MSU LlBRARiES W v RETURNING MATERIALS: P1ace in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped be1ow. STUDIES ON THE CHEMICAL AND PHARMACOTOXICOLOGICAL PROPERTIES OF POLYBROHINATBD BIPHENYLS BY Cynthia Doninka Hillie A THESIS Submitted to Michigan State University in partial fulfillment of the requirements A for the degree of MASTER OF SCIENCE Department of Biochemistry 1984 \ ' x \x V o l \) 5/- 3/ '7 ‘7 ‘ ABSTRACT STUDIES ON THE CHEMICAL AND PHARHACOTOXICODOGICAL PROPERTIES OF POLYBROHIHATHD BIPHEHYLS BY Cynthia Dominka Hillis Synthesis and separation techniques were develOped and employed to obtain several PBB congeners. The structures were determined employing mass-spectrometry and 1H-NHR Spectroscopy. PBB congeners purified were 2.3.3'.4'-tetra-, 3.4,3'.4'-tetra-. 3.5.3',5'-tetra-, 3.4.5.3',5'-penta- and 3.4.5.3'.4'.5'-hexa- bromobiphenyl. Several chemical properties of these pure PBB congeners were also studied. Various PBB congeners of Firemaster were photolyzed under laboratory conditions. Rates of photolysis and UV-absorption spectras were determined and several photOproducts identified. The photolysis mixture of 2.4.5.2"4'.S'-hexabromobiphenyl, three major photoproducts (2:405:3'14'-o 2.4.5.2'.5'-penta- and 3:4:3'.4'—tetra-bromobiphenyl) 2:4:S.2'.4':S'-hexabromobiphenyl and 3.4.5.3'.4'.S'-hexabromobiphenyl were evaluated for their acute effects on rat liver microsomal drug metabolizing enzymes and for various toxic effects. typified by 2.3.7.8- tetrachlorodibenzo-p-dioxin (TCDD). The effects of an equimolar dose of 3.4.5.3‘.4'.5'— hexabromobiphenyl or 3.4.3'.4'-tetrabromobiphenyl were compared over time. Results of enzyme induction, tissue levels and TCDD- like toxic responses suggest that 3:4:5.3'c4'.5'- hexabromobiphenyl is more toxic than 3:4.3'.4'-tetrabromobiphenyl even though i vitro binding studies revealed that 3:4:3':4'- tetrabromobiphenyl served as a better ligand for the TCDD- receptor. TO HY PARENTS ii ACKNOWLEDGEMENTS I would like to express my appreciation to Dr. Steven D. Aust for his support and guidance as my academic advisor. I am truly grateful for his tolerance and friendship which he has shown me throughout my graduate studies and thank him for taking the time to teach his students more than just science. I also wish to express my thanks to Dr. Stuart D. Sleight and Dr. Jack T. Watson for serving as members of my guidance committee. I wish to thank several people including Dr. Rick Jensen and Dr. Margit Rezabek (histOpathology studies). Betty Baltzer (mass spectrometry studies) and Dr. Klaas Hallenga (la-NMR spectral studies) for providing data and results that have become part of this research. I consider myself very fortunate to have worked with so many peOple in Dr. Aust's laboratory and elsewhere at Michigan State University. I would like to acknowledge the members of Dr. Aust's laboratory for providing advice. assistance and friendship. I particularly wish to thank Paula Bank. Cathy Custer. Todd Mouser and Dick Mills. and especially David Erickson and Debi Metcalf for providing invaluable assistance. advice. support and friendship. I am forever indebted to my parents. Helen and Andrew Millie. for their continued love. support and guidance throughout this research for without it. this thesis would not be. iii TABLE OF CONTENTS Page LIST OF TABLES e e e e e a a e e e a e e e e e e e e e e a a a e e e a e a a e a a e a e e e e a a e 0 Vi 1 LIST OF FIGURES e e e e e e e e e e e e e e e e e e e e e e e a e e e e e e a e e e e e e e e e e e 0 Vi i 1 LIST OF ABBREVIATIONS 0.0.0.0....OOOOOOOOOOOOOOOOOOOO0.0... Xi INTRODUCTION 0......OO...00......O0............OOOOOOOOOOOO 1 LITERATURE REVS!" O O I O O O O I O I O O O O O O O I O O O O O O O O O I I O O O O O O O O O O O O 7 Hepatic Microsomal Monooxygenase System .............. 7 Induction of Hepatic Microsomal Drug Metabolizing enzymes 0....IOOI.00............OOOOOOOOOO...0....OO 1‘ Consequences of the Induction of Microsomal Drug- ".tab01121ng Enzymes O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 19 Halogenated Aromatic Hydrocarbons and Polycyclic Aromatic Compounds : An Examination of a Mechanism of Toxicity and Enzyme Induction ................... 22 Toxic Responses ................................. 23 Biochemical Responses ........................... 24 Enzyme Induction and Role of Receptor ........... 25 Structure Activity Relationships ................ 37 Induction of Drug Metabolizing Enzymes by and Toxicity of Polybrominated Biphenyls ........................ 44 CHAPTER 1 3 THE SYNTHESIS. PURIFICATION AND CHEMICAL CHARACTERIZATION OF SEVERAL POLYBROMINATED BIPHENYL CONGENERS 0......-OO......OOOOOOOOOOO......OOOOOOOOOOO 63 AbstraCt ............................................. 64 Introduction ......................................... 64 Materials and Methods ................................ 67 Materials .......................................... 67 Gas Chromatography ................................. 67 as Chromatography-Mass Spectrometry ............... 68 H-NMR SpectroscOpy ................................ 68 UV-Absorption Spectrosc0py ......................... 69 Synthesis of Several PBB Congeners ................. 69 Purification of PBS Congeners ...................... 71 I. General Scheme for Purifying 2.3.3'.4'-TBB and 3:4-TBB eeeeeeeeeeeeeeeeeeeeeeeeeeseaeeeeeeeeeeae 71 Neutral Alumina Absorption Chromatography ....... 74 Recrystallization in Mexane ..................... 7S II.General Scheme for Purifying 3.4.S-HBB. 3.4.5.3'.5'-Pentabromobiphenyl and 3.5-TBB ...... 75 Reverse Phase Thin Layer Chromatography (RP-TLC) ... 76 Melting Point Determination ........................ 79 Results .............................................. 79 iv Synthesis and Purification of PBB Congeners ........ Synthesis of 2.3.3'.4'-TBB and 3.4-TBB ............ Purification of 2.3.3'.4'-TBB and 3.4-TBB ......... Purification of 3.4.S-HBB. 3.4.5.3'.S'—PBB and 3.5-TBB Structural Characterization of PBB Congeners ...... Several Chemical and Chromatographic PrOperties of PBB Congeners ............................... High Resolution Gas Chromatographic Analysis of Firemaster BP-6 ................................ Discussion . CHAPTER 2 : CHARACTERIZATION OF THE PHOTOLYSIS OP 2.4.5.2'.4'.5'-HEXABROMINATED BIPHENYL .............. Abstract ... Introduction Hat.ria18 and ".thOds 0............OOOOOOOOOOOOOOOOOO Chemicals Gas Chromatography ................................ Gas Chromatography-Mass Spectrometry .............. UV-Absorption Spectroscopy ........................ Photochemical Procedures .......................... Results .... . O0.0.0.0....0............OOOOOOOOOOOOOOO. Identification of PhotOproducts ................... Photoreactivity of HBB ............................ Photoreactivity of Primary PhotOproducts .......... Initial Photolysis Rates for various PBB Congeners Ultraviolet Spectrosc0py .......................... Discussion . CHAPTER 3 : MICROSOMAL ENZYME INDUCTION BY AND TOXICOLOGY OF THE PHOTOLYSIS PRODUCTS OF 2.4.5.2'.4'.S'-HEXA- BROHINATED BIPHENYL eeaeaeeeeeeeeeaeeeeeeeeaeeeeaeeee Abstract ... Introduction Materials and Methods ............................... Chemicals Animals and Treatments ............................ Isolation of Microsomes ........................... Tissue Collection and HistOpathology .............. Enzyme Assays and Tissue Levels of PBB ............ Photochemical Procedures .......................... R.'u1t‘ O0......OI.......-0............OOOOOOOOOOOOO. Discussion . CHAPTER 4 : TOXICITY OF 3.4.5.3'o4'.5'-HEXABROMINATED BIPHENYL AND Abstract ... Introduction 3.4.3'.4'-TETRABROMINATED BIPHENYL ..... Materials and Methods ............................... Chemicals V 79 8O 85 88 95 110 120 121 127 128 128 130 130 131 131 131 132 132 132 137 143 146 150 150 160 161 162 164 164 165 166 166 166 174 177 188 193 194 195 197 197 Animals ............................................ 198 Isolation of Microsomes ............................ 199 Tissue Collection and Histopathology ............... 199 Preparation of Receptor ............................ 200 Receptor Binding ................................... 200 Enzyme Assays and Tissue Levels of PBB ............. 202 Results .............................................. 202 Organ Weights ...................................... 202 Histopathology ..................................... 205 Competitive Receptor Binding Studies ............... 205 AHH Induction ...................................... 211 In vivo Metabolism Studies ......................... 216 Extrahepatic AHH Induction and Organ Weights ....... 216 Discussion............................................ 223 GENERAL DISCUSSION seeseeaeeoeoeaeeeeeeaeeeeeee eeeeeeeeeeee 227 REFERENCES eeeee eeeeeaeeee eeeeeeeeeeeeeeeeeeeee eeeaa aaaaaaa 237 APPENDIX : LIST OF REFERENCES .................... ......... 254 vi Table 1 10 11 12 13 LIST OF TABLES Page Proton Chemical Shifts and Coupling Constants of PBB cong.n.t‘ 0......OI...0.0.0.0..........OOOOOOOOOOOOO 109 Relative GC Retention Times (tR) of Pure PBB cong.n°t' O....0.0............OOOOOOOOOOOOOOOOO0.... 111 R: Values of PBB Congeners on Reversed Phase TLC ..... 112 Melting Points for Various PBB Congeners ............. 114 UV-Spectral Data of PBB Congeners .................... 117 HBB Photolysis Rates Under Various Concentrations .... 140 Initial Photolysis Rates for Various PBB Congeners ... 149 UV-Spectral Data for Individual PBB Congeners and Ph°t°1yzed HBB O......OOOOOOOOOOOOOOOOOO0.00.0000... 158 Body and Organ Weights of Rats Administered Various PBB Congeners .. ..... ............................... 181 Several Hepatic Microsomal Enzyme Parameters of Rats Administered Various Treatments .................... 183 PBB Concentration in the Liver and Adipose Tissue of Treated Rats ...-0.0.0....0............OOOOOOOOOOOOO 185 Body and Organ Weights of Rats Administered HBB ...... 221 Bthoxyresorufin-O—Deethylase Activity in Various Tissues Of Rats Administ.r.d HBB ......OOOOOOOI.00.0.0000... 222 vii LI ST OF FIGURES Figure Page 1 Gas Chromatographic Blution Profile and Structures of Polybrominated Biphenyls in a Synthetic Mixture 73 2 Gas Chromatographic Blution Profile and Structures of Polybrominated Biphenyls in a Commercial "ixtur. 0000.........O..........OOOOOOOOOOOOOOC0.0 78 3 A Scheme for the Formation of 3,4-Dibromoaniline ... 82 4 The Scheme of the Mechanism of the Gomberg-Hey R.acti°n ......OOOOOIOO......OOOOOOOOOOOOOOOOOOOO. 84 5 Neutral Alumina Chromatography Elution Profile of a Synthetic Mixture containing 2.3:3':4'-TBB and 31‘I3.I4.-TBBeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeeee 87 6 Gas Chromatographic Elution Profiles of Pure 2.3.3'.4'-TBB and 3:4,3'o4'-TBB from a Synthetic "ixtur. ...0.0.0....O......OOOOOOIOOOOOOOOOOOIO... 90 7 Neutral Alumina Chromatography Elution Profile of a Commercial Mixture containing 3.5.3'.S'-TBB: 3141513.15'-P336nd 3I4IS-HBBeeeeeeeseeeeeeeeeeeee 92 8 Gas Chromatographic Elution Profiles of Pure 3:5:3':5'-TBB, 3:4:S:3',5'-PBB and 3.4.5-HBB from acometCial Mixtur. 0.00............OOOOOOOOOOOOO 94 9 1H-NMR Spectrum and Structure of 2:3,3’:4'-Tetra— BromObiph.ny1 0.0.........OOOOOCOOOOOOOOOOOO ...... 98 10 1H-NMR Spectrum and Structure of 3.4.3'c4'—Tetra- Bronchiph.ny1 O0.0.0....O......O...’0.000.000.0000 100 11 1H-NMR Spectrum and Structure of 3.5,3'a5'-Tetra- BrOMObiph.ny1 0......00.0.0000.........OOOOOOOOOOO 103 12 1H-NMR Spectrum and Structure of 3.4.5,3',5‘-Penta- BromObiph.ny1 ..........0.........OOOOOOOOOOOOOOO. 106 13 1H—NMR Spectrum and Structure of 3,4:S,3'94'.S'-Hexa- Br°m°biph.ny1 ...O....O.........OOOOOOOOOOOOOOOOOO 108 14 UV—Absorption Spectra of 2.3.3‘o4'-TBB. 3.4.3':4'-TBB: 3:513'15'-TBB: 3I‘ISI3'IS.-PBB and 3:415:3'14'I5'- HBB 00.00.000.000....O..0.........OOOOOOOOOOOIOOOO 116 viii 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 High Resolution Capillary Chromatogram of Firemaster BP-G 0....O00............OOOOOOOOOOOOCOOOOC0...... GC Elution Profile and Structures of PBB Congeners in th. Ph°t°1yz.d HBB Hixtut. ......OOOOOOOOOOOOOO (A) GC-MS (Total Ion Current) of the Photolyzed HBB "ixtur. 0.0.0.0...0.00..........OOOOOOOOOOOOOOOOOC (B) 70 ev Mass Spectrum of Scan No. 186 in the Photolyzed HBB Mixture (C) 70 ev Mass Spectrum of a Pure Pentabrominated BiphOHYI (2t4lSIZ'IS.-PBB) GC Elution Profile and Structures of the Pure PBB Congeners in the Photolyzed HBB Mixture .......... Photochemical Degradation of HBB (0.32 9!) over time and Formation of Products Photochemical Degradation over time and Formation Photochemical Degradation over time and Formation of of of of UV-Absorption Spectra of the “BB! 2I4ISI3.I4.-PBB and 3I4I3'l‘.-TBB eeeeeeeeeee 2:41502.05'-PBB (0.29M) PrOducts ......COOOOOOO 2:415:3'14'-PBB (0.27m!) Products .............. Photolyzed HBB Mixture. UV-Absorption Spectra of 2.4.5,2'o5'-PBB: 2.5,2',5'- TBB! 21412.15'-T335nd 2I5I3.I4.-TBB eeseeeseeeeee The linearity of the deethylation of ethoxyresorufin With Varying Protein Concentrations of Rat Micro- somes Isolated from Rats Treated with Either (A) MC or (B) PB ..................................... Lineweaver—Burke Plot for the Deethylation of Ethoxy— Resorufin by Rat Microsomes Isolated from Livers of Rats Treated with either MC or PB ............. GC Elution Profile and Structures of PBB Congeners in the Photolyzed HBB Mixture .................... Effects of Vehicle and Various Treatments on Hepatic Structure Fourteen Days After Treatment .......... GC Elution Profile of PBB congeners in Liver (A). Adipose (B) and Photolyzed Mixture (C) ........... Absolute Liver Weights from Rats Treated with Either 21.3 umoles/kg Body Weight of HBB or TBB or corn 011(contr01) 00.0.0.0...............OOOOOOOOOOOOO ix 120 134 136 136 136 139 142 145 148 152 154 170 172 176 179 187 203 30 31 32 33 34 35 36 37 38 Absolute Thymus Weights from Rats Treated with Either 21.3 umoles/kg Body Weight of HBB or TBB or corn 011(contr01) O.......OOOOOOOIOO0.0.0.0...00...... Effects of Vehicle on Rat Liver Structure Fourteen Day‘ Aft‘r Tr.atment 0.00.0.0.........OOOOOOOOOOOO Effects After Effects After SUCI'OIO of TBB on Rat Liver Structure Fourteen Days Tr.atm.nt O0.0.0.0.0........................ of HBB on Rat Liver Structure Fourteen Days Tr.atm.nt ......OOOOOOOOOOOOOOOOOO0.0.0.0... Density Gradiens Detection of Specific High Affinity Binding of [ Hl-TCDD to a component in the 105.000 x g Supernatent of Rat Liver ......... Comparative Competition of TBB and HBB for Specific Binding of [ Hl—TCDD to Sprague-Dawley (SD) Rat and 36 House H.patic R.c.pt°r .0 ......OOOOOOOOOOOO Ethoxyresorufin—O-Deethylase (EROD) Activity in Liver Microsomes Isolated From Rats Treated With 21.3 umoles/kg Body Weight of Either HBB or TBB or corn 011(contr01) .0...I.......OOOOOCO......OOOOOOOOOO Concentration of HBB or TBB in the Adipose Tissue of Rats Treated with 21.3 umoles/kg Body Weight of the Respective congeners 0.0.0.0.0....0.00.00.00.00... Concentration of HBB or TBB in the Livers of Rats Treated With 21.3 umoles/kg Body Weight of the Resp.ctiv. congeners 00............OOOOOOOOOOOOOO. 204 206 207 208 210 213 215 218 220 LIST OF ABBREVIATIONS AHH BNF butylated hydroxytoluene DNA c ECD GC HZ ip MC NADH NADP+ NADPH NMR PAPS PB PBB PCB PEG PPm RER aryl hydrocarbon hydroxylase B-Naphthaflavone 2.6-di-tert-buty1-p-cresol deoxyribonucleic acid extinction coefficient electron capture detector gas chromatography Hertz (cycles per second) intraperitoneal cOUpling constant 3-methylcholanthrene B-nicotinamide adenine dinucleotide. reduced form B-nicotinamide adenine dinucleotide phosphate B—nicotinamide adenine dinucleotide phosphate. reduced form nuclear magnetic resonance phosphoadenosinephosphosulfate phenobarbital polybrominated biphenyls polychlorinated biphenyls polyethylene glycol parts per million relative thin layer chromatographic mobility rough endOplasmic reticulum xi RNA SD 808 SER TCDD TLC Tris UDP UV ribonucleic acid standard deviation sodium dodecyl sulfate smooth endoplasmic reticulum retention time 2.3.7.8-tetrachlorodibenzo-p-dioxin thin layer chromatography tris (hydroxymethyl) aminomethane uridine-S'-diphosphate ultraviolet xii INTRODUCTION Polybrominated biphenyls (PBB) were used as fire retardants and were manufactured in the early 1970s under the tradename Firemaster (Michigan Chemical Company. St. Louis. MI). In 1973 at the feed mill Operated by the Farm Bureau Services. Inc. at Battle Creek in Michigan. 500 to 1000 pounds of PBB were accidently mixed with cattle feed when it was mistaken for the mineral sUpplement. magnesium oxide. Magnesium oxide. whose tradename was Nutrimaster. was also manufactured by Michigan Chemical Corporation who also produced Firemaster. Both products had similar physical appearance and were normally packaged in pre-printed bags with their trade mark names. The details of the mix-Up are not completely known or understood but apparently a temporary shortage of the pre—printed bags resulted in both Nutrimaster and Firemaster being packaged in brown bags with the trade name stenciled at the tOp. How a few bags of Firemaster consequently ended Up at the feed mill operated by the Farm Bureau Services is unknown. The consequences of the contamination in Michigan have been enormous since the error went undetected for a number of months. As a result most Michigan residents consumed contaminated milk. beef and other products and some estimates report nearly 90% of Michigan's residents have measurable PBB levels in their bodies. Thousands of cattle. hogs and sheep along with over 1.5 million chickens were killed in addition to the destruction of tons of feed and dairy products. Human health effects associated with PBB are variable and not completely understood. Neurological disorders. stomach discomfort. lethargy and musculoskeletal symptoms are reported among the ailments of afflicted farm families. However. an unambiguous relationship between these human illnesses and PBB can not be made since many of these people. understandably. may have been emotionally and psychologically distressed over the contamination and loss of their farm as well as fear of the unknown effects of PBB on their families and themselves. Thus. a clear cause and effect relationship between PBB and human health effects have not been established. Long-term effects however may become apparent in years to come. Some workers occupationally exposed to PBB have been diagnosed as hypothyroid but again no clear relationship was observed (Bahn 25 21.. 1980). The possible transfer of PBB in milk to babies nursing contaminated mothers is of concern to the public since PBB may affect normal growth and development. The toxicological and biochemical effects of the crude Firemaster mixture have been studied extensively in the rat. This laboratory along with others found that the Firemaster mixture caused a mixed-type induction of liver microsomal drug metabolizing enzymes similar to that seen after the coadiministration of phenobarbital (PB) and 3-methylcholanthrene (MC) (Troisi. 1975: Dent 25 51.. 1976a; 1976b). These effects have also been observed for the polychlorinated biphenyls (PCB). PBB and PCB have been found to belong to a class of toxic polyhalogenated aromatic hydrocarbons which is typified by the most toxic member. 2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is considered to be a strict MC-type inducer and produces a variety of toxic responses which include liver hypertrophy. thymic involution. porphyria. loss of body weight and appetite. and chloracne. The extent of most of these effects are species and sex dependent. The potency of the compounds to elicit these responses vary as well i.e. TCDD is roughly 104 times more toxic than the PBB mixture. Although the mechanism of toxicity of these compounds remains unknown. there is good evidence to suggest that the responses seen are mediated by a soluble binding protein. referred to as the TCDD- or Ah-receptor. The first events to occur are believed to be the passive transfer of the compound across the cell membrame and the highly specific binding to the Ah receptor. Following an apparent translocation of the inducer-receptor complex into the nucleus or. binding to a receptor nuclear in origin. a pleitropic reSponse is evoked. PBB are highly stable and resistent to microbial degradation. Sunlight degradation has been shown to occur although not rapidly. Firemaster is a powder or granular material and lipOphilic in nature. being insoluble in water (11 ug/kg) and reasonably soluble in organic solvents and fat. It has an average bromine content of six bromines. Various congeners of the mixture have widely different chemical prOperties and these differences seem to be influenced mainly by the number of bromines and their positions on the biphenyl molecule (see chapter one). GC analysis of Firemaster reveals at least twelve to four- teen major PBB congeners of which ten have been structurally identified. All congeners present have at least one bromine orthg to the bridge carbon of the biphenyl. Nine of these PBB congeners. which total nearly 97% of Firemaster. have been purified and examined for some of the toxicological effects in rats. A reconstituted mixture. prepared with the nine purified and characterized congeners. was found to elicit relatively the same response as the original mixture which contaminated Michigan. indicating that these congeners may account for the biological effects of the mixture. or that the mixture did not contain some minor. toxic component. The first objective of the research described in this thesis was to develop a method for synthesizing and purifying PBB congeners which. according to present-day structure-activity rules. would be the most toxic congeners. Then the structures of the PBB congeners were determined and certain chemical. and chromatographic prOperties of these congeners were examined and compared. Most of these properties were shown to be mainly influenced by the total number of bromines. extent of bromination ggghg to the biphenyl bridge as well as bromination in the mega and 2253 positions. Then through the employment of capillary chromatography. Firemaster was analyzed and found to contain minute amounts of non-ggthg brominated biphenyl congeners. These studies are all presented in chapter one of this thesis. Since PBB can be degraded quite readily by UV-light and therefore to some extent by sunlight. the composition of Firemaster can change. giving rise to lower brominated congeners. Since toxicity of PBB is related to the number and positions of bromines. a non-toxic congener can in theory give rise to a toxic congener. This aSpect is studied in chapters two and three of this thesis. In chapter two. the main congener of Firemaster. in its pure form. was irradiated with UV-light. Rates of degradation and formation of products was determined. Rates of degradation of secondary photoproducts was also performed to acquire some kinetic data in order to help elucidate the mechanistic pathway of the photolysis reaction. ‘The UV-spectra of the photolysis mixture as well as the purified components were studied as well as the rates of debromination. In chapter three. the photolysis mixture along with three purified photoproducts were evaluated for their effects on rat liver microsomal drug metabolizing enzymes. on the histopathology of the liver as well as on the size of certain organs. The main purpose was to identify the congeners of the photolysis mixture responsible for the toxicity observed and for the MC-type induction. Relationships between PBB structure. TCDD-like toxicity. microsomal enzyme induction and PBB metabolism are considered. In chapter four. how metabolism of PBB affects microsomal enzyme induction and influences toxicity is addressed. A metabolized and a non-metabolized PBB congener are compared for their effect on liver and thymus size. microsomal enzyme induction. on the histopathology of the liver and the ability of these two congeners to bind to the TCDD receptor. The results indicated that although in 21539 binding studies may give an indication of the ability of a compound to be toxic. it does not take into consideration the ability of a compound to be rapidly metabolized. The results also indicate that a PBB congener which is metabolized rapidly. decreases in toxicity over time after an acute dose. A second eXperiment was performed using a non- metabolized PBB congener and its ability to induce AHH activity in extrahepatic tissues was investigated. LITERATURE REVIEW A literature review will be presented concerning the hepatic microsomal drug-metabolizing enzymes and the present-day concepts concerning the genetic system (s) controlling the induction of these enzymes in response to various xenobiotics. with emphasis on polyhalogenated aromatic hydrocarbons. The most accepted mechanism by which certain polyhalogenated aromatic hydrocarbons cause a characteristic toxic response and the induction of enzymes will also be presented. Hepatic Microsomal Monooxygenase System In mammalian systems. the liver is the most intensively studied organ with regards to the metabolism of xenobiotics. Several other organs are known to be active in this reSpect. however the specific activity of enzymes and the range of substrates metabolized is not as large as in the liver. Therefore this discussion will be limited primarily to the hepatic microsomal drug-metabolizing enzymes. Microsomes are derived from the endoplasmic reticulum of a cell as a result of homogenization and isolated by differential centrifugation (Claude. 1969). The microsomal fraction obtained is known to be contaminated with adsorbed cytoplasmic proteins and ribosomes. Therefore the microsomes are usually homogenized i]: a buffer of high ionic strength containing a chelating agent. *which is effective in disrUpting and removing both ribosomes and adsorbed proteins. and pelleted by ultracentrifugation to obtain a more pure membrane fraction (Welton and Aust. 1974; Kuriyama 25 21.. 1969). The microsomal monooxygenase system contains an NADPH- dependent electron transport chain which requires NADPH and dioxygen (molecular 02) to catalyze the hydroxylation of a wide variety of both endogenous and exogenous substrates. This enzyme complex. embedded in the endoplasmic reticulum. requires three components for proper function and electron flow (Strobel 25 21.. 1970). The first component is NADPH-cytochrome P-4SO reductase. a flavoprotein that contains one mole each of FMN and FAD per mole of enzyme. which transfers reducing equivalents from NADPH to the terminal oxidases. referred to as just cytochrome P-450 (Lu :5 $1.. 1969). It is believed that the sequence of electron transfer in this system is as follows: NADPH—> FAD-> FMN-> P-450 (Vermilion 35 21.. 1981). The reductase is also referred to as NADPH-cytochrome c reductase. since cytochrome c can function as an artificial electron acceptor for this enzyme (Gillette 35 51.. 1972). It is commonly accepted that multiple forms of endogenous cytochrome P—450 hemOproteins exist in liver microsomes. based on the diverse catalytic activities for a variety of substrates as well as the electrophoretic. spectral and immunological evidence (Welton and Aust. 1974; Thomas 25 31.. 1976; Guengerich. 1977: Guengerich. 1979; Lu and West. 1980; Nebert 35 21.. 1981). The cytochrome P-450 is characterized as a hemoprotein with protoporphyrin Ix as the prosthetic group (Omura and Sato. 1964a; Omura and Sato. 1964b). The NHz-terminal sequence analysis of two cytochrome P-450s demonstrated that they had 31 identical residues and that the terminal end is highly hydrophobic suggesting that this NHz-terminal portion of the P-450 maybe involved in membrane anchoring (Waxman and Walsh. 1982). The association of NADPH-cytochrome P-450 reductase with cytochrome P-450 hemOprotein is often referred to as the microsomal mixed- function oxidase system (Conney. 1967: Gillette 35 21.. 1972). The only other component necessary for prOper and functional activity of this complex is phosphatidyl choline (Strobel 25 51.. 1970). This component is not directly involved in electron transfer but is thought to be involved in the COUpling of the reductase and the cytochrome P-450 and in binding of the substrate to the cytochrome (Lu 3; 21.. 1974; Lu. 1976). Cytochrome P-450. like other hemoproteins. has a unique absorption spectrum in the visible region. However. unlike most cytochromes. it is named. not from this absorption maximum of the reduced form but from the unique wavelength of the absorption maximum of the carbon monoxide (CO) derivative of the reduced form. specifically 450 nm (Garfinkel. 1958; Klingenberg. 1958). The mixed-function oxidase system in hepatic microsomes performs oxidations in which one atom of molecular oxygen is reduced to water while the other is incorporated into the substrate. The term ”monooxygenases” has also been used for enzymes which catalyze this type of reaction (Ullrich. 1978). The mechanism by which cytochrome P-450 is believed to function is as follows. The initial step consists of binding of the substrate to the oxidized form of cytochrome P-450. followed by the one electron reduction catalyzed by the NADPH-cytochrome P— 450 reductase which forms the reduced cytochrome-substrate 10 complex. Oxygen then binds to the reduced cytochrome-substrate complex. and the activated oxygen complex is reduced further. After the transfer of one atom of oxygen to the substrate and the other to form water. dismutation reactions occur which lead to the formation of a hydroxylated product. water and oxidized cytochrome P-450. The reaction mechanism of this cytochrome P- 450 dependent microsomal mixed-function oxidase system is summarized below (Mason. 1957: Posner 25 21.. 1961: Gillette gt al.. 1972; Ullrich. 1978). ROH H20 Fe+3 2H"' +3 3 *3 (RH)E:e —02] (RH) Fe r a- (RH)[Fe—o;_] 02+!)ng 02 (RH)[F;2'OJ (Modified from white. R.E. and Coon. M.J. (1980). In Annual Review of Biochemistry. Vol. 47. pp.3lS-356.) Hundreds of endogenous and exogenous substrates for this enzyme system exists. Some of the endogenous substrates include steroids. indoles. biogenic amines. prostaglandins. thyroxine. vitamins and fatty acids (Gillette. 1966: Wada 25 al.. 1968; Tephly and Mannering. 1968; Nebert 25 21.. 1981). The exogenous 11 substrates include polycyclic hydrocarbons such as benzo(a)pyrene and anthracenes; polyhalogenated aromatic hydrocarbons such as polychlorinated and polybrominated biphenyls. defoliants and insectides; numerous aromatic amines such as the dyes. nitro aromatics. aminoazo and diazo compounds; alkaloids; safrole derivatives: antioxidants. other food additives and many ingredients of foodstuffs as well as xanthines. flavones and organic peroxides that occur in food (Nebert 35 21.. 1981: Kappas and Alvares. 1975; Conney and Burns. 1972; Kuntzman. 1969). The majority of xenobiotics which enter the body are lipOphilic. The metabolism of these xenobiotics usually results in increasing their water solubility and thus facilitate their elimination. This process is generally believed to consist of two phases. phase I reactions and phase II reactions (Hodgson and Dauterman. 1980). Phase I reactions consists of introducing a reactive polar groUp into the compound. thus producing a suitable substrate for phase II reactions. Phase II reactions include all conjugation reactions in which the polar grOUp on the xenobiotic is complexed with an endogenous compound. for example glutathione. glycine or glucuronic acid. to form a highly-water soluble conjugate which can be more readily excreted in the bile or urine. Phase I reactions include the already mentioned cytochrome P-450 dependent mixed-function oxidations. Other reactions include epoxidations and aromatic hydroxylations. Epoxides formed in turn can be hydrated by epoxide hydrase to form trans- dihydrodiols which are less reactive than the parent epoxide 12 (Oesch. 1973: Lu and Miwa. 1980). Epoxides formed may be highly unstable or somewhat stable and since they are electrophilic they may react irreversibly with nucleophiles in the cell such as protein moieties. DNA or RNA to generate adducts. The proximate carcinogens produced from the metabolic activation of benzo(a)pyrene are believed to be isomers of benzo(a)pyrene 7.8- diol-9.10—epoxide. which arise from the 7.8-epoxide via epoxide hydrase (Osborne. 1979; Bresnick 25 21.. 1977; Wood 25 1.. 1976; Gurtoo and Bejba. 1974). These isomers are both mutagenic and carcinogenic. Epoxide hydrase in this case metabolized a chemical to a more toxic chemical. In other cases. epoxide hydrase reactions can act as a detoxification step. The epoxides formed can also rearrange nonenzymatically to the phenols or interact with epoxide hydrase as in the case of naphthalene metabolism (Jerina gs al.. 1970). They can also interact with cytosolic enzymes. such as glutathione-S-epoxide- transferases. to yield a glutathione conjugate (Sims and Grover. 1974: Jerina and Daly. 1974). The reactions of these enzymes are believed to act as a detoxification step since an inverse relationship exists between the level of glutathione and hepatotoxicity of electrOphilic intermediates (Mitchell 35 21.. 1975). As with epoxide hydrases. not all glutathione transferase reactions act as a detoxification step since some dihalo-ethanes are metabolically activated to a mustard-like glutathione conjugate which is more mutagenic than the parent compound (Rannug 25 31.. 1978; Hill 25 21.. 1978). Other phase I reactions include various types of dealkylations. oxidations and oxidative deaminations. 13 Xenobiotics which contain functional groups such as hydroxyl. amino. epoxide. carboxyl or halogens or are metabolites of phase I reactions. can undergo conjugations. also referred to as phase II reactions. Usually the endogenous conjugating agent is derived from a carbohydrate. a protein source or a sulfur component. Glucuronic acid. derived from UDP- glucuronate. may be conjugated with an aglycone by the action of UDP-glucuronyltransferases (Dutton. 1975). Sulfotransferases uses the sulfating agent PAPS to form sulfate conjugates (Aitio. 1978). Methyltransferases and glutathione-S-transferases are also considered to be phase II reactions as well as acylation reactions. The combination of phase I and phase II reactions are therefore responsible for the metabolism of hundreds of xenobiotics. It should be pointed out that these metabolic reactions are not all detoxifications since many xenobiotics are metabolized to highly reactive products which are responsible for the toxic effects. These include the activation of carcinogens. mutagens and hepatotoxicants. Some xenobiotics. namely the polyhalogenated aromatic hydrocarbons. are toxic in themselves and are not usually metabolically activated by these enzymes. This may in part explain the long half lives and distribution of these toxicants throughout the body. namely the adipose tissue (Tuey and Matthews. 1980; Tuey and Matthews. 1977; Lutz 25 al.. 1977). These xenobiotics are however relatively good inducers of the hepatic microsomal drug—metabolizing enzymes. the subject of the next section. 14 Induction of Hepatic Microsomal Drug Metabolizing Enzymes Several hundred compounds of diverse chemical structure have been shown to induce the hepatic microsomal drug-metabolizing enzymes. This phenomenon is believed to be wide spread. quite nonspecific and increases the ability of the enzymes to metabolize xenobiotics as well as endogenous compounds (Conney and Burns. 1972; Sher. 1971; Conney. 1967). The compounds which cause induction include drugs. insecticides. polycyclic hydrocarbons and many others. all of which have certain factors in common in that they are all organic and lipOphilic. Some heavy metals and anesthetic gases haven been shown to inhibit or depress certain microsomal enzymes (Kappas and Alvares. 1975; Ivanetich and Bradshaw. 1977). The many inducers of mixed-function oxidase activity are classified into two principal classes. one exemplified by phenobarbital (PB) and contains many other drugs and compounds of diverse chemical classes. and the other exemplified by 3~methy1- cholanthrene (MC). benzolalpyrene and many polycyclic hydrocarbons. Many inducers require either relatively large dose levels or repeated dosing to be effective. Some chemicals can induce at very low doses. such as the most potent inducer known of the MC-type 2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) (Poland 25 21.. 1979). In the liver. PB-type inducers cause a marked proliferation of the smooth endoplasmic reticulum (ER). increase the liver size and induce certain microsomal drug-metabolizing enzymes. The two major components of the monooxygenase system which are induced 15 are NADPH-cytochrome P-450 reductase and one or more forms of cytochrome P-450. The cytochromes P-450 induced by PB have approximately the same spectral characteristics as that in the liver of an uninduced animal. in that the absorption maxima of the reduced-CO complexes occur at 450 nm though the specific content is 2.5 fold greater. These cytochromes also show basically the same reduced cytochrome-ethyl isocyanide difference spectral 455/430 nm absorbance ratio of 0.5. as seen with the uninduced animal. The apparent molecular weight(s) of the major cytochrome P-450(s) present in PB-induced microsomes is 50.000- 51.000 as shown by SDS-polyacrylamide gel electrOphoresis (SDS- PAGE) (Guengerich. 1977; Guengerich and Martin. 1980). Other forms of cytochrome P—450 invariably induced in microsomes from PB-treated rats have subunit molecular weights of 50.000. 47.000. and 52.000 as judged by SDS-PAGE (Guengerich gt 21.. 1982). A wide range of oxidative activities is induced including N- demethylation of aminOpyrine. ethylmorphine and benzphetamine. O- demethylation of p-nitroanisole and pentabarbital hydroxylation (Conney. 1967; Parke. 1975). Among other enzymes induced are microsomal epoxide hydrase (Bresnick 25 al.. 1977). chloramphenicol UDP—glucuronyltransferase (Bock _5 _l.. 1973) and glutathione-S-transferase (Kaplowitz 25 21.. 1975). Induction by polycyclic hydrocarbons. in contrast. causes hardly any proliferation of the smooth ER. thus liver size increase is minimal. Induction is characterized by a hypsochromic shift of the xmax of the reduced cytochrome-CO complex from 450 nm to 448 nm. Another spectral change is seen 16 in the reduced cytochrome-ethyl isocyanide difference spectral 455/430 nm ratio which increases nearly 3 fold above control. There appears to be at least two forms of cytochrome P-450 induced by MC-type inducers as indicated by SDS-PAGE results. The results indicate the existence of two isozymes with apparent molecular weights of 54.000 and 56.000-57.000 (Atlas 25 al.. 1977; Guenthner and Nebert. 1978). The former isozyme is termed cytochrome P-448 or P-450BNF-B since this protein shows the reduced cytochrome-CO complex having a xmax - 448 nm (Guenthner and Nebert. 1978; Guengerich _£ 21.. 1982). Acetanilide 4- hydroxylase activity is also associated with this isozyme (Atlas and Nebert. 1976; Novak and Vatsis. 1982). The latter isozyme is termed cytochrome P1-450 or P-4SOBNF/ISF-G and seems to have associated with it the aryl hydrocarbon hydroxylase (AHH) activity. usually measured as the rate of hydroxylation of benzo(a)pyrene (Guengerich 25 al.. 1982; Guenthner and Nebert. 1978; Nebert and Gielen. 1972). The cytochrome P-450 hemOproteins induced by MC and TCDD have a much narrower substrate specificity than those induced by PB. Ethoxy-O- deethylase activity. a measure of AHH activity (Burke 25 al.. 1977). 2-biphenyl hydroxylation and epoxidation activities (Parke. 1975) have also been associated with the hemOproteins induced by MC. NADPH-Cytochrome P-450 reductase as well as pentabarbital hydroxylation are not affected by MC-type inducers. However. p-nitrophenol UDP—glucuronyltransferase (Bock 25 31.. 1973) and DT—diaphorase (Lind and Ernster. 1974) are induced. Not all inducers readily fall in one or the other of these two classes. Some oxidations can be induced by either a PB- or 17 MC-type inducer such as aniline hydroxylations (Parke. 1975). warfarin metabolism (Kaminsky 25 21.. 1983) and biphenyl hydroxylation (Creaven and Parke. 1966; Billings and McMahon. 1978). Biphenyl is metabolized mainly to the 4-OH derivative by PB microsomes and to the 2—OH derivative by MC microsomes. however the formation of the other metabolite also occurs in each case but to a lesser extent. Also. while benzo(a)pyrene hydroxylase activity is induced mainly by MC-type inducers. PB will induce this activity but to a lesser extent (Parke. 1975). The results are partly explained by the observation that the purified cytochrome P-450 hemOproteins possess different but often overlapping substrate specificities (Coon and Vatsis. 1978; Lu and West. 1980) and exhibit both pronounced regio- and stereo- selectivities toward the substrate and product formed (Lu and West. 1980; Vatsis 25 al.. 1980). Polyhalogenated biphenyl mixtures appear to have the ability to induce both types of induction simultaneously. This mixed- type induction is also observed when both PB and MC are coadministered (Dent 35 al.. 1978). The mixed-type induction caused by polybrominated and polychlorinated biphenyl (PBB and PCB. respectively) mixtures may be due to the presence of inducers of both types in the mixture or the presence of congeners each by themselves having the ability to elicit both responses simultaneously (Moore 25 al.. 1978b; Dannan 25 al.. 1978a; Moore 25 al.. 1979; Dannan 35 al.. 1982b). It has been commonly accepted that the mechanism of induction of the mixed-function oxidase activity is indeed an 18 induction involving g5 2939 synthesis of protein and not the activation of protein already synthesized. since it is prevented by inhibitors of protein synthesis. such as puromycin and cyclo- heximide (Dehlinger and Schimke. 1972; Haugen 25 _l.. 1976). The employment of suitable inhibitors of RNA and DNA synthesis showed that inhibitors of RNA synthesis. such as actinomycin D. blocked enzyme induction whereas hydroxyurea. an inhibitor of thymidine incorporation into DNA. had no effect (Parke. 1975). Thus it appears that DNA synthesis is not required and the effects of the inducer are at the level of transcription. These observations imply that the inducers may act as derepressors of regulator or other genes. This theory is better argued with the case of polycyclic aromatic hydrocarbon inducers. Genetic work done primarily with the mouse. using two strains. one ”responsive” to the effects of aromatic hydrocarbons and the other ”nonresponsive” to them. revealed that the induction of AHH activity (or P1-450 or P-450BNF-B) is due to a dominant gene locus. the Ah locus (Nebert and Gielen. 1972). Recently. a relatively specific antibody to P -450 was developed (Negishi and 1 Nebert. 1979) and used to size P1-450 mRNA present in livers from MC-treated B6 mice (responsive mouse) (Negishi and Nebert. 1981). A cDNA clone derived from a 23s mRNA from the MC-treated B6 mouse liver was then isolated and characterized (Negishi 25 21.. 1981). This cDNA clone was then used to show that P1-450 induction is correlated with large increases in P -450 (23s) mRNA. that it 1 involves a large molecular weight precursor mRNA (Tukey 25 al.. 1982). and that it requires an interaction of membrame-bound polysomes with P -450 mRNA (Chen and Negishi. 1982). 1 19 Consequences of the Induction of HicrosomCl_Drug: Hetabolizing Enzymes Induction of microsomal drug—metabolizing enzymes by various compounds can have physiological. pharmacological. toxicological. and carcinogenic consequences. The administration of compounds that induce microsomal drug— metabolizing enzymes can alter the physiologic action of normal body constituents. Treatment of rats with PB and certain insecticides increase the hydroxylation of androgens. estrogens. progestational steroids and adrenocortical steroids (Conney and Klutch. 1963; Conney 25 31.. 1966; Levin gt al.. 1968; Conney and Burns. 1972) as well as the biosynthesis and breakdown of cholesterol (Parke. 1975). Administration of barbiturates to animals also enhance the enzymatic glucuronidation of bilirubin by liver microsomes. stimulates bile flow and accelerates the in 3132 metabolism of bilirubin (Catz and Yaffee. 1962; Roberts and Plaa. 1967). Treatments of animals with PB also effects the deiodination of thyroxine (Stanbury at 21.. 1960). the demethylation of the methylated purines as well as alter metabolism of vitamins D and K (Conney. 1967; Mountain gt 31.. 1970; Breckenridge. 1975). Enzyme induction can also markedly alter the pharmacological action of drugs subsequently administered. One classic example is that of the action of the muscle relaxant drug. zoxazolamine. in that pretreatment of rats with either PB or 3.4-benzpyrene 20 greatly alters the half—life and thus the action of zoxazolamine (Conney 23 21.. 1960). In control rats the half-life is 9 hours whereas it's 48 and 10 minutes after PB or 3.4-benzpyrene administration. respectively. However. 3.4-benzpyrene administration has no effect on the pharmacologic action of hexabarbital whereas PB enhances it. indicating that polycyclic hydrocarbons are specific enzyme inducers and do not stimulate the activity of all drug-metabolizing enzymes (Conney. 1967). Enzyme induction by PB is believed to also result in shortening the half—life of anticoagulants such as bishydroxycoumarin and warfarin (Conney and Burns. 1972; Breckenridge. 1975). Certain drugs including alcohol stimulate their own metabolism through enzyme induction and this perhaps explains why individuals who habitually consume large amounts of alcohol tend to show a tolerance to barbiturates and other sedatives when sober and enhanced sensitivity when inebriated (Conney and Burns. 1972; Breckenridge. 1975). Induction of hepatic drug-metabolizing enzymes can also increase or decrease the incidence of toxicity of certain xenobiotics. For example. bromobenzene causes hepatic and lung necrosis and eventually death. However. pretreatment with MC decreases its toxicity while PB pretreatment potentiates its toxicity. When rats are pretreated with MC. the enzymes induced convert the majority of bromobenzene to the less reactive and non-toxic 2.3-epoxide. PB. however. enhances the 3.4—epoxidation of bromobenzene which is highly reactive and depletes reduced glutathione levels and ultimately binds covalently to cellular macromolecules thus increasing its hepatotoxicity (Gillette gt 21 al.. 1974; Parke. 1975). Carbon tetrachloride (CC14) toxicity is similarly influenced by MC and PB pretreatment. PB enhances CCl4 hepatotoxicity. probably by increasing CCl formation which may 3 be involved in peroxidative damage of membrane lipids (Parke. 1975) while MC has a protective effect. For certain insecticides whose toxicity depends on metabolic activation. various inducers can greatly increase their toxicity. An example is the organophosphate guthion. which is relatively non-toxic until it is metabolized to an active cholinesterase inhibitor. MC pretreatment substantially increases its toxicity while pretreatment with SKF 525-A (B-diethylaminoethyldiphenylprOpyl— acetate). an inhibitor of the drug—metabolizing microsomal system. results in decreased toxicity (Murphy and DuBois. 1958). Several examples are known in which enzyme induction can modify chemical carcinogenesis. The potent liver carcinogen. 3'— methyl—4-dimethylaminoazobenzene. does not produce hepatomas when fed to rats simultaneously with MC or other polycyclic hydrocarbons (Richardson 2; 21.. 1952; Miller 25 21.. 1958). In contrast to the inhibition of chemical carcinogenesis. SKF 525-A enhances the carcinogenicity of 9.lO-dimethyl-l.2-benzanthracene (Wheatley. 1968). Also. some polycyclic aromatic hydrocarbons do not contain highly reactive electrOphilic sites and are not carcinogenic themselves but need to be metabolically activated. One of the most widely studied of these compounds is benzlalpyrene which is metabolized by the mixed-function oxidase system and epoxide hydrase into various dihydrodiols. triols. tetrols. phenols. quinones. epoxides. diol epoxides. and 22 conjugates (Yang 33 31.. 1976. 1977. 1978; Sims 25 31.. 1974). Some of the metabolites and reactive intermediates. such as diol epoxides and epoxides. can be highly mutagenic and carcinogenic (Jerina and Daly. 1974; Wood 33 31.. 1976; Yang 33 31.. 1978). Thus. it is possible that MC or PB pretreatment would alter the metabolic profile observed. Halggenateg Aromatic Hydrocarbons and Polycyclic Arogatic Eggpounds: An Examination of a Mechanism of Toxicgty and Enzyge Induction Chlorinated dibenzo-p—dioxins and dibenzofurans as well as PCB and PBB. collectively termed the polyhalogenated aromatic hydrocarbons (PHAH) are usually considered together. because they i) are structurally related i.e. approximate isostereomers ii) produce a similar and characteristic toxic syndrome and iii) are believed to act through a common mechanism. Polycyclic aromatic hydrocarbons such as MC also cause similar toxic and biochemical reSponses and are believed to act through the same mechanism. Numerous reviews and symposia have covered the chemistry. analytical detection. animal toxicology. environmental consequences. commercial production and epidemiologic investigations of these compounds. The reader is referred to several of these sources for a comprehensive coverage of the various aspects of this vast and complex subject (Lee and Falk. 1973; Kimbrough. 1974; Hutzinger _3 31.. 1974; Brinkman and Reymer. 1976; Lucier and Hook. 1978; Nicholson and Moore. 1979; Kimbrough. 1980; Kahn and Stanton. 1981; Poland and Knutson. 23 1982). In this section I will briefly summarize the toxic effects produced by these compounds and attempt to more closely examine the biochemical responses. Structure-activity relationships for toxicity and biochemical responses as well as the most current and widely accepted mechanism by which these compounds elicit their toxic responses and the results and observations which sUpport it will be presented. I will focus primarily on the work with TCDD since it is the most thoroughly studied and potent compound. and therefore serves as the prototype of the PHAHs. Toxic Responses A variety of toxic effects are produced by PHAHs. The toxic effects observed after administration. however. vary with the dose. length of eXposure and most important with the species of animal used. In different Species the acute oral LD (pg/kg) 50 of TCDD varies over a 5000-fold range : LDso for guinea pig - 1; male rat - 22; female rat - 45 (Schwetz 33 31.. 1973); monkey < 70 (McConnell t al.. 1978); mouse - 114 (Vos et 1.. 1974; a McConnell _3 31.. 1978); bullfrog > 500 (Beatty 33 31.. 1976); hamster - 5000 (Olson 33 31.. 1980a; Henck 33 31.. 1981). The variation in Species Specificity has been shown not to be due to different rates of metabolism (Olson 33 31.. 1980b; Gasiewicz and Neal. 1979). Although there are species. age and sex differences. the toxic responses to TCDD and related compounds include a wasting syndrome (progressive weight loss). lymphoid involution. hepatotoxicity. skin disorders (chloracne and hyperkeratosis). carcinogenesis. chick edema syndrome and embryotoxicity and/or teratogenesis (reviewed in Poland and Knutson. 1982; Parkinson 24 and Safe. 1981). The actual mechanism (s) for toxicity and the ultimate cause (s) of death however remains unknown. giochegica1 Re33onse3 A variety of biochemical responses are evoked by TCDD and related compounds and are as varied as the toxic effects. The most well characterized and commonly studied biochemical effect is the induction of hepatic microsomal enzymes. such as cytochrome P —450 and will be the subject of the next section. A 1 number of biochemical studies have been conducted in order to elucidate the mechanism of toxicity and the determinant of death due to TCDD. Since TCDD is so extremely toxic to certain animals it has been suggested that TCDD may be affecting some fundamental process in animal cells leading to a general dysfunction of those cells. From biochemical studies however it was concluded that liver DNA synthesis. ATP synthesis. protein synthesis. the oxidation-reduction state of cells. the action of glucocorticoids. and the absorption and metabolism of amino acids. carbohydrates and fatty acids from intestine do not appear to be the mechanism through which TCDD exerts its toxicity (reviewed by Neal 33 31.. 1979). Among other biochemical effects elicited by TCDD and related halogenated aromatic hydrocarbons are endocrine effects which include reproduction impairment (Murray 33 31.. 1977) and decreased serum thyroxine concentration (Bastomsky. 1977; Bastomsky. 1974). A decrease in hepatic storage of vitamin A (retinol) (Thunberg 33 31.. 1979; 1980; 1979) porphyria (Goldstein 33 31.. 1976a; Goldstein 33 31.. 1973) and effects on lipid metabolism (Poli 33 al.. 1980; Thompson. 25 1981) are also observed. Enzyme Induction and Role og Recegtor As mentioned in an earlier section . Nebert and coworkers (1972a. 1972b) as well as Thomas 33 31 (1972) have shown that certain inbred strains of mice respond to the induction by MC of hepatic cytochrome P -450 and its associated AHH activity. while 1 other inbred strains fail to respond. The strain responsive to MC is C57Bl/6 (B6) and the prototype-non-responsive strain is DBA/2 (D2). In genetic crosses and backcrosses between B6 and D2. responsiveness. or the induction of AHH activity by polycyclic aromatic hydrocarbons. was found to be inherited as a simple autosomal dominant trait. The genetic locus that controls the induction by polycyclic aromatic hydrocarbons of AHH and numerous other drug-metabolizing enzyme activities is termed the Ah locus (for Aromatic hydrocarbons). The ability of TCDD to induce rat hepatic AHH activity nearly 30.000 times that of MC (Poland and Glover. 1974) prompted researchers to examine the effects of TCDD on the B6 and D2 mice. TCDD induced hepatic AHH activity in all mice tested. regardless of their response to MC (Poland 33 31.. 1974). Thus. D2 mice. which were nonresponsive to MC. did respond to the more potent inducer TCDD and therefore appeared to possess the structural and regulatory genes necessary for the expression of AHH activity. The failure of D2 mice to respond to MC suggested that they did not recognize MC. It was postulated that a mutation in the D2 mice resulted in a defective recognition or in a receptor site which had a diminished affinity for MC and other polycyclic 26 aromatic hydrocarbons but which still recognized TCDD (Poland 33 al.. 1974). This hypothesis suggested then that D2 mice would have less sensitivity to TCDD than B6 mice. Dose-response curves for induction of hepatic AHH activity in D2 and B6 mice revealed that B6 mice were more sensitive to TCDD (EDSO- l x 10"9 mol/kg) than D2 mice (ED 2 l x 10"8 mol/kg). supporting this hypothesis 50 (Poland and Glover. 1975). Similar results were obtained in cell cultures from B6 and D2 fetal mice (Niwa _3 _1.. 1975). The receptor was first detected 13 31333 in both B6 and D2 hepatic 105.000 x g sUpernatant fraction (also commonly referred to as the cytosol fraction) through the use of high specific activity [3Hl-TCDD with a charcoal-dextran binding assay (Poland _3 31.. 1976a). The reversible binding of [3Hl-TCDD to specific binding sites (a pool of high affinity. saturable sites) occurred with a high affinity (KD- 0.27 x lo-gm) in B6 cytosol and maximal binding capacity (n) of roughly 8.5 x 10-l4mol of TCDD/mg cytosolic protein. The KD compared quite well with the 13 3133 potency of TCDD to induce AHH activity. Much lower specific binding of [BHI-TCDD was observed in D2 hepatic cytosol. and in fact it was so low a KD could not be estimated for D2 mice (Poland 33 31.. 1976a). The failure to demostrate specific binding in D2 mice was believed to be due to the presence of a receptor with reduced affinity rather than a diminished receptor concentration since the dose-reSponse curve for AHH induction in D2 mice was shifted to the right. while. the maximum response was not lower than that for B6 mice. There was an excellent correlation between the binding 27 affinities (KD) of 23 halogenated dibenzo-p-dioxin and dibenzofuran congeners and their potency (ED ) to induce AHH 50 activity (Poland 33 31.. 1976a). However. several polycyclic aromatic hydrocarbons. which induce AHH activity. showed a less stringent correlation. MC and other polycyclic aromatic hydrocarbons showed a high affinity for the receptor (for MC. HD- 1.7 x 10-9m) in contrast to their weak potency 13 vivo. This suggested that the ability of these compounds to induce AHH activity may be diminished 1 vivo by rapid metabolism in contrast to TCDD (Poland 33 31.. 1976a). Thus. no well-defined structure-activity relationship between AHH induction and receptor binding could be ascertained for the polycyclic aromatic hydrocarbons. Compounds which did not induce cytochrome Pl-450 and its associated AHH activity but induced a different pattern of cytochrome (s) P-450. such as a PB and pregnenolone 16a- carbonitrile. did not compete for binding. Several endogenous compounds. such as steroid hormones and thyroxine. also did not compete for binding and implied that TCDD bound to a distinct cytosol Species (referred to as either TCDD- or Ah—receptor) separate from these. The Ah receptor is principally. if not completely. composed of protein since it has been shown to be heat labile and is inactivated by trypsin. chymotrypsin and proteinase K. whereas it is unaffected by ribonuclease. deoxyribonuclease. lipase and neuraminidase (Poland 33 31.. 1976a; Okey 33 31.. 1979). Rat hepatic Ah receptor was characterized by gel filtration and ultracentrifugal sedmentation as having a Stokes radius of 6.6 nm. a sedimentation coefficient of 5.0s and a calculated 28 molecular weight of 136.000 (Carlstedt-Duke 33 31.. 1981). Mouse hepatic Ah receptor was also characterized by ultracentrifugal sedimentation as having a Stokes radius of 7.5 nm. a sedimentation coefficient of 7.5s and a calculated molecular weight of 245.000 (Hannah 33 31.. 1981). A recent study by Gasiewicz (1983) reported. using a gel-exclusion HPLC method. that although small differences may exist. qualitatively the molecular weights of the Ah receptor from different species appeared to be quite similar. ranging between 220.000 - 280.000. The Ah receptor has also been identified in rat and mouse cytosolic fractions of liver. lung. thymus and kidney (Carlstedt— Duke. 1979; Mason and Okey. 1982). The Ah receptor has been believed to mediate the nuclear uptake and binding of TCDD in mouse liver and assumed to be an early step in the sequence of events resulting in the increased synthesis of cytochrome P -450 (Greenlee and Poland. 1979). 13 1 3133 experiments revealed that hepatic nuclear Uptake of [3H]- TCDD in B6 mice administered [3H1—TCDD was at least 5 times as much as that in D2 mice and this uptake was diminished by coadministration of unlabelled TCDD. but not by inactive congeners i.e. PB. 13 31333 experiments showed that the specific Uptake of [3H1-TCDD by B6 hepatic nuclei was greater when incu- bated with charged cytosol from the same strain than when incu- bated with hepatic cytosol from D2 mice. The capacity 1_ 31333 of unlabelled polycyclic aromatic hydrocarbons and halogenated aromatic congeners to compete for [3Hl-TCDD nuclear uptake reflected their ability to compete for specific binding sites in 29 cytosol as well as their potency as 13 3133 inducers of AHH activity (Greenlee and Poland. 1979). Okey and coworkers (1979) reported similar results employing a sucrose density gradient (SDG) analysis following dextran-charcoal treatment method. This assay was considered more reliable in characterizing the receptor than DEAD-cellulose column chromatography or the published dextran-charcoal adsorption assay since it separates the class of high affinity. low capacity binding sites from the nonsaturable binding sites (Okey 33 31.. 1979). Again. the receptor was shown to bind polycyclic aromatic hydrocarbons which induce AHH activity and was detectable in responsive strains. not detectable in nonresponsive strains and detectable in heterozygous strains (B6 x D2 backcross). Administration of [3HJ-TCDD to B6 mice resulted in the appearance 2 to 18 hours after treatment of a hepatic nuclear inducer-protein complex that sedimented at 5.75 on SDG of high ionic strength (cytosolic receptor normally sedi- ments at 8-108 on SDG of low ionic strength). This peak however was not observed in similarly treated D2 mice. A more recent study . using the SDG assay in a vertical tube rotor (VTR) which decreased the centrifugation time from 16 hours to 2 hours (Tsui and Okey. 1981) reported a detectable nuclear Ah receptor in D2 mice administered [3H1-TCDD although it was much lower than that from B6 mice (Mason and Okey. 1982). The failure of earlier efforts to detect the nuclear Ah receptor in D2 mice was suggested to be due to the instability of the ligand-receptor complex formed and that during the long centrifugation spin (16 hours) dissociation occurred (Mason and Okey. 1982). . Other reports include one by Okey and coworkers (1980). who showed a 30 temperature-dependent cytosol to nucleus translocation of the Ah receptor also occurred in continous cell culture lines following treatment of [3HI—TCDD. Tukey and coworkers (1982) studied the cytosolic and nuclear Ah receptor in livers from B6 and D2 mice given a dose of [3H1-TCDD. The receptor was detectable in the cytoplasm and nucleus in B6 mice 4 hours after treatment whereas the receptor was not measurable in D2 cytosol but found in the nucleus at levels one fourth to one fifth of those in BG mice. An excellent correlation was found between the amount of [3H]- TCDD-Ah receptor complex in the nucleus and the quantity of P1- 450 mRNA (23s) induced in the mice (Tukey 33 31.. 1982). Other assays for quantitating the Ah receptor include tryp— sin treatment of cytosol followed by isoelectric focusing (Carlstedt-Duke 33 31.. 1978). a detergent-washing procedure with purified nuclei (Greenlee and Poland. 1979). gel permeation and anion exchange chromatography (Hannah 33 31.. 1981) and a hydroxyl apatite adsorption assay (Gasiewicz and Neal. 1982). MC. in the form of [BHI-MC of high specific activity. has also been shown to bind to macromolecular components in rodent hepatic cytosols (Smith 33 31.. 1977: Sarrif 33 31.. 1976). A component which sediments at 4-5s (on SDG) in rat hepatic cytosol was shown to bind [3H1—MC and with a very high binding capacity (Tierney 33 31.. 1980). When hepatic cytosol was fractionated by Sephadex chromatography and the [3H1—MC bound protein (4-5s) was incubated with rat liver nuclei. translocation of radioactivity to the nucleus was observed. It was suggested that this component. which may be different than the Ah receptor due to 31 different molecular weights and sedimentation coefficients and shown to be different than the estradiol receptor. may be involved in the process of AMH induction (Tierney 33 31.. 1980). Other studies revealed that some macromolecules which bind [3M]- MC have prOperties similar to those of the Ah receptor (characterized with [afll-TCDD) but that the majority of [BHI-MC bound in mouse hepatic cytosol was associated with components which do not appear to be involved in regulation of ARM activity (Hannah 33 31.. 1981). Okey and Vella (1982) reported that [3M]- MC and [BHI-TCDD do bind to the same Ah receptor site (8-10s region on SDG). However. they also reported that [3H1-MC bound to another macromolecular site (4-5s region on SDG) and that the capacity of unlabelled TCDD and other congeners to inhibit [3H]- MC binding to the 4-5s region did not correlate with their ability to induce AHH activity (Okey and Vella. 1982). The component (3) which sediment at 4-5s was found in both B6 and DZ mice cytosol. appearing not to segregate with the Ah locus. They therefore concluded that the component (5) which sediment at 4-5s did not appear to be involved in the regulation of ARM (Okey and Vella. 1982). These studies appear to support a model which is similar to the molecular mechanism of steroid hormone action. The current model postulates a two-step mechanism through which the Ah receptors control the expression of the cytochrome P -450 gene: 1 an interaction between polycyclic aromatic hydrocarbon or halogenated aromatic ligands and a ”cytosolic" receptor. followed by an apparent temperature-dependent "translocation“ of the ligand-receptor complex from the cytOplasm to the nucleus. where 32 a pleiotropic response is evoked which involves the activation of a number of structural genes including cytochrome P -450 and its 1 associated AHH activity. Among the other coordinately expressed enzyme activities are UDP-glucuronosyltransferase (Owens. 1977). DT-diaphorase (Beatty and Neal. 1976). ornithine decarboxylase (Nebert 33 31.. 1980) and B-aminolevulinic acid synthetase (Poland and Clover. 1973). Induction of several of these enzyme activities. namely UDP-glucuronosyltransferase and DT-diaphorase. have been shown to segregate with the Ah locus in inbred strains of mice (Owens. 1977; Nebert 33 al.. 1980). A schematic representation of this prOposed model is shown below. TCDD fig TCDD (55;: TCDD—® TCDjf@ W Nucleus mRNAs (— . AHH ACTIVITY A B C Gymwmsm T A recent paper by Whitlock and Galeazzi (1984) however 33 prOposed a slightly different mechanism based on new findings. Employing mouse hepatoma (Hepa lclc7) cells. a cell line previously shown to contain a normal functioning Ah receptor (Legraverend 33 31.. 1982). they showed that dilution of the cell homogenate prior to centrifugation. markedly influenced the distribution of bound [BHI-TCDD between the nuclear and cytosolic fractions. In studying the effect of homogenate dilution on the distribution of unoccupied TCDD receptors (untreated cells) between cytosol and nuclear fractions in Mepa lclc? cells. they found that in undiluted cell homogenate roughly 80% of the bound [BHI-TCDD was associated with the nuclear fraction. However following an 8-fold dilution of the homogenate. roughly 80% of the bound [3ul-TCDD was associated with the cytosolic fraction. Also. intermediate distributions were seen when intermediate dilutions were used and analogous changes in receptor distribution was observed when different cell dilutions were used. The results indicated that the [3H1-TCDD-binding species in these cells were subject to redistribution among subcellular fractions during homogenation and centrifugation. and the appearance of the TCDD receptors in the cytosolic fraction may be a consequence of redistribution and may not reflect their location in the intact cell. Competition experiments revealed that the nuclear [3H1-TCDD—binding species had the expected specificity for TCDD receptors in that unlabelled known ligands for the TCDD receptor competed for specific binding whereas chemicals known not to be ligands did not compete. Also. through Scatchard analysis. they showed that the nuclear and cytosolic species bOth had an apparent KD - 1 nM. They also showed similar 34 results in experiments where they incubated intact cells at 0° C o C with [BHI—TCDD and then performed similar dilution and 37 experiments. Again. dilution of the homogenate had a marked effect on the amount of TCDD receptor complexes associated with nuclei. In undiluted homogenate from cells incubated at 0° C. 60-80% of the ligand-receptor complexes were nuclear. whereas an 8—fold dilution of the homogenate caused almost all the ligand- receptor complexes to appear in the cytosol. The results from intact cells incubated at 37° C with [3H1-TCDD were somewhat different in that the effect of dilution was less compared to that seen with cells incubated at 00 C. In undiluted homogenate of cells incubated at 37° C. roughly 90-100% of the ligand- receptor complexes was nuclear. however after an 8-fold dilution. 80% of the ligand-receptor complexes remained associated with the nuclei. These results suggested that the binding of TCDD- receptor complexes to nuclei might be stronger in cells incubated with [3H1-TCDD at 37° C than in cells incubated at 0° C. Whitlock and Galeazzi (1984) then performed receptor release experiments where they varied the ionic strength to test this hypothesis. They found that higher ionic strength was required to release TCDD-receptor complexes from nuclei from cells exposed to [Bul-TCDD at 37° C whereas lower ionic strength was effective in releasing TCDD-receptor complexes from nuclei from cells incubated at 0° C or in releasing unoccupied receptors. The results implied that TCDD-receptor complexes bind more strongly than do unoCCUpied receptors and that the TCDD—receptor complexes undergo a temperature-dependent event which strengthens their 35 binding. The authors concluded that their results were compatible with the current two-step model for the mechanism of action of TCDD. in that the first event is the binding of TCDD to a receptor but that this occurs primarily in the nucleus. thus imposing a constraint on the nature of the second. temperature- dependent translocation step. They suggested that the second step is not a translocation since the first step occurs in the nucleus and that a temperature-dependent event may further strengthen the binding of the TCDD-receptor complex to the nucleus (Whitlock and Galeazzi. 1984). A schematic representation of this proposed model is also shown below. TCDD a TCDD :1 CVKwM3m AHH ACTIVITY A B c etc. This observation that the TCDD receptor appears to be n“Clear in origin is not an anomaly. Recent studies by two different groups (King and Greene. 1984: Welshons 33 1.. 1984) 36 have shown that the unoccupied estrogen receptor. shown by numerous reports to be cytoplasmic. is not a cytOplasmic protein in the intact cell but a nuclear one. The unoccupied vitamin D receptor and thyroid hormone receptor have also been reported to be nuclear (Halters 33 31.. 1980; Samuels and Tsai. 1973). Thus. more research needs to be done in order to address the implications of a nuclear TCDD receptor and its molecular function (s) and association (s) with the genes it regulates. However. certain aspects of the receptor remain unchanged. There are still two identifiable states 3 an active (ligand-associated) state and an inactive (unoccupied) state. and the TCDD receptor- 1igand complexes are nuclear regulatory elements. wherever they originate in the cell. A common model for the mechanism of toxicity by TCDD and similar congeners has been hypothesized by Poland and coworkers (Poland 33 31.. 1979; Poland and Glover. 1980; Poland and Knutson. 1982). It should be noted that this model. or any model. was based on toxicity studies employing a limited number of congeners from several classes of PHAHs or polycyclic aromatic hydrocarbons. The structure-toxicity relationships therefore are generally limited when compared to the structure-activity relationships for AHH induction and Ah receptor binding. Assuming this limitation. there generally appeared to be qualitatively and quantitatively a good correlation between receptor binding. AHH induction and toxic responses for these compounds. Of course. situations existed where large discrepancies occurred and it had been suggested that metabolic 37 inactivation 1 vivo maybe responsible (Poland 33 al.. 1976a). These observations suggested that the parent compound was responsible for toxicity. that the initial event in the action of these compounds was the stereospecific recognition and binding by the Ah receptor and that the toxicity observed was somehow mediated through this receptor (Poland 33 31.. 1979; Okey 33 31.. 1979). 8tructure-activit3 Relationshigs As mentioned earlier a large number of PHAMs and polycyclic aromatic hydrocarbons have been studied for their ability to induce AHH activity and bind to the receptor. The $050 for AHH induction for a large number of these compounds was determined in the chick embryo liver (Poland _3 31.. 1976a: Poland and Clover. 1973) and the hepatic receptor affinity in the B6 mouse (Poland _3 _1.. 1976a). The receptor affinity for TCDD does not appear to vary greatly in different species (Gasiewicz. 1983) whereas the LDso (toxic potency) especially of TCDD. varied greatly with species. This imposed a constraint on a direct comparison between receptor affinity and [D50 or a pathological response. Therefore. the structure-toxicity relationship of congeners and receptor affinities was usually related by a comparison of congeners in rank-order (i.e. TCDD is a better ligand for the receptor than octachlorodibenzo-g-dioxin (OCDD) and therefore TCDD» should elicit a greater toxic response than OCDD in any species). In this section. I will attempt to summarize the structure— activity relationships for several classes of PHAHs for receptor 38 affinity. AHH induction and toxicity. The first class of PHAMs are the halogenated dibenzo—E—dioxins. of which 75 possible isomers exist. and the dibenzofurans. of which 135 possible isomers exist. These compounds are found as trace contaminants in certain halogenated industrial mixtures and are not synthesized for commercial purposes. The ability of various chlorinated dibenzo-g—dioxin and dibenzofuran isomers to bind to the TCDD receptor 13 31333 correlates well with their ability to induce AHH activity (Poland 33 31.. 1976a). These congeners have been found to possess several structural similarities. One is that they have halogen atoms in three or four of the lateral ring positions (2.3.7. and 8) and at least one unsubstituted ring position (Poland 33 31.. 1976a; Poland and Clover. 1973). Bromine was also shown to be slightly more active than chlorine as a substituent. followed by fluorine and nitro groups. which were considered inactive (Kende 33 _1.. 1974). The relative lethality (LDSO) of several of these congeners in the guinea pig. mouse and chick embryo corresponded quite well with their potency as inducers of hepatic AHH (Poland and Clover. 1973; McConnell 33 31.. 1978; Moore 33 31.. 1979: Bradlaw and Casterline. 1979). A discrepancy existed between the binding of 2.3.7- trichlorodibenzo-E—dioxin to the TCDD receptor (Poland 33 31.. 1976a) and its activity in the chick embryo and guinea pig (reviewed in Goldstein. 1980). It was suggested that 13 3133 metabolism may decrease its potency. A number of other studies have shown that sructure-activity relationships between the toxic responses caused by halogenated dibenzo-g-dioxins. and 39 dibenzofurans correlates well with the criteria for binding and enzyme induction (Schwetz 33 31.. 1973: Moore 33 31.. 1979; Poland and Knutson. 1982). Polyhalogenated biphenyls are another class of PMAHs. PCB are industrial chemicals which have usually been produced as commercial mixtures due to their heat resistance properties (Sissons and Welti. 1971). The related PBB have been manufactured as fire retardants. Commercial mixtures of PCB and PBB contain a number of isomers. which vary with degree and positions of halogenation. Early studies reported that these mixtures induced hepatic microsomal monooxygenases typically induced by both PB and MC and were thus referred to as mixed-type inducers (Alvares _3 31.. 1973; Dent 33 31.. 1976a; 1976b: Alvares and Kappas. 1977). For example. Aroclor 1254 (a PCB mixture containing 54% chlorine) induced AMM activity. typically induced by MC or TCDD. as well as ethylmorphine-N-demethylase. typically induced by PB (Ryan 33 31.. 1979). The P-450s induced by this mixture were isolated and found indistinguishable from a mixture of hemOproteins from PB and MC treated rats by spectral. electrOphoretic. catalytic and immunologic evidence (Ryan 33 31.. 1979). These results suggested that this PCB mixture induced forms of cytochrome P-450 inducible by PB as well as those inducible by MC or TCDD. Subsequent work with various pure PCB congeners have shown that the positions and degree of halogenation govern the type of induction observed and the extent of the toxicological and biochemical response (Bcobichon and Comeau. 1975: Goldstein 33 al.. 1976b; 1977). Initial studies employing mostly symmetrical —— 4O PCB congeners suggested that there appeared to be two distinct grOUps of inducers : a PB-type. typified by 2.4.5.2'.4'.5'- hexachlorobiphenyl (-HCB). and a MC-type. typified by 3.4.5.3'.4'5.'-HCB (Goldstein 33 31.. 1977: Goldstein. 1979). In other studies with a limited number of PCB congeners. it appeared that the only congeners that induced AHH activity and bound to the TCDD receptor were 3.4.3'.4'-tetrachlorobiphenyl (-TCB). 3.4.5.3'.4‘.5'-HCB and 3.4.5.3'.4'.5'-hexabromobiphenyl (-HBB) (Poland and Glover. 1977: Goldstein 33 31.. 1977: Goldstein. 1980). These results suggested that biologically active biphenyls i) had halogens in at least adjacent 3333 and 3333 positions on each ring of the biphenyl and ii) lacked 33333 halogens which were considered to lead to marked nonplanarity. These halogenated biphenyls produced biological effects similar to TCDD. but were at least loo-fold less active. Various studies however had reported 2.3.4.2'.4'.5'-HCB. 2.3.4.2'.3'.4'-HCB and 2.4.5.2'.4'.5'—MCB to be mixed-type inducers in themselves (Stonard and Greig. 1976; Alvares and Kappas. 1977). Goldstein and coworkers (1979) later reported that the 2.4.5.2'.4'.5‘-MCB used by Alvares and Kappas (1977) was contaminated with 44 ppm 2.3.7.8-tetrachlorodibenzofuran. a potent MC-type inducer. and thus was considered a strict PB-type inducer. This was also confirmed by a study using the brominated analogue by Moore and coworkers (1978). However. more recent studies by Parkinson and coworkers (l980a.l981) reported results which supported the mixed-type induction seen with 2.3.4.2'.3'.4'-MCB and 2.3.4.2'.4'.5'-MCB. using highly pure congeners. 41 Other investigations using a much larger series of bromo- and chlorobiphenyls have modified the structure-activity relationship. Dannan and coworkers (1978a) reported that 2.4.5.3'.4'.5'-HBB caused a mixed-type induction as well as some TCDD-like responses. Several PCB congeners containing a single chlorine 33333 to the bridge carbon were also shown to possess mixed—type induction properties (Parkinson 33 31.. 1979. 1980b. 1980c). Other PBB congeners which possessed a bromine 33333 to the bridge carbon. were also shown to be mixed-type inducers and caused a TCDD-like toxicity (Dannan 33 31.. 1982b; Dannan 33 31.. 1982s). The results suggested that the presence of an 33333 halogen does not completely abolish MC-type induction or toxicity. In addition to the MC-type induction the compounds also appeared to contain some PB-type inducing properties. and therefore are considered mixed-type inducers. Several studies suggested that 3333 substitution was not an absolute requirement for a congener to cause a PB-type induction (Goldstein 33 31.. 1977). 2.3.5.2°.3'.s'-nca and 2.3.5.6.3'.5'— HCB. both 3333-unsubstituted. caused strong PB-induction (Kohli _3 31.. 1979; Parkinson 33 31.. 1980c). However. in the case of 2.3.6.2'.3'.6'-MCB and 2.3.5.6.2'.5‘-MCB. no or very little PB- type induction was observed (Goldstein 33 31.. 1977: Parkinson 33 31.. 198°C). The type and extent of induction appears to be influenced by the amount of halogenation 33333 to the bridge carbon of the biphenyl system. The extent of induction. however. may be influenced by 3333 and 33333 substitution as well. The induction of ARM activity (MC-type) is believed to be mediated by the 42 binding of these congeners by the TCDD receptor. The binding affinities of 16 pure PCB congeners to the TCDD receptor has been reported (Bandiera 33 31.. 1982). The most active agonists were 3.4.3'.4'-TCB. 3.4.5.3'.4'—pentachlorobiphenyl (-PCB). and 3.4.5.3'.4'.S'-MCB. which are strong AHH inducers and considered approximate isostereomers of TCDD (Poland and Glover. 1973; Goldstein. 1979: Parkinson 33 31.. 1980c). Several mono-33333 substituted congeners. which were weaker AHH inducers and considered mixed-type inducers. exhibited less affinity for the TCDD receptor (Parkinson 33 31.. 1980b. 1980c). Bandiera and coworkers (1982) also reported that although PB was not bound to the TCDD receptor. 2 PB—type inducers as well as 2 congeners which are neither PB- nor MC-type inducers were bound but with apparent affinities 600-1000 times less than that of 3.4.5.3'.4'- PCB. Por all the PCB congeners tested. the relative order for binding was considered to correlate well with the rank-order for induction of AMH activity in cell culture (Parkinson 33 31.. 1980b; Sawyer and Safe. 1982). A limited number of biochemical studies have also shown that the relative binding of certain PCB congeners to the TCDD receptor appeared to correlate quite well with their TCDD-like toxicity (Poland and Glover. 1977; Poland 33 31.. 1979). Several of the mono-33333 chlorinated congeners. shown to be mixed-type inducers. also seemed to elicit an intermediate TCDD-like response (Yoshihara 33 31.. 1979; Parkinson 33 31.. 1980b. 1980c). As seen with the chlorinated dibenzo-g-dioxins and dibenzofurans. toxicity studies seemed to indicate a correlation 43 between the ability of PCB congeners to cause a TCDD-like toxicity and its ability to induce AHH activity. Thus. the approximate isostereomers of TCDD. 3.4.3'.4'-TCB. 3.4.5.3'4'—PCB and 3.4.5.3'.4'.5'-HCB. are much more toxic than those congeners. such as 2.4.5.2'.4'.5'-MCB. which are considered to be PB-type inducers (McKinney t al.. 1976; Yoshimura 33 31.. 1979: Biocca _3 _1.. 1981). The approximate isostereomers are planar or can assume a near planar configuration and their molecular structures can roughly fit into a rectangle 3 x 10 X with halogens occupying all four corners (Poland and Glover. 1977). This is a rough approximation for structure-activity relationships since several exceptions exist. The most severe limitation of this model is the case of the polycyclic aromatic hydrocarbons. While both PMAH and polycyclic aromatic hydrocarbons bind to the receptor and induce AHH activity. only the PHAH elicit a TCDD-like toxicity. It has been postulated that enzyme induction is an early event and that toxicity is a later event and requires persistent receptor occupation and gene expression (Poland and Knutson. 1982). Polycyclic aromatic hydrocarbons are metabolized at a significant rate and thus do not persist in the cell. A tissue which expresses TCDD toxicity. contains the Ah receptor and has a low capacity to metabolize polycyclic aromatic hydro- carbons is the thymus and it has been shown that MC produced a dose-related thymic involution. supporting this hypothesis (Poland and Glover. 1980; Poland and Knutson. 1982). The structure-activity relationship for toxicity and receptor binding are quite similar and support the prOposal that PHAH congeners exert their toxicity through the Ah receptor. 44 However. if toxicity is mediated through the receptor then toxicity should segregate with the Ah locus. the gene that determines the receptor (Poland and Knutson. 1982). Work which supported segregation of toxicity with the Ah locus has been done with the responsive and nonresponsive strains of mice. BS. D2 and B6 x D2 P1 (reviewed by Poland and Knutson. 1982). It was found that like AHH induction and the Ah receptor. thymus involution. teratogenesis and hepatic porphyria all segregated with the Ah locus. Thus in summary. two lines of evidence. namely i) the correlation of the structure-activity relationship for toxic potency and receptor binding and ii) the segregation of three toxic responses evoked by TCDD with the murine Ah locus. support the hypothesis that toxicity of PHAM congeners is mediated through their binding to a receptor. Induction of Drug Metabolizing an33es b3 and Toxicit3 of Polybrog1nated Bighenxls The research that will be described in this thesis is concerned with various prOperties of polybrominated biphenyls (PBB) and therefore a review of the research on the effects of PBB is given separately. The initial research on the effects of PBB on hepatic microsomal drug metabolizing enzymes has been done primarily using the crude PBB mixture. Firemaster. which allegedly contaminated Michigan. This mixture has been found to contain at least twelve major PBB congeners (>l%). ten of which have been identified (Jacobs 33 31.. 1976; Sundstrom 33 al.. 45 1976a: Moore 33 31.. 1978a; Moore and Aust. 1978; Dannan 33 31.. 1982a). Other studies have reported and identified approximately ten other minor PBB congeners (Orti 33 31.. 1983: Robertson 33 31.. 1983a). At least twenty non-PBB trace impurities as well as 200 ppm brominated naphthalenes have been detected in the Firemaster PF-l mixture (Hass 33 31.. 1978). For the purpose of this thesis. a brief review of several of the TCDD-like toxic responses of the PBB mixture. namely Firemaster BP-6. will be discussed first. It should be noted that the mechanism (s) by which these effects are expressed remains largely unknown. All the animal species studied to date respond to significant amounts of PBB by weight loss or reduced weight gain which may be accompanied by a decrease in food intake (Sleight and Sanger. 1976: Corbett 33 31.. 1978; Lambrecht 33 31.. 1978; Ringer. 1978; Gupta and Moore. 1979; Praker. 1980: Gupta 33 31.. 1983a). Other characteristic responses include atrOphy of the lymphatic tissues (namely the thymus. spleen and bursa). hepatomegaly. and in certain cases. death (Ringer. 1978; Gupta and Moore. 1979: Praker. 1980: Gupta 33 31.. 1983b). Among the organs effected by PBB. the liver has been reported to enlarge (hepatomegaly) in the following species studied. the rat. mouse. chick. cow and monkey (Sleight and Sanger. 1976; Ringer and Polin. 1977: Moorhead _3 31.. 1978: Kasza 33 31.. 1978; Corbett 33 31.. 1978: Allen 33 31.. 1978: Gupta and Moore. 1979; Praker. 19803Akoso 33 31.. 1982a; Dharma t al.. 1982). Hepatomegaly is believed to occur due to hepato- ”* cellular hypertrOphy and ultrastructurally. is mainly due to 46 extensive proliferation of the endoplasmic reticulum (ER) particularly smooth (Sleight and Sanger. 1976; Corbett 33 31.. 1978; Gupta and Moore. 1979). Among other characteristic hepatic ultrastructural changes include increased numbers of cytoplasmic lipid droplets. increased liposomes. disorganized rough BR and formation of concentric arrays of laminated BR membrames (also referred to as myelin figures or whorls) around lipid droplets (Sleight and Sanger. 1976; Kasza 33 _1.. 1978: Gupta and Moore. 1979). Induction of neOplastic nodules in rats six months after a single oral dose of l g Piremaster/ kg has been reported (Kimbrough _3 31.. 1978). These neOplastic nodules appeared to develop into hepatocellular carcinomas when rats were killed nearly two years after a similar dose of PBB was given to rats (Kimbrough _3 31.. 1981). The mechanism by which PBB can induce hepatic nodules is largely unknown. however. the ability of PBB to serve as promoters in experimental hepatocarcinogenesis in rats has been investigated (Jensen 33 31.. 1982). Dietary concentrations of 10 and 100 ppm of Firemaster were found to promote hepatocarcinogenesis. as assayed by measuring enzyme altered foci exhibiting gamma glutamyl transpeptidase activity (Jensen 33 31.. 1982). In a lifetime study. Firemaster was also shown to increase the incidence of atypical hepatocellular foci. neOplastic nodules. and hepatocellular carcinomas in rats exposed to 10 mgPBB/kg/day for 6 months (GUpta 33 31.. 1983b). Aside from the liver. the immune system has also been shown to be a target of PBB toxicois. AtrOphy of the thymus has been 47 shown histologically to be due to a marked loss in the cortex area due to lymphocyte depletion (Sleight and Sanger. 1976: Gupta and Moore. 1979; Praker. 1980). Firemaster has also been shown to decrease bursal weights in cockerels fed 10 and 100 ppm PBB for 28 days (Dharma 33 31.. 1982). Upon histological examination it was observed that depletion of lymphocytes occurred and was most likely responsible for the decreased weights. Immunological alterations were observed when mice and rats were administered 22 daily oral doses of Firemaster at levels ranging from 0.03 to 30 mg/kg body weight (Luster 33 31.. 1978). Cell mediated immunity was severely depressed in both rats and mice given the largest dose whereas humoral immune function. as determined by measuring the antibody production. serum immunoglobulin levels. and splenic lymphocytes response to mitogens were depressed only in mice. (Luster 33 31.. 1978). In a chronic study. several immunological responses were depressed in mice and rats given 10 mg PBB/kg/day for 6 months (Luster 33 31.. 1980). It was suggested that although PBB induced immune alterations in rodents. the effects were most apparent at exposure levels that caused other significant toxic responses (Luster 33 _1.. 1980). Praker (1980) reported a dose-dependent reduction in B- and T-helper cells in female mice fed diets containing 0. l. 10. or 100 ppm Firemaster for 30 days. which indicated that the immune system could be dramatically affected. Ho effects on T-cell dependent delayed type hypersenstivity was observed (Praker. 1980) which agreed \vith earlier results (Praker and Aust. 1978). The effects of PBB upon some immune functions of swine during gestation and Lactation have been investigated as well (Howard 33 31.. 1980). 48 During the last half of gestation and the following 4 weeks of lactation. sows were fed Firemaster in the diet at levels of 0. 100 or 200 ppm. Significant reduction in mitogenic response of peripheral blood lymphocytes of PBB fed sows and their four week old offspring were observed. whereas no significant changes in bactericidal activity of whole blood in either sows or offspring were seen. The mechanisms of the suppression of some immune functions by PBB were not known (Howard 33 31.. 1980). In a study where mice were fed diets containing 5 or 167 ppm Firemaster for either 3 or 6 weeks. results suggested that PBB minimally affected antibody production against T-dependent antigens or on host defense to protozoan parasites yet caused a selective depletion of serum immunoglobulin isotypes (Loose 33 31.. 1981). Therefore. it appears that PBB is immunosuppressive in most laboratory animals but at dosages which evoke other toxic responses. Also. variable results have been observed where PBB was shown to affect the cell mediated immunity in one case (Luster 33 31.. 1980) and the humoral immune function in another case (Praker. 1980). These variable effects may be time— dependent since normal proliferative processes of B- and T- lymphocytes are not the same (Vos 33 31.. 1980). Among other effects which have been shown to be related to Firemaster or PBB congeners are an effect on the thyroid. proliferation of the bile duct. and porphyria (Akoso 33 I. H 0 \ 1982b; GUpta 33 31.. 1983a: Gupta 33 31.. 1981). PBB has also been shown to affect the metabolism of estrogens (Bonhaus 33 31.. 49 1981) and testosterone (Newton 33 31.. 1982) as well as have an effect on the levels of hepatic and serum vitamin A (Akoso 33 31.. 1982a; Darjono _3 31.. 1983). The effect most widely studied has been enzyme induction and is discussed below. Early studies have shown that induction of hepatic cytochrome P-450 and other activities occurred after the administration of Firemaster. Japanese quail fed 100 ppm of PBB showed a decrease in egg production and hatchability as well as induction of hepatic microsomal enzymes induced by PB and MC (Babish 33 31.. 1975). A decrease in egg production and hatchability was also seen in chickens given Firemaster (Polin and Ringer. 1978). Cecil and coworkers (1975) reported that administration of Firemaster to Japanese quail decreased pentobarbital sleeping times. Firemaster was also shown to induce hepatic cytochrome P-450 and to be weakly teratogenic in mice (Corbett 33 31.. 1975). Beaudoin (1977) also reported that a single high dose of Firemaster caused teratogenic effects in rats. Sleight and Sanger (1976) reported increased liver weights. hepatic histOpathological changes and induction of hepatic microsomal enzymes in rats fed diets containing Firemaster. The first study which showed that Firemaster caused a mixed typed induction of hepatic microsomal enzymes in rats was performed by Troisi (1975). The study reported that both a single dose of 90 mg/kg or a diet of 10 ppm of PBB greatly increased liver weights. cytochrome P—450. cytochrome P-450 reductase activity. aminOpyrine demethylation as well as benzo(a)pyrene hydroxylation. The pattern of induction caused by 50 Firemaster was similar to that caused by co-administration of PB and MC. suggesting that Firemaster caused a mixed—type induction. The induction of several enzymes was also still elevated 10 days after treatment. Similar conclusions were reported by Dent and coworkers (1976a). A later report confirmed these findings and also showed the elevation of several hepatic microsomal enzymes was still persistent at 14 days after treatment (Dent 33 31.. 1976b). The effect of Firemaster on rat liver. kidney. and mammary gland was also investigated. The mammary and hepatic benzolalpyrene hydroxylase activity (or AHH activity) both increased following PBB administration. however epoxide hydrolase activity was elevated only in the liver and depressed in the mammary gland (Dent 33 31.. 1977a). It was suggested that the induction of mammary AHH activity and depression of mammary epoxide hydrolase may increase the susceptibilty of the mammary gland to a second toxic agent. which may be metabolized to a reactive intermediate (i.e. arene oxide of benzo(a)pyrene) which normally epoxide hydrolase would convert to a more stable and less toxic compound. In another study. both hepatic and renal microsomal AHH activity was induced in rats fed diets containing 100 ppm Firemaster for 3 months. however renal epoxide hydrolase was depressed to 14% of control values whereas hepatic levels were elevated (McCormack 33 31.. 1978). It was suggested that the PBB did not cause an impairment of renal function. however again it was postulated that increased susceptibilty of the kidney to a second agent could occur. 51 The effects of Firemaster on neonatal rats were also investigated. Dannan (1978) reported that nursing pups appeared ten times more senstive than their mothers. who were fed 1 ppm PBB for 18 days. since the mothers were unaffected whereas their pups had a mixed-type induction of liver microsomal enzymes. It was suggested that compounds responsible for the induction in Firemaster was transmitted via the milk. A later study. which confirmed this. reported that PBB congeners found in the milk of lactating rats resembled those in Firemaster although in slightly different proportions (Dannan 33 31.. 1978). PBB may be transmitted also via the placenta. since a mixed-type induction of hepatic enzymes was observed in microsomes isolated from livers from pups exposed to PBB 13 33333 but nursed by untreated dams (Dent 33 31.. 1978a). In another study. body weight gain and hepatic concentrations of vitamin A were reduced in rats by pre- and postnatal exposure to 100 ppm Firemaster (McCormack 33 31.. 1982) In two studies by Dent and coworkers (1977b; 1978) the pattern of induction of cytochrome P-450 hemoproteins caused by Firemaster over time was reported. In the earlier study. cytochrome P-450 and two PB-inducible activities. ethylmorphine- N-demethylation and epoxide hydrolase. were maximal at day 2 'whereas MC-inducible activities. ethoxycoumarin-O-deethylation and benzolalpyrene hydroxylation. were maximally induced at day day'4 after treatment with 150 mg Firemaster/kg body weight (Dent 33_339. 1977b). Through inhibitor studies. it was suggested that the characteristics of cytochrome P-450 and its associated enzyme activities changed from PB-type to MC-type with time after PBB 52 administration. In the second study. the time-course changes in ethylisocyanide spectral properties. reaction kinetics. inhibitor effects. and SOS-PAGE microsomal profiles were studied (Dent 33 31.. 1978). Anomalous results were obtained in that the kinetic data suggested that the PBB caused a mixed-type induction early after administration and then returned to an induced control state whereas the electrophoretic data indicated that early after PBB administration. induction was dissimilar to PB induction and then returned to an induced control state. The authors suggested that PBB may represent a new class of inducing agents since they appeared to have some but not all of the mixed-type induction properties of both PB and MC. Firemaster was also shown to produce hyperkeratosis using the rabbit ear test (Kimbrough 33 31.. 1977a). A dose of 60 mg/rabbit ear produced hyperkeratosis while similar reactions were elicted by tetrachloro— or a tetrabromdibenzofuran at much smaller doses. 20.5 or 17.5 pg/rabbit ear. respectively. In another study. in an attempt to characterize the hyperkeratotic agent. Firemaster was fractionated into a polar and a nonpolar fraction by alumina column chromatography and each fraction was tested for an effect (Kimbrough 33 31.. 1977b) The polar fraction produced a pronounced hyperkeratosis while the nonpolar fraction produced a very mild reaction. It was concluded that the polar fraction contained chemicals with acnegenic properties. Hass and coworkers (1978) performed a similar study. Firemaster was fractionated by florisil chromatography and a polar and nonpolar fraction obtained. The polar fraction. S3 constituting nearly 0.17% of the original mixture. was found to contain twenty or more non-PBB contaminants but no brominated di- benzofurans or dibenzo-g-dioxins were detected. The polar fraction was found to be toxicologically inert to guinea pigs and mice at a dose of 1 mg/kg (equivalent to 500 mg of Firemaster) and 15 mg/kg. respectively. The acnegenic effects of Firemaster. polar and nonpolar fractions were also tested. The polar fraction did not produce hyperkeratosis. whereas Firemaster and the nonpolar fraction. both of which were found to contain nearly 200 ppm naphthalenes. produced a reaction. It was concluded. since such low levels of brominated naphthalenes were present. that the brominated biphenyls were responsible for the toxic effects of Firemaster (Hass 33 31.. 1978). A later study by Goldstein and coworkers (1979) reported a similar conclusion. 2.3.6.7-Tetrabromonaphthalene. predicted to be the most potent MC-type inducer of the bromonaphthalenes. given at a dose equivalent to the concentration of bromonaphthalenes in Firemaster had no effect. Thus the bromonaphthalenes in Firemaster were unlikely to account for the MC—type induction effects (Goldstein 33 31.. 1979). The major component (56%) of Firemaster was determined to be 2.4.5.2'.4'.5'-HBB by two separate reports (Jacobs 33 31.. 1976; Sundstrom 33 31.. 1976). 2.4.5.2'.4'.5‘-HBB was purified to >99% pure from Firemaster and found to be a strict PB-type inducer (Moore 33 31.. 1978b). A dose of 90 mg/kg of 2.4.5.2'.4'.S'-HBB increased microsomal protein. cytochrome P-450. as well as two PB-inducible activities. NADPH-cytochrome P-450 reductase and aminOpyrine demethylat ion. Induction of benzo(a)pyrene S4 hydroxylation by 2.4.5.2'.4'.5'-HBB was similar to that caused by PB. but was fivefold lower than that by MC. SDS-PAGE results of 2.4.5.2'.4’.5'-HBB-induced microsomes revealed that heme and protein profiles were similar to PB-induced microsomes but markedly different from those of control. It was concluded that 2.4.5.2'.4‘.5‘-HBB contributed to the PB—type induction caused by Firemaster (Moore 33 31.. 1978b). Another major component (27%) of Firemaster was identified as 2.3.4.5.2'.4'.5‘— heptabromobiphenyl (Moore and Aust. 1978). This compound was later shown to also be a PB-type inducer (Moore 33 31.. 1979). The first report of a mixed-type inducer present in Firemaster was by Dannan and coworkers (1978). 2.4.5.3'.4'.5'- HBB. which was detected and structurally identified by Moore and Aust (1978). was found to increase liver weight and hepatic microsomal protein and induce hepatic NADPH-cytochrome P-450 reductase. aminopyrine demethylation as well as benzo(a)pyrene hydroxylation and 3-nitrophenol—UDP-glucuronyltransferase activity in rats given a single dose of 90 mg/kg and sacrificed one week later. The hepatic microsomal cytochrome P-450 CO difference spectral maximum was shifted from 450 to 448.5 nm. It was concluded that 2.4.5.3'.4'.5'-HBB caused a mixed—type induction similar to that caused by both PB and MC and may contribute to the MC-like aspects of Firemaster (Dannan 33 31.. 1978). In studies by Robertson and coworkers (1980; 1981). two other components of Firemaster. namely 2.4.5.3'.4'-PBB and 2.3.4.5.3'.4'-HBB. were synthesized and found to also be mixed- 55 type inducers. 2.3.4.5.3‘.4'-HBB was also shown to competitively displace [3H1-TCDD from the protein believed to be the TCDD- receptor (Robertson 33 31.. 1981). A later study by Dannan and coworkers (1982b) confirmed that 2.4.5.3'.4'-PBB was a mixed-type inducer and produced a TCDD-like response. Among the toxic responses evoked by this chemical was decreased body. splenic and thymic weights as well as an effect on the immune system in that splenic T- and B-helper cells were substantially impaired at. a dose of 90 mg/kg. Ultrastructural changes in the hepatocytes of a treated rat included proliferation of the smooth ER and appearance of lipid droplets and concentric membrane whorls. It was suggested that a single 33333 halogen may weaken but not abolish the toxicity of PBB congeners (Dannan 33 31.. 1982b). This was also later confirmed by another study in which 2.3.4.5.3'.4'-HBB. previously identified as a mixed-type inducer (Robertson 33 31.. 1981). was purified from Firemaster and tested for toxic responses (Dannan 33 31.. 1982c). At 1 week after administration of 90 mg/kg. 2.3.4.5.3'.4'—HBB decreased body. splenic and thymic weights and severely affected the structure of the thymus. Hepatic ultrastructural changes caused by 2.3.4.5.3'.4‘-HBB included marked disorganization of the ER membrame which gave rise to concentric membrane whorls around one or more of the lipid vacuoles. Two other compounds also studied. 2.3.4.2'.4'.5'-HBB and 2.4.5.3'.4'.5'-HBB. showed similar ultrastructural changes but to a lesser degree. and did not affect body or any tissue weight. except for increasing liver weight (Dannan 33 31.. l982c). In a following study (Dannan 33 31.. l982d). a reconstituted mixture was formulated to resemble 56 Firemaster BP-6 in order to elucidate if the nine congeners purified from Firemaster (totalling nearly 97% of the mixture) accounted for the induction by and toxicity of the Firemaster. Hepatic microsomes. isolated from rats given 90 mg/kg of either Firemaster PP-l. BP-6 or the reconstituted mixture and killed seven days later. all had equivalent amount of induction of several enzyme activities. Body weights were unaffected by any treatment whereas thymic weights were depressed by the reconstituted mixture. It was concluded that the toxic effects of Firemaster were due to these congeners and that some minor trace contaminant would not significantly contribute to the toxicity (Dannan 33 31.. l982d). The induction of specific isozymes of P-450 by a single dose (90 mg/kg) of Firemaster or several of its purified congeners has been investigated using immunoelectrophoretic assays (Dannan et al.. 1983). P-450 and P-450 __ __ BNF-B BNP/ISF-G' two isozymes preferentially induced by MC (or BNF). were invariably induced by 2.4.5.3'.4'-PBB. 2.4.5.3'.4'.5'-HBB. 2.4.5.2'.3'.4'-HBB. 2.3.4.5.3'.4'-HBB as well as 3.4.5.3'.4'.5'-HBB. P-450 isozymes. typically induced by PB. were invariably induced by all congeners present in Firemaster. The Firemaster mixture induced both MC- and PB-inducible forms of P-450. although not to the level as in the case of the purified PBB congeners (Dannan 33 31.. 1983). The authors suggest that the results support the idea that the presence of one or more 33333 substituents does not hinder c0planar ring configuration and MC-type induction. The results also suggest an inverse relationship between the number of ortho 57 hl dc ' " " a ogens an po ency of induction of P ‘SOBNP-B and P 45°BNP/ISFG as well as a direct correlation between the number of ortho halogens and the induction of P-450PB_B. P-450PB_D. and to a lesser extent P-450 and P-450 (Dannan 33 31.. 1983). PB-C PB/PCN-E Similar results were seen by Parkinson and coworkers (1983). The ability of several PBB congeners. purified from Firemaster. to compete with [3Hl-TCDD for binding to the TCDD receptor has been investigated using the charcoal-dextran assay (Dannan. 1981). 2.4.5.3'.4'-PBB and 2.4.5.2'.3'.4'-HBB were found to compete with [3Hl-TCDD for binding with KD-l.3x10-8M and 1.5x10-7M. respectively. These two congeners. as mentioned earlier. both induce AHH activity and evoke a TCDD-like toxic response. It was suggested that these congeners may be considered a derivative of 3.4.3'.4'-TBB. which is expected to be among the most toxic congeners (Robertson 33 31.. 1982). The 13 3133 and 13 31333 metabolism of PCB and PBB congeners has been studied and will be briefly reviewed. PCB congeners have been shown to undergo metabolism by the microsomal mixed- function oxidase enzyme system into a number of products or intermediates. some of which can covalently bind to cellular macromolecules (Shimada and Sato. 1978; Shimada and Sato. 1980; Shimada and Sawabe. 1983). The i vivo and in vitro metabolism of chlorobiphenyls and identification of metabolites have been performed (reviewed by Sundstrom 33 31.. 1976c). The structural requirements for in vitro metabolism of ten dichlorobiphenyls have been investigated using microsomes isolated from rats given different pretreatments (Kennedy 33 31.. 1980; 1981). Biphenyls with unsubstituted ortho positions were metabolized the fastest 58 by microsomes isolated from rats treated with B-naphthoflavone (an MC-type inducer). whereas 33333 substituted biphenyls were metabolized the fastest with microsomes isolated from PB—treated rats. The mixed function system along with epoxide hydrolase were thought to be involved in metabolizing the various dichloro- biphenyls into monohydroxy and dihydrodiol metabolites through arene oxide intermediates (Kennedy 33 31.. 1981). The PBB metabolism research has been conducted using 13 31333 techniques where hepatic microsomes were incubated with PBB congeners and the necessary cofactors to support NADPH-dependent cytochrome P-450 catalyzed metabolism. The results of 13 31333 metabolism using microsomes isolated from control. PB- or MC- treated rats suggest that structure-activity relationships do exist (Dannan 33 31.. 1978a; Moore 33 31.. 1980). The initial experiments revealed that only two PBB congeners from Firemaster were metabolized when incubated with microsomes isolated from PB- or PBB-treated rats. namely 2.4.5.2'.S'-PBB and 2.3.6.2'.4'.5'— HBB. No metabolism could be observed with control microsomes or microsomes from MC—treated rats. The congeners metabolized had two adjacent nonhalogenated carbons on at least one ring. 13 vitro metabolism studies with PCB congeners also indicated that nonhalogenated meta and 3ara positions were required for metabolism using PB-induced microsomes (Kato 33 al.. 1980; Shimada and Sato. 1980; Shimada and Sawabe. 1983). PBB congeners in Firemaster with unsubstituted ortho and meta positions did not appear to be metabolized by any microsomes. whereas two PCB congeners. 2.4.2'.4'-TCB and 3.4.3°.4'-TCB. were 59 metabolized by MC-induced microsomes (Shimada and Sato. 1980; Shimada and Sawabe. 1983). It was hypothesized that adjacent nonhalogenated carbons. one of which is 3333. was required for metabolism (Dannan 33 31.. 1978a). In 13 31333 metabolism studies using seven model PBB congeners (di- through pentabromobiphenyls). which were believed not to be present in Firemaster. structure-activity relationships were further tested using only PB-induced microsomes (Moore 33 31.. 1980). The results further suggested that PBB congeners could be metabolized 13 31333 by PB-induced microsomes only if they possessed a 3333 unsubstituted carbon. since significant rates of metabolism were not observed if both 3333 carbons were halogenated (Moore 33 31.. 1980). During the course of this research. 13 31333 metabolism studies were conducted in this laboratory employing a large number of pure PBB congeners and induced microsomes. The results of these studies suggest that structure-activity relationships also exist for MC-induced microsomes (Mills 33 31.. 1984). Briefly. the results showed that MC pretreatment was found to increase the 13 31333 microsomal metabolism of pure PBB congeners which possessed adjacent nonhalogenated 33333 and 3333 carbons on at least one ring and that pentabromination or higher appeared to prevent metabolism. Similar results as mentioned above were observed with PB-induced microsomes. in that adjacent nonhalogenated 3333 and 3333 positions are required on at least one ring and that higher bromination does not appear to prevent metabolism (Mills 33 31.. 1984). The distribution and excretion of 2.4.5.2'.4'.5'-HBB. the 60 major component of Firemaster. has been investigated and the results suggested that the PBB was readily absorbed from the intestine. initially distributed throughout the body and eventually stored in the adipose tissue and was not subject to appreciable metabolism (Matthews 33 31.. 1977). It was suggested that in modeling the pharmacokinetics of this congener. present and future tissue concentrations and their relationship to exposure could be estimated. The results of another study using Firemaster suggested that 2.4.5.2'.5'-PBB was cleared more rapidly than any other PBB congener in the mixture (Domino 33 31.. 1980) which agrees with the 13 31333 metabolism studies (Dannan 33 31.. 1978a). Several studies have been performed in an attempt to reduce body burden of PBB. without much success (McConnell 33 31.. 1980: Kimbrough _3 _1.. 1980). Several pure congeners have been tested for their ability to serve as tumor promotors. Several congeners have been shown to inhibit metabolic cooperation in Chinese hamster cells and thus share a prOperty associated with tumor promotors (Tsushimoto 33 31.. 1982). It appeared that PBB congeners possessing an 33333 bromine could act as a tumor promotor and inhibit cell-cell communication. A recent paper by Jensen and coworkers (1983) reported the hepatic tumor-promoting ability of 3.4.5.3'.4'.5'- HBB as assayed by measuring enzyme altered foci. The results suggested that induction of MC-type enzymes can occur without toxicity or tumor-promoting ability and that the tumor promoting ability of 3.4.5.3'.4'.5'-HBB was most likely the result of hepatic degeneration and necrosis (Jensen 33 al.. 1983). An earlier study (Jensen 33 31.. 1982) had shown that 2.4.5.2'.4'.5°-HBB. a PB-type inducer which has 2 33333 bromines was a strong tumor promotor at a dietary concentration that was not hepatotoxic. The research that will be described in this thesis is concerned with studying the relationships between the chemical and 13 3133 and 13 31333 pharmacotoxicological preperties of polybrominated biphenyls. The objectives are to develop methods of synthesis and purification of 3.4.3'.4'-TBB and 3.4.5.3'.4‘.5'-HBB. It has been suggested that the non-33333 substituted PBB congeners with bromines in the lateral positions (3333 and 3333 positions to the bridge carbons). namely 3.4.3'.4'-TBB. 3.4.5.3'.4'-PBB and 3.4.5.3'.4'.5-HBB. should be the most toxic PBB congeners (Robertson 33 31.. 1982). Preliminary chemical studies (see chapter one) have indicated that the non-halogenated 33333 congeners all possess similar chemical prOperties which differ only with the total number of bromines and positions of bromines. The detection of these congeners in the Firemaster mixture or in a photolyzed mixture (which could occur in the environment) would suggest contribution of these congeners to toxicity if present in relatively high amounts and if indeed these congeners had strong biologic potency. Biological and toxicological effects of the photolyzed mixture have been compared to purified congeners in order to assess what congeners were responsible for the enhanced biologic potency of the photolyzed mixture. These compounds have also been suggested to be deposited predominantly in the adipose tissue and are considered to be extremely resistant to metabolism 62 and elimination. with the exception of two congeners (Dannan 33 1.. 1978b: Matthews and Kato. 1979). Therefore. i vivo studies in comparison to 13 31333 studies are essential for understanding the pharmacotoxicological properties of PBB congeners. The manner of administration. the observations to be made and the time at which the observations are made are also extremely important. especially if a PBB congener is metabolized at an appreciable rate. in studying the pharmacotoxicological properties of these chemicals. CHAPTER 1 THE SYNTHESIS. PURIFICATION AND CHEMICAL CHARACTERIZATION OF SEVERAL POLYEROHINATED IIPHENYL CONGENERS 63 64 ABSTRACT A procedure for the synthesis of 2.3.3'.4'- and 3.4.3.'.4'- tetrabromobiphenyl(-TBB)s is described as well as their purification by repeated alumina chromatography and recrystallization. The structures of these PBB congeners were determined by mass spectrometry and 1H-NMR spectrosc0py. A method for purifying 3.5.3'.5'-TBB. 3.4.5.3'.5'- pentabromobiphenyl(-PBB). and 3.4.5.3'.4'.5'-hexbromobiphenyl(- HBB) from a commercial mixture has also been developed and entails repeated alumina chromatography. Two non-33333 brominated biphenyls. 3.4.3‘.4'-TBB and 3.4.5.3'.4'.5'-HBB. have been tentatively identified as minor components ((1%) of Firemaster BP-6 and PP-l. commercial preparations of polybrominated biphenyls (PBB). by capillary chromatography. The UV-absorption spectra have been recorded for all these PBB congeners. Certain chemical and chromatographic prOperties have also been studied. and these have been compared and related to the structures of the congeners. The results of these studies seem to indicate that the total number of bromines and the positions of the bromines. especially the 33333 positions. largely determine the physio-chemical properties of the PBB congeners. INTRODUCTION Polybrominated biphenyls (PBB) have the empirical formula C r with n - l - 10. The number of possible PBB lZHlO-nB n congeners (209) depends upon the number and positions of the 65 bromine. PBB were initially used as flame retardants for use in hard plastics and perhaps would not be so intensely studied if some 500-1000 pounds of PBB had not been accidently mixed with cattle feed in Michigan in 1973 (Carter. 1976). The industrial process used for the manufacture of PBB consists of a Priedal- Crafts type reaction in which the bromination of biphenyl is carried out with bromine in the presence of chloride (e.g. bromine chloride) under an organic solvent and a catalyst (usually aluminum chloride) under anhydrous conditions (Brinkman and deKok. 1980). The PBB mixtures so produced contain bromine contents ranging from six to ten bromines per molecule. Two of these mixtures. Firemaster BP-6 and Firemaster PP-l (a pulverized form of Firemaster BP-6 containing 2% calcium polysilicate as an anti-caking agent) have an average bromine content of six bromines (DiCarlo 33 31.. 1978). The synthesis of nearly 50 individual mono- through hexa- brominated biphenyl congeners has been reported in the literature (VanRoosmalen. 1934: Sundstrom _3 _1.. 1976b). Some of the reactions used for PBB synthesis include phenylation of aromatic substrates. other aryl condensations. and direct bromination of biphenyl. A number of other PBB congeners. mainly mono- through tetra-substituted biphenyls. are commercially available in small quantities (< 100 mg) from Ultra Scientific. Inc. (formerly RPR Corp.). The mixture Firemaster PF-l (believed to be of lot no. FH7042) which contaminated Michigan. has been widely studied and found to contain at least twelve PBB congeners. ten of which have 66 been identified (Dannan 33 31.. l982a; Moore. 1978: Moore and Aust. 1978: Moore 33 31.. 1978a; Sundstrom 33 31.. 1976a: Jacobs _3 31.. 1976). It has also been reported that at least twenty other non PBB trace impurities. including 200 ppm brominated naphthalenes. could be detected by GC-MS analysis but that no brominated dibenzofurans or dibenzo-3-dioxins were detected at (0.05 ppm (Hass 33 31.. 1978). Of the ten PBB congeners identified in the mixture. all had at least one bromine 33333 to the bridge carbon and only recently during this research a study has reported the detection of non-33333 brominated PBB congeners in Firemaster BP-6 and Firemaster FP-l (Orti 33 31.. 1983). Mon- 33333 brominated PBB congeners. especially those with bromines present in the lateral positions (3333 and 3333). are of great interest since they are believed to be the more toxic PBB congeners (Moore 33 31.. 1980). In order to perform biological studies on these compounds. relatively large quantities of absolutely pure compounds are required. Thus. it was necessary to devise synthetic methods and purification schemes for obtaining rather large amounts of non-33333 brominated PBB congeners in order to study the relationships between their chemical preperties and biochemical affects. The structures of several purified congeners from synthetic and commercial mixtures were identified. and some chemical and chromatographic preperties of these PBB congeners were studied. Two non-33333 brominated biphenyls were also detected in Firemaster BP—6 and Firemaster PP-l. The results of these chemical characterization studies are the subject of this chapter. 67 MATERIALS AND METHODS Firemaster BP-6 (lot 6224-A) and Firemaster PP-l (lot PH7042) were gifts from Michigan Chemical Corporation (St. Louis. Michigan) and Farm Bureau (Lansing. Michigan). respectively. Neutral alumina (activity grade I) was purchased from Sigma Chemical Company (St. Louis. Missouri). A crude 3.4.5.3'.4'.5'- hexabromobiphenyl (3.4.5-HBB) mixture was purchased from Ultra Scientific. Inc. (formerly RPR Corp.) Hepe. Rhode Island. Hexane (n-hexane. b.p. 68—690) and all solvents were of glass-distilled grade for pesticide analysis. from Burdick and Jackson Laboratories. Inc.. Muskegon. Michigan. 3% OV-l on Gas Chrom 0. 100/120 mesh was obtained from Applied Science Laboratories. Inc.. State College. Pennsylvania. 3-Bromoaniline and 3— dibromobenzene were purchased from Aldrich Chemical Company Inc.. Milwaukee. Wisconsin. OV-l HP 25m capillary column was purchased from Hewlett Packard (Avondale. Pennsylvania). Kieselguhr G-TLC plates (0.25 mm thickness) was purchased from Altech. Inc.. Newark. Delaware. Gas Chromat33ra3h3 PBB mixtures and fractions were routinely analyzed on a Varian 3700 gas chromatograph (GC) (1.8m x 2mm ID glass column packed with 3% OV-l on Gas Chrom 0 100/120 mesh) equipped with a 63 pulsed Ni electron capture detector (ECD) and a Hewlett Packard Model 33805 integrator recorder. N2 was the carrier gas and the column flow rate was maintained at 30 ml/min at whatever. column temperature employed. A Hewlett Packard (25m x 0.83mm ID. WCOT 68 OV-l) capillary column was employed to analyze PBB mixtures and pure PBB congeners and was maintained at 2450 with He (linear velocity (LV) - 45 cm/sec) as the carrier gas and N (30 ml/min) 2 as the purge gas. A Varian universal direct injector system was employed with the capillary column. Standard curves for each purified PBB congener were constructed by plotting the GC-ECD response (log area counts) versus various amounts (log gram) of each PBB congener. Linear regressions on the data points for each curve were obtained and then the slope. intercept and correlation coefficient were calculated. The absolute amount of each congener in various fractions was then calculated using these standard curves. It should be mentioned that these standard curves were constructed concurrently while analyzing the samples to account for any electronic drift the ECD may have undergone. Gas Chromat3gra3h3-Mass S3ectrometry (GC-M31 Molecular weights of purified PBB congeners were determined by GC-MS (Hewlett Packard 5840A/5985) at 70 ev and a source temperature of 200°. employing a 1.8m x 2mm glass column packed with either 3% OV-l or SE-30. I thank Betty Baltzer of Michigan State University Mass Spectrometry Facility for performing most of the analyses. AH-NMR 32.6% romet t! 250 MHz 1H-NMR spectra of purified PBB congeners were obtained using a Bruker WM-250 spectrometer at ambient 69 temperature. Samples analyzed were prepared by dissolving the PBB congener in either CDCl3 or as the case of 2.3.3'.4'-TBB. deuterated DMSO (approximately 1% w/v) with tetramethylsilane (TMS) as the internal standard. I thank the Chemistry Department of Michigan State University and Dr. Klaas Hallenga for obtaining the spectra. UV-Absor3tion S33ctrosc333 The UV-absorption spectra of purified PBB congeners were recorded on a Perkin-Elmer 559A UV/VIS spectrophotometer. The sample of a PBB congener in UV—grade hexane was scanned from 390 to 190 nm and the extinction coefficients (s) were calculated for the major UV-absorption bands (Ana ). X S nthesis of Several PBB Con eners The synthesis of two PBB congeners. namely 2.3.3'.4'- tetrabromobiphenyl (-TBB) and 3.4.3'.4'-TBB (3.4-TBB). will be described. Several possible synthetic routes exist for these congeners. however the route employed produced basically only these two congeners which represented >90% of the reaction mixture obtained. The following procedure was used. 0.02 Moles of 3-bromoaniline was added to acetic acid (2 ml) and acetic anhydride (2 ml): and the solution boiled for approximately 30 minutes under reflux. The solution was then added to water (50 ml) under mechanical stirring. cooled and the solid bromoacetanilide filtered. Bromine (3.7 g) in acetic acid (6 ml) was added dropwise to the bromoacetanilide (5 g). which was in warm acetic acid (12 ml) 70 while stirring mechanically. The solution was stirred for 30 minutes after all the bromine had been added. The reaction mixture was then poured into cold water (40 ml containing sodium bisulfite) in a thin stream with stirring. The solid (dibromoacetanilide) was filtered and washed with the solution of sodium bisulfite again if the yellow color had not disappeared. The solid was dried as much as possible for the following step. The dibromoacetanilide (4.7 g) was dissolved in ethanol (8 ml) and boiled under reflux. Concentrated hydrochloric acid (6 ml) was added to the boiling solution and the reflux continued for an hour. During this time the dibromoaniline hydrochloride crystals began to separate. The mixture was diluted with water (40 m1). cooled and then with vigorous stirring a 5% sodium hydroxide solution was added until alkaline. The 3.4- dibromoaniline crystals (m.p. 80-810) were then filtered and washed with water (the crystals could be recrystallized as necessary from dilute alcohol) and its structure confirmed by 1H-NMR spectrometry. The 3.4-dibromoaniline was dissolved in the minimum amount of water (Sg/6m1 water) and heated until the dibromoaniline dissolved. Concentrated hydrochloric acid (3 ml) was carefully added with mechanical stirring. The solution was then cooled in an ice/salt bath to below 50 C. While the solution was stirring and kept below 50 C. it was diazotized with a solution of sodium nitrite (lg/1.5 ml water; keeping water to a minimum). The mixture was stirred for 30 minutes at this low temperature. The solution was then filtered and the filtrate (solution of 71 diazonium salt) was added to cold vigorously stirring dibromobenzene (30 m1) followed by the addition of a solution of sodium acetate (Sg/lOml water). The reaction mixture turned to an orange color and was stirred at room temperature for one to two days. The reaction was followed by GC analysis of the products. The organic phase was then washed several times with water. dried over magnesium sulfate and filtered. The dibromobenzene was removed by distillation and was reused. The final reaction mixture was analyzed by GC (Figure l) and the molecular weights determined by GC-MS. on both the crude mixture and the purified congeners. I thank Shoshana Weiner for developing and performing parts of the synthesis. Purification of P93 Congeners Several techniques were used for resolving PBB congeners of the synthetic mixtures and commercially obtained mixtures. In general. one of the purification techniques was by itself sufficient to purify any of mentioned congeners. The following two sections have been divided so as to describe the purification schemes employed for two different mixtures of PBB congeners. I. General Scheme for Purif3ing 2.3.3'.4'-T33 and 3.4-TBB The synthesis previously described yielded a mixture of tetrabrominated biphenyls. 2.3.3'.4'-TBB and 3.4-TBB. which represents approximately 90% of the mixture. and a small amount (>5%) of a tribrominated biphenyl (Figure l). 2.3.3'.4'-TBB and 3.4-TBB were purified from the mixture by one or more of the following procedures. 72 FIGURE 1 - GAS CHROMATOGRAPHIC ELUTION PROFILE AND STRUCTURES OF POLYBROMENATED BIPHENYLS IN A SYNTHESIS MIXTURE. The mixture. 2.1 ng. was injected into an electron capture (63Ni) GC equipped with a 3% OV-l column. Injector port. column and detector temperatures were 260°. 240°. and 330° C respectively: attenuation - 10 x 16 (Amp/mv). RESPONSE _.—zs-.--_-—.i 1.- 's.‘—1:-'_.‘.J;.‘-'-;.‘..'_':'__;:_:'_’.:.r_“"—":_—_ * -.—--...-.-.... _ 73 TIME FIGURE 1 74 Neutral Alumina Chromat3gra3h3 Approximately 300-600 mg of the crude mixture can be used to start each purification. The column used most often and with best resolution was 30 x 1.9 cm ID. fitted with a Teflon stOpcock and a glass fritted disk. The column was first positioned perfectly vertical and then flushed with methanol. acetone and then hexane (approximately 200 ml of each) to ensure cleanliness and proper flow through the glass fritted disk. The column was filled with fresh hexane and with stepcock Opened. powdered neutral alumina (activity grade 1. 1009/9 PBB) was added. After packing. approximately 500 m1 of hexane was passed through the column. The PBB were dissolved in the minimum amount of hexane and the final concentration was approximately 1 - 2 mg/ml. The solution of PBB was carefully applied to the column so as not to disturb the top of the column bed. The column was eluted with hexane at a flow rate of nearly 2 ml/min. Fractions were collected at 10 minute intervals and the GC analysis was performed on every fifth fraction (or more or less as needed). When less than 0.1 mg of PBB was eluting. the column was rinsed with approximately 500 ml of chloroform and collected in a l 1 round bottom flask and analyzed as well. GC Standard curves for the ECD response to PBB (log area counts vs log gram PBB) were constructed concurrently with the analyzed fractions. Fractions of similar compositions were pooled and from these. 3.4-TBB was purified by recrystallization or repeated alumina chromatography to greater than 99% pure and 2.3.3'.4'—TBB was purified to greater than 99% pure by repeated alumina chromatography. It should be noted that the 75 concentration of the enriched PBB congener solution applied to the column after the initial fractionation increases to approximately 3 — 10 mg/ml. most likely due to the absence of polymers which are separated on the first column. Recr3stallization in Hexane Recrystallization of partially purified fractions from hexane was effective at separating the two tetrabrominated biphenyls when the percentage of 3.4-TBB was greater (>50%) than that of 2.3.3'.4'-TBB (see later). Between 50 and 125 mg of an enriched fraction of 3.4-TBB was transferred into either a 50 m1 screwcapped culture tube or a 50 ml glass stoppered erylenmyer flask and dissolved in 10-25 m1 of hot (SO-60°) hexane. Samples in the culture tubes were heated and vortexed frequently to achieve a saturated solution and samples in the erylenmeyer flasks were heated while stirring. The saturated solutions were then allowed to cool slowly to room temperature in a covered Dewar flask filled with hot (40-50°) water and then to recrystallize in the covered flask for 2 to 3 days. The crystals were dissolved and recrystallized further (usually l-3X) until >99% 3.4-TBB was obtained as assayed by GC. II. General Scheme gor Purifying 3.4.5-H3B. 3.4.5.3'.5'- Pentabromobi3hen3l and 3.5.3'.5'-T§3 A crude commercial mixture of 3.4.5-HBB (approximately 60%) was found to be significantly contaminated with 3.4.5.3‘.5'- pentabromobiphenyl (-PBB) (20%) and 3.5.3'.5'-TBB (3.5-TBB) (15%) 76 (Figure 2). Repeated alumina chromatography proved to be the most successful means of purification for these congeners. Approximately 300 mg of this crude mixture was chromatographed on neutral alumina. The procedure used was basically the same as described in section I. however several parameters were altered. The ratio of 200 g alumina/ g PBB was employed. Also. the concentration of the PBB sample applied to the column was lower (1.0 - 0.2 mg/ml) due to the presence of higher brominated congeners and thus the running time of the column was increased as well. Fractions of similar compositions and purities were pooled and from these 3.4.5-HBB. 3.4.5.3'.5'-PBB and 3.5-TBB were purified by repeated alumina chromatography (1-3X). It is impor- tant to note that at least 2 to 3 columns must first be run with crude mixture to collect enough material that is enriched in the desired congener before an enriched fraction is chromatographed. Thus. approximately six neutral alumina columns must be run to collect nearly 150-200 mg of the pure (>99%) congener. Reverse Phase Thin Layer Chromatography (RP-TLC) Kieselguhr G-TLC plates were prepared for reverse-phase chromatography as described by DeVos and Feet (1971). The plates were impregnated with paraffin oil (8% in petroleum ether) and air dried before using. The PBB congeners (4 ug in hexane) were applied to the plate using a 10 ul Hamilton syringe. The plates were then develOped in a paraffin oil-saturated solvent mixture of acetonitrile : methanol : acetone : water (20 x 20 : 9 : l). The plates were redeveloped two more times after they were allowed to air-dry between successive developments. The dry 77 Figure 2 - GAS CHROMATOGRAPMIC ELUTEON PROFI3E_AND_QTRUCTURES OF POLYBROMINATED BIPMENYES IN A COMMERCIAL MIXTURE The mixture. 2.0 ng. was injected into an electron 63N capture ( i) GC equipped with a 3% OV-l column. Injector port. column. and detector temperatures were 260°. 280°. and 330° C respectively: attenuation - 10 x 16 (Amp/my). 78 Br Br Br Br Br Br Br mmzoammm TIME Figure 2 79 plates were then sprayed with a solution of silver nitrate (0.859 silver nitrate/ 100 m1 ethanol (95%) to which 0.5 m1 ammonium hydroxide was immediately added before usage) and intermittently steamed for 15 seconds. For visualization of the plates. the plates were irradiated with a UV-lamp for approximately 30 minutes. The PBB congeners appeared as dark brown spots. R E values were then calculated for each PBB congener. Melting Point Determination The melting points were determined with a Hoover Unimelt capillary melting point apparatus (Arthur H. Thomas Company. Philadelphia. Pa.). w gynthesi3 and Purification o3 PBB Congeners There were at least two objectives for synthesizing and purifying PBB congeners. The first was to obtain several PBB congeners whose low levels ((0.2%) can be detected in commercial mixtures such as Firemaster FF-l and BP-6 (discussed later) but purification of these congeners from such a mixture would not be feasible. The second objective was to test the chemical and biological properties of several PBB congeners and to expand and more closely define structure-activity relationships. Studies such as these require relatively large amounts of highly pure (>99%) compounds. which can be obtained through synthetic and purification methods. 8O S3nthesis of 2.3.3'.4'-TBB and 3.4-TBB The organic synthesis employed was a simple ring substitution of an aromatic amine followed by the Gomberg-Hey reaction. The substitution of the amine was the acetylation of 3- bromoaniline. and then the substitution by bromine to the 3333 position (which is the major product) and then the hydrolysis of the amide back to the amine (Figure 3). The Gomberg-Hey reaction is the production of aqueous diazonium salts. which when in alkali. decompose to form a free radical. which attacks the aromatic halogenated hydrocarbon. which is present as the solvent. to form the halogenated biphenyl (Figure 4: Gomberg and Bachmann. 1924; Ruchardt 33 31.. 1970). The synthesis was performed twice with only difference being one reaction was allowed to run 3 days to obtain more 3.4-TBB. By increasing the reaction time there appeared to be a 10% increase in the amount of 3.4-TBB produced. However. the increased reaction time did not appear to greatly affect the overall yield which was l.8-2.0 g (the percent yield based on dibromoaniline was approximately 40% (the dibromobenzene being in excess)). The resulting mixture was reddish-brown in color and had a coarse texture. The composition of the mixture was shown to contain two tetrabromobiphenyls (>90%) and a small amount of a tribromobiphenyl ((5%) when analyzed by GC-MS and GC techniques (Figure l). 81 Figure 3 - A SCHEME FOR THE FORMATION OF 3.4-DIQROMOANIEINE. The substition of the aromatic amine. m- dibromoaniline with bromine to form 3.4- dibromoaniline. CH3COZH (CH3CO)20 Figure 3 83 Figure 4 — A SCHEME OF THE MECHANISM OF THE GOMEERG-HEY REACTION. This involves the diazotization of 3.4- dibromoaniline. conversion of the diazonium salt to the 3.4-dibromobenzenediazoacetate. decomposition of the diazoacetate to yield a free radical which reacts with 3-dibromobenzene to yield 2r3t3'14'-TBB and 3:413.I4.-TBB. Figure 4 38, 85 Purification of 2.3.3'.4'-T3B and 3.4-TBB 2.3.3'.4'-TBB and 3.4-TBB could be purified from the resulting synthesis mixture (Figure 1) by repeated alumina chromatography and recrystallization as in the case of 3.4.-TBB. Figure 5 shows the elution profile when 320 mg of the synthetic mixture was chromatographed on alumina (not shown are the elutions of minor components which eluted at various times contaminating the two major components). Fractions 65-125 yielded approximately 120 mg of 99% pure 2.3.3'.4'-TBB. Fractions 245-455 yielded approximately 70 mg of 95% 3.4-TBB. At least 20 mg of 99% pure 3.4-TBB can be recovered from this fraction by repeated recrystallization in hexane. However. if this fraction is pooled with another fraction of similar composition and rechromatographed by similar alumina chromatography. nearly 45% can be recovered as >99% pure 3.4-TBB (elution profile not shown). Fractions 126-170 contained nearly 10 mg of material of which 95% was 2.3.3'.4'-TBB. and this material was pooled with fractions from other columns and rechromatographed. Nearly 55% of this material was recovered as >99% pure 2.3.3'.4'-TBB. After fraction 490. 500 m1 of chloroform was passed over the column and collected in a 1 1 round bottom flask. When assayed by GC. the chloroform rinse was found to contain nearly 36 mg of material of which 95% was 3.4.— TBB. This material was not combined with the 70 mg of 95% 3.4.- TBB collected from fractions 245-455 since it was found to be contaminated with a later eluting compound. The chloroform rinses from columns were usually pooled together and rechromatographed by similar alumina chromatography. The 86 FIGURE 5 - NEUTRAL ALUMENA CHROMATOGRAPHY EEUTION PROFILE OF A SYNTHETIC MIXTURE CONTAINING 2.3.3'.4'-TBB AND 3.4.3'.4'-T333 320 Mg of the synethic mixture (Figure l) was applied to a 30 x 1.9 ID cm column packed with 320 g of neutral alumina. The column was eluted (2 ml/min) with hexane. and 20 ml fractions were collected and analyzed by GC. The amount of each congener in the fractions was determined from standard detector response curves. Milligrams 87 Br Br - H. Br Br -o—»— ems Br v I60 :60 200 “2'50 3'00 3:50 450 450 560 Fraction Number Figure 5 congeners purified to greater than 99% pure are shown in Figure 6. Purification of 3.4.5-HBB. 3.4.5.3'.5'-PBB and 3.5,3'.5'-TBB The purification of 3.4.5-HBB. 3.4.5.3'.5'-PBB and 3.5-TBB from a mixture (Figure 2) was achieved primarily by repeated alumina chromatography. Figure 7 shows the resulting elution profile when 370 mg of the mixture (Figure 2) was chromatographed on alumina. Fractions 75-120 yielded approximately 25 mg of 99% pure 3.5-TBB. Fractions 150-190 yielded nearly 37 mg of material of which 90% was 3.4.5.3'.5'-PBB. This material was usually pooled with another fraction of similar composition and rechromatographed by comparable alumina chromatography. Nearly 25% of this material was recovered as >99% pure 3.4.5.3'.5°-PBB (elution profile not shown). Fractions 330-610 yielded approximately 70 mg of 96% pure HBB. This fraction was also pooled with other fractions of comparable composition and rechromatographed. Approximately 50% of this material was recovered as >99% pure HBB. After fraction 610. 500 ml of chloroform was passed over the column and collected. When assayed by GC this chloroform rinse was found to contain nearly 10 mg of material of which 92% was 3.4.5-HBB. Again. as with the purification of 2.3.3'.4'-TBB and 3.4-TBB. the chloroform rinses from the columns usually were pooled together and rechromatographed by similar alumina chromatography. The congeners purified to >99% pure from the commercial mixture are shown in Figure 8. 89 FIGURE 6 - 933 CHROMATOGRAPHIC ELUTION PROFILES OF PURE 2.3.3'.4'-TBB AND 3.4.3'.4'-TBB FROM A SYNTHETIC MIXURE. The mixture. 2.1 ng. and purified congeners. 1-2 ng. were injected into an ECD-GC equipped with a 3% ov-l column. Injector port. column and detector temperatures were 260°. 240°. and 330°. respectively: attenuation - 10 x 16 (Amp/mv). 90 Figure 6 8r HEW B 8)- Br 3 5:8. / 8. .c lilii!) M mmzoammm LJL KM} L TWME 91 FIGURE 7- NEUTRAL AQUMINA CHROMATOGRAPHY EEUTION PROFILE OF A COMMERCIAL MIXTURE CONTAINING 3.5.3'.5'-T§§3 3.4.5.3‘.5'-PBB AND 3.4.5-HBB. 370 Mg of the commercial mixture (Figure 2) was applied to a 30 x 1.9 ID cm column packed with 740 g of neutral alumina. The column was eluted (2.5ml/min) with hexane and 20-25 m1 fractions were collected and analyzed by GC. The amount of each PBB congener in the fractions was determined from standard detector IOSPODSO CUI'VOS e 92 13:52 5:08.... Omm 000 Omm 00m Omv 00¢ Own 00m CON CON Om. CO. L _ L _ b _ _ . CF _ _ - J.» Figure 7 swmfinuw FIGURE 93 8 — GAS CHROMATOGRAPHIC ELUTION PROFILES OF PURE 3.5.3'.5'-TBB. 3.4.5.3'.5'-PBB AND 3.4.5-HBB FROM A COMMERCIAE MIXTURE. The mixture. 2.0 ng. and pure congeners. l-2 ng. were injected into an ECD-GC equipped with 3% Ov-l column. Injector port. column and detector temperatures were 260°. 280°. and 330° c. reSpectively: attenuation - 10 X 16 (Amp/mv). RESPONSE 8' 8! ...—a “...... :— 94 a: E? v F :4 ‘P mf 8' 8 e—6~fke >4 a E o TIME Figure 8 95 structural Characterisation of Pg! congeners Structure assignments have been made for five PBB congeners considering the following assumptions derived from 1H-NMR spectral studies on a number of PBB congeners (Dannan. 1981: Moore and Aust. 1978: Moore. 1978). Chemical shifts of 6.9-7.2 ppm. 7.3-7.7 ppm and 7.8-8.0 ppm are indicative of protons adjacent to 0. l and 2 bromines. respectively. The chemical shifts for protons adjacent to both a bridge carbon and a brominated carbon fall into three categories which depend on the number of total 95559 bromines. Chemical shifts between 7.55- 7.60 ppm for the ortho protons are usually indicative of the presence of only one ortho bromine. Chemical shifts between 7.45-7.49 ppm for the ortho protons are usually indicative of the presence of two ortho bromines. while with three ortho bromines chemical shifts of 7.37 ppm and 7.38 ppm have been reported (Moore. 1978). Coupling constants also comprise three categories. Two protons that split each other with coupling constants of 7-9 Hz and 2-3 Hz indicate protons ortho and meta to each other. respectively. Unsplit signals (J99%). The NMR splitting pattern and coupling constants (Figure 9) indicated 96 three QEEDQ and maaa cOUpled protons. the one at 7.78 ppm being adjacent to a bromine. Thus. two of these protons are on one ring. while the other is on the other ring. The SEEDS split proton at 7.82 ppm (J-8.2 Hz) and the aaaa split proton at 7.71 ppm (J-2.l Hz) are also adjacent to a bromine. The only possible position for the SEEDS split proton would be in the EQEE position. since it can't be an gaaaa proton and a gaaa proton would be unlikely since another aaahg coupled pattern should be observed. The maaa split proton at 7.71 ppm has to be an QEEEQ proton since if it were in any other position it would be between two bromines and thus eXpected to be more downfield. The 95559 and maaa split proton at 7.34 ppm would be on the same ring as the QEEEQ split and maaa split protons in the aaaag position. giving rise to a 3.4-dibromo pattern on this ring. The other two 95552 and maaa split protons at 7.78 ppm and 7.34 ppm on the other ring therefore must be in the gaaaa and paaa positions with the ortho. ortho split proton at 7.39 ppm (J-7.5 Hz) inbetween at the maaa position giving rise to a 2.3-dibromo pattern on the second ring. Therefore the structure of this congener was deter— mined to be 2.3.3'.4‘-tetrabromobiphenyl. gaaa splitting (J<0.S Hz) can also be seen in Figure 9. The second major eluting peak in Figure l is also a tetra- bromobiphenyl as indicated by GC-Ms analysis. The NMR splitting pattern and cOUpling constants (Figure 10) indicated two 252E2- coUpled protons one of which was also aaaa-coupled. Therefore. one ring must have three protons which are split QEEDQ' maaa and ortho and meta and since no other signals exist the molecule has 97 FIGURE 9 - ifl-NMR SPECTRUM AND STRUCTURE OF 2.3.3'.4'-TBTRA- BROMOBIPHBNYL. The gas chromatographic profile of purified 2.3.3'.4'- T38 is shown in the insert. RESPONSE f. Br :1 r 138.... 9 Br 98 Figure 9 7.780 7.8I7 7.7I2 é é 3 f2 RETENTION TIME (min) I 7.342 7.386 7.297 99 FIGURE 10- lM—MMR SPECTRUM AND STRUCTURE OF 3.4.3'.4'-TETRA— BROMOBIPMENYL. The gas chromatographic profile of purified 3.4.3'.4'- T88 is shown in the insert. 100 Figure 10 N N no .b RESPONSE 7.320 ’:§EE§===: :;§§==: _-_—-——- —— ’ SP Br \ u 3 e é Ié RETENTION TIME (min) CHCI3 7.9 7.8 7.7 7.6 7.5 7.4 7.3 PF”n 7.2 7. l 101 symmetrical rings. Three possible substitution patterns can be postulated for the ring where the two bromines are at carbons 2 and 4. 2 and S. or 3 and 4. The 25222 and maaa-split proton at 7.32 ppm suggests the proton is adjacent to a bromine which would rule out the first possibility. but this would mean the 2222- split proton at 7.78 ppm (J-2.2 Hz) be an 25322 proton. This can not be since it is far too downfield. thus the second possibility is deleted. The first possibility is still ruled out since the gaaaa-split proton at 7.68 ppm (J-8.4 Hz) arises from a proton adjacent to a bromine. The QEEEQ and maaa split proton at 7.32 ppm appears to be slightly more downfield than eXpected for a proton not adjacent to any bromines but it can be seen that a 3.4-dibromo substitution is the only possible structure that can be rationalized. Thus the structure of this congener is 3.4.3'.4'-tetrabrombiphenyl. The GC-MS analysis of the first major congener (>30%) eluting peak in Figure 2 indicated four bromines. A highly purified sample (>99%) of this congener. whose GC profile is shown in the inset of Figure ll. gave rise to three proton NMR signals. indicating a symmetrical compound. A doublet centered at 7.58 ppm (J-l.7 Hz) and a triplet centered at 7.68 ppm (J-l.7 Hz) arise from protons maaa to each other. The chemical shifts imply the presence of only 3 95559 protons and that the protons are all adjacent to a bromine. A tetrabromobiphenyl is not able to exist in this configuration. Since no other signals exist. it can be rationalized that the triplet centered at 7.68 ppm is a proton adjacent to two bromines. The only possible substitution pattern of the two rings is the symmetrical arrangement of bromines in 102 FIGURE 11- lH-NMR SPECTRUM AND STRUCTURE OF 3.5.3'.S'-TETRA- BROMOBIPHENYL. The gas chromatographic profile of purified 3.5.3'.S'- T83 is shown in the insert. 103 Figure ll l i i Br Br i 7.580 - 31, E g i Br Br a Q U) ll] K .¥ 23 4 is '8 7.534 RETENTION TIME '(min) CHCI3 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7. I PPm 104 the 3 and 5 positions. Therefore the structure of this congener is 3 . 5 . 3 ' . 5 ' -tetrabromobiphenyl . GC-MS analysis of the second major eluting peak (>25%) in Figure 2 indicated five bromines and thus contains five protons. Th. NMR spectrum (Figure 12) shows a singlet centered at 7.72 ppm. a doublet at 7.58 ppm (J-l.7 Hz). and a triplet centered at 7.70 ppm (J-l.7 Hz). The last two signals arise from protons._m_e_aa to each other and thus be on the same ring. The chemical shifts indicate that one proton is adjacent to two bromines. therefore the only substitution pattern for such a ring would be for the bromines to occupy the 3 and 5 positions. The second ring must contain the remaining two protons. Since only a singlet exists and no splitting is observed. the two protons must occupy equiva- lent positions. The chemical shift (7.70 ppm) implies that they are protons adjacent to only one bromine. The only possible substitution pattern for this ring would be for the remaining bromines to occupy the 3.4. and 5 positions. The structure of this congener is therefore 3.4.5.3'.S'-pentabromobiphenyl. The GC-Ms analysis of the last major eluting peak in Figure 2 indicated six bromines. of which forty-two possible isomers can exist. A highly purified sample (>98%) of this congener. whose GC Prefile is shown in the inset of Figure 13. gave rise to only 00° 319nal. a singlet having a chemical shift at 7.70 ppm. which 1‘ iridicative of protons adjacent to one bromine. This signal must be due to protons in the 9.5.59.2 positions. which are OqUivalent and thus give rise to one signal. The only possible strUCture for this congener is 3.4.5.3'.4'.S'-hexabromobiphenyl. Coupling constants and chemical shifts are summarized in Table I. 105 FIGURE 12 - lM-MMR SPECTRUM AND STRUCTUREkOF 3.4.5.3'.5'-PENTA- BROMOBIPMENYL. The gas chromatographic profile of purified 3.4.5.3'.S'-PBB is shown in the insert. 106 Figure 12 3' Br { Br Br 7.720 ... Q E 3 7.702 ‘r I I 7.582 ‘I 5'; \w\aJI RETENTION TIME (min) CHCI3 7.9 7.8 7.7 7.6 7.5 7.4 7.3 - 7.2 7. I PPm 107 FIGURE 13 - lfl-NMR SPECTRUM AND gTRUCTURE OF 3.4.5.3'.4'.5'-MEXA- QROMOBIPHENYL. The gas chromatographic profile of purified 3.4.5.3'.4'.S'-MBB is shown in the insert. Figure 13 7.7l9 108 3' Br I. I In”... I Br Br RESPONSE 2 4 6 B Ito RETENTION TIME (min) CHCIS . 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7,| PPm 109 any odh.b .0..N.0.N AscozaenosOunoxezI.m..v..n.m.v.n 5.” so.~. n and ~os.s .v R.” i.e.... n and ~om.n .o..~ R.H 1....No n ..s anu.s o.~ HacosasnosounoucomI.m..n.m.v.n R.“ iv.~c n 103 oao.n .... o.H .m.~s n is. nmm.n .o.o..~.~ ascotasnoeotnouuoeI.m..n.m.n Ammo o~n.h .o.o v.o 1o.m. n its one.» .m.m ~.~ .o.~o n .uo .on.n .~.~ AacocnwnOEOHneuueal.e..n.v.n its. na~.h .o ~.o i.e..m. n .6. use.» .m H.~ i.e..~o n in. ~Hn.h .~ ~.R Ao.ms n Ammo ~vn.n o m.~ Ao.vo a sub mmn.p m m.h Am.eo n .063 ooh.h v ascotasnOEOunouuoeI.v..n.n.~ “are uuceuecou mauaaoou uc0u0um AEQQV uuficm Aeuueezu COuOum aueceucou mum no euceuecou usuansou was euuuzm Aequecu cOHONE A fldfldb 110 Several Chemical and Chromatggaaphic Properties of PBS Congeners The GC retention times (tr) of several PBB congeners are shown in Table 2. It can be seen that the retention time (tr) for the elution of the P38 congeners from the gas chromatograph (GC) is proportional to the total number of bromines. The three tetrabrominated biphenyls have smaller retention times than either the penta- or the hexa-brominated biphenyl. The presence of an aaaaa bromine on 2.3.3'.4'-TBB as compared to 3.4-TBB decreased the tr and suggests that QEEEQ bromination also influences PBB elution. In comparing 3.4-TBB and 3.5-TBB. it is interesting to note that maaa bromination decreases tr. whereas Eaaa bromination increases it. Pure PBB congeners were also compared for their thin layer chromatographic relative mobility (R ) under reverse-phase con- f ditions (Table 3). The effect of the number of developments was investigated in order to observe whether resolution could be improved. It appeared that 2.3.3'.4‘-TBB could be slightly separated from 3.4-TBB. however none of the congeners in the commercial mixture were successfully resolved from each other. The chromatographic mobility of PBS congeners under these conditions seem to be somewhat influenced by the total number of bromines and the presence of QEEEQ bromines (Table 3). The presence of two maaa bromines on one ring appears to retard the congener (i.e. 3.5-TBB). when compared to the presence of a paaa and maaa bromine on one ring (i.e. 3.4-TBB). which appears to increase mobility. The inability to resolve any congener of the commercial mixture may be related to limited solubility in the mobile phase. since these con—geners in the pure form. appeared 111 TABLE 2 Relative GC Retention Times (tR) of Pure PBB congeners Relative t PBB Congeners (minutes)R 2.3.3'.4'-TBB 1.62 3.5.3'.S'-TBB 1.62 3.4.3'.4°-TBB 2.14 3.4.5.3'.5'-PBB 3.70 3.4.5.3'.4'.S'-HBB 8.80 112 TABLE 3 R Values of PBS Congeners on Reversed Phase TLC F PBB Congener Times DevelOped 1X 2X 3X 2.3.3'.4'-TBB 0.494 0.687 0.835 3.4.3'.4'-TBB 0.461 0.626 0.824 3.5.3'.S'-TBB 0.309 0.368 0.676 3.4.5.3'.5'-PBB 0.309 0.368 0.682 3.4.5.3'.4'.5'—HBB 0.309 0.405 0.694 113 to be smeared on the TLC plate. The melting points of pure PBB congeners (>99%) are shown in Table 4. The presence of an aaaaa bromine appears to decrease the melting point of tetrabromobiphenyls. Also as the total bromine content is increased. the melting points increase as well. The UV-absorption spectrum of PBS congeners have two characteristic absorption maxima. The main band commonly occurs in the 'r ----- > f * electron transitions. The K-band occurs in the region of 250 to 270 nm and includes contributions of both phenyl rings and is believed to arise from the conjugation of the biphenyl system. It is generally accepted that the intensity of the K-band is proportional to the extent of interaction between the two phenyl rings and thus the coplanarity of two rings. Thus the degree of 95559 bromination would appear to play a major role in determining the UV-absorption spectral characteristics of PBS congeners. In 25322 unsubstituted congeners. such as 3.5-TBB. 3.4—TBB. 3.4.5.3'.5'-PBB. and 3.4.5-HBB. the UV—spectra show a strong K-band at 255. 266. 261 and 271 nm. respectively (Figure 14. Table 5). The K—band appears to undergo a bathochromic shift when bromines in the maaa positions are changed to bromines in the paaa and aaaa positions (3.5-TBB vs 3.4-TBB). Also. as additional bromines are added at gaaa and E2£2 positions to the bridge carbons. a bathochromic shift is also seen (Figure 14. Table 5). Substitution in the 23222 position. as with 2.3.3'.4'- TBB. diminishes the intensity of the K-band (Figure 14). The main UV—absorption band appeared at 215 to 227 nm. This band also seemed to undergo a slight bathochromic shift as 114 TABLE 4 Melting Points for Various PBS Congeners 2.3.3‘.4'—rss 103-104°c 3.4.3'.4'-rss 178-179°C 3.5.3'.S'—TBB 191-192°c 3.4.5.3'.s'-psa 195-195°c 3.4.5.3'.4'.5'-HBB 248-250°C 115 Figure 14 —UV-ASSORPTION SPECTRA OF 2.3.3‘.4'-TSB. 3.4.3'.4'- TBB. 3.5.3'.S'-TBB. 3.4.5.3'.5'-PBB AND 3.4.5.3'.4‘.5'-MBB. Spectra of the PBS congeners were recorded in hexane using concentration of 5 ppm. 116 A Absorbonce Figure 14 3H: B'H: I90 240 290 340 390 wavelength (nm) 117 TABLE 5 UV-Spectral Data of PBS Congeners PBB MAIN BAND K-BAND CONGENER Lo s Lo s A 9 A 9 max max -1 -1 -1 —1 (nm) (l.mole .cm ) (nm) (l.mole .cm ) 2.3.3'.4'-TBB 215 4.65 250 3.4.3°.4'-TBB 219 4.75 266 4.48 3.5.3'.S'-TBB 220 4.78 255 4.19 3.4.5.3'.5'-PBB 224 4.75 261 4.27 3.4.5.3'.4'.5'-HBB 227 4.74 271 4.33 118 additional bromines were added at aaaa and papa positions to the biphenyl bridge. The main UV-absorption band of 2.3.3'.4'-TBB ( x'max- 215 nm) underwent a slight bathochromic shift when the QEEDQ bromine is absent along with and additional papa bromine as seen with 3.4-TBB (Table 5). Also. the main UV-absorption band of 3.5-TBB ()Tmax- 220 nm) underwent a bathochromic shift when additional para bromines are added to yield 3.4.5.3'.5'-FBB ( k max' 224 nm) and 3.4.5-HBB ()Lm‘x- 227nm). The extinction coefficients of the main UV-absorption band appeared to be similar for all the PBS congeners whereas the K-band extinction coefficients appeared more variable. being the greatest for 3.4- TBB (Table 5). High Resolution Gas Chromatographic Analysis of Firemaster gag The analysis of Firemaster BF-6 by high resolution capillary GC using an ECD is shown in Figure 15. The peaks are numbered according to Moore and Aust (1978) and Dannan (1981). 3.4-TBB and 3.4.5-HBB are shown on the chromatogram. The relative abundance of these two PBB congeners in Firemaster BP-6 according to electronic integration (direct measurement) are 0.05% (3.4- TBB) and 0.03% 3.4.5-HBB. 119 FIGURE 15 - HIGH RESOLUTION CAFILLARY CMROMATOGRAM_OF__FIREMABTER 22:2- The mixture (1.0 ng) was injected into an ECD-CC equipped with a 25 m WCOT OV-l capillary column as described in Materials and Methods. Injector port. column. and detector temperatures were 290°. 240°. o and 330 C. respectively; attenuation - 10 X 16 (Amp/mv). 120 ' Figure 15 SSH-9‘ l2 ‘2— I... II 881-12“; - ——————_—~._.—_ IL ESNOdSBH TIME 121 DISCUSSION 2.3.3'.4'-TBB and 3.4-TBB were synthesized by the usuage of the Gomberg-Hey reaction and resulted in the mixture shown in Figure 1. Amount of each congener formed was 635 2.3.3'.4'-TBS and 27% 3.4-TBB. giving a 2‘2 ratio of products formed of 2.3. (212 ratio is the ratio of 2.3.3'.4'-TSB. formed by 9 attack on the p-dibromobenzene by the dibromobenzene radical. to 3.4-TBB. which was formed by p attack). These results agree with those of Meingarten (1961). who reported. using phenyl and a series of chlorophenyl radicals and p-dichlorobenzene and employing the Gomberg-Hey reaction. that the 21p ratio of the products formed was roughly 2. Using recrystallization and alumina columnn chromatographic methods (Figure 5). these two congeners were successfully purified to >99% pure. as assessed by GC. Using MS- GC and 1M-NMR spectroscOpy. the structures of these two congeners were confirmed. The commercial mixture . shown in Figure 2. was found to contain three major congeners. Through repeated alumina column chromatography these compounds were purified to >99% pure and using GC-MS and 1H—NMR spectroscopic techniques. the structures of these congeners were found to be 3.5-TBB. 3.4.5.3'.S'-FSB and 3.4.5-388. The patterns of 1H-MMR chemical shifts and coupling constants are very similar to those seen with other brominated or chlorinated biphenyls (Dannan. 1981: Moore and Aust. 1978: Moore. 1978; Welti and Sissons. 1972). Deshielding effects were felt by protons that were adjacent to one or two bromines. Also. the 122 chemical shifts of SEEDS protons appeared to decrease in the presence of bromines QESDQ to the bridge carbon. The chemical shifts of all protons ranged between 7 and 6 ppm. Signals with chemical shifts near 7 ppm were due to protons that were not adjacent to any bromines. whereas chemical shifts near 7.5 ppm and 8 ppm were due to protons adjacent to l and 2 bromines. respectively. The reduction in the GC retention time with paaaa bromination is presumably due to a corresponding decrease in the interaction of the congener with the stationary phase. In a study with alkyl substituted biphenyls. the retention times were also decreased by increasing the size and number of the 25392 alkyl groups and these effects were attributed to steric hinderance. or the decreasing planarity of the biphenyl with a concomitant reduction in the conjugation of the biphenyl system. which results in less interaction with the non—polar stationary phase (Beaven aa al.. 1957). The increased OC retention times of the non-aaaap brominated biphenyls with increasing bromination in the papa and BEE! positions suggest greater interaction with the non-polar stationary phase (methyl silicone. OV-l). The overall conjugation of the biphenyl system may have increased. however in a study by Hutzinger and coworkers (1974). interaction with the stationary phase increased as the total number of chlorines increased . Similar results were also reported by Dannan (1981). Thus. whether or not increased conjugation is responsible for the increased retention times is not clear. Also. the gas chromatographic separation of polyhalogenated biphenyl congeners 123 may be dependent on their volatility (boiling point). which may be inversely proportional to the degree of halogenation. The melting points of PBS congeners appear to be directly prOportional to the degree of bromination. The positions of bromines also appears to influence the melting point since the lack of 25222 bromines tended to increase it. Attempts to resolve PCB mixtures by reverse-phase TLC. where the mobile phase is relatively more polar than the stationary phase (Kieselguhr saturated with paraffin oil) have been made (DeVos and Feet. 1971: Stalling and Huckins. 1973). Only one study attempted to investigate the relationships between PBB structures and their chromatographic mobility (Dannan. 1981). Using similar TLC conditions. the results in this chapter suggest that 25522 bromines affect mobility as well as the total number of bromines. However. the non-paaaa brominated biphenyl congeners appeared to interact with the non-polar stationary phase and appeared independent of the total number of bromines. except 3.4-TBB. which was in contrast to the GC results. This may have been due to the limited solubility of these congeners in the mobile polar phase. Alumina chromatography and recrystallization have been two techniques previously used for purifying sufficient quantities of several PBB congeners from mixtures (Bairstow as al.. 1978: Moore _a a;.. 1978b; 1979; Dannan a5 al.. 1982a). These techniques proved useful in this research for purifying PBB congeners. With non-aaaap brominated biphenyls. the congeners eluted in order of increasing bromine content (molecular weight). 95322 bromination appeared to decrease the retention volume (VR) as in the case of 124 2.3.3'.4'-TBB and 3.4-TBB. It has been reported that Lipidex- 5000. a lipophilic Sephadex derivative. was useful in purifying certain PBB congeners from enriched fractions (Dannan aa al.. 1962a). The solvent system employed. however. was not suitable for the non-aaaaa brominated congeners of the commercial mixture. which were minimally soluble. The synthetic mixture. which is initially dark red and coarse. was also minimally soluble in the solvent system. However. further investigations in this laboratory proved Lipidex-SOOO chromatography to be useful in purifying enriched fractions of 2.3.3'.4'-TBB and 3.4-TBB which were fractionated by alumina chromatography first (unpublished results). The introduction of various types of halogens into the positions on the biphenyl system resulting in steric effects have been studied by several researchers (Pickett aa al.. 1936: Beaven and Hall. 1956; Beaven. 1958). These studies have employed UV- absorption spectroscopy techniques. The intensity of the K-band (250 to 270 nm). which includes contributions of both phenyl rings. is generally accepted to be proportional to the coplanarity and conjugation of the two rings. The presence of aaaaa halogens appear to diminish the intensity of the K-band which is believed to be due to the restriction of free rotation about the bridge carbons due to the SEEDS substituent which results in loss of coplanarity between the two rings. Qaaaa substitution also caused a hypsochromic shift of the maxima which is believed to occur due to increasing the energy of the excited state relative to that of the ground state (Beaven. 1958). The 125 effects of aaaa and papa substituents on the K-band have also been studied and showed to cause a bathochromic shift of the maxima (Pickett aa 2l°' 1936; Beaven. 1958). The UV-spectra of the PBB congeners studied in this chapter agree with these results. 2.3.3'.4'—TBB. which contains one SEEDS bromine. showed a small K-band in the region of 250 nm. With no ortho bromines. a distinct K-band was observed for all the other PBB congeners. Meta and para bromines caused a bathochromic shift of the maxima of the K-band as well. Thus. the aaaap-unsubstituted congeners are coplanar and conjugation of the biphenyl system is retained. The ability of PBB and PCB congeners to be toxic has been related to the extent of 25222 bromination. Since PBB congeners which are unsubstituted in the 25522 position and have bromines in the lateral positions (3 and 4 positions) are hypothesized to be the most toxic members. it was of interest to determine whether or not these congeners were present in Firemaster at appreciable amounts. The fractionation of Firemaster by GC using packed columns allows minimal separation of the congeners present in small amounts. The advantages of capillary chromatography over packed column gas chromatography are discussed elsewhere (Farrell. 1980). The results. though not conclusive. suggest the presence of 3.4-TBB (<0.05%) and 3.4.5-MBB ((0.03%) in Firemaster BP-6. The detectionof 3.4-TBB is dubious. since a standard of 2.4.5.2'.4'-PBB was not obtainable and this congener is known to coelute with 3.4-TBB (see chapter two). GC-MS employing a capillary column to determine the molecular weight did not prove successful since good resolution could not be obtained. The 126 detection of 3.4.5-HBB by GC-MS capillary column chromatography was also insufficient. The amounts of these two congeners in Firemaster are extremely low. The formation of these two congeners could occur through the photolysis of Firemaster. Since 3.4-TBS is considered to be the more toxic . it was of interest to determine if indeed 3.4-TBB formation occurred through photolysis of 2.4.5.2'.4'.5'—HBB. which is the major congener of Firemaster and if it could contribute to any enhanced toxicity observed. This subject is discussed in the later chapters of this thesis. CHAPTER 2 CHARACTERIZATION OF THE PHOTOLYSIS OF 2.4.5.2'.4'o$'- NEXABROMINATED BIPNENYL 127 128 ABSTRACT 2.4.5.2’.4'.S'-Hexabrominated biphenyl (HBB). was irradiated with UV—light in hexane with stirring. and photolysis monitored by gas chromatography (GC). HBB decomposed at an average rate of 0.66 a 0.02‘pmoles/minute and the reaction appeared zero-order from 0.159 to 1.59 mM HBB. Several PBB congeners were identified as photoproducts of HBB. 2.4.5.2'.5'-Pentabrominated biphenyl (-PBB). formed by papa debromination. accumulated at a higher rate than did 2.4.5.3'.4'-PBB. formed by appaa debromination. 2.4.5.2'.4'-PBB was formed by papa debromination. 3.4.3'.4'- Tetrabrominated biphenyl (-TBB) was found as a secondary photOproduct formed by appap debromination of 2.4.5.3'.4'—PBB. 2.5.2'.5'-TBB and 2.4.2'.S'-TBB were formed by debromination of 2.4.5.2'.S'-PBB papa and papa. respectively. 2.5.3'.4'-TBB could be formed by either appap debromination of 2.4.5.2'.S'-PBB or papa debromination of 2.4.5.3'.4‘-PBB. Rates of degradation and accumulation of the penta- and tetra-brominated biphenyls were also studied. The UV-spectra of the HBB photolysis mixture as well as the purified components were studied and are also reported. INTRODUCTION Polybrominated biphenyls (PBS) and polychlorinated biphenyls (PCB) have been shown to be susceptible to photodegradation. The photoproducts observed were consistent with a reductive dehalogenation pathway where preferential cleavage of the appag halogen occurred (Ruzo ap al.. 1976: Ruzo t 1.. 1975; Ruzo and 129 Zabik. 1975; Bunce ap _l°' 1975: Ruzo _p _a.. 1974a; Safe and Hutzinger. 1971). PBS were found to be more photoreactive than PCB. which was attributed to the lower bond energy of the C-Br bond (Ruzo and Zabik. 1975). The photodegradation of these compounds are also believed to proceed from triplet excited states since rates of photolysis were found to be slightly retarded in air-saturated solutions as compared to degassed solutions (Ruzo ap al.. 1976; Magner. 1967). Although it is assumed that photodecomposition of PBS and PCB proceeds with preferential loss of the appap substituent. a complete analysis of the photoproducts has not been reported. Robertson and coworkers (1983a) have reported. using Firemaster BP-6. that the reductive debromination of 2.4.5.2'.4'.5'- hexabromobiphenyl (HBB). the major component of Firemaster. did not follow a stepwise preferential loss of appap bromines. The present study was intended to investigate the photodegradation of pure HBB by monitoring photOproducts formed and their photodegradation to help elucidate the mechanistic pathway involved in photolysis. The toxicity of polyhalogenated biphenyls appears to be dependent upon the number and position(s) of the halogens (Poland and Knutson. 1982) and therefore a non-toxic PBB congener. such as HBB. can in theory yield toxic congeners through photolytic debromination. Thus. it was necessary to identify the photoproducts of BBB before evaluating the toxicity of the photo- lyzed HBB mixture (this subject is examined in the next chapter of this thesis). The rates of photodegradation. the products 130 formed during photolysis. and the UV-spectra of a number of PBB congeners containing 4-6 bromines are also reported. MATERIALS AND METHODS Hexane (n-hexane. b.p. 68-69o) and all other solvents were of glass-distilled grade from Burdick and Jackson Laboratories. Inc.. Muskegon. Michigan. The source of 3% OV-l on Gas Chrom 0. 100/120 mesh. was Applied Science Laboratories. Inc.. State College. Pennsylvania. Lipidex-SOOO was obtained from Packard Instruments Company. Inc.. Downers Grove. Illinois. Neutral alumina (activity grade I) was purchased from Sigma Chemical Company. St. Louis. Missouri. Two tetrabromo biphenyls (-TBB). 2.4.2'.S'- and 2.5.2'.5'—TBB. were purchased from Ultra Scientific. Inc.. Hope. Rhode Island and 2.5.3'.4'-TBB was a gift from Dr. Michael Mullin. US—EPA. Bethesda. Maryland. A WCOT OV-l capillary column (25 m) was purchased from Hewlett Packard. Avondale. Pennsylvania. HBB. 2.4.5.2'.5'—pentabromobipheny1 (-PBB). and 2.4.5.3'.4'-PBB were purified from Firemaster BP-6 (lot no. 6224-A) which was manufactured by Michigan Chemical Corp.. St. Louis. Michigan. The details of the purification methods. which involved repeated recrystallization and chromatography on both alumina and Lipidex-SOOO. can be found in Dannan ap al.. 1982a. 3.4.3'.4'-TBB was synthesized and purified by recrystallization and column chromatography on alumina as described in the first chapter of this thesis. 131 Qaa Chromatpgraphy L99) Photodegradation reactions were monitored using a Varian 3700 gas chromatogram (1.8 m x 2 mm ID glass column packed with 3% OV-l on Gas Chrom 0 100/120 mesh) equipped with a pulsed 63Ni electron capture detector (ECD) and a Hewlett Packard Model 3380A integrator recorder. Nitrogen was the carrier gas and the column flow rate was maintained at 30 ml/min. Standard curves for each purified PBB congener were constructed by plotting the GC-ECD response (log area counts) versus various amounts (log gram) of each PBB congeners. The absolute amount of each congener in various fractions was then calculated using these standard curves. The standard curves were obtained concurrently with sample analyses. Gas Chromatogpaphy—Mass Spectrometry (GC-MS) The molecular weights of PBS congeners present in the photolyzed HBB mixture were determined by GC-MS (LKB 2091 and a Hewlett Packard 5840A/5985) at 70 ev in the BI mode and a source temperature of 200°. The GC column was a 1.8 m x 2 mm glass column packed with 3% OV-l. UV-Absorption SpectroscOpy The ultraviolet spectra were obtained from a Perkin-Elmer 559A UV/VIS Spectrophotometer. The samples were dissolved in UV-grade hexane and scanned from 390 to 190 nm and the extinction coefficients (c) calculated for the major UV-absorption bands ("max)' 132 Photochemical Procedures Samples were dissolved in hexane at concentrations of 0.159 to 1.59 mM and irradiated with a UV source (germicidal UV lamp having a peak energy output at 254 nm) while either in a beaker stirring 12.5 cm from the source (surface area - 23.8 cmz) or in quartz cuvettes. 20 cm from the source (1 cm2 surface area). Photolysis was monitored over time by removing an aliquOt from the photolyzed mixture and determining the amount of each PBB congener from standard detector response curves. The rates of photolysis were calculated by using log A° - log A - Kt (where A - amount of parent compound at time t and A0 - intial amount of parent compound) and plotting log A against t. determining the slope to ascertain the value of the rate constant. K. The rates were also calculated from the zero-order expression: Ao - A - Kt and found to be the same. RESULTS agentippcatpaa pp Photgproducts pp app A hexane solution of H88 (1.59 mM) irradiated for 200 minutes while stirring. yielded a mixture containing at least eight major photoproducts (Figure 16). Six photoproducts were identified by comparing their gas chromatographic properties with authentic compounds of known structure. The results were then confirmed by analyzing the mixture by GC-Ms. A reconstructed total ion current chromatogram of the GC-MS analysis of the photolyzed HBB mixture (Figure 17a) revealed that scan no. 186 (Figure 17b) constituted mainly a pentabrominated biphenyl. The photodebromination of HBB can give rise to only three possible 133 Figure 16 - GC_§&UTION PROFILE AND STRUCTURE OF_PBB_OONGENERS IN IEE PHOTOLYZED HBB MIXTURE. A sample of the mixture (0.5 ng) was injected into an ECD-GC equipped with a 3% OV-l column. Injector port. column. and detector temperatures were 270. 240. and 310°C. respectively: attenuation - 10 x 32 (A/mv). 134 Figure 16 Br Br Br Br Br Q Q Br Br Br Br H// ”M Br Br EH Br Br MI\\d\./©.d B "Q BBHNB mmzoammm .... Q . I2 (min) TIME 135 Figure 17 - (A) GC—MS (total ion current) of the photolyzed HBB I mixture. (B) 70ev Mass spectrum of scan no. 186 in I the photolyzed HBB mixture. (C) 70 ev Mass spectrum of a pure pentabrominated biphenyl (2.4.5.2‘.5'-PBB)- _—_.____ 136 Figure 17 T11 Scon#l86 III III II I - f “y J LI ‘ rt.“ w. ‘ ‘ 5.~“.‘,- -. 5" - --\~ - w-v‘fi “ “ ”wake-v " MnA’J 'v-no‘. «3' ‘ *vw—mvxd-ae' I‘- "I” J rr‘l’jIIrITTrT‘I'TITT]IIIIIITTTITTTI‘IrITTT TTTTIITT11r1TT-II'I'I'I 25 SU 75 100 125 150 175 200 '25 250 2.c 300 Scan Number [_LJ 1 1 I NJ} I 1 II- I i 1 ul IL 1 HIIJ I b) vvlrvvvavvrv vav vvv‘lrvvr fiY—V ~vvv 'TT'VVI' Ifil 7T7 I_ -00 2 3 320 330 340 £90 360 370 330 290 300 310 330 3-30 34U 35C 3E0 3 W 350 - I I _4 I I I . II II ..4 M ‘ IIIII " "I 1.: .I TNII'LIIAIV 1.1. . . fl va W a - v 'VIIIIITIV H - III“ I I frI . I H V V Y 7' I WIY V W" H V 'VWVI'TVV V'V W I 330 390 40; 410 420 430 440 4E0 4e0 4. 0 420 490 500 $10 530 530 940 5E0 E. m/z l L J_1 I 'I L 1 iii I'ILIIJ . - in . “I“! J JIIIII - . . .1 . ’ 'ifi'I "'IVTY I I'VVFI""I 'T'I' I'Y' I' "I ' I ' 7T_ V I ' I"' bijjin 3153 33:1 -‘_-Zi_1 340 75.3 3:153 3ft} 3:30 1'9st 3:0st 31 I 3' ...... II I IIII 111:1 . ALI-I I.- LIIII '- 31: W77 vvvv fiv v VT v I v fi‘ 1 w v vv fi 4 I V VI '7 .0 J90 400 410 420 430 440 450 460 470 430 490 Siii 510 FI :0 F' -0 F40 FF0 Stu m/z 137 pentabrominated biphenyls, namely ZO‘ISI3.I4.-I 2:4.5.2'.4'- and 2.4.5.2',5'-PBB. by 95559. 5252 and 22£2 debromination. respectively. Since 2.4.5.3'.4'— and 2.4.5.2'.5'-PBB were identified by comparison with authentic standards: the only possible structure for the compound in scan no. 186 is 2.4.5.2'.4°-FBB. formed through 2252 debromination. However: 3.4.3'.4'-TBB. a secondary photoproduct. could not be separated from 2.4.5.2'.4'-PBB on a packed glass column. The results of the mass spectrum (Figure 17b) of scan no. 186. when compared to the mass spectra of a pure pentabrominated biphenyl (Figure 17c): seem to indicate some contamination with a tetrabrominated biphenyl. Although not conclusive. these results appear to indicate the presence of a tetrabrominated biphenyl. Since an authentic sample of 3,4.3'.4'—TBB appeared to co-elute with 2.4.5.2'.4'-PBB, and in theory can be formed by photolytic debromination of HBB. it appeared to be present in the mixture as well. Figure 16 shows the GC pattern and structure of identified products of the mixture after irradiation of H88 with UV—light. The GC elution profiles of the eight photoproducts in their purified form are shown in Figure 18. Photoreactivity 2; fig; The initial rates of photolysis of BBB did not change significantly from 0.65‘pmole/min when the concentration of HBB varied from 0.159 to 1.59 mm (Table 6). After 15 minutes of irradiation the quantity of products identified did not equal the .amount of HBB degraded (Figure 19). This could be due to rapid further debromination of photoproducts or the formation of 138 Figure 18 - OC_§LUT§ON PROFIQES AND STRUCTUR!8_OF_TH! FUR! Pg; CONGBNERS IN THE PHOTOLYZED HBB MIXTURE. Approximately 0.2 - 0.4 ng of each of the congeners was injected into an ECD-CC equipped with a 3% OV-l column. GC conditions were the same as Figure 16. Figure 18 RESPONSE C? E (E J \9 ©, 2? TIME 140 TABLE 6 * HBB Fhotolyeie Ratee Under Verioue Concentrations Concentration of HBB Rate -l -l . -l (mM) (mg.ml ) (pmole.min ) (mg.m1n ) 0.159 0.1 0.65 0.41 0.319 0.2 0.66 0.42 1.59 1.0 0.68 0.43 * Irradiated in an Open beaker stirring 12.5 cm from the light source. 141 Figure 19 -PHOTOCH§HICAL DEGRADATION or Hg; (0.3g_gg) ovg§__r;!§ Ag? FORMATION OF PRODUCTS. The photochemical reaction was performed in open beaker while stirring 12.5 cm from the source. Aliquots from the irradiated mixture were taken at each time point and the amount of each PBB congener was determined from standard detector response curves obtained concurrently with eample analysis. Injector port. column. and detector 0 temperatures were 270°. 240°. and 310 C. reSpectively. 142 Figure 19 mmh-.¢._m.v.m + mmn.-.v..m.m.¢.m mma..¢..m.m.¢.m mmafimdmm mmz-.m.w._~.n¢.m ITIOII 32:55 zo_._.<_o 0.97). 150 Ultraviolet Spectroscopy The UV spectrum of PBS congeners features two absorption maxima. one is the main band in the region of 201 to 221 nm and the second is referred to as the K band (250 to 270 nm). The UV- spectra of the PBS congeners are shown in Figure 22 and 23. 3.4.3'.4'-TBB showed a strong K-band withAunx at 267 nm (Figure 22). Increased substitution by bromine at the 2 and 2' positions caused this band to diminish. as seen with 2.4.5.3'.4'-PBB. and to completely disappear. as seen with HBB. While the UV-spectrum of HBB showed no K-band. the spectrum of the photolyzed HBB mixture indicated a small bathochromic shift. Figure 23 shows the spectra of other PBB congeners formed through the photolysis of HBB. The spectra of those congeners which have 2 ppgpp bromines showed no K-band while the spectrum of 2.5.3'.4'-TBB. which has only one 95559 bromine. indicated a bathochromic shift to the region of 253 to 270 nm. Also. when a bromine is removed from 2.4.5.2'.S'-PBB. from either the 2252 or £253 position to form 2.5.2'.5'-TBB and 2.4.2'.5'-TBB. respectively. the main band under went a hypsochromic shift from k max at 215 nm to a Xmax of 207 nm. respectively. 151 Figure 22 - UV-AggORPTION SPECTRA OF THE PHOTOLYZBD HBB MIXTURE: HBB: 214,513.14'-PBB AND 31413.14'-TBB- SPOCCI'O were recorded in hexane at 5 ppm concentration. 152 Figure 22 Absorbonce 2’ é é) 2" 9 © 9 @ PHOTOLYZED HBB MIXTURE *fi l I I I I90 240 290 340 390 wavelength (nm) 153 Figure 23 - UV-ABSORPTION SPECTRA OF 2.4.5.2'.5'—PBB. 2.5.2‘.5'- TBB. 2.4.2'.S'-TBB. and 2.5.3'.4'-TBD. Concentrations of all samples were 5 ppm in hexane when spectra were recorded. 154 Figure 23 I... H’ a, 3,; He: Absorbonce I I l I I90 240 290 340 390 wavelength (nm) 155 DISCUSSION The results of this study indicate that HBB readily undergoes photolytic debromination to yield a number of lower brominated congeners. 2.4.5.2'.5'-PBB. formed by pppg debromination accumulated at a higher rate than either 2.4.5.3'.4'-PBB. formed by pgphp debromination. or 2.4.5.2'.S'- PBB. formed by 9352 debromination. 2.4.5.2'.5'-PBS and 2.4.5.3'.4'-PBB seem to degrade photolytically at almost the same rates (0.77 pmoles/min and 0.76 pmoles/min. respectively) and therefore it appears that p253 debromination of BBB occurs preferentially. This is in agreement with Robertson and coworkers (1983) who reported that in the reductive debromination of HBB. 95559 debromination did not occur preferentially. The photolysis rates appear to follow zero-order kinetics over the concentration range used. This observation agrees with the results of Ruzo and coworkers (1976). As seen with HBB. it appeared that p253 debromination of 2.4.5.2'.5'-PBB. which forms 2.5.2'.5'—TBB. is preferred over ppppp and mgpg debromination. forming 2.5.3‘.4'-TBB or 2.4.2'.5'- TBB. respectively. Initial rates of photolysis of 2.5.3'.4'-TBB and 2.4.2'.5'-TBB were slower than that for 2.5.2'.5'-TBB. Therefore the accumulation of 2.5.2'.5'-TBB during the photolysis of 2.4.5.2'.5'—PSB is due to preferential p253 debromination. In the case of the irradiation of 2.4.5.3'.4'-PSB however. p255 and ppphp debromination. forming 2.5.3'.4'-TBB and 3.4.3'.4'—TBB respectively. appeared to occur preferentially and equally since 2.5.3‘.4'-TBB and 3.4.3'.4'-TBB accumulated in equivalent 156 amounts. The main UV absorption band (201—221 nm) of PBS corresponds to the w’-———€> 1""transitions while the K-band (250 to 270 nm) is attributed to the conjugated biphenyl system with contributions of both rings. Since the K-band includes contributions of both phenyl rings. it is generally accepted that the intensity of the K-band is proportional to the coplanarity of the two rings. The effects of substituents on the K—band have been studied and it was reported that the pppg and 2252 substituents cause a bathochromic shift and ggppg substituents cause a hypsochromic shift with diminished value (Pickett pp 31.. 1936; Beaven. 1958). It is accepted that in the highly hindered ppppp-substituted biphenyls. there is restriction to free rotation about the bridge carbons which results in the loss of coplanarity between the two phenyl rings. While the UV- spectrum of HBB. which contains two ppppp bromines. revealed no K—band. the loss of an ppppp bromine to form 2.4.5.3‘.4'-PBB indicated a bathochromic shift to the 250-260 nm region. In losing the pgppg bromine from 2.4.5.3'.4'-PBB to form 3.4.3'.4'- TBB. the UV-spectra of 3.4.3'.4'-TBB shows a strong K-band with max at 267 nm. indicating a c0p1anar congener. Since the irradiation of HBB produces a mixture of lower brominated congeners. some of which are cOplanar. the small bathochromic shift seen in the UV-spectrum of the photolyzed HBB mixture may be due to the presence of coplanar congeners. However. the bathochromic shift is very slight and therefore may be a reflection of the small amounts of coplanar congeners actually formed. 157 The spectra of the congeners formed by photolysis of HBB which have 2 ppphp bromines showed no K-band. as seen by 2.4.5.2'.5'-PBB for example. while the spectrum of 2.5.3‘.4'—TBB which has only one ppphp bromine. indicated a bathochromic shift to the region of 253 to 270 nm. Also. when a bromine is removed from 2.4.5.2'.5'-PBB. from either the p25: or mpg: position to form 2.5.2'.5'-TBB and 2.4.2'.5'-TBB. respectively. the main band under went a hypsochromic shift from xmax at 215 nm to a Ikmax of 201 nm and 207 nm. respectively. The UV-spectra data of all these PBB congeners are summarized in Table 8. The results presented here indicate that the reductive debromination of HBB does not appear to precede through a stepwise preferential loss of ppppp bromines. Although earlier work reported that PCB and PBS undergo preferential loss of pgggp bromines. these studies were performed with lower brominated congeners (i.e.. tetrabromobiphenyls and lower) (Ruzo pp al.. 1974a; Ruzo pp al.. 1974b; Ruzo pg 21.. 1975). It appears that ppppp debromination is also not preferential in the photolysis of 2.4.5.2°.5‘—PBB. The debromination of BBB leads to some coplanar congeners such as 2.4.5.3'.4'-PBB and 3.4.3'.4'—TSB. Patterson and coworkers (1981) have reported enhanced hyperkeratosis by a photolyzed Firemaster mixture and suggested that this may be due to the presence of these two congeners. Robertson and coworkers (1981) also reported increased biologic activity of a photolyzed Firemaster mixture. again suggesting that it was due to the presence of these two photoproducts. This study showed that these 158 TABLE 8 UV Spectral Data for Individual PBB Congeners and Photolysed HID PBB MAIN DAND’ K BAND Log 4 Log e X x max _1 _1 max _1 _1 (nm) (l.mole .cm ) (nm) (l.mole .cm ) 2.2'.4.S'-TBB 207 4.78 2.2'.5.S'-TBB 201 4.82 2.5.3'.4'-TBB 219 4.65 253-27o 3.3'.4.4'-TBB 219.5 4.67 267 4.40 2.3'.4.4'.5—PBB 221 4.68 250—260 2.2'.4.5.5'-PBB 215 4.71 HBB 217 4.75 Photolyzed HBB 215.5 159 two congeners accumulate during the irradiation of HBB but at low rates. In the next chapter the toxic effects of a photolyzed HBB mixture and several of its purified photoproducts are reported. CHAPTER 3 NICROSONAL ENZYME INDUCTION BY AND TOXICOLOGY OF THE PHOTOLYSIS PRODUCTS OP 2.4.5.2'04'oS'-HEXADRONINATED DIPHENYL 160 161 ABSTRACT The irradiation of 2.4.5.2'.4'.5'-hexabromobiphenyl (HBB) by UV-light created a mixture of lower brominated polybrominated biphenyl (PBB) congeners. Three photoproducts. 2.4.5.3'.4'- pentabromobiphenyl (—PBB). 2.4.5.2'.5'-PDS and 3.4.3'.4'-tetra- bromobiphenyl (3.4—TDB) as well as H83 and the photolyzed HBB mixture. were administered to rats in a single ip injection (90 mg/kg. except 3.4-TBB. which was given at 2 mg/kg) two weeks before sacrifice. All treatments except 3.4-TBB induced NADPH- cytochrome P-450 reductase and aminopyrine-N-demethylase activities while all treatments except HBB induced ethoxy— resorufin-O-deethylase and UDP-glucuronosyl transferase activities. Thymus to body weight and spleen to body weight ratios were unchanged compared to control for all treatments whereas an increase in the liver weights was observed for all treatment groups. Histologic examination revealed that the photolyzed HBB mixture caused moderate to severe hepatocyte enlargement. Results of tissue analysis for the pure PBB congeners indicated that 2.4.5.2'.5'-PBB and 3.4-TBS were metabolized i vivo. The results revealed that the photolyzed HBB mixture caused a mixed-type induction of hepatic drug metabolizing enzymes. This is most likely due to the effect of H88 and toxic congeners formed during the irradiation of H88. 2.4.5.3'.4'-PBB. which is toxic and apparently not metabolized. is believed to be the major congener contributing to the increased toxicity of the photolyzed HBB mixture since 3.4-TBB. was metabolized and appeared not to be as potent an inducer of 162 aryl hydrocarbon hydroxylase (AHH) activity. INTRODUCTION The accidental contamination of Michigan (DiCarlo pp 21.. 1978) and its people (Wolff pp 21.. 1982) by a mixture of polybrominated biphenyls (PBB). commercially known as Firemaster FF—l. has stimulated an enormous amount of research on their toxicological and biological effects. Firemaster contains at least twelve to fourteen major PBB congeners of which ten have been identified structurally (Moore 25 21.. 1978a: Moore and Aust. 1978; Moore pp 31.. 1980: Dannan pg 31.. 1982a). Dent and coworkers (1976a.b) found that the Firemaster mixture caused a mixed-type induction of liver microsomal drug metabolizing enzymes similar to that seen after the coadministration of phenobarbital (PB) and 3-methylcholanthrene (MC). The effects of Firemaster mixture at high dosages (Parkinson and Safe. 1981) resemble that of 2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD) and other polyhalogenated aromatic hydrocarbons (PHAH) and include a variety of toxic responses. Among these toxic responses are hepatotoxicity. thymic involution. porphyria. chicken edema. and loss of body weight (Sleight and Sanger. 1976; Gupta and Moore. 1979: Polin pp 21.. 1979; Fraker. 1980: Gupta pp 21.. 1981). Firemaster also suppresses the immune response in mice and rats (Fraker. 1980: Fraker and Aust. 1978; Dannan pp 21.. 1982b). Although the mechanism of toxicity of these compounds remains unknown. there is good evidence which suggests that the responses seen are mediated by a binding protein. referred to as the TCDD 163 receptor (Poland pg 21.. 1976a; Poland and Glover. 1980). The receptor is believed to bind a coplanar compound which causes a pleiotropic response (Greenlee and Poland. 1979). Nine PBB congeners totaling nearly 97% of Firemaster have been purified and examined for some of their toxicological effects in rats (Moore pp al.. 1978b. 1979;.Dannan pp 21.. 1978b: Besaw pp pl.. 1978: Dannan pg 21.. l982b. l982c). Four congeners in the mixture were found to be toxic and potent inducers of aryl hydrocarbon hydroxylase (AHH). an enzyme activity typically induced by MC. and classified as mixed-type inducers. The remaining five congeners which comprise nearly 82% of Firemaster were found to be strict PB-type inducers. A reconstituted mixture. prepared with nine purified and characterized congeners. was found to elicit relatively the same biological response as the original mixture which contaminated Michigan. indicating that these congeners seemed to account for the biological effects of the mixture. or that the mixture did not contain some minor. very toxic component (Dannan pp 21.. l982d). However. PBB have been shown to be quite susceptible to photochemical debromination (Ruzo pp 21.. 1976: Ruzo and 2abik. 1975). Thus it follows that since HBB is the major component of Firemaster. in theory it could undergo photolysis to produce a significant amount of photoproducts. Since the toxicity of PBS congeners is dependent upon the number and position(s) of bromines. a non-toxic congener. such as HBB. can theoretically be converted to toxic congeners. In chapter two of this thesis seven major photoproducts of HBB were identified and reported. Two congeners. namely 2.4.5.3'.4'-pentabromobiphenyl (-PBB) and 164 3.4.3'.4'-tetrabromobiphenyl (3.4-TBB). are considered potent inducers of AHH (Robertson pp 31.. 1980) and have been found to be minor components of Firemaster (Moore 35 1.. 1978a; Robertson _5 _1.. 1983a: Orti pp 21.. 1983; Chapter one of this thesis). In this present study. the biologic and toxic activities of Firemaster FF-l. HBB. a photolyzed HBB mixture and three of its photOproducts. namely 2.4.5.3'.4'-PBB. 2.4.5.2'.5'-PBB and 3.4- TBB were compared. Firemaster was used in this study as an index for mixed-type induction while 3.4.5.3'.4'.5'-hexabromobiphenyl (3.4.5-HBB) was used as a ”reference" congener since it is reported to be a strict MC-type inducer (Dent pp 31.. 1976a.b; Poland and Glover. 1977: Render pp 21.. 1982). The assessment of the metabolism of the photolysis HBB mixture and photoproducts was also performed in an attempt to correlate 1p vitpp metabolism with 12 vivo tissue levels of the various PBB congeners. MATERIALS AND NETHODS Chemicals HBB. 2.4.5.2'.S‘-PBB and 2.4.5.3'.4'-PBB were purified from Firemaster BP-G as described in Dannan pp 21.. (l982a). 3.4-TBB was synthesized and purified by recrystallization and column chromatography on alumina as described in Chapter one of this thesis. 3.4.5—HBB was purified in a similar manner also described in Chapter one. The purity of all PBB congeners was >99% as determined by GC chromatography using a Varian 3700 gas 6 chromatograph equipped with a 3N1 ECD. Resorufin was purchased from Eastman Organic Chemicals. 165 Rochester. NY. 7-Ethoxyresorufin was purchased from Pierce Chemical Co.. Rockford. IL. Crude 3.4.5-HBB (65%) was obtained from Ultra Scientific. Hope. RI. Cytochrome c (horse heart. type III). NADP. NADPH. isocitrate. isocitrate dehydrogenase. MC. sodium PB and UDP-glucuronic acid were purchased from Sigma Chemical Co.. St. Louis. MI. Aminopyrine was purchased from Aldrich Chemical Co.. Milwaukee. WI. Firemaster BP-6 (Lot No. 6224-A) and Firemaster FF-l (Lot No. FH7042) were gifts from Michigan Chemical Corp.. St. Louis. MI. and Farm Bureau. Lansing. MI.. respectively. Animals and Treatments Outbred male Sprague-Dawley rats (75-1009) were purchased from Harlan Sprague-Dawley. Inc.. Haslett. MI. The animals were allowed an acclimation period of at least 48 hours before any treatments. PBB congeners and MC were dissolved in polyethylene glycol (PEG) (5-20 mg/ml). PB was dissolved in water. Six rats per each group received an ip injection of 90 mg/kg body weight of Firemaster FF—l. HBB. photolyzed HBB mixture. 2.4.5.3'.4'-PBB or 2.4.5.2'.5'-PBB. Rats in the sixth and seventh groups received an ip injection of 2 mg/kg body weight of either 3.4-TBB or 3.4.S-HBB. Rats in an eighth group (controls) were administered 4 ml PEG/kg. Rats in the last three groups received an ip injection of PS (250 mg/kg administered over 3 consecutive days before sacrifice). MC (40 mg/kg administered over 2 consecutive days before sacrifice). or PB and MC coadministered as above. All rats were given free access to feed and water 166 except for the night before sacrifice when feed was removed. Isolation pg Microsomes Rats were killed by decapitation and the livers perfused with cold 1.15% KCl containing 0.2% nicotinamide ;_ situ. Liver microsomes were isolated from each rat according to previously described procedures (Pederson and Aust. 1970). Microsomal pellets were then resuspended in 0.3 M sucrose buffer containing 0.1! tetrasodium pyrophosphate. pH 7.5 and repelleted by ultracentrifugation at 105.000 x g for 90 min. The resulting pellet was resuspended in a 50 m! Tris-HCl buffer (pH 7.5 at 25°C) containing 50% glycerol and 0.01% (w/v) butylated hydroxytoluene and stored at -20°C under argon. Tissue gellection and Histopathology The rats were first weighed and then killed by decapitation. Liver. kidney. thymus. brain and spleen weights were recorded for each rat. Tissue samples from the liver. spleen. kidney. thyroid. thymus. trachea. lung. adrenal gland. pancreas. bladder. brain. stomach and heart were fixed in 10% buffered formalin for histOpathological examination. The formalin-fixed tissues were automatically processed (Histomatic. Model 166. Fisher Scientific Company. Pittsburg. PA). embedded in paraffin. sectioned at 6 um and stained with hematoxylin-eosin. Enzype Assays and Tissue Levels of PBB The concentration of microsomal protein was determined by the method of Lowry pp pl. (1951). Cytochrome P-450 was assayed 167 by its reduced carbon monoxide saturated difference spectra in 10% glycerol using an extinction coefficient of 91 pM-1 cm-1 between Amax and A490 according to the method of Omura and Sato (1964a). NADPH-cytochrome P-450 reductase activity was assayed by the rate of cytochrome c reduction at 550 nm using an extinction coefficient of 21 95-1 cm-1 in accordance to the method of Pederson pp $1.. (1973). UDP-Glucuronosyl transferase activity was determined by the method of Lucier 25 21. (1977). Reaction mixtures contained 0.8 mg p-nitrOphenol. 1.4 m! UDP-glucuronic acid. 10 m! MgClz. 150 a! Tris-HCl (pH 7.4 at 37°). 0.2 ul of Triton X—lOO/mg microsomal protein (0.3—0.6 mg). in a final volume of 1.4 ml. Reaction mixtures were prepared by adding triton x-100 and microsomes to a small volume of Tris buffer. vortexed. and preincubated batchwise at 37° C for five minutes. A solution containing p-nitrophenol. Tris and MgCl was then added (0.9 m1). 2 Reactions were initiated by the addition of UDP-glucuronic acid. vortexed and placed back in water bath. After 15 minutes. five ml of 0.2 M sodium glycine pH 10.4 was added and absorbance at 403 nm was determined. Blanks contained microsomes and all other components except UDP-glucuronic acid. Rates were calculated using an extinction coefficient of 18.0 25'1 cm'l. Aminopyrine-N-demethylase was determined by measuring the production of formaldehyde as previously described (Moore 25 al.. 1978b). The reaction mixtures (5 m1 total volume) contained 509! 0.5 a! NADP+. Tris-HC. pH 7.5 (at 37° C). 5 mg MgCl 5 a! MnCl 2' 2' 5 pg DL-sodium isocitrate. 0.5 units of isocitrate dehydrogenase. 168 20 pg aminOpyrine. and 2.5 mg microsomal protein. Reaction mixtures were prepared on ice in 20 ml beakers. The reaction was initiated by the addition of 10 ul of isocitrate dehydrogenase to the beaker which was then placed in a shaking Dubnoff metabolic shaker at 37°. At 4.7. and 10 minutes. 1 ml aliquots were removed and added to 1 ml of 10% trichloroacetic acid (TCA) and vortexed. After several minutes to ensure protein precipitation. 2 ml of Nash reagent (2 g ammonium acetate. 0.05 g acetic acid and 0.02 g 2.4-pentanedione) was added to each aliquot and heated at 60° for 10 minutes for reaction to occur. After cooling. samples were centrifuged for 15 minutes and the absorbance of the sUpernatant was measured at 412 nm against a blank consisting of 1 ml 10% TCA. 1 ml buffer. and 2 ml Nash reagent. Extinction -1 and dilution factor of 4 were used coefficient of 7.8 mM’1 cm to calculate the formaldehyde content. Ethoxyresorufin-O-deethylation was assayed as described by Burke and Mayer (1974) with the following modifications. The reaction mixtures (1 m1 total volume) contained 66 pg Tris-HCl buffer pH 7.8 (at 37°). 3.3 pg MgClz. 0.3 pg NADP+. 3 pg DL- soduim isocitrate. 0.3 units of isocitrate dehydrogenase and 2.5 pg ethoxyresorufin. The following microsomal protein concen- trations were routinely used 3 liver microsomes from MC—induced rats. 2-10 pg/ml; liver microsomes from control or PB-induced rats. 0.1-0.4 mg/ml. Using these protein concentrations. the reaction was found to be linear up to 10 minutes (Figures 24A and B). 2.5 pg Ethoxyresorufin was used as the substrate (concentration since the apparent Km's for either PB- or MC- induced microsomes were roughly 0.24 and 0.19 pg (Figure 25). II inf-*- _me 169 Figure 24 -The linearity of the deethylation of ethoxyresorufin with varying protein concentrations of rat microsomes isolated from livers of rats treated with either (A) MC (0). or (B) PB (O)- 170 Figure 24 A. 0J4~ .5 033‘ SE \ 3 B 0.2 " EE (1' 0.l- I 1 5 I0 PROTEIN (pg) E3 (12 . £5 EE ‘\ (.0 a, I 15 (ll - E: C I V r I00 200 300 460 PROTEIN (pg) 171 Figure 25 - Lineweaver-Burke plots for the deethylation of ethoxyresorufin by rat microsomes isolated from livers of rats treated with either MC(O) or PB(. ). 172 Figure 25 301 O 20- O _L V I0- I I I0 20 ml- 173 Blanks and standards were made for each protein concentration range used. Reaction mixtures were prepared on ice in 13 x 100 mm test tubes. in the absence of ethoxyresorufin. and then preincubated at 37 o for 5 minutes in a Dubnoff metabolic shaking incubator. The reaction was initiated by the addition of ethoxy- resorufin. vortexed and returned to the water bath. Ethoxy- resorufin was not added to blanks or standards. After 5 minutes. 1 ml of ice cold acetone was added to all samples. vortexed and placed on ice. Buffer (1 ml) was added to all samples and at this time ethoxyresorufin was added to both blanks and standards. Reaction mixtures used for standards had lpg resorufin (in buffer) and were used to calibrate the assay. The fluorescence of resorufin was determined employing a Perkin—Elmer Fluorimeter. using an excitation wavelength of 530 nm and an emission wave- length of 590 nm. with a slit arrangement of 10 nm (entrance slit) and 20 nm (exit slit) to decrease Raleigh scattering. Portions of liver and abdominal adipose tissue were analyzed for the PBB congeners. Standard procedures for extraction and analysis for PBB in tissues were followed (Thompson. 1977) with the following modifications. Frozen tissue was allowed to thaw. One-half gram of tissue was homogenized 4 times. each with 15 ml toluene. Each 15 ml extract was combined and centrifuged for 10 minutes. The last 15 ml homogenate was heated in a 80° C water bath for 15 minutes. centrifuged. and combined with prior extracts. The samples was diluted to 100 ml and a 20 ml aliquot was removed to determine lipid content. The sample was then evaporated to a volume of 5-10 ml and then passed over a column of activated florisil topped with anhydrous soduim sulfate. The 174 sample flask was rinsed with toluene several times which was also passed over the column. The column was rinsed 10 ml of toluene after sample. The sample was evaporated to nearly 5 ml and redissolved in a known volume. The amount of each PBB congener was determined by GC analysis using standard curves which were constructed concurrently with sample analysis by plotting the ECD-GC response (log area counts) versus various amounts (log gram) of each EBB congener. Photochemical Procedures HBB was dissolved in hexane at a concentration of 1.59 pg and was irradiated with a UV source (germicidal UV lamp having a peak energy output at 254 nM) in a beaker while stirring. 12.5 cm from the source (surface area exposed - 23.8 cmz) for 3.5 hours. The resulting solution was slightly cloudy due to the formation of polymers (Chapter two of this thesis). The solution was centrifuged at 4.000 rpm for 15 min. The supernatant was removed and the pellet resuspended in four volumes of hexane and recentrifuged. This procedure was performed two times and all the resulting supernatants were combined and analyzed by GC. The GC elution profile of the resulting mixture which was administered to rats is shown in Figure 26. 175 Figure 26 - GC EEUTION PROPIEE AND STRUCTURES OF PBB CONGENERS IN THE PHOTOLYZED HBB MIXTURE. The mixture (0.8 ng) was injected into an ECD-GC equipped with a 3% OV-l column. Injector port. column. and detector temperatures were 290. 240 and 300°C.respectively. 176 Figure 26 Bf Bf Br Br mm) ....- “ Br“ BrHBr n9) VII, Br Br Br Br 3)% I 5mm NW RESPONSE \99 (E TIME (min) 177 RESULTS The photolysis of HBB resulted in the formation of lower brominated biphenyls. The mixture of the photolyzed HBB which was administered to rats in this study is shown in Figure 26. Eight photOproducts of HBB have been previously identified (see Chapter two of this thesis) and therefore only the structures of those photoproducts studied individually in this report are shown (Figure 26). All treatment groUps were evaluated for their effects on the histologic appearance of selected organs and for their induction effects on several hepatic drug metabolizing enzymes. HBB. 2.4.5.3'.4'-PBB and 2.4.5.2'.5'-PBB were previously characterized as a strict PB-type. a predominantly MC-type. and a PB-type inducer. respectively (Moore pp pl.. 1978: Dannan pp 21.. l982b. 1982c). 3.4.5-HBB was used as a “reference" compound since it has been reported to be a strict MC-type inducer (Poland and Glover. 1977; Render pp 21.. 1982). Histological sections from the bladder. brain. adrenal glands. stomach. spleen. thymus. thyroid. trachea. pancreas. heart. lungs and kidney were apparently normal for all treatment groups. The liver was the only organ apparently changed histologically. 3.4—TBB caused the least histological changes in the liver. although liver weight 'was significantly increased (Table 9). Rats administered this congener had some swollen hepatocytes and less sinusoidal space but like control livers. no cytoplasmic vacuoles were apparent (Figure 27B). Livers of rats treated with 2.4.5.2'.5'-PBB also appeared quite normal histologically (not shown) which is similar 178 Figure 27 - EFFECTS OF VEHICLE AND VARIOUS TREATMENTS ON HEPATIC STRUCTURE 14 DAYS AFTER TREATMENT. Sections of liver from a control rat administered an i.p. injection of PEG (3 ml/kg) (A). and from a rat treated with 2 mg/kg (i.p.) 3.4-TBB (B) or 90 mg/kg (i.p.) photolyzed HBB mixture (C). H and E stain) magnification. 430 x. 179 180 Figure 27 C 181 TABLE 9 Body and Organ Heights of Rats Administered Various PBB CongenersA Average Daily Body Weight 4 95 Body Weight Treatment Gain(g) Liver Spleen Thymus Control 5.74:0.61 3.91:0.15 0.37:0.02 0.40:0.08 Firemaster 4.76:0.64 5.87:0.90B 0.3510.03 0.3910.02 Photolyzed B see Mixture 5.14:0.66 6.44:0.96 o.4s:o.o7 0.37:0.04 HBB 4.89:0.35 6.29:0.698 0.37:0.06 0.43:0.06 2.4.5.3'.4'- a pee 4.20:1.08 5.9510.93 0.34:0.03 0.3410.04 2.4.5.2'.s'- a pee 4.79:0.22 4.73:0.37 0.34:0.03 0.35:0.02 3.4-ras 4.74:0.40 4.75:0.34B 0.40:0.05 0.35:0.05 3.4.5-HBB 4.53:0.50 5.57:0.363 0.37:0.07 0.3410.o7 A Six rats per treatment were administered a single ip injection of 4 ml PEG/kg (Control) or either of the PBB congeners or Mixture at 90 mg/kg (except 3.4-TBB and 3.4.5-HBB at 2 mg/kg) two weeks before sacrifice. Data are expressed as mean 1 SD. BSignificantly different from mean of control using Student's t-test (p < 0.05). 182 to earlier findings (Dannan pp pl.. l982c). Rats pretreated with 2.4.5.3'.4'-PBB or HBB had enlarged and vacuolated hepatocytes (not shown) which is also in agreement with earlier results (Dannan pp 21.. 1982a: Moore 25 p;.. 1978). The photolyzed HBB mixture caused the most severe histologic changes in the liver (Figure 27C). Hepatocytes were greatly enlarged and mild vacuolation was also observed. The body and organ weights of rats administered the various PBB congeners are shown in Table 9. All treatments significantly increased the liver to body weight ratio. No significant change was observed for body weight gain. spleen to body weight ratio or thymus to body weight ratio (Table 9). Levels of several hepatic drug metabolizing enzymes were evaluated 'in order to characterize the type of induction caused by the congeners and mixtures. Cytochrome c reductase activity and cytochrome P-450 content were invariably increased by all treatments. except by 3.4-TBB and MC (Table 10). The activity of the reductase was almost doubled by 3.4.5-HBB. 2.4.5.3'.4'-PBB. Firemaster. HBB. the photolyzed HBB mixture. PB. and PB plus MC. 2.4.5.2'.5'-PBB also significantly increased reductase activity. but not to the extent caused by the other treatments. All treatments except 3.4-TBB induced cytochrome P-450 by 2-4 fold. and 3.4.5-HBB. 2.4.5.3‘.4'-PBB. and MC shifted the absorption maximum of the CO difference spectrum (xma ) toward 448 nm. X While HBB and 2.4.5.2'.5'-PBB both had no effect on ‘xmax' the photolyzed HBB mixture. like Firemaster. had an intermediate effect ( )‘max - 449 nm). It is also of interest to note that while the total content of cytochrome P-450 was not increased by 183 was mesao> Docuo Has Ado.o A a. douucou 0v Deduewe «AHo.o V DC umeulu m.ucev:um mafia: AODUCOU 0» uceueuuwv adaeuwuewueum cueuODQ OE c«E\ueAOEco cueu0un mE\eeaoecu 000590: NUDE: DOflwHOIOU .0 09.3 IUCOEUOGHF“ I I I I .owv. os+sos o.n+ ~.os A.~+m.es ms+oo~ om.o+aa.~ mesam.v.n a a o a 3:: as New onn.su o~.n ~.~ap.vs smegma ss.oun~.~ mmer.n I I I I .one one s +~m ns.o+ va.o ao.~+n.- avn+o- on~.o+ao.~ I.m..~.m.v.~ I .ocv. one 0 .1 e a I e I. e l e I s s s \ onn+~ou nu ~+ m - om H+n as oo~+aon eon.”mw" ~ .4 .n n c m a Hem onH.oH «v.0 an.nH~.sn nemusun Doe.onmv.n on: I I I I .owvs ouauxs: no: en+ovu v.m+ o.o~ o.~+n.o~ om+~mn mm.o+oo.~ moasaouosm o n a a a so... I . I . . I . I . I . so asses on +n~s as H+ o ~H no n+~ ~n oon+onn Doolmuww n u em on +os~ no.~+ c.n~ oo.m+s.sn onm+mvn nonmmuwm.v u: + as on~Hon~ ao.~M v.- m.oHn.~H m~usn~ a-.ou~o.u u: .omvo n Ham o>~.ou Ho.o oo.oHH.on o~muomn oma.ouvn.n mm .om'e o Hem no.6” A~.o m.~Ho.~s o~unvs oo.oumo.s souucou 2:: 33.2 A . eueueuuceuu euoaxnueoc ous~>£ueeoc eueuusflou omvlm uceEueeuP Aweocowausamlmo: Iolwwusu080u>x0:9u U IZIocflu>QOCME< u GEODw00u>UI=mou MUCOEUQOHH < OH fldfl‘fi e:0«ue> Teueueucuaflt sued no eueueEouem eE>Ncm AeSOeouuut nausea: Heue>em 184 3.4-TBB. it did shift the x'max from 450 nm to 449 nm. Aminopyrine-N-demethylase activity. which is usually induced by PB. was almost doubled by all treatments except. MC. 3.4— TBB and 3.4.5-HBB which caused no change in this activity (Table 10). 2.4.5.3'.4'-PBB and 2.4.5.2'.5'-PBB both increased this activity by nearly the same amount. which was roughly 1.5 x. In order to assess the extent of MC-type induction. aryl hydrocarbon hydroxylase (AHH) activity was measured by using the fluorometric assay for ethoxyresorufin deethylation to resorufin (Burke and Mayer. 1974). The development of the assay was given in Materials and Methods. The apparent Km's for microsomes isolated from rats treated with either MC or PB did not appear to be different (Figure 25). MC induced this activity about 100 fold while PB increased the activity only about 4-fold. While HBB was the least effective inducer of this activity. increasing it only 2-fold. the photolyzed HBB mixture increased it 80-fold (Table 10). All other treatments increased AHH activity by 60-100 fold except 2.4.5.2'.5'-PBB (3 fold) and 3.4-TBB (17 fold). Similar results were seen for microsomal p-nitrOphenol-UDP-glucuronosyl transferase activity (Table 10). The lg 2129 metabolism of the photolyzed HBB mixture was assessed by measuring the tissue levels of the PBB congeners. Table 11 shows that HBB and 2.4.5.3'.4'-PBB were present in relatively the same concentration. while the level of 2.4.5.2'.5'-PBB. also given at the same dose. was nearly 3-fold lower. Also. while 3.4—TBB and 3.4.5-HBB were given at the same dose (2 mg/kg). 3.4.5-HBB adipose levels were 17 fold higher than the 3.4-TBB adipose levels and 3.4-TBB was not detectable in PBB Concentration in the Liver and Adipose Tissue of Treated Rate 185 TABLE 11 A Treatment Liver Adipose (ppm per lipid basis) (ppm) Control NDB ND use 77:28 461 1215 2.4.5.3'.4'—PBB 130:7? 341 1214 2.4.5.2'.5'-PBB 29:25 250 1 87 3.4-rse ND 0.41 1 0.19 3.4.5-HBB 47:33 5.7 + 3.0 ASix rats per treatment were administered a single ip injection of 4 ml/kg PEG (Control) or either of the PBB congeners at 90 mg/kg (except before sacrifice. ND - Not Detected. 3.4—TBB and 3.4.5-HBB at 2 mg/kg) two weeks Data are expressed as mean 1 SD. 186 Figure 28 - GC EQUTION PROFIgE OF PIBs IN LIVER (A).__ADIPOSI (D) AND THE PHOTOLYZED HBB MIXTURE (C). C represents the GC response to 0.8 ng photolyzed HBB mixture injection. Figure 28 RESPONSE I I I I I 1 CHILI 87 lJVER I I I we I ADPOSE i I I I II I "I I. IF I 1 l I FPHOTOLYZED HBB MIXTURE I I ' I I I I '. II I ? l .'= I Z 3 I II I 5 ' I ‘I II I I I " kk‘LJI Q) Li 14 \g—J \¥—\_ 1 -i.-. _ ...L_____. 4 8 I2 TIME (min) 188 liver (Table 11). Figure 28 shows a GC analysis of both adipose tissue and liver extract from a rat pretreated with the photolyzed HBB mixture. It can be seen that 2.4.5.2'.5'-PBB is markedly decreased in adipose and absent from liver. Earlier eluting peaks are decreased in both adipose and liver. The peak corresponding to 3.4.-TBB and 2.4.5.2'.4'—PBB. which coelute (see Chapter two of this thesis) is decreased in adipose but increased in liver. This increase may be due to the lack of metabolism of 214(512'14'-PBB. DISCUSSION HBB. the major component of Firemaster. readily underwent photolytic debromination to yield a mixture of lower brominated congeners (see Chapter two of this thesis). HBB induced hepatic microsomal drug metabolizing enzymes typically induced by PB (Moore pp pl.. 1978). The photolyzed HBB mixture also induced enzymes which are classically induced by MC. By the enzymic and spectral properties of the microsomes from animals pretreated with the photolyzed HBB. the mixture has been characterized as a mixed-type inducer. like Firemaster (Dent pp pl.. 1976b). The photolyzed HBB mixture increased the liver weight and caused swelling and mild vacuolation of the hepatocyte. Two of the three major photoproducts used in this study. namely 2.4.5.3'.4'-PBB and 3.4-TBB. induced AHH activity as well as UDP-glucuronosyl transferase activity. although 2.4.5.3‘.4'— PBB was given at a higher dose than 3.4-TBB. 3.4-TBB did not appear to be as potent an inducer as 3.4.5-HBB. although the rats 189 received 4 pmol/kg of 3.4-TBB and 3 ‘pmol/kg of 3.4.5-HBB. Although 3.4-TBB caused an increase in liver weight. it did not cause a significant histopathological change in the liver. This congener also did not significantly increase the content of cytochrome P-450 but it did shift the Amax from 450 nm to 449 nm. Thus it appears to act like an MC-type inducer. in causing a hypsochromic shift of the xmax' however it seems to be less effective than 3.4.5-HBB. Similar results were seen by Yoshimura and coworkers (1979) who reported that after a dose of 10 mg/kg of 3.4-TCB (the chlorinated analog of 3.4—TBB). the xmax did not differ from control (449.5 nm compared to control 450.0 nm) whereas 10 mg/kg of 3.4.5-HCB shifted the xmax to 448.5 nm. Poland and Glover (1977) reported 3.4.5-HBB to be a strict MC-type inducer since it can exist in a coplanar configuration and compete with [3H1-TCDD to bind to the TCDD receptor. Based on the structure-activity relationships for polyhalogenated aromatic hydrocarbons. it appeared that 3.4-TBB should serve as a better ligand for the TCDD receptor and therefore should be a more potent inducer of AHH than 3.4.5-HBB (reviewed in Poland and Knutson. 1982). The pp 3132 metabolism of the photolyzed HBB as well as the purified congeners was studied to determine if 3.4- TBB was metabolized and if this may account for the discrepancies in the enzymatic data. The results of the tissue analyses for the PBB congeners in the liver and adipose tissue from rats pretreated with them suggested that 3.4—TBB was metabolized to some extent since it was not detected in the liver. and the adipose levels were 17 fold lower than 3.4.5-HBB. which was given at the same dose. 2.4.5.2'.5'-PBB also appeared to be 190 metabolized since it was 3 fold lower in concentration when compared to either HBB or 2.4.5.3'.4'-PBB. The concentrations of the PBB congeners in the photolyzed HBB mixture administered to rats also appeared to change over time. The results of the liver and adipose tissue level analyses of the photolyzed HBB mixture were not conclusive. however. since 2.4.5.2'.4'-PBB coelutes with 3.4—TBB (see Chapter two of this thesis). The results of the liver and adipose tissue level analyses also suggested that the lower brominated congeners. 2.5.3'.4'-TBB. 2.5.2'.5'-TBB. and 2.4.2'.5'-TBB. were also metabolized pp 2129 as evidenced by their absence. From the pp gpppp metabolism studies using microsomes from rats pretreated with various inducers it appeared that structure—activity relationships existed for the metabolism of PBB congeners and they differed with the different types of microsomes used. since different profiles were observed (Mills pp _p.. 1984). Metabolism of 2.4.5.2'.5'—PBB was only observed with microsomes isolated from rats pretreated with PE and not by microsomes isolated from MC-pretreated rats or control rats. The inverse was found for 3.4-TBB. which was metabolized only by microsomes isolated from rats pretreated with MC and not with PB. The structure-activity relationship for metabolism of PBB congeners by microsomes from different sources is the subject of a communication (Mills _p pl.. 1984). and it appears that microsomes from PB-treated rats require adjacent pppp and pppp positions on either ring to be free of halogens whereas for MC- pretreated microsomes. adjacent ortho and meta positions free of bromines are required. 191 The results of the 1 vivo metabolism studies are in agreement with several other reports in that 3.4-TCB (the chlorinated analog) has been reported to be metabolized by both male and female rats as well as female monkeys and is cleared relatively fast (Abdel-Hamid pp pp.. 1981). McNulty and coworkers (1980) also reported that female monkeys fed a 3.4—TCB diet recovered rapidly when they were taken off the diet. How metabolism of 3.4-TBB affects toxicity and enzyme induction will be reported in chapter four of this thesis. In assessing the toxicity of the photolyzed HBB mixture as well as its metabolism and enzyme induction. the results of this study suggest that the increased biologic and toxic response of this mixture when compared to pure HBB and three photOproducts is due to 2.4.5.3'.4'—PBB and to a lesser extent 3.4-TBB. since the latter is metabolized and the former is apparently not. The results of this study are in agreement with a study by Patterson and coworkers (1981) who reported that a photolyzed HBB mixture had a hyperkeratotic effect whereas HBB itself did not. They suggest that the formation of 2.4.5.3'.4'-PBB and 3.4-TBB during the irradiation of HBB are responsible for the hyperkeratosis induced by the mixture. In a similar study. Robertson and coworkers (1981) reported that the photolysis of Firemaster yielded products which increased the biologic potency of the commercial mixture. It was difficult. however to determine which photoproduct(s) was(were) responsible. since they could not detect the expected increases in the concentration of 2.4.5.3'.4'-PBB and 3.4-TBB in the photolysis mixture (Robertson 192 _p pp.. 1981). The formation of these two congeners is expected to be high if indeed the photolysis of HBB proceeds through a sequential loss of pppgp bromines (Ruzo pp pp.. 1976; Ruzo pp pp.. 1975; Bunce pp pl.. 1975). However. several workers have reported. using Firemaster or HBB. that the stepwise preferential loss of pppgp bromines from HBB to give 2.4.5.3'.4'-PBB and ultimately 3.4-TBB is not followed (Robertson pp pp.. 1983a: see Chapter two of this thesis). It is therefore concluded that the photolyzed HBB mixture does cause a mixed—type induction and that this enhanced biologic effect is due mostly to 2.4.5.3'.4'-PBB and to a lesser extent to 3.4-TBB. since the latter and most of the other photOproducts present are metabolized. CHAPTER 4 TOXICITY OF 3.4.5.3'.4'.5'-REXABROMINATED BIPMENYL AND 3.4.3'.4'-TETRABROMINATED BIPMENYL 193 194 ABSTRACT Immature male rats were given a single equimolar dose (21.3 pmoles/kg body weight) of 3.4.5.3'.4'.5'-hexabromobiphenyl (HBB) or 3.4.3’.4'-tetrabromobiphenyl (TBB) and sacrificed at various times up to fourteen days after treatment. Liver micro- somal aryl hydrocarbon hydroxylase (AHH) activity for the TBB treatment group was maximal at day 2 and then steadily decreased. whereas this activity was induced in one day and remained high for the HBB treatment group. Tissue levels of HBB appeared to be unchanged over time whereas tissue levels of TBB decreased in a biphasic manner. HBB caused moderate to severe hepatic changes while TBB treated rats had only mild hepatic changes. The relative binding of TBB by the liver receptor for 2.3.7.8- tetrachlorodibenzo-p-dioxin (TCDD) was about ten times that for HBB. The results suggest that even though the receptor binding affinities imply that TBB should be more toxic than HBB. it is less toxic than HBB because it is metabolized. These results also suggest that receptor binding and AHH induction do not accurately reflect toxicity for polyhalogenated aromatic hydrocarbons which are metabolized. presumably because continued occupation of the receptor and persistent induction of some enzyme activity are required for toxicity. The ability of HBB to induce AHH activity in extrahepatic tissues was also investigated. AHH activity was induced in kidney. lung. small intestine as well as in the liver. 195 INTRODUCTION Polybrominated biphenyls (PBB) belong to a class of toxic polyhalogenated aromatic hydrocarbons (PHAH) which is typified by the most toxic member. 2.3.7.8-tetrachlorodibenzo-p-dioxin (TCDD). This class of chemicals includes the polychlorinated biphenyls (PCB). the polychlorinated dibenzofurans and the polychlorinated dibenzo-p-dioxins. These chemicals produce a similar and characteristic toxic syndrome and a variety of biochemical responses in mammalian systems (Poland and Knutson. 1982). Among the toxic responses reported are progressive weight loss. hepatotoxicity. lymphoid involution. chloracne and gastric lesions (reviewed by Poland and Knutson. 1982)- Some of the biochemical responses include endocrine effects. porphyria. effects on lipid metabolism and induction of microsomal enzymes (Poland and Knutson. 1982). Although the mechanism of toxicity for these PHAH remains unknown. there is good evidence which suggests that both the toxic and biochemical responses are mediated by a binding protein. referred to as the TCDD receptor (Poland _p _p.. 1976a; Greenlee and Poland. 1979; Poland and Glover. 1980). One of the enzymes which is coordinately expressed is a form of cytochrome P-450 with aryl hydrocarbon hydroxylase (AHH) activity (Poland pp al.. 1976a). The ability of a PHAH congener to be a ligand for the receptor is usually assessed by determining its ability to induce AHH activity. It has been postulated that enzyme induction is an early event and that toxicity occurs later and requires a persistent receptor occupation and gene expression (Poland and Knutson. 1982). This 196 type of 'hyperinduction” has been seen in rats given a single dose of TCDD. where AHH activity remained induced for over 35 days. reflecting the prolonged biological half-life of TCDD (Poland and Glover. 1973). In chapter three of this thesis it was observed that 3.4.3'.4‘-tetrabromobiphenyl (TBB) was not as potent an inducer of AHH activity as was 3.4.5.3'.4'.5'-hexabromobiphenyl (HBB). Since both have no pppgp bromines. they can exist in a coplanar configuration and thus should bind to the receptor with almost equal affinities. The tetrachloro analogue (TCB) has been shown to be a better ligand for the receptor than the hexachloro analogue (HCB) (Bandiera pp pp. 1982: Poland and Glover. 1977). pp gpppp metabolism studies showed the disappearance of TBB but not HBB when incubated with microsomes isolated from rats treated with 3-methylcholanthrene (MC) (Mills pp pl.. 1984). Therefore. it was of interest to perform a time course experiment. in which rats were given an equimolar dose of either TBB. a congener which appears to be metabolized pg ppppp. or HBB. a congener which is not. or very slowly. metabolized. to examine how metabolism may effect enzyme induction and thus influence toxicity. Also. the affinity of TBB and HBB for the TCDD receptor was determined by their competition with [3H1-TCDD for specific binding sites as measured by sucrose density gradient analysis. Many tissues 1 vivo have been shown to contain the TCDD receptor (Carlstedt-Duke. 1979; Carlstedt-Duke pp pl.. 1979). However. whether these tissues respond to a PBB congener which binds to the receptor by induction of AHH activity has not been investigated. Several tissues which have been shown to contain 197 the receptor and respond to TCDD with induction of AHH activity show no toxic response and it has been postulated that while the receptor may be essential for toxicity. it may not be sufficient (Poland and Knutson. 1982). Therefore it was of interest to investigate whether or not HBB could induce AHH activity in extrahepatic tissues. METHODS Chemicals [BHI-TCDD (50 Ci/mmol) was purchased from KOR IsotOpes. Cambridge. MA. The radiolabeled TCDD contained greater than 85% of the TCDD congener and the remainder consisted of di- and tri- chlorodibenzo-p-dioxin congeners. which are poor ligands for the cytosolic receptor (Poland pp pl.. 1976a). 2.3.7.8- Tetrachlorodibenzofuran (TCDF) was purchased from Cambridge IsotOpes. Cambridge. MA. Sucrose (Ultra Pure Crystalline) was purchased from Schwarz-Mann Inc.. Spring Valley. N.Y. HEPES. dextran. nicotinamide. MC. NADP. NADPH. isocitrate. isocitrate dehydrogenase. and dithiothreitol were purchased from Sigma Chemical Co.. St. Louis. MO. Resorufin was purchased from Eastman Organic Chemicals. Rochester. N.Y. 7-Ethoxyresorufin was purchased from Pierce Chemical Co.. Rockford. IL. Methylated albumin (bovine serum) (methyl-14C. 0.017 Ci/mg) was purchased from New England Nuclear. Boston. MA. Glycerol. charcoal (Norit A). toluene and p-dioxane were purchased from Fisher Scientific Co.. Pittsburgh. PA. Crude HBB ((65%) was obtained from Ultra 198 Scientific. Hope. R.I. TBB was synthesized and purified by recrystallization and column chromatography on alumina as previously described in chapter one. HBB was purified in a similar manner. The purities were. determined by GC chromatography using a Varian 3700 gas chromatograph equipped 63Ni ECD and found to be >99%. with a Animals Outbred male Sprague-Dawley rats (75-100 9) and inbred male C57BL/6NH (B6) mice (5-6 weeks) were purchased from Harlan Sprague-Dawley. Inc.. Haslett. MI. The animals were allowed an acclimation period of at least 48 hours before any treatments. TBB and HBB were dissolved in corn oil at a concentration of 4-5 mg/ml. Three rats in each groUp were administered each congener by gavage at a dose of 21.3 pmoles/kg body weight (10 mg/kg for TBB and 13.3 mg/kg for HBB) and were killed on various days after treatment. Control rats were administered corn oil at a dose of 1% body weight. All rats were given free access to feed and water except for the night before sacrifice when feed was removed. To study whether HBB could induce extrahepatic AHH activity. four rats were administered HBB by gavage at a dose of 10 mg/kg (l6‘pmoles/kg) 7 days before sacrifice. Three rats were given corn oil at a dose of 4% body weight (control) and sacrificed 7 days later. All rats were given free access to feed and water except for the night before sacrifice when feed was removed . 199 Isolation pp Microsomes Rats were killed by decapitation. and the livers were perfused with cold 1.15% KCl containing 0.2% nicotinamide pp situ. Liver microsomes were isolated individually from each rat according to previously described procedures (Pederson and Aust. 1970). Microsomal pellets were then resuspended in 0.3g sucrose buffer containing 0.1g tetrasodium pyrophosphate. pH 7.5 and repelleted by ultracentrifugation at 105.000 x g for 90 minutes. then resuspended in a 0.05g Tris-HCl buffer (pH 7.5 at 25°C) containing 50% glycerol and 0.01% (w/v) butylated hydroxytoluene and stored at -20°C under argon (Welton and Aust. 1974). Rats administered 10 mg/kg HBB or corn oil were killed 7 days later by decapitation. Livers. kidneys. and lungs were perfused with cold 1.15% KCl containing 0.2% nicotinamide pg situ. The first proximal third of the small intestine. brain. and thymus were removed and rinse with perfusate. All tissues were weighed. Microsomes were isolated from individual rats. except for thymus samples which were pooled. as previously described (Pederson and Aust. 1970). Microsomes from all these rats were not washed. Tissue Collection 29g Histopatholpgy At each time point. rats were weighed and killed. Liver and thymus weights were recorded for each rat. Tissue samples from the liver. spleen. kidney. urinary bladder. thymus. trachea. lung; -adrenal. pancreas and small intestine were fixed in 10% buffered formalin for histological examination. The formalin- fixed tissues were automatically processed (Histomatic. Model 166. Fisher Scientific Company. Pittsburg. PA). embedded in 200 paraffin. sectioned at 6 um and stained with hematoxylin—eosin. Preparation pp Receptor 36 mice or Sprague-Dawley rats were killed by cervical fracture and the liver was perfused with cold lOpg phosphate- buffered saline (0.85% NaCl solution) i situ via the inferior vena cave. The livers were weighed. placed in HEDG buffer (25pg HEPES. 1.5pg EDTA. lpg dithiothreitol. and 10% glycerol. pH 7.6; 3 ml/g of liver). minced and homogenized. The homogenate was centrifuged at 10.000 x g for 20 minutes and the resulting supernatant was filtered through a layer of cheesecloth and ultracentrifuged at 105.000 x g for 60 minutes at 0°C. Surface lipid was carefully removed with a Pasteur pipette and the supernatant removed with a second Pasteur pipette without disturbing the pellet. The resultant supernatant usually con- tained about 15-20 mg protein/ml. All protein concentrations were determined by either the method of Lowry (Lowry pp al.. 1951) or Gornall pp pp.( 1949) with bovine serum albumin as the standard. Supernatants from livers of 3 mice of the same age were pooled and used for receptor binding studies within an hour. whereas supernatant from individual rat livers was used. Receptor Binding The method used to quantitate the receptor was sucrose density gradient analysis following dextran-charcoal treatment OOkey pp pp.. 1979). This assay has been shown to be more nmliable in separating a class of high-affinity. low-capacity sites from non-saturable binding than dextran-charcoal adsorption 201 methods or DEAE-cellulose column chromatography. Samples for sucrose density gradient analysis were prepared by incubating 1 ml of supernatant (5-6 mg/ml protein) with lopg [BHI-TCDD for 1 hour at 0-4°C in a shaking water bath. [3H1—TCDD was added in 10 pl of p-dioxane/ml of sUpernatant. In competitor experiments. an equal amount of p-dioxane containing the desired concentration of competitor was added. A control containing [3H1-TCDD plus 10 pl p-dioxane was prepared to determine total binding. Another control which contained [3Hl-TCDD plus 200x excess of TCDF was prepared to determine non-specific binding. After the 1 hour incubation. the unbound and loosely bound [3H]- TCDD and competitor were removed by adding the incubation mixture to a test tube containing dextran-charcoal (10 mg of charcoal/mg of dextran pelleted from HEDG buffer by centrifugation at 4.000 x g for 15 minutes). The dextran-charcoal was mixed with the sUpernatant by vortexing and the mixture incubated at 0-4°C for 15 minutes before centrifugation at 4.000 x g for 15 minutes. Aliquots of supernatant were taken both before and after dextran- charcoal treatment for determination of total and bound radioactivity. Three hundred microlitres of the charcoal-treated supernatant were layered onto linear (5 to 20%) sucrose density gradients prepared in HEDG buffer. Gradients were centrifuged at 48.000 rpm at 2°C for 16 hours in a Beckman SW 60 Ti rotor (gav - 235.000). After centrifugation. 45 fractions of 0.1 ml each were collected into 20 m1 glass scintillation vials. Radioactivity in each fraction was determined by liquid scintillation counting and corrected for counting efficiency. To determine approximate 202 sedimentation values. methylated [l‘Cl-labeled bovine serum albumin was added to the supernatant as an internal sedimentation marker (4.6 S): sedimentation coefficients were then calculated by the method of Martin and Ames (1961). Enzzpe Assaxs ppg Tissue Levels pp :35 Induction of AHH activity was assessed by assaying for ethoxyresorufin-0-deethylase activity according to the method of Burke and Mayer (1974) with slight modifications as described in chapter three and cytochrome P-450 was assayed by the method of Omura and Sato (1964a). All protein concentrations were determined by the method of Lowry pp p; (1951). Portions of liver and abdominal adipose tissue were stored at -20°C until analyzed for HBB and TBB. Standard procedures for extraction and analysis for PBB in tissues were followed (Thompson. 1977) as described in chapter three. RESULTS Organ Weights The time dependent change in the absolute liver weights for rats treated with either HBB or TBB is shown in Figure 29. The liver weights from rats given HBB were significantly higher (p < 0.05) than the liver weights from TBB treated rats after day 6. The thymus weights from rats treated with HBB were significantly lower than those from rats given TBB also after day 6 (Figure 30). The thymus weights of animals in both treatment groups were less than in control animals. Body weights for bOth treatment 203 (g) WEIGHT LIVER ilkk I234 6 8 IO l4 TIME (days) Figure 29 - Absolute liver weights from rats treated with either 21.3 pmoles/kg body weight of HBB (0) or TBB (0). or corn oil (control. D ). +Significantly different from mean of control using Student's t-test (p < 0.05). * Treatment groups significantly different from each other using Student's t-test (p < 0.05). 204 ,1 0.6«- U) I— . . a; «» a] 0.4-- , . - ~{# 3 I‘.‘ ° * $2 I \‘e #95 2 I ‘l I . II E I l.— 0.2 " . .* Io I4 NI» 0]. .b 0') (1) TIME (days) Figure 30 - Absolute thymus weights from rats treated with either 21.3 pmoles/kg body weight of HBB (0) or TBB (0). or corn oil (control. D ) . +Significantly different from mean of control using Student's t-test (p < 0.01). R Treatment groups significantly different from each other using Student's t-test (p < 0.05). 205 groups were not significantly different from controls (data not shown). Histopatholggy The liver had the most remarkable histologic changes related to treatment. Livers from control animals showed normal architecture of the hepatic lobule. with distinct sinusoidal spaces (Figure 31). Rats administered TBB had mild diffuse hepatocellular swelling and decreased sinusoidal space in the midzonal and periportal areas (Figure 32). The most dramatic histologic changes in the liver were in rats given HBB (Figure 33). The hepatocytes were moderately to markedly swollen. especially in the peripherolobular regions. with subsequent obliteration of the sinusoidal spaces and loss of hepatic cord architecture. CytOplasmic vacuolation of the centrilobular hepatocytes was also seen as well as a moderate increase in the number of binucleated hepatocytes. Rats administered either TBB or HBB had severe involution of the thymus and an apparent equal loss of thymocytes from both the cortical and medullary regions. Lung. spleen. kidney. trachea. adrenal. pancreas. small intestine and urinary bladder in all groups had no significant abnormalities. gpppetitive Recpptor Binding Studies Incubation of rat hepatic 105.000 x g supernatant with 10 pg [3Hl—TCDD for 1 hour at 5° C produced a specific binding peak which was detectable by sucrose density gradient analysis. The peak sedimented at approximately fraction 30 (an average of 8.1s) 206 Figure 31 - sewer or cog on. op RAT LIVER s'rauc'runs souarssn DAYS AFTER TREATMENT. Section of liver from a control rat. Normal architecture of the hepatic lobule is seen. with distinct sinusoidal spaces in the central vein area. Hematoxylin and eosin stain: x 430. 207 Figure 32 - EFFECT OF TBB ON RAT LIVER STRUCTURE FOURTEEN DAYS AFTER TREATMENT. Section of liver from a rat killed 14 days after treated with 21.3 pmoles/kg body weight of TBB. The central vein area has swollen. vacuolated hepatocytes and decreased sinusoidal space. Hematoxylin and eosin stain; x 430. 208 Figure 33 - EFFECT OF HBB ON RAT LIVER STRUCTURE FOURTEEN DAYS AFTER TREATMENT. Section of liver from a rat killed 14 days after treatment with 21.3 pmoles/kg body weight of HBB. The centrilobular and midzonal hepatocytes are swollen and contain variably sized vacuoles. Sinusoidal space is decreased. Hematoxylin and eosin stain: x 430. 209 Figure 34 - Sucrose density gradient detection of specific high affinity binding of [3Hl-TCDD to a component in the 105.000 x g supernatant of rat liver. 105.000 X g supernatant (8.5 mg/ml) from Sprague-Dawley rat was incubated with 10 pg [3Hl-TCDD in the absence of competitor (D---D) and in the presence of 200 X TCDF (I--—I ). or 0.1 pg HBB (e---O). or 1.0 pg HBB (O---O). Following dextran-charcoal treatment. gradients were centrifuged and fractionated as described (see Materials and Methods). (H CD (D C) I N C) C) C) I IOOO- [3H]TCDD BOUND (dpm) TOP 210 Figure 34 IO nM I°HITCDD + O.IpM HBB + m pM HBB + 2.0 PM TCDF IIII IO 20 30 4O 5O FRACTION NUMBER 211 under these experimental conditions (Figure 34). The concentration of the receptor was found to be approximately 30 fmol/mg cytosolic protein and remained approximately the same throughout these experiments. Figure 35 shows the competition by HBB and TBB for the specific binding of [3Hl-TCDD to mouse and rat hepatic receptor. TBB was found to be the more potent competitor and completely inhibited [3Hl-TCDD binding at a concentration of 1 pg. whereas a concentration of 10 ‘pg was required for the HBB to completely inhibit [3Hl-TCDD binding. The ECso (the concentration of competitor effective in displacing 50% of [3Hl-TCDD specifically bound) values for TBB and HBB are approximately 0.10 pg and 1.3 pg in the B6 mouse. respectively; and 0.05 pg and 0.5 pg in the Sprague-Dawley rat. respectively. Another [3H1-TCDD binding peak was also observed. which sedimented at approximately fraction 15 (average 4.4 8) under these experimental conditions. The peak basically remained unaltered by treatment with large amounts of PBB congener competitors (Figure 34). This suggests that it represents nonspecific binding. Although 200 X TCDF appeared to decrease this peak . this observation was not consistent from animal to animal. ggg Induction The time course of the induction of microsomal ethoxyresorufin-o-deethylase (EROD) activity in rats by HBB and TBB is shown in Figure 36. This activity was significantly (p < 0.001) elevated above control for both treatment .groups. However. after day 6. the EROD activity for HBB treated rats was 212 Figure 35 -Comparative competition by TBB (0. I ) and HBB (O. D ) for specific binding of [BHI-TCDD to Sprague-Dawley (SD) rat (0.0) and B6 mouse (0.. ) hepatic receptor. The specific binding of [3Hl-TCDD was determined by sucrose density gradient analysis following charcoal- dextran treatment. The concentration of [BHI-TCDD was 10 pg and concentration of TBB and HBB are given on the abscissa. 213 m..o. Figure 35 a): o..o_ mmoos we mmeéd mmooz mm: 81-3.. 2m .o.m\ mmeim Em .o.m\ mmxoi mo..._._.ma_200 LO Zo_hEmmo mma 253 29.3.2 Ea mEE IO 6 TIME (days) 234 O 219 Figure 38 - Concentration of HBB (O) or TBB (0) in the livers of rats treated with 21.3‘pmoles/kg body weight of the respective congeners. L.D. - Limit of Detection. 220 Figure 38 Pun. _ _ _ _ “__ r_ _ _‘ _ _ “___,_ _ F _ _ m~__ _ _ L _ nu II IJ lOO—; om>mmmmo man. 253 203.22 Ea mEE . _ . . _ . _ _ _ _ _ _ _ _ _ _ _ _ _ b q IO 6 |234 0 (days) TIME 221 TABLE 12 Iody and Organ Heights of Rats Administered “BOA Tissue Tissue Weight (grams) Cont: :01 “BB Body 180.31 8.7 l70.0112.l Liver 5.5910.4s 3.0510.”B Thymus 0.6210.16 04510.05B Kidney 1.7610.06 1.4710.1oB Lung 1.23:0.14 1.30:0.06 Small Intestine 1.54:0.15 1.5310.ll Brain 1.63:0.02 l.6210.02 AThree rats were administered corn oil (4% body weight)and four received 10 mg/kg HBB one week before sacrifice. BSignificantly different from mean of control using Student's t- test (p < 0.05). 222 TABLE 13 Ethoxyresorufin-O-Deethylese Activity in Various Tissues of Rats Administered HBBA Tissue EROD (nmoles/min/mg protein) Control HBB Liver o.215:o.oqe 29.333332B Thymus 0.038 0.040 Kidney >0.00l l.70l:O.450 Lung 0.038:0.028 o.204:o.ozzB Small Intestine >0.00l 0.081:0.032 Brain 0.04410.016 0.02110.009 AThree rats were administered corn oil (4% body weight)and four received 10 mg/kg HBB one week before sacrifice. BSignificantly different from mean of control using Student's t- test (p < 0.05). 223 DISCUS$ION Results of this study demonstrate that. at an equimolar dose. HBB is more toxic than TBB as evidenced by greater thymic involution. increased liver weights and more extensive hepatic histOpathological changes. However. in 21559 binding studies revealed that the TCDD receptor binds TBB with greater affinity than HBB. suggesting that TBB should be roughly ten times more toxic than HBB. TBB. however. has been shown to be metabolized in 31559 whereas HBB was not and TBB appeared to induce its own metabolism (see Chapter three and Millis 25 al.. 1984b). Also. TBB. but not HBB. rapidly disappeared from liver and adipose tissue suggesting in gigg metabolism of Tab but not of H38. These results suggest that the metabolism of PBB congeners greatly decrease their toxicity. The results also suggest that receptor binding affinities may not accurately reflect toxicity of those PHAH congeners which may be metabolized. The ability of PHAH congeners to induce AHH activity is often used to assess the relative toxicity of these chemicals. Both T88 and H38 have been reported to be potent inducers of hepatic microsomal AHH activity (Robertson :5 al.. 1983b; Robertson gt al.. 1982; Poland and Clover. 1977: Render :5 al.. 1982: and Jensen 2; al.. 1983). In this study. early after administration of TBB. hepatic AHH activity was higher than in liver microsomes from animals treated with HBB. However. after 2 days. AHH activity remained elevated in liver microsomes from animals treated with HBB while it declined in microsomes from TBB-treated rats. Also. the absorption maximum of the CO 224 difference spectrum of cytochrome F-4SO shifted from 448 nm to 449 nm after day three in microsomes isolated from TBB-treated rats whereas it remained at 448 nm even at day ten for the H33 treatment (data not shown). The time course for the induction of microsomal metabolism of TBB correlates well with that seen for AHH activity (Hills 25 al.. 1984). The results of this study suggest that the i vivo concentration of TBB had decreased sufficiently to cause its dissociation from the receptor. and therefore a decrease in gene eXpression. These results support the prOposal of Poland and Knutson (1982). in that persistent receptor ocCUpation by the ligand is required for gene eXpression and toxicity. The results also indicate that toxicity may not be accurately estimated by determining the chemical's ability to induce AHH activity if the PEAR is metabolized. From this study it is obvious that AHH activity in microsomes from livers of animals treated with TBB depends Upon the time after the administration of the chemical at which the observations are made. In one study by Robertson and coworkers (1983b) it was reported that at a relatively high dose (150 pmole/kg). TBB caused significant reduction in growth rate. thymic atrOphy and liver enlargement in rats in two weeks. The results of this study are in agreement with those of Abdel-Hamid and coworkers (1981) who reported that TCB. the chlorinated analog of TBB. was cleared rapidly in rats. In other studies using TCB. McNulty and coworkers (1980) reported that female monkeys fed a TCB—diet rapidly recovered when they were taken off the diet. Yoshimura 225 and coworkers (1979) reported that although TCB was considered an MC-type inducer. a dose level of 10 mg TCB/kg (34‘pmol TCB/kg) was insufficient to induce significant amounts of cytochrome P- 448 in the rat 4 days after treatment or to shift the absorption maximum of the CO difference spectrum of cytochrome P-450 from 450 nm to 448 nm. Even at a relatively high dose of 50 mg TCB/kg (170 pmol/kg). they considered TCB to be much less potent an inducer than 3.4.5.3'.4'-pentachlorobiphenyl. which is considered the most potent HC-type PCB inducer. It is possible that these principles may be applicable to any ligand for the TCDD receptor which may be metabolized. including polycyclic aromatic hydrocarbons such as "C. Therefore. the manner of administration of the chemical. the observations to be made. and the time at which the observation is made must be carefully considered in studies designed to test the relative toxicity of chemicals which may produce a TCDD-like toxicity. For example. a single injection may be appropriate for testing HBB toxicity in animals whereas chronic exposure would be more appropriate for TBB. Simply determining relative binding affinity by the TCDD receptor in 31559 is inapprOpriate if the ligand is rapidly metabolized i vivo. Likewise it may be inapprOpriate to study enzyme induction in cells in culture when determining the relative effectiveness of the inducer unless the relative rates of metabolism are also considered. The results of this study also demonstrate that HBB could induce extrahepatic AHH induction. The TCDD receptor has been «detected in liver. lung. kidney and thymus and at much lower (concentration in testes. skeletal muscle. and brain (Carlstedt- 226 Duke. 1979: Carlstedt-Duke _5 al.. 1979). Induction of AHH activity in liver. lung. and kidney appear to correlate with the presence of the TCDD receptor. Induction of renal and small intestine AHH activity are in agreement with other studies which used either Firemaster or NC. respectively (HcCormack 25 al.. 1978; 1979: Nebert and Gelboin. 1969). A lack of induction in the thymus and brain was observed. Poland and Glover (1980) reported using TCDD. that thymuses from 86 mice had very low but inducible levels of AHH activity. It could be that the dose of HBB was not high enough to induce AHH activity although thymic weights were effected or that thymic involution may somehow affect enzyme induction. The results agree with Poland and Knutson (1982) who postulate that although the receptor may be essential for toxicity it may not be essential for toxicity and that tissues which develop a toxic response may express an additional set of genes which may effect a cellular process. 227 GENERAL DISCUSSION Several relatively important questions have been answered by the research presented in the thesis. Several aspects of the toxicity caused by PBB congeners have been addressed as well as how certain factors influence toxicity. Several aspects may be important to the public and environment of Michigan as well as of interest to biochemists and toxicologists who are pursing an understanding of the mechanism (s) of polyhalogenated aromatic hydrocarbons toxicity. The most important issue addressed was the influence of metabolism on the toxicity of PBB congeners. Several biological studies have been performed using certain PBB or PCB congeners and measuring or observing one or more of the biochemical and toxicological effects normally caused by TCDD. As discussed in the Literature Review. TCDD-like morphological and functional changes have been observed in the liver and lymphatic tissues of animals which received Firemaster or a HC—like PBB congener. Hepatic changes included proliferation of the SER. increased formation of lipid droplets which are surrounded by concentric arrays of laminated BR membrames and porphyria (Sleight and Sanger. 1976; Gupta and floore. 1979: Dannan 25 al.. l982b. 1982c: Gupta _5 al.. 1983a). The Firemaster mixture evokes numerous biochemical responses in the liver including the induction of microsomal drug metabolizing enzymes that are normally induced by TCDD and NC and other enyzme which are typically induced by PB and thus Firemaster is classified as a mixed-type inducer (Troisi. 1975; Bent t al.. 1976a. 1976b). Firemaster and 228 several pure PBB congeners have also been shown to cause immunotoxic responses such as a loss of cell-mediated immunity. humoral immune function and thymic atrOphy (Luster 25 al.. 1978; Fraker. 1980: Dannan 25 al.. l982b. 1982c. l982d). In all cases. the amount of Firemaster or PBB congener required to evoke one or more of these TCDD-like responses has been several orders of magnitude higher than that of TCDD. It is generally accepted that the initial event in the mechanism of toxicity of TCDD and similar compounds is the binding of these compounds to the TCDD receptor. The presence of the receptor-ligand complex in the nucleus results in a pleiotrOpic response which includes the initiation of the synthesis of specific mRNAs and protein. There appeared to be good correlations between the binding to the TCDD receptor. induction of enzymes typically induced by TCDD and the TCDD-like toxic responses (Poland and Glover. 1977. 1980: Poland _5 al.. 1979: Greenlee and Poland. 1979; Poland and Knutson. 1982). The classification of PBB and PCB congeners as HC-. PB- and mixed—type inducers has been made using a number of congeners and depend upon their type of microsomal enzyme induction profile and biological effects (Goldstein 25 al.. l977. 1979: Dannan gt al.. 1978a. l982b. l982c. 19826; Poland 25 al.. 1979). The ac- type inducers include few congeners. one of which is 3.4.5.3'.4'.5'-hexahalobiphenyl. Several congeners in this group have been shown to bind to the TCDD-receptor. strictly induce NC- type microsomal enzymes and evoke TCDD-like toxic response (8) (Poland and Glover. 1977; Render :5 al.. 1982: Jensen 25 al.. 229 1983). The second grOUp. referred to as PB-type inducers and represented by 2.4.5.2'.4'.S'-hexahalobiphenyl. have been shown not to bind to the TCDD receptor. strictly induce PB-type enzymes. and do not evoke a TCDD-like toxic response and thus are considered not toxic (Poland 25 al.. 1979: Moore 25 al.. 1978b). Congeners with a halogen at a carbon 9:529 to the bridge gives rise to the last group. namely the mixed-type inducers. These congeners are considered to possess a PB-ring and a MC-ring and represented by 2.4.5.3'.4'.S'-hexahalobiphenyl. They are considered intermediate to the two groups and thus moderately toxic (Dannan at al.. 1978a. 1982b. 1982c; Parkinson 23 al.. 1979. 1980b. 1980c; Robertson :5 al.. 1980. 1981). The presence of these mixed-type inducers in Firemaster has been suggested to be responsible for the TCDD-like aspects of Firemaster since no ortho-unsubstituted PBB congeners were detected (Dannan 25 al.. l982d; Hass 25 al.. 1978; Dekok _g _l.. 1977). Trace impurities of similar chlorinated compounds are present in certain crude mixtures of PCB. and are considered responsible for some of the TCDD-like effects of those mixtures (Hutzinger 25 al.. 1974). This thesis has provided answers to questions regarding the presence and toxicity of ortho unsubstituted PBB congeners in Firemaster. 3.4.3'.4'-TBB (TBB) and 3.4.5.3'.4'-PBB. like 3.4.5.3'.4'.5'-HBB (HBB). is supposed to be among the most potent TCDD-like congeners since their chlorinated analogues are also the most potent of all PCB congeners (Goldstein 25 al.. 1977: Yoshimura t al.. 1979: Robertson 2; al.. 1982). They are however. expected to be nearly 100 fold less potent then TCDD (Poland and Glover. 1977: Poland 25 al.. 1979; Dannan. 1981). 230 Two PBB congeners. namely TBB and HBB. were found to comprise nearly 0.05% and 0.03%. respectively of Firemaster. Certain chemical features of PBB appear similar. whereas their chemical properties vary widely. Differences in their chemical properties should reflect the differences in their biological effects. This is supported by the fact that correlations appear to exist between their chemical and biological properties. An example is the UV-absorption spectral characteristics of TBB and HBB. TBB has a larger K-band with a greater extinction coefficient than does HBB. suggesting that TBB be more coplanar than HBB. Results of the in gitgg TCDD receptor binding studies suggest that TBB is bound 10 x more avidly than HBB. Thus. correlations between a biological property and chemical property do exist. The formation of TBB through the irradiation of 2.4.5.2'.4'.S'-HBB (2.4.S-HBB). the major congener of Firemaster. was lower than expected and the results suggested that the reductive debromination of 2.4.5-HBB did not proceed through ggghg debromination preferentially. This was in agreement with Robertson and coworkers (1983a). who reported using Firemaster. that the amount of TBB was lower than expected and suggested that the stepwise preferential loss of ortho bromines from 2.4.5- HBB to give 2.4.5.3‘.4'-PBB and ultimately TBB is not followed. Studies using sunlight have shown that 2.4.5-HBB degrades readily and produced a very similar GC chromatogram to that attained in this research (Patterson 25 al.. 1981; Chapter 2. Figure 16). It has been suggested that photochemical process may contribute to the breakdown and removal of PBB and PCB from the environment 231 (Ruzo 25 al.. 1974; Ruzo 25 al.. 1975; Ruzo and Zabik. 1975: Ruzo _5 _l.. 1976). The enhanced toxicity of and enzyme induction by the photolyzed 2.4.S-HBB mixture was studied and the congeners responsible for it elucidated. The photolyzed 2.4.5-MBB mixture induced microsomal hepatic enzymes which are classically induced by coadministration of MC and PB. or Firemaster and caused severe histological changes in the liver. The photolyzed HBB mixture acted very similar to Firemaster.being slightly less potent (Table 9 and 10). 2.4.5—HBB. itself. as well as 2.4.5.2'.5'-PBB. previously classified as PB-type inducers. induced PB—type enzymes (Table 10). 2.4.5.3'.4'-PBB. produced through ortho debromination of 2.4.5-HBB. showed strong MC-type as well as PB- type induction. TBB. formed through 95552 debromination of 2.4.5.3'.4'-PBB. produced no PB-type induction and only weak MC- type induction when compared to HBB (Table 10). TBB caused some swelling of hepatocytes but overall did not appear to cause any significant hepatic histological changes. Analysis of hepatic and adipose tissue for PBB levels revealed that TBB and 2.4.5.2'.5'-PBB appeared to be metabolized. whereas 2.4.5.3'.4'- PBB and HBB were not. In giggg metabolism agreed with these findings (Mills 23 al.. 1984). It was concluded that the photolyzed 2.4.5-BBB mixture caused a mixed—type induction and that this enhanced biologic effect was due mostly to 2.4.5.3'.4‘- PBB and to a much lesser extent to TBB. since the latter and most of the other photoproducts present were metabolized. The observation that TBB was a weak MC-type inducer and was memabolized was of interest. The chlorinated analogue (TCB) had 232 been shown to be a better ligand for the TCDD receptor than the chlorinated analogue of HBB (HCB). suggesting that TCB should be more toxic than HCB. Yoshimura and coworkers (1979) however reported that TCB appeared to be a much less potent inducer of hepatic microsomal cytochrome P-448 than HCB. Thus it was of interest to investigate if metabolism of PBB congeners could affect enzyme induction and influence toxicity. The results of the in 21559 binding of TBB and HBB to the TCDD receptor revealed that TBB was roughly 10 x better as a ligand than HBB in both the mouse and rat (Figure 35). An equimolar dose of TBB and HBB was administered to rats and various parameters were then studied over time. Liver weights were significantly increased in rats administered HBB. whereas no increase was seen with TBB treated rats. Hepatic histological changes were much more pronounced in HBB treated rats than TBB treated animals. Thymic involution was evident in both treatment groups. however after day 8. thymic weights in HBB treated rats were significantly lower than those in TBB treated rats. Hepatic microsomal AHH induction was maximal at day 2 in TBB treated rats and then declined over time. whereas it remained elevated in HBB treated animals. Further studies revealed that the in vitro metabolism of TBB correlated very well with the extent of AHH induction (Mills 33 al.. 1984). It was also found that the isozyme . cytochrome P-4SOBNF-B appeared to be responsible for the metabolism of TBB. since the amounts of this isozyme correlated with the amount of AHH activity and rates of in 21559 metabolism of TBB. Similar decreases were seen in hepatic and adipose tissue levels of TBB 233 whereas HBB remained relatively constant. It appeared that TBB was metabolized i vivo at a rate significant to reduce the in 3139 concentration of TBB sufficient to cause its dissociation from the receptor and thus cause a decrease in gene expression. These results support the prOposal of Poland and Knutson (1982). in that persistent receptor occupation by the ligand is required for gene expression and toxicity. which was based on studies using MC. a polycyclic aromatic hydrocarbon which is metabolized. These studies also show that the observations to be made. both in gigg and in 31559. and the time at which these in 3132 observation are made should carefully be considered in toxicity studies. Clearly the time at which the amount of induction. or a toxic response is measured. could result in false conclusions with a congener such as TBB. which is metabolized relatively fast. Also. in vitro studies. such as TCDD-receptor binding studies. can also lead to a false positive result since it does not account for metabolism. as was the case of TBB. From these studies. it is suggested that TBB and HBB in Firemaster are not present in significant amounts to contribute to the toxicity of Firemaster. especially since the former is metabolized. This agrees with a study by Dannan and coworkers (1982d). who reported that reconstituted mixture of Firemaster elicited the same response as Firemaster. It was concluded that 2.4.5.3'.4'-PBB. 2.3.4.2'.4'.5‘-HBB. 2.4.5.3'.4'.5'-HBB and 2.3.4.5.3'.4'-HBB were responsible for the toxicity of and MC-type induction by Firemaster and that some minor trace component would not significantly contribute to the toxicity. The likelihood of cOpurifying a trace toxic component with a congener. such as 234 2.3.4.5.2'.4'.S'-hepatbromobiphenyl (congener g). which coelutes on a GC packed column with HBB. is strong and if HBB were present in significant amounts to add to the observed toxicity. then congener g. when tested for enzyme induction. should have induced AHH activity. which it did not (Moore 25 al.. 1978b). Thus. it does not appear likely that HBB would contribute to the toxicity of Firemaster at the levels detected. More research devoted to the biochemical mechanism (s) of toxicity by TCDD and similar planar halogenated aromatic hydrocarbons such as PBB must be performed in order to understand the toxic changes in the animal. The hypothesis that TCDD evokes its toxic effects by initially binding to the TCDD receptor and then binding of this receptor-ligand complex to a component in the nucleus. most probably chromatin . is also worthy of further investigation and can help explain the results of this research (Poland 25 al.. 1979; Poland and Knutson. 1982). The similarities between the initial action of TCDD and the mechanism of action of steroids and other hormones has not gone unnoticed. However. while hormone stimulation of gene expression normally is under strict regulation in reference to extent and duration. the effects of gene expression of the TCDD-receptor complex may be more persistent. The toxic effects of TCDD and similar agonists may be caused by their sustained occupancy of a biological receptor which normally is involved in binding a hormone and regulating one or more enzymes for very brief periods of time. In effect. the TCDD-receptor complex may interact with the same nuclear sites which normally the hormone-receptor interacts with. 235 However. compared to the hormone-receptor complex. the TCDD- receptor complex could have a stronger binding affinity to nuclear sites. which would result in a more sustained gene expression than normal and this response eventually leads to toxicity. as would be the case for HBB. The amount of TCDD- receptor complex bound to the nuclear sites should remain high over time. whereas the hormone-receptor should have a much shorter occupational time. A shorter occupational time could also be rationalized for a congener which is metabolized fast. such as TBB. These hypothetical events are summarized in the scheme shown below. ” ----------------------- HBB II I ,’ Nuclear I’ Receptor-Ligand I Level I . I TBB foxicify ”'- -------------- III-I..- HBB ’I I, I I .' 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Acad. Sci. 332. 179-192. APPENDIX 254 APPENDIX LIST OF PUBLICATIONS Manuscripts submitted Cynthia D. Millis. Richard A. Mills. Stuart D. Sleight. and Steven D. Aust.Microsoma1 Enyzme Induction by and Toxicology of the Photolysis Products of 2.4.5.2'.4’.5'-Hexabrominated Biphenyl. Submitted to Fund. Appl. Toxicol. Cynthia D. Millis and Steven D. Aust . Characterization of the Photolysis of 2.4.5.2‘.4'.5'-Hexabrominated Biphenyl. Submitted to Fund. Appl. Toxicol. Cynthia D. Millis. Richard A. Mills. Stuart D. Sleight and Steven D. Aust. Toxicity of 3.4.5.3'.4'.5'-Hexabrominated Biphenyl and 3.4.3'.4'-Tetrabrominated Biphenyl. Submitted in Toxicol. Appl. Pharmacol. Richard A. Mills. Cynthia D. Millis. Ghazi A. Dannan. F. Peter Guengerich. and Steven D. Aust. Studies on the Structure- Activity Relationships for the Metabolism of Polybrominated Biphenyls by Rat Liver Microsomes. Submitted to Toxicol. Appl. Pharmacol.