PCB in the phosphate buffer mixture for 5 min at room temperature before addition of cytochrome c and NADPH. The final concentrations of PCBs, dissolved in DMSO, were as follows: PCB 47, 1.6 uM; PCB 52, 0.8 pM; PCB 54, 0.8 uM with microsomes of PB-treated rats; and PCB 47, 15 uM; PCB 52, 15 pM; PCB 77, 15 pM with microsomes of MC-treated rats. In control incubations, microsomes were preincubated with an equivalent volume of DMSO. 2. Results In order to determine whether PCBs affected NADPH-CYP reductase activities, microsomes were preincubated with PCBs prior to the determination of NADPH-CYP reductase activities. In the absence of PCBs, the NADPH-CYP reductase activities in hepatic microsomes from PB- and MC-treated rats were 474 and 376 nmol reduced cytochrome C/min/mg microsomal protein, respectively. As shown in Table 16, preincubation of microsomes with PCBs did not have an effect on NADPH-CYP reductase activities. 130 Table 16. N A D P H - C Y P reductase activity in the presence of P C B s Preincubation Reaction Rate (nmol reduced cyt. c/min/mg microsomal protein) Microsomes of PB-treated rats No PCB 474 1.6 uM PCB 47 476 0.8 uM PCB 52 467 0.8 uM PCB 52 467 Microsomes of MC-treated rats No PCB 376 15 uM PCB 47 348 15 uM PCB 52 352 15 uM PCB 77 381 Data are the average of the absorbance change between 1 and 2 minutes and 2 and 3 minutes. 3. Reference Strobel HW and Dignam JD (1978) Purification and properties of NADPH-cytochrome P-450 reductase. Methods Enzymol 52: 89-96. 131 

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