1 . 1 oooooo 9.. 1111111111.. 111111“ .1 7 7 - l 1 . k '3: \ . ' . 1 l '1 ' 11112111151 - 1» ' '1: ’-. .31.. ' q .1' . .’ | ' . ‘ I ' z ’1. 1.1 .;11;11_1:'?§1"111111‘1.;;.:::3 :"' h. ‘ _ 1' ‘1 to” ‘. ”ll . M . . 3 1': , I ' . .3 1”,. ‘ 1111111111 .,1 . . a't. ‘ 14 . . ‘1 .1113 -. "1‘ ,1 'v .v - .. I 1~1.1'v‘"g".;.1:11.".!"111-' ‘ “ 1 11111131111119}, 11 .nv 1 . .1 . , 1 1 ~ 1. . .‘w' 11 ‘.| [1'1111' 11, “13?.” 4 :11. 111‘ ,fi {2%, n.-‘1 :2: :' .1. 1 , 2 1::.- .' 11.11.§ 1-1111‘121‘1 . , - "1., .. . ' 31 . ' "‘1 1“ I ‘1 1 I I ..z ' 3 ' {‘1‘ |t‘§;;: I11. '1' .1 t 1.111 3‘ " ' ‘4“ "A. 1o 1 ‘ i 1 .,‘_. x 111 - . o '12.;fi .11 :‘.' '11' -"‘ :‘V i ‘1 no '1‘“... _.:i. :1"- 1.}! ‘ 'IA' 3'., 1' 1 . 11' ‘1 11" LL 7 : .' . ‘ ‘ 1 I. 1 I ,,,,, :1-1“. , 1 .11111111111’1111'1w11l‘ .1111“ . 111131111111 11111‘1111111111111l1 1" ‘.‘..1. ‘ M11311 1'1 1W 1 1 11 11111.11 11111111|11“111 111111 111111 1 1111111111111,~11'| '1 I1 1 1 1111'” 1.. '1 '11. . 1111111111111 1.1119111. 1:11.11111 111' ‘ 1' 111112111111111111111111111111111111111111111 11111111 ”‘1; 1131 11111111111 1“ 11111 11 .1111 11.11111. 11 11‘. 1.114111 1111 1 111 1 .1111 11~‘«‘\.’~ fiu' l I! ll ll lfllll ll Ill l II! III Ill l I/ 9 10315 This is to certify that the thesis entitled CHEMISTRY AND BIOCHEMICAL PHARMACOLOGY OF POLYBROMINATED BIPHENYL CONGENERS presented by Robert William Moore has been accepted towards fulfillment of the requirements for Ph.D. degree inBiochemistry “fig ééX/J/f Major professor Date log/W457 /7 7g 07639 “ “’99 OFT, ‘ 2 34?. A “.392 515333 9 ‘0 3:, CHEMISTRY AND BIOCHEMICAL PHARMACOLOGY OF POLYBROMINATED BIPHENYL CONGENERS By Robert William Moore A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry l978 ABSTRACT CHEMISTRY AND BIOCHEMICAL PHARMACOLOGY OF POLYBROMINATED BIPHENYL CONGENERS By Robert Hilliam Moore The objectives of this research were to identify the chemical structures of the components of polybrominated biphenyl mixtures (P885), to determine if P88 congeners can be bioactivated into metabolites capable of binding covalently to protein and DNA, and to determine the effects of pure PBB congeners on rat liver microsomal drug metabolizing enzymes. PBBs are complex mixtures which can contain thirty or more compon- ents. however, only the structure of the major Firemaster component, 2,2',4,4',5,5'-hexabromobiphenyl, had been identified. Two PBB mixtures have been fractionated by alumina chromatography and recrystallization, and the resultant purified congeners were analyzed by their lH-NMR. 13C-NMR. and infrared spectra, and by gas chromatography-mass spectro- metry. The major components of Firemaster. in order of their elution by gas chromatography, are 2,2',4,5,5'-penta-, 2,3',4,4',5-penta, hexa-, 2,2'.4,4',5.5'-hexa-, 2,2',3,4,4',5'-hexa-, 2,3'.4.4',5,5'~ hexa-, hepta- and hexa-. 2,2',3,4,4',5,5'-hepta-, hepta-. hepta-, octa-, and 2,2',3,3',4,4',5,5'-octabromobiphenyl. Similarly, the major components of a higher molecular weight PBB mixture are 2,2',4,4',5,5'- hexa-. 2,2',3.4,4',5,5'-hepta-. hepta-, octa-, 2,2',3,3',4,4',5,5'- octa-, 2,2',3,3',4,4'.5,5',6-nona-, and 2,2',3,3',4,4',5,5',6,6'-deca- bromobiphenyl. The possibility that PBB components can covalently bind to cellular Robert William Moore macromolecules was investigated by aerobically incubating 1"C-PBBs with rat liver microsomes and NADPH. The substrate consisted almost exclu- sively of 2,2',4,4',5,5'-hexabromobiphenyl (H886) and 2,2',3,4,4',5,5'- heptabromobiphenyl (H887). Following exhaustive organic extractions, less than 0.05% of the radioactivity was found in the microsomes, regardless of whether control microsomes, or the microsomes from 3-methylcholanthrene-, phenobarbital-, or PBB-pretreated rats were used. When DNA was included in these incubation mixtures, no binding of P885 or their metabolites could be detected. It thus appears that the two major Firemaster components are not metabolically activated into reactive species. Parallel incubations demonstrated the covalent binding of 3H-benzo[a]PYrene metabolites to DNA, and that the micro- somes isolated from PBB-pretreated rats enhanced this binding six-fold. H886 and H837 comprise 56 and 27%, respectively, of the Firemaster mixture of P885. The effects of H886, H887, and of the suspected trace component 2,2'-dibromobiphenyl (DBB) on liver microsomal drug metaboliz- ing enzymes were examined. Rats were injected i.p. with 90 mg/kg of these compounds. and sacrificed at intervals up to twenty-two days later. H886 and H887 increased liver weights, and strongly induced microsomal protein, NADPH-cytochrome P450 reductase. cytochrome P450. aminopyrine demethylation, and epoxide hydratase. Both caused only small inductions in benzo[a]pyrene hydroxylation and p-nitrophenol-UDP- glucuronyltransferase, and neither shifted the cytochrome P450 spectral . maximum from 450 nm. These results, and the results of SDS-polyacryl- amide gel electrophoresis. demonstrate that H886 and H887 affect micro- somes in a manner very similar to phenobarbital, and that their Robert William Moore inductions are distinct from those caused by either 3-methylcholanthrene or P885. 088 had little if any effect on any parameter examined. a result which demonstrates that not all brominated biphenyls are micro- somal inducers. While H886 and H887 are both strictly phenobarbital-type inducers of liver microsomal drug metabolizing enzymes, PBBs cause a mixed-type induction of these enzymes. Seventeen percent by weight of Firemaster remains uncharacterized; one or more of these components must be responsible for the 3-methylcholanthrene-like aspects of the induction caused by the P88 mixture. ACKNOWLEDGMENTS I wish to thank my thesis advisor, Steve Aust. for his guidance and suggestions. It is also a pleasure to acknowledge other members of the lab for their help, advice, and friendship. including Bruce Svingen, Fred O'Neal, John Buege, Tom Pederson. and especially Ann Welton. I have collaborated on PBS-related research (not presented in this Thesis) with Cathy Troisi and Ghazi Dannan, to whtl trust has been our mutual benefit. .I have been extremely fortunate to have been assisted at various times by Jane Albright, Fred “Results" Askari. Debi Metcalf, and Poonsin Olson. I am grateful to all four, whose help was uniformly excellent and valuable, but especially so to Poonsin, because she helped the longest, and through the most difficult parts. ’ A number of individuals are deserving of special thanks. John Dent and Kevin McCormack provided preprints of their papers, Larry Besaw provided part of the heptabromobiphenyl and assisted with some of the animal work, Joelle Andre performed the GC-MS analysis. the MSU Chemistry Department (Frank Bennis) determined the 1H-NMR spectra, John O'Conner and John Pierce determined the l3C-NMR spectra, and Paul Kindel lent us his GC thereby making this whole project possible. A very special thanks goes to Stuart Sleight of the Department of Pathol- ogy, who performed all the pathological analyses of the experimental animals. His work has added greatly to the value of the research we have performed. ii y I thank NIH for a predoctoral traineeship, and a variety of people in and out of the Department for their friendship, and for advice and assistance on matters large and small. Lastly. Bob Bernstein, Ed Faleski, Rosemary Schraer, and the members of The Intergalactic Society provided crucial support, moral and otherwise, when my chances for a career as a scientist were quite precarious and when support was needed the most. In particular. without Bob's encouragement, this Thesis would not have been possible. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . ORGANIZATION . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTION . .'. . . . . . . . . . . . . . . . . . . . . LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . . . . Microsomes . . . . . . . . . . . . . . . . . . Microsomal Drug Metabolizing Enzymes . . . Induction of Microsomal Drug Metabolizing Enzymes . Consequences of Induction of Microsomal Drug Metabolizing Enzymes . . . . . . . . . . . . . . . . Polyhalogenated Biphenyls and Chemical Carcinogenesis . . Induction of Microsomal Drug Metabolizing Enzymes by PBBs Conclusions . . . . . . . . . . . . . . . . . . . . . . . CHAPTER I: PURIFICATION AND CHARACTERIZATION OF POLYBROMINATED BIPHENYL CONGENERS . . . . . . Abstract . . . . . . . Introduction . . . . Materials and Methods . Materials . . . . . . Gas Chromatography . . Gas Chromatography-Mass Spe Determination of Spectra . . . . . . Other Determinations . . . . . . Fractionation of Firemaster and 088 Mixtures General Procedures . . . . . . . . . . . . . . Purification of Individual Bromobiphenyl Congeners Nomenclature . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . Characterization of Peak 1 . Characterization of Peak 2 Characterization of Peak 4 Characterization of Peak 5 Characterization of Peak 6 . O O O O C O O O 0 iv 0 O O O O O O O Page vi vii Characterization of Peak 8 Characterization of Peak ll Characterization of Peak 12 Characterization of Peak 13 Discussion CHAPTER 2: BIPHENYLS T0 PROTEIN AND DNA Animals . . . . . . . . Isolation of Microsomes . . . Preparation of Reaction Materials Incubation Conditions . . . . . Binding of1 Abstract . . . . . . . . . . . . . Introduction . . . . . . . . Materials and Methods , . . . . . Materials . . . . . . . . . . . STUDIES ON THE COVALENT BINDING OF POLYBROMINATED “C- P885 to Microsomal Macromolecules Binding of 1'‘C- P835 and 3H- Benzo[aIPYrene to DNA Analysis of 1"Cu PBBs for Metabolites Other Methods . . . . . . . . . . . . Results . . . . . . Discussion CHAPTER 3: Abstract . . . . . . . . . Introduction . . . . . . . Materials and Methods . . . Materials . . . . . . Preparation of Brominated Biphe y 5 Animals . . . . . . . . . . Necropsy Examination . . . Isolation of Microsomes . . Aminopyrine Demethylation . Benzo[a]pyrene Hydroxylation Epoxide Hydratase . . . . . . UDP-Glucuronyltransferase . . SDS-Polyacrylamide Gel Electrop Other Assays Results . Discussion 06 i §~ 00.30.000.030... ooomoooooooooooo o o o (no 0 o o o o 0 do REFERENCES 0 O C O O O O O O O O O O O 0 APPENDIX: LIST OF PUBLICATIONS . . . . . EFFECTS OF PURE POLYBROMINATED BIPHENYL CONGENERS ON RAT LIVER MICROSOMAL DRUG METABOLIZING ENZYMES O O O I O O O O O O O O O O 102 103 104 '105 105 107 107 108 108 108 109 111 112 113 115 116 139 158 165 Table 1 LIST OF TABLES Page Determination of the Number of Bromines Present in Polybrominated Biphenyl Congeners . . . . . . . . . . . 43 Proton Chemical Shifts in Polybrominated Biphenyl Congeners . . . . . . . . . . . . . . . . . . . . . . . 77 Effects of Microsomal Enzyme Induction on the In Vitro NADPH-Dependent Covalent Binding of 1“C-PBB Metabolites to Microsomal Macromolecules . . . . . . . . . . . . . . Effects of Microsomal Enzyme Induction on the In_Vitro NADPH-Dependent Covalent Binding of ll‘C-PBB and 3H- Benzo[a]pyrene Metabolites to DNA . . . . . . . . . . . 94 Summary of the Effects of Pure Polybrominated Biphenyl Congeners on the Type of Induction of Microsomal Drug Metabolizing Enzymes . . . . . . . . . . . . . . . 152 vi Figure 1 10 11 12 13 14 LIST OF FIGURES Gas Chromatographic Profiles and Numbering System for the Components of Polybrominated Biphenyl Mixtures 1H-NMR Spectrum and Structure of 2,2',4.5,5'-Penta- bromobiphenyl (Peak l) . . . . . . . . . . . . . . . . 1H-NMR Spectrum and Structure of 2,2',4,5,5'-Penta- bromobiphenyl (Peak 1) . . . . . . . . . . . . . . . . 1H-NMR Spectrum and Structure of 2.3',4,4',5-Penta- bromobiphenyl (Peak 2) . . . . . . . . . . . . . . . . 1H-NMR Spectrum and Structure of 2,2',3,4,4'.5'- Hexabromobiphenyl (Peak 5) . . . . . . . . . . . . . . 1H—NMR Spectrum and Structure of 2,3',4,4',5,5'- Hexabromobiphenyl (Peak 6) . . . . . . . . . . . . . . . 13C-NMR Spectrum of 2,3',4,4'.5,5'-Hexabromobiphenyl (Peak 6) O O O O O O O O O C O C O O O O O C O C O O 0 1H-NMR Spectrum and Structure of 2,2',3,4,4',5,5'- Heptabromobiphenyl (Peak 8) . . . . . . . . . . . . . 13C-NMR Spectrum of 2,2'.3,4,4',5,5'-Heptabromobiphenyl (Peak 8) O O O O O O O I O O O C O O C O O O O I O O 0 1H-NMR Spectrum of Peak 11 . . . . . . . . . . . . . . . 1H-NMR Spectrum and Structure of 2,2'.3,3',4,4',5,5'4 0ctabromobiphenyl (Peak l2) . . . . . . . . . . . . . 13c-NMR Spectrum of 2.2',3,3i,4,4',5,5'-Octabromobiphenyl (Peak 12) O O 0 O C O C C O O I O C O O C O O C O C . 1H-NMR Spectrum and Structure of 2,2',3.3',4,4',5,5',6- Nonabromobiphenyl (Peak 13) . . . . . . . . . . . . . Infrared Spectra and Structures of Five Pure Polybromin- ated Biphenyl Congeners . . . . . . . . ... . . . . . vii Page 42 47 50 53 56 57 59 61 62 65 67 69 7O 72 Figure Page 15 Gas Chromatographic Profile of Polybrominated Biphenyls (Firemaster) and Structures of Its Components . . . . . 74 16 Gas Chromatographic Profile of Polybrominated Biphenyls (Octabromobiphenyl) and Structures of its Components . . 75 17 Gas Chromatographic Profiles of ll‘C-PBBs and Firemaster P885 0 O O O O O O O O O O O I O O O C O O O O O I O O O 91 18 Distribution of Radioactivity Following Thin Layer Chromatography of the Organic Extracts of Microsomal Incubations with 1“C-PBBs . . . . . . . . . . . . . . . 97 19 Gas Chromatographic Profiles and Structures of 088, H886, andHBB7coo00000000000000.00000‘01‘8 20 Effect of H886 on Liver Structure . . . . . . . . . . . . 121 21 Effects of 90 mg OBB, H886. and HBB7/kg on Liver Weight . I23 22 Effects of 90 mg 088, H886, and HBB7/kg on Microsomal PrOtein oooooooooooooooooooooooo126 23 Effects of 90 mg 088, H886, and HBB7/kg on NADPH- Cytochrome P450 Reductase . . . . . . . . . . . . . . . 129 24 Effects of 90 mg 088, H886. and HBB7/kg on Cytochrome P450 0 O O I O O O O O O O O O O O O I O O O O A. O O O O 131 25 Effects of 90 mg DBB. H886, and HBB7/kg on Aminopyrine DEMthyTaCIOn 00000000000000.000000133 .26 Effects of 90 mg 088, H886, and HBB7/kg on Benzo[a]PYrene HYdroxy1ation . O O C O C . C C C O C . C . C Q . . . O 136 27 Effects of Polybrominated Biphenyl Congeners and Other Xenobiotics on the Protein and Heme Profiles of Microsomes Subjected to SDS-Polyacrylamide Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . 133 28 Effects of 90 mg 088, H886, and HBB7/kg on Epoxide Hydratase ooooooooooooooooooooooo14] 29 Effects of 90 mg 088, H886, and HBB7/kg on UDP- Glucuronyltransferase . . . . . . . . . . . . . . . . . 143 viii LIST OF ABBREVIATIONS butylated hydroxytoluene 2,6-ditert-butyl-p-cresol C control Ci Curie cpm counts per minute DBB 2.2'-dibromobipheny1 DCB 2.2'-dichlorobipheny1 dimethyl-POPOP l.4-bis[2-(4-methy1-5-phenyloxazolyl]- benzene DNA deoxyribonucleic acid HBB6 2.2',4,4',5,5'-hexabromobipheny1 H887 2,2',3,4,4',5,5'-heptabromobiphenyl HCB6 2,2',4,4',5,5'-hexadhlorobiphenyl HCCH l,2.3,4,5.6-hexach1orocyclohexane Hz Hertz (cycles per second) i.p. intra peritoneal MC 3-methylcholanthrene NADH B-nicotinamide adenine dinucleotide, reduced form NADP+ B-nicotinamide adenine dinucleotide phosphate NADPH B-nicotinamide adenine dinucleotide phosphate. reduced form NMR nuclear magnetic resonance ix 038 Pb PBBs PCBs PCN PEG PPm PPO RNA 505 SE TCA TLC‘ Tris UDP octabromobiphenyl PBB mixture phenobarbital polybrominated biphenyls polychlorinated biphenyls pregnenolone-lGa-carbonitri1e polyethylene glycol parts per million 2,5-diphenyloxazole ribonucleic acid sodium dodecyl sulfate standard error of the mean trichloroacetic acid thin layer chromatography tris (hydroxymethyl)aminomethane uridine 5'-diphosphate ORGANIZATION Research has been perfbrmed on the chemistry of P88 mixtures, the binding of P88 metabolites to macromolecules, and the effects of P885 and purified PBB components on microsomal drug metabolizing enzymes. Each of these topics is described in a separate Chapter, and is pre- sented in a format similar to that used in most scientific journals. These Chapters are preceded by a Literature Review in which microsomal drug metabolizing enzymes, and the effects of P885 on these enzymes, are discussed. All references cited in these four sections are pre- sented together at the end of the Thesis. A brief history of P88 con- tamination incidents, with a separate list of general references, is included in the Introduction. The Introduction does not deal strictly with science, and unlike the other sections of this thesis, it is not fully referenced. Unless stated otherwise, the term PBBs refers to industrial mix- tures of polybrominated biphenyls, including whatever non-polybrominated biphenyl contaminants may be present. In Chapters 2 and 3, the terms PBBs and Firemaster are used interchangeably. The terms cytochrome P450 or cytochrome P450 hemoproteins, Unless specifically stated otherwise. refer to the sum total of all such microsomal hemoproteins, whether or not Amax is at 450 nm. The term MC (or HBBG, etc.)-induced microsomes is frequently used. This refers to the microsomes isolated from organs of animals which had been pretreated with MC (or other agents) before sacrifice. INTRODUCTION Polybrominated biphenyls (PBBs) were manufactured in response to a growing need for flame retardant textiles, plastics, electronic parts, ‘ and other components. They have, unfortunately, made their way into the environment, with serious consequences. A major contaminatiOn be- gan in 1973, when PBBs were mistaken for a mineral supplement (magne- sium oxide) and added to cattle feed in Michigan. Because the error was undetected for a number of months, most residents of the state con- sumed contaminated milk, beef, and other products, and built up small amounts of PBBs in their blood, fat, and milk as a result. It appears that 500 to 1000 or more pounds of P885, averaging 75% bromine by weight, entered the food chain. The consequences of the contamination in Michigan have been enor- mous. Thirty-thousand cattle, 6000 hogs, 1500 sheep, and 1,500,000 chickens have died as a result, although nearly all of these were de- liberately sacrificed because they were too heavily contaminated to be sold. In addition, 850 tons of feed, and several dozen tons of dairy products have been destroyed. At least $30,000,000 in damages have been paid, with numerous cases still in court or awaiting trial. PBBs have been a cause of widespread concern among the general Michigan popUlation, particularly among women contemplating breast feeding their babies, and among the most heavily exposed farm population. The fact that it was a dairy farmer, Rick Halbert, who was the motivating force 2 3 behind the eventual identification of P885 as an environmental contami- nant does not speak well for a number of institutions, governmental or otherwise. To put it mildly, most people in positions of authority have not distinguished themselves by their responses to the P88 prob- lem. Fortunately, the preponderance of scientific evidence now indi- cates that PBBS are not highly toxic, and in that sense the residents of Michigan have been very lucky, because there is little reason to believe that a more toxic environmental contaminant would have been re- moved from the food supply any more quickly. In 1977, PBBS were found in human hair, fish, plants, soil, and water in the vicinity of P88 manufacturing plants in New York and New Jersey. More recently, PBBs have been reported in fish in the Ohio River. The Environmental Protection Agency was also reported to be "urgently" examining fish, human hair, and breast milk around plants in Illinois, California, Mississippi, Ohio, Tennessee, and Pennsylvania where PBBs are used in large amounts. Since PBBs are also manufactured in Europe, it does not seem unreasonable to expect environmental con- tamination to be discovered there as well. Because thousands of tons of P885 have been manufactured, and because of their stability and per- sistence, it is likely that the effects of those PBBs already in the environment will be felt for a number of years, and that future inci- dents of contamination will occur as a consequence of the disposal of materials containing PBBS. PBB research in Dr. Aust's lab began in the spring of 1975. The initial experimentswere designed to determine the effects of P885 on rat liver microsomal drug metabolizing enzymes. Work done mainly by Cathy Troisi, with my collaboration, demonstrated that PBBs are excellent inducers of these enzymes (see Catherine Troisi, Masters Thesis, Michigan State University, 1975). At about the same time, I began to more fully investigate the properties of P885, both chemically and bio- logically. Ghazi Dannan and I initiated a collaborative project de- signed to determine the effects of P885 on drug metabolizing enzymes in lactating and nursing rats. The results demonstrated that PBBs could exert their full range of effects by being transmitted through ratS' milk, and that nursing pups were more sensitive to the effects of P885 than were the dams. The research described in this Thesis was then initiated. PBBs were found to be a mixture of many different chemicals, and while nu- merous useful studies can be and have been done with mixtures of chem- icals, such studies only rarely elucidate the mechanisms by which bio- logical effects are manifested. The example provided by vitamin 0 re- search serves as a perfect case in point. For several decades, little progress in this area was made, until DeLuca, Norman, and others frac- tionated vitamin D preparations, identified the components. and began to investigate the biological effects of these purified components. It is now known that most of the effects once attributed to the major compounds are actually caused by trace quantities of very potent de- rivatives, and the mechanisms by which vitamin 0 acts are consequently much more thoroughly understood. , Nearly every research project on P885 has been conducted knowing only the general chemical formula for P885 and the structure of the major component of the mixture. However, as demonstrated in Chapter 3, and by the metabolic investigations by Ghazi Dannan, not all brominated biphenyls have the same biological effects or metabolic fates, nor 5 shoUld they be expected to. Fractionation and characterization of P88 components, and research on the purified components, is vital to any attempt to understand the pharmacokinetics and pharmacodynamics of P885, or to doing something as fundamental as assaying the proper com- ponent(s) when evaluating the extent of environmental contamination. Because such knowledge is central to all biological studies, the chemical structures of P88 components had to be identified. The first objective of this research was therefore to find out what PBBs consist of. In several cases, structures were elucidated by analyzing nearly homogeneous preparations. Other congeners, while enriched 10-30-fOld, were still not homogeneous, but structural identification was still achieved. The second objective was to determine some of the biological properties of purified PBB components. This work primarily concerns the effects of P88 congeners on liver microsomal drug metabolizing enzymes. The relative levels of these enzymes is of more than just academic interest, because changes in these levels affect the pharma- cology, toxicology, and carcinogenicity of a variety of other exogenous compounds. Enzyme induction also alters steroid metabolism, which can result in a variety of secondary responses. Even the immune system may be affected by induction; polychlorinated biphenyls are immunosup- pressive, perhaps by inducing adrenal drug metabolizing enzymes, thereby increasing the serum levels of corticosteroids and causing atrophy of the thymus. Although with one exception the secondary ef- fects of microsomal enzyme induction by either P885 or their purified constituents were not evaluated, it is hoped that such research will be conducted in the future. 6 To this date, the only chemical structure reported in the litera- ture for PBB components is that of the major congener. Chapter 1 pre- sents the structures of eight additional congeners. Several other con- geners have been partially characterized. Chapter 2 describes the results of a brief set of experiments de- signed to determine whether or not several of the P88 congeners can be. enzymatically activated into metabolites capable of binding to cellular macromolecules including DNA. Chapter 3 represents the beginning of biological experiments with pure PBB components. The two major congeners in Firemaster, which to- gether comprise greater than 80% of the mixture of P885 which contami- nated the Michigan food chain, were isolated from crude PBB mixtures, and their effects on liver microsomal drug metabolizing enzymes were examined. The effects of a third, rapidly metabolized congener were also determined. The results serve to underscore the point that PBBs consist of a variety of chemicals whose biological effects can differ widely. Additional purified congeners will be tested in the near fu- ture in Dr. Aust's lab for their effects on microsomal drug metabolizing enzymes. References on the history of P88 contamination incidents are given below. Anonymous (1977). Time, May 10, 75-76. Anonymous (1977). Chem. and Eng. News §§_(26), 16. Anonymous (1977). The Lancet II, 19-21. Carter, L.J. (1976). Science 122, 240-243. Culliton, B.J. (1977). Science 191, 849. Dunckel, A.E. (1975). J. Am. Vet. Med. Assoc. 161, 838-841. 7 Jackson, T.F., and Halbert, F.L. (1974). J. Am. Vet. Med. Assoc. 165, 437-439. Kay, K. (1977). Environ. Res. 11, 74-93. Mercer, H.D., Teske, R.H., Condon, R.J., Furr, A., Meerdink, G., Buck, W., and Fries, G. (1976). J. Toxicol. Environ. Health 1, 335-349. Stadtfeld, C.K. (1976). Audubon 18, 110-118. LITERATURE REVIEW Introduction This Thesis is concerned with the chemistry and biochemical phar- macology of polybrominated biphenyl congeners. Although a massive literature exists concerning the closely related polychlorinated bi- phenyls (PCBs), I will not attempt to review it here, except for cer- tain relevant aspects. The book by Hutzinger gg_a1, (1974) is an ex- cellent presentation of the chemical aspects of PCBs, and reviews of the toxicity of PCBs and related compounds by Kimbrough (1974) and by Fishbein (1974) are also extremely useful. An additional source of information concerning PCBs, their contaminants, and related molecules may be found in Volumes 1 and 5 of Environmental Health Perspectives (1972, 1973), a journal published by the National Institute of Environ- mental Health Sciences which presents the results of various symposia on environmental chemicals. Four years ago, virtually nothing was known about polybrominated biphenyls (PBBS). The contamination incident in Michigan has provided the impetus for a large amount of research on P885, and the literature is now expanding rapidly. Again, no effort will be made to review all papers dealing with P885, although a considerable number will be dis- cussed. On October 24 and 25, 1977, a workshop on the Scientific Aspects of Polybrominated Biphenyls-PCB was held at Michigan State University, and virtually every research group to have investigated P885 8 9 presented their results. The proceedings of this conferenceare sched- uled to be published in the April 1978 issue of Environmental Health Perspectives, and the interested reader is referred to this volume for information concerning the entire range of research on P885. 1 The literature on the chemistry of PC85 is relatively straight- forward, while that concerning P885 is essentially nonexistent. Both will be presented as needed in Chapter 1, which deals strictly with chemistry. In order to fully understand the results presented in Chapters 2 and especially 3, which deal with the biochemical aspects of the research, some knowledge of microsomal drug metabolizing enzymes is required. This Literature Review is therefore concerned with the nature of these microsomal enzymes, their inducibility by P885 and other chemicals, and the biological consequences of induction of micro- somal drug metabolizing enzymes. Microsomes Microsomal drug metabolizing enzymes have been found in many mam- malian tissues, however, the vast majority of research on these en- zymes has been with liver. This discussion will therefore deal pri- marily with hepatic microsomes. . Microsomes are formed during tissue homogenization as the endo- plasmic reticulum breaks up and reseals as small spheres. This process appears to not alter the enzymatic properties of the constituents. Following homogenization, the microsomes are isolated by differential centrifugation (Claude, 1969). Microsomes are often rehomogenized in a buffer containing a chelating agent in order to detach ribosomes and adsorbed proteins, then repelleted by ultracentrifugation to obtain a 10 more pure membrane preparation, designated washed microsomes (Walton and Aust, 1974a; and many others). Microsomal Drug Metabolizing Enzymes Microsomes contain an NADPH-dependent electron transport chain which catalyzes the hydroxylation of a wide variety of substrates, both endogenous and exogenous. The first component is NADPH-cytochrome P450 reductase, a flavoprotein which transfers reducing equivalents from NADPH to the terminal oxidases. Because of its ability to reduce exog- enous cytochrome c, it is often referred to as NADPH-cytochrome c re- ductase (Gillette gt_a1,, 1972). There appear to be at least eight different terminal oxidases in rat liver microsomes (Guengerich, 1977); these are the cytochrome P450 hemoproteins. The combination of NADPH- cytochrome P450 reductase with the cytochrome P450 hemoproteins is often referred to as the microsomal mixed-function oxidase system (Conney, 1967; Gillette 31_a1,, 1972). i More than two hundred substrates with diverse structures can be metabolized by the cytochrome P450 hemoproteins (Conney, 1967), in a variety of overall reactions, all of which incorporate one atom of oxygen from molecular oxygen into the substrate as a hydroxyl group or epoxide. The other atom of oxygen is incorporated into water (Gillette ‘91 11,, 1972; Gunsalus e__a1,, 1975). The cytochrome P450 substrates include endogenous compounds such as fatty acids and steroids, as well as numerous exogenous foreign compounds (xenobiotics) such as drugs and environmental pollutants (Conney, 1967; Kuntzman, 1969; Gillette at 31,, 1972; Conney and Burns, 1972). In many cases, the hydroxylated product is unstable, and so the net reaction appears to be something 11 different, such as an N-demethylation (Brodie et_ 1., 1958); Gillette ._£H_l:9 1972). The eight or more cytochrome P450 hemoproteins differ in their substrate specificities (Guengerich, 1977), which in part accounts for the capacity of the system to metabolize so many diverse substrates. Many of the cytochrome P450 metabolic Products can in turn serve as substrates for several additional enzymes. Epoxides can either rearrange nonenzymatically to form phenols, or they can be converted into far less reactive dihydrodiols by the action of epoxide hydratase. Both the formation (by cytochrome P450 hemoproteins) and cleavage (by epoxide hydratase) of epoxides are of considerable interest to persons investigating chemical carcinogenesis, because many epoxides are un- stable electrophilic compounds which can covalently bind to protein, RNA, and DNA. Epoxides can also be conjugated with reduced glutathione, both nonenzymatically and as catalyzed by a family of glutathione-S- epoxide transferases (Sims and Grover, 1974; Jerina and Daly, 1974). Some hydroxylated cytochrome P450 metabolites are conjugated with glucuronic acid by the action of a family of enzymes called UDP- glucuronyltransferases (Dutton, 1975; Parke, 1975). Conjugations to different sugars, such as galacturonic acid, have also been reported (Vessey and Zakim, 1973), although these appear to be minor pathways. In general, both the mixed-function oxidases and the above-mentioned enzymes convert hydrophobic molecules into more hydrophilic derivatives, which can then be more easily excreted in the bile or by the kidneys. 12 Induction of Microsomal Drug Metabolizing Enzymes One interesting property of the microsomal drug metabolizing en- zymes is the ability of compounds (often substrates) to induce the enzymes which catalyze drug metabolism. The two prototype inducers are the barbiturate phenobarbital (Pb) and the carcinogen 3-methyl- cholanthrene (MC), each of which alone causes a distinct and character- istic pattern of induction. Within the past several years, evidence has been presented for the existence of an additional distinct inducer, pregnenolone-lEe-carbonitrile (PCN). Because of the differing biologi- cal implications of induction by these prototype compounds, much re- search effort has gone into characterizing the type of microsomal in- duction caused by various agents. Pb causes liver enlargement, and a large increase in the quantity of microsomal protein. NADPH-cytochrome P450 reductase is strongly induced, and the total amount of cytochrome P450 is greatly increased. The spectral maximum in the reduced carbon monoxide difference spectrum is at 450 nm, and there is no change in the ratio of the 455 nm/430 nm peaks of the reduced ethyl isocyanide difference spectrum. Pb induces the cytochrome P450-catalyzed N-demethylation of benzphetamine, amino- pyrine, and ethylmorphine, and the aliphatic hydroxylation of hexobar- bital, pentobarbital, and the 16 a position of testosterone. These activities are at most only slightly induced by MC (Conney, 1967; Parke, ‘ 1975). Pb also strongly induces epoxide hydratase (Bresnick gt_a1,, 1977), the UDP-glucuronyltransferase-catalyzed glucuronidation of chloramphenicol (Bock a; a1,, 1973), and one or more 45,000 dalton microsomal hemoproteins (Welton and Aust, 1974b). Induction by MC causes none of these effects. 13 MC has little effect on liver weights, and causes a smaller in- crease in microsomal protein than does Pb. It has no effect on NADPH- cytochrome P450 reductase, and while it induces the total amount of cytochrome P450, the magnitude of the induction by NC is smaller than that caused by Pb. MC shifts Amax in the reduced carbon monoxide dif- ference spectrum to 448 nm, and increases the ratio of the 455 nm/430 nm peaks in the reduced ethyl isocyanide difference spectrum. Unlike the strong inductions caused by Pb, MC has little effect on the cyto- chrome P450-cata1yzed metabolism of benzphetamine, aminopyrine, ethyl- morphine, hexobarbital, pentobarbital, or the 16 a position of testos- terone (Conney, 1967; Parke, 1975). Unlike Pb, MC strongly induces the aromatic hydroxylation and epoxidation of benzo[a]pyrene, the hydroxyla- tion of biphenyl at the 2 position (Parke, 1975), and the O-deethylation of ethoxyresorufin (Burke gt_a1,, 1977) and ethoxycoumarin (Thomas e1_ a1,, 1976). MC also has no effect on epoxide hydratase (Bresnick, 1977), it induces the conjugation of p-nitrophenol but not chlorampheni- col by UDP-glucuronyltransferases (Bock g;_a1,, 1973), and it induces one or more 53,000 dalton microsomal hemoproteins (Welton and Aust, 1974b). Both MC and Pb also increase the metabolism of a variety of other cytochrome P450 substrates, such as p-nitroanisole (Conney, 1967), but assays of these activities provide no information as to the nature of the microsomal induction. The effects of PCN on microsomal drug metabolizing enzymes have not been well characterized. It induces NADPH-cytochrome P450 reduc- tase, and appears to cause partial inductions of several cytochrome P450-catalyzed activities, but these are all also induced by either MC 14 or Pb. PCN causes a slight shift in the reduced carbon monoxide dif- ference spectrum towards 449 nm (Lu gt_a1,, 1972; Birnbaum g1_a1,, 1976). Its only unique feature is the induction of the 50,000 dalton hemopro- tein, which is the hemoprotein which predominates in control microsomes (Birnbaum _t.a1,, 1976). For the remainder of this thesis, the PCN- type induction of microsomal enzymes will be ignored, and microsomes will be considered to be control, MC, Pb, or mixed-type (see below). It is hoped, however, that future research on drug metabolism will in— clude the development of assays specific for each different cytochrome P450 hemoprotein, so that more refined analyses of the nature of induc- tions caused by various compounds, such as halogenated biphenyls, can be made. A One term which should be defined is "mixed-type inducer." This refers to an inducing agent which causes the responses in microsomes seen fbllowing the administration of both MC and Pb. Hexachlorobenzene. 2,2',3,3',4,4'-hexachlorobiphenyl, and 2,2',3,4,4',5'-hexachlorobiphenyl are the only single chemicals reported to cause a mixed-type induction (Stonard and Nenov, 1974; Stonard, 1975; Stonard and Greig, 1976), whereas mixtures of chemicals can easily cause such an induction pro- vided that both MC- and Pb-type inducers are present. Consequences of Induction of Microsomal Drug1Metabolizing Enzymes The induction of microsomal drug metabolizing enzymes can have pharmacological, toxicological, physiological, and Carcinogenic conse- quences. Microsomal enzymes can both activate and inactivate drugs, and changes in the levels of these enzymes can alter the pharmacological 15 effects of numerous drugs. Phenobarbital treatment decreases the plasma concentrations of the oral anticoagulant bishydroxycoumarin, hydrocorti- sone (cortisol) administration enhances the elimination of antipyrine, and cigarette smoking decreases the plasma concentration of phenacetin; these and numerous other interactions are attributed in largepart to the induction of microsomal enzymes. Many drugs also stimulate their own rates of metabolism through induction, which in part explains cer- tain cases of drug tolerance (Breckenridge, 1975). The toxicity of certain compounds can be altered by enzyme induc- tion. Bromobenzene is capable of causing hepatic necrosis and death, and pretreatment of rats with Pb enhances bromobenzene toxicity, while MC decreases the toxicity. In Pb-pretreated rats, the major metabolite is 3,4-bromobenzene epoxide, which is capable of depleting reduced glutathione levels and covalently binding to cellular macromolecules. When rats are pretreated with MC, bromobenzene metabolism is not mark- edly accelerated, the less reactive 2,3-bromobenzene epoxide is the major product, and toxicity is reduced (Gillette e1_a1,, 1974; Parke, 1975). The hepatotoxicity of carbon tetrachloride is likewise influ- enced by the nature of microsomal enzymes. Rats pretreated with Pb are far more susceptible to CClB hepatotoxicity than are control rats, while MC induction of microsomes has a protective effect. When rats 'are given a sublethal dose of carbon tetrachloride, then challenged with what would normally be a lethal dose, no rats die, because the first dose destroyed much of the cytochrome P450 responsible for bio- activating the molecule into its reactive intermediates (Recknagel and Glende, 1973). . 16 Enzyme induction can have a variety of physiological consequences, particularly on the metabolism of steroids. Pb induces both the bio- synthesis and breakdown of cholesterol, the hydroxylation of cholic ‘acids, the 6 B—hydroxylation of cortisol, and the hydroxylation of testosterone, estrogens, and progesterone. A variety of secondary ef- fects can result from these changes (Parke, 1975). Of perhaps the greatest interest and importance are the effects of microsomal induction on chemical carcinogenesis. Polynuclear aro- matic hydrocarbons are not themselves carcinogenic. They are, however, bioactivated by cytochrome P450 hemoproteins into reactive intermedi- ates, such as epoxides and dial epoxides, which can be highly mutagenic and carcinogenic (Sims and Grover, 1974; Jerina and Daly, 1974). The dependence of mutagenicity on activation for many compounds can clearly be demonstrated, for example, by the Ames test for mutagenicity. Bac- teria incubated with most carcinogens are not mutated unless both NADPH and microsomes, preferably induced microsomes, are included in these incubations (Ames gt_a1,, 1973). Studies of the effects of induction on chemical carcinogenesis have yielded mixed results. Depending on the type of induction and the chemical being tested, either protection against or increased sus- ceptibility to a secondary chemical carcinogen can be observed (Jerina and Daly, 1974; Parke, 1975). While PBBs have not been tested in this regard, PCBs have been shown to protect against some carcinogens and enhance the effects of others. These results will be discussed below. 17 Polyhalogenated Biphenyls and Chemical Carcinogenesis Both PCBS and P885 are complex mixtures consisting primarily of halogenated biphenyls, but other halogenated compounds including naph- thalenes and dibenzofurans can often be present in small amounts. (Fishbein, 1974; Hutzinger 91_a1,, 1974; Kay, 1977). Because no re- search has been performed on the relationship between purified or par- tially purified halogenated biphenyls and chemical carcinogenesis, this discussion concerns the effects of these mixtures as a whole, including their impurities. Multiple adenomatous nodules were observed in the livers of rats fed approximately one gram of PCBS (Kanechlor-400) over a four hundred day period. These nodules, which were found only in the female rats, were considered to be benign neoplastic lesions (Kimura and Baba, 1973). A wide variety of morphological changes were seen in the livers of male and female rats fed up to 1000 ppm PCBS (Arochlors 1254 and 1260) for eight months, however, no cancerous or precancerous changes were observed (Kimbrough 23.21;. 1972). In a similar investigation, clus- ters of pancreatic-type cells were observed in the livers of a number of rats fed 500 ppm PCBS (Arochlor 1254) for six months, but again, no cancerous or precancerous changes were seen (Kimbrough, 1973). How- ever, when nearly two hundred female rats were fed 100 ppm PCBS (Arochlor 1260) for twenty-one months, different results were obtained. Hepato- cellular carcinomas were found in 14% of the PCB-fed rats, but in only 0.6% of the control animals. Neoplastic nodules were found in the livers of 79% of the treated rats, but in none of the controls. While not proof of carcinogenicity, neoplastic nodules are a part of the 18 spectrum of responses to hepatocarcinogens. Ninety-nine percent of the PCB-treated rats had areas of hepatocellular alteration, compared with an incidence of 16% in the controls. No such PCB-dependent effects . were seen in other organs. This study showed PCBs to have a hepato- carcinogenic effect in rats (Kimbrough e1_a1,, 1975). Neoplastic nod- ules have also been observed in the livers of both male and female rats given a single one gram dose of P885 (Firemaster FF-l) by gavage (Kimbrough g1_a1,, 1977). In both cases in which the effects of PCBS on mice were examined, evidence of carcinogenicity was obtained. Ito e1_a1, (1973) found nod- ular hyperplasia and well-differentiated hepatocellular carcinomas in the livers of male mice fed 500 ppm PCBs for thirty-two weeks. These neoplasms were only observed using a PCB mixture averaging five chlo- rines per biphenyl (Kanechlor 500); when mixtures averaging three or four chlorines per molecule were used (Kanechlors 300 and 400, respec- tively), no neoplasms were seen. Kimbrough and Linder (1974) found hepatomas in nine of twenty-two male mice feed 300 ppm PCBS (Arochlor 1254) for eleven months, while none were found in mice fed a control diet. In addition to these investigations with laboratory animals, there is tentative epidemiological evidence for an increased incidence of malignant melanoma among human workers heavily exposed to PCBS (Bahn g1_ 21,, 1976; Lawrence, 1977; Bahn gt_a1,, 1977). It is not known which components of these PCB mixtures are car- cinogenic, nor whether these components require metabolic activation in order to initiate carcinogenesis. 19 No studies have yet appeared on the effects of P885 on the car- cinogenicity of secondary agents. Several such studies have been re- ported with PCBs, the results of which are presumed to reflect altered levels of microsomal drug metabolizing enzymes. Uchiyama gt a1. (1974) surgically implanted MC-impregnated threads into the uteri of mice. Dietary PCBS (Kanechlor 400) had no effect on the MC-induced changes in the cervical epithelium, although dietary DOT (a microsomal inducer) increased the severity and frequency of precan- erous and cancerous tissue changes. Ito egba1, (1973) fed male mice 250 ppm of a PCB mixture averaging five chlorines per molecule (Kanechlor 500) for twenty-four weeks. Al- though twice this dose for thirty-six weeks caused nodular hyperplasias and well-differentiated hepatocellular carcinomas, neither of these neoplasms were observed with this regimen. The B isomer of l,2,3,4,5,6- hexachlorocyclohexane (HCCH) was fed simultaneously with PCBS at levels up to 250 ppm, and while B-HCCH caused no effects by itself at any dose tested, mice fed both PCBs and this compound had a significant incidence of both nodular hyperplasia and hepatocellular carcinoma. The a isomer alone was carcinogenic at high dietary levels, and simultaneous feeding of PCBS with a-HCCH at this level increased the incidence of carcino- mas. Nodules were not seen at lower doses of a-HCCH unless PCBs were fed simultaneously. PCBs have also been shown to decrease the carcinogenic effects of compounds. Makiura gt a1, (1974) investigated the effects of dietary PCBS (Kanechlor 500) on the incidence of carcinogenesis caused by the hepatic carcinogens 3'-methyl-4-dimethylaminoazobenzene, N-Z-fluorenyl- acetamide, and diethylnitrosamine. While hepatocellular carcinomas 20 developed in a majority of rats given these three compounds, either alone or in pairs, rats fed PCBS in addition to these carcinogens de- veloped only a few tumors. PCB treatment alone (500 ppm for twenty weeks) caused no liver tumors." PCBS also protected against the devel- opment of nodular hyperplasias, oval cell infiltration, and bile duct proliferation. Induction of Microsomal Drug Metabolizing Enzymes by PBBs With one very recent exception (Poland and Glover, 1977), all pre- vious research concerning the effects of brominated biphenyls on micro- somal drug metabolizing enzymes has been conducted using mixtures of P885, which are known to contain brominated naphthalenes (Kay, 1977) and possibly also brominated dibenzofurans, in addition to a large num- ber of brominated biphenyl congeners. While this research has provided much useful information concerning microsomal induction, it provides no clues as to which of the components induce microsomal enzymes, or of the nature of the inductions caused by the individual components. Re- search on the effects of pure brominated biphenyl congeners will be presented in Chapter 3. It appears that Farber and Baker (1974) were the first to demon- strate microsomal induction by what they called "hexabromobiphenyl." Since this research has only appeared in abstract fOrm, it cannot be determined exactly what they studied, but it was probably the Firemaster mixture of P885. Rats were fed 0, 0.5, 5, 50, or 500 ppm "hexabromo- biphenyl" for thirty days. Liver weights, microsomal protein, NADPH- cytochrome P450 reductase, cytochrome P450, and two of the Pb-inducible cytochrome P450-catalyzed drug metabolism activities were induced. By 21 comparison with a PCB mixture averaging five chlorines per molecule, it was concluded that "hexabromobiphenyl" was at least five times as potent as PCBS as a microsomal inducer. Cecil e1__1, (1975) gave Japanese quail a single oral dose of PBBS, at 100 mg/kg body weight, and determined pentobarbital sleeping times at intervals thereafter. Pentobarbital is inactivated by hydroxylation, a reaction catalyzed by one or more Pb-inducible enzymes, so what they were studying was the extent of the Pb-type induction caused by PBBs. Pentobarbital sleeping times were found to be markedly reduced. Babish g1_a1, (1975) fed Japanese quail 0, 10, 20, or 100 ppm PBBs for nine weeks. Cytochrome P450, aminopyrine demethylation (Pb-induci- ble), and three activities inducible by both Pb and MC were all eleva- ted, some by as little as 10 ppm PBBs. Babish g1.a1, (1976) and Farber g1_a1, (1976) have published ab- stracts showing that PBBS can cause at least partial microsomal induc- tions in rats and dogs, respectively. Corbett §1_a1, (1975) noted that 'PBBs induced cytochrome P450 and a 55,000 dalton hemoprotein in mouse liver, and Sleight and Sanger (1976) found that as little as 1 ppm P885 in the diet of rats could induce microsomal drug metabolizing enzymes. The first demonstration that PBBs are a mixed-type inducer was carried out in Dr. Austls laboratory in 1975. Both a single 90 mg/kg i.p. injection and a diet of 10 ppm PBBs caused major increases in liver weight, microsomal protein, NADPH-cytochrome P450 reductase, cytochrome P450, aminopyrine demethylation, and benzo[a]pyrene hydroxyl- ation, all within a week. PBBs caused greater increases in liver weights, microsomal protein, and cytochrome P450 than did either MC or Pb when either was given alone at a maximally effective dose. Induction 22 by PBBs was distinct from that caused by either MC or Pb alone, but was quite similar to simultaneous injections of both prototype com- pounds, thereby demonstrating that PBBs cause a mixed-type induction. A single injection of 90 mg PBBs/kg was as effective as five daily in- jections of this magnitude, and inductions were still pronounced ten days after a single injection (the effects of MC or Pb were more tran- sient) (Troisi, 1975). Subsequent analyses of these microsomes (un- published) demonstrated that p-nitrophenol-UDP-glucuronyltransferase was induced (an index of MC-type induction), and that hemoproteins at 53,000 daltons (MC-type) and especially 45,000 daltons (Pb-type) were induced by PBBS. Complement fixation assays, using an antibody pre- pared against the 45,000 dalton hemOprotein, showed that this hemopro- tein was induced to comparable extents by both Pb and P885. Dent, Gibson, and co-workers have characterized many aspects of the microsomal induction caused by PBBS, in liver as well as in kidney and mammary gland, and a review of this work and research in other laboratories is in press (Dent, 1978). The initial experiment (Dent gt_a1,, 1976a) was a dietary dose- reSponse investigation, in which PBBs were shown to be potent inducers of hepatic microsomal drug metabolizing enzymes. The pattern of induc- tion included the characteristics of microsomes induced both by Pb and 'by MC. Enzymes assayed included the Pb—responsive NADPH-cytochrome P450 reductase, epoxide hydratase, and ethylmorphine demethylation, as well as the MC-responsive ethoxycoumarin-O-deethylation and benzo[a]- pyrene hydroxylation, all of which were strongly induced. Cytochrome P450 was also induced, and Amax in its reduced carbon monoxide differ- ence spectrum was lowered by 1.4 nm. 23 Dent _t_a1, (1976b) also investigated the effects of a single i.p. injection of PBBS, at either 25 or 150 mg/kg body weight. The rats were killed at various times thereafter, and their microsomes were com- pared with the liver microsomes isolated from rats pretreated with MC, Pb, or MC plus Pb. The assays listed above were performed, with essen- tially the same results. FEB-pretreated and MC plus Pb-pretreated rats both had mixed-type inductions of their hepatic microsomal drug metabo- lizing enzymes. McCormack e1_a1, (1977) fed 100 ppm P885 to rats for three months, then analyzed hepatic and renal drug metabolizing enzymes and renal function. Enzyme activities responsive to both MC and Pb (biphenyl 4-hydroxylation) and to MC alone (biphenyl 2-hydroxylation and benzo- [aprrene hydroxylation) were induced in both liver and kidney. One interesting result was that while hepatic epoxide hydratase, a Pb- responsive enzyme, was induced, the renal activity was decreased to 14% of the control values. Since epoxide hydratase is normally con- sidered beneficial, in that it converts reactive epoxides formed by the cytoChrome P4505 into more stable dihydrodiols, it was postulated that this combination of enzyme activities may increase the suscepti- bility of the kidney to toxicological damage caused by a second agent. Renal function was not affected by the P88 treatment,.however. . The composition of microsomal drug metabolizing enzymes varies with age, and neonates typically have much lower levels of these enzymes . than do adults (Conney, 1967; Parke, 1975). McCormack E£.él: (1978) therefore investigated the effects of a 150 mg/kg i.p. injection of P885 on the hepatic and renal drug metabolizing enzymes in neonatal rats. The rats were examined at intervals up to sixty—three days 24 later, at which time they were seventy days old. In liver, peak activ- ities of ethoxyresorufin-O-deethylation, biphenyl-2-hydroxylation, and benzo[a]pyrene hydroxylation were reached before the peak activities of hexobarbital hydroxylation, glutathione S-epoxide transferase, and ‘ epoxide hydratase were attained, indicating that the MC-like aSpects of .the mixed-type induction were fully expressed somewhat more rapidly than were the Pb-like aspects. Pb-responsive renal activities were either non-detectable (hexobarbital and biphenyl hydroxylation) or not induced (epoxide hydratase), while both benzo[a]pyrene hydroxylation and ethoxyresorufin O-deethylation were strongly induced. Dent e1.a1, (1977a) examined the effects of PBBS on drug metabolism in liver and mammary gland in lactating rats. The animals were fed 50 ppm PBBS from day 8 of gestation through day 14 postpartum, and P885 increased liver weights, microsomal proteins, benzo[a]pyrene hydroxyla- tion, and epoxide hydratase. Mammary benzo[a]pyrene hydroxylation was increased, while epoxide hydratase was reduced by 50%. This combina- tion of activities has also been observed in the kidney (McCormack gt a1,, 1977), and could play a role in the toxicity of xenobiotics being secreted into milk, since the enzymes forming epoxides are elevated and the enzyme to cleave them is depressed. The pups from this study were cross fostered in order to examine the effects of prepartum and postpartum exposure to PBBS (Dent g1_a1,, 1977b). Benzo[a]pyrene hydroxylation and epoxide hydratase were induced in pups exposed fin Uggrg only, as well as in pups exposed only during lactation. Pups exposed both transplacentally and by nursing were more strongly induced than were pups exposed by either of these routes alone. 25 Another investigation of drug metabolizing enzymes in FOB-fed lac- tating rats and their nursing pups has been carried out by Moore g1_ 31,, 1976, 1978). Lactating rats were fed 0, 0.1, 1.0, or 10. ppm PBBS for the eighteen days following delivery, at which time mothers and pups were sacrificed. Pups nursing from mothers fed 10 ppm PBBS showed significant increases in liver weights and microsomal protein, and both mothers and pups had increased cytochrome P450, aminopyrine demethyla- tion, benzo[a]pyrene hydroxylation, and p-nitrophenol-UDP-glucuronyl- transferase. Pups nursing from mothers fed 1.0 ppm had increases in microsomal protein, cytochrome P450, aminopyrine demethylation, and benzo[a]pyrene hydroxylation, while their mothers were unaffected. PBBS caused a mixed-type induction in both the lactating rats and their nursing pups, therefore, components of the P88 mixture responsible for both the MC- and Pb-like aspects of the induction must be transmitted through milk. The neonates were approximately ten-fold more sensitive to the effects of PBBS in their mothers' diets as were the dams. The approximate no-effect level for microsomal induction in nursing rat pups was 0.1 ppm PBBs in the diet of the adult. Dent gt a1, (1977c) have demonstrated that PBBS cause a mixed-type induction Of liver microsomal drug metabolizing enzymes in mice as well as rats. Ethylmorphine demethylation and epoxide hydratase peaked forty-eight hours after a 150 mg/kg i.p. injection of P885, while ethoxycoumarin O-deethylation and benzo[a]pyrene hydroxylation peaked ninety-six hours after injection. These activities, plus the ethyl isocyanide difference spectra, showed the Pb-like induction to occur more rapidly in mouse hepatic microsomes than did the maximal MC-like induction. This shift was also verified by following the toxicity of 26 bromobenzene at various time points after PBB administration (Roes g1_ 31,, 1977). In mice, Pb decreases the median time to death following an injection of bromobenzene moreso than does MC, and MC also alters the slope of the time-lethality curve. When bromobenzene was injected at various times after PBBS were given, the responses to it went first from the Pb-type reaction to the MC-type response. In order to better characterize the nature of the PBB-inducible cytochrome P450 hemoproteins, Dent _1“a1. (1977d) examined the ethyl isocyanide spectral properties, reaction kinetics, inhibitor effects, and SDS-polyacrylamide gel electrophoretic profiles of liver microsomes induced by treating female rats with 150 mg PBBS/kg. Evidence was ob- tained for both a shift from Pb- to MC-, and from MC- to Pb-type induc- tions. Also, a 58,000 dalton hemoprotein unique to MC-induced micro- somes was observed. This hemoprotein was found in the microsomes of rats pretreated with MC plus Pb, but not in PBB-induced microsomes, and .so it was concluded that PBBs have some but not all of the properties of a mixed—type inducing agent. This hemoprotein appears to be absent from male rats, however, regardless of whether or not the animals were pretreated with microsomal inducing agents (Welton and Aust, 1974b). Conclusions The papers cited above all agree, or are consistent with the idea, that PBBS cause a mixed-type induction of rat (and mouse) liver micro- somal drug metabolizing enzymes. PBBS, however, are a mixture of at least thirty different chemicals, and these papers provide no informa- tion as to which PBB components are responsible for or are capable of causing the induction. Indeed, with one very recent exception to be 27 discussed in Chapter 3, no biological effects of any sort have been shown to result from any individual brominated biphenyl. In order to begin to understand the biological effects of bromi- nated biphenyls, and to better understand the effects of P88 mixtures, brominated biphenyls were purified from two crude PBB mixtures, and the structures of a number of the congeners were determined. These results are presented in Chapter 1. The results of experiments designed to examine the covalent binding of the two major PBB components to macro- molecules are presented in Chapter 2. Chapter 3 represents the begin- ' ning of experiments designed to elucidate the biological effects of pure brominated biphenyls. The effects of three pure congeners on rat liver microsomal drug metabolizing enzymes were determined. CHAPTER 1 PURIFICATION AND CHARACTERIZATION OF POLYBROMINATED BIPHENYL CONGENERS 28 ABSTRACT Polybrominated biphenyls (PBBS) are complex mixtures which can contain thirty or more components. Sundstrbm EL 21: (1976a) and Jacobs g1_a1, (1976) have shown the major congener of the Firemaster mixture of P885 to be 2,2',4,4',5,5'-hexabromobiphenyl, however, no other struc- tures have been published. The molecular weights of the more prominent components of two different PBB mixtures have been determined by gas chromatography-mass spectrometry. A number of congeners have been purified from these mixtures by chromatography and recrystallization, and characterized by 'H-NMR spectroscopy. When possible, the 13C-NMR spectra, infrared spectra, and melting points of these congeners were also determined. By these analyses, the structures of eight additional polybrominated biphenyl congeners have been determined, these are: 2,2',4,5,5'-penta-, 2,3',4,4',5-penta-, 2,2',3,4,4',5'-hexa-, 2,3',4,4',5,5'-hexa-, 2,2',3,4,4',5,5'-hepta, 2,2',3,3',4,4',5,5'-octa-, 2,2',3,3',4,4',5,5',6-nona-, and 2,2',3,3',4,4',5,5',6,6'-decabromobi- phenyl. INTRODUCTION Polybrominated biphenyls (PBBs) were industrially synthesized fbr use as flame retardants. Although the exact procedures used in the manufacturing process have not been published, the reaction probably 29 3O involves the addition of molecular bromine to biphenyl in the presence of a catalyst such as FeC13, heat, and/or pressure. Depending upon the choice of reaction conditions, different compositions can be obtained in these mixtures, and two such mixtures have been utilized in these investigations. One was the product Firemaster, which contaminated much of the Michigan food chain beginning in 1973 (Carter, 1976), and which averages six bromines per molecule. The other mixture is of an unknown origin, although it has a gas chromatographic profile very similar to that reported by Zitko (1977) for "octabromobiphenyl" (OBB), manufactured by Dow Chemical. More than thirty components can be seen when Firemaster is frac- tionated by column chromatography and assayed by gas chromatography. 088 is much simpler, although at least eleven components may likewise be visualized. Different biological consequences may be expected to result from exposure to the components of these and similar mixtures, and while much useful information can be gained by studying the effects of PBB mixtures on biological systems, an understanding of these ef- fects at the molecular level requires studies with the individual puri- fied compounds. Differences in the biological fates of P88 components may also be expected to occur, as indeed has been shown by Dannan (1978). PBBs have not been well characterized chemically. The molecular weights of the more prominent congeners have been published (Fries and Marrow, 1975; Jacobs g1.a1,, 1976; Zitko, 1977). The structure of the major component of Firemaster has been identified as 2,2',4,4',5,5'- hexabromobiphenyl (Sundstrdm g1_a1,, 1976a; Jacobs _1__1,, 1976), and brominated naphthalenes and a methyl brominated furan have also been found in Firemaster (Kay, 1977). SundstrOm g1_a1, (1976b) have also 31 compiled chemical information about a large number of brominated bi- phenyls having four or fewer bromines. Because of the importance of determining the biological effects of pure PBB components, and of elucidating the structure-function relation- ships for both the pharmacokinetics and pharmacodynamics of these com- ponents, it was decided to attempt to purify and structurally identify as many of them as possible. The results of these chemical characteri- zations are presented in this Chapter. MATERIALS AND METHODS Materials Glass-distilled chloroform, ethyl acetate, acetonitrile, and non- spectro hexane, all suitable for pesticide analysis, were purchased from Burdick and Jackson Laboratories, Muskegon, Michigan. Hexane was purified before use by passing it over a column of basic alumina. Neu- tral and basic alumina were obtained from Sigma Chemical Co., St. Louis, Missouri. Deuterochloroform, 99.8 atom % D, tetramethylsilane, and 088 were purchased from Aldrich Chemical Co., Milwaukee, Wisconsin. 2,2'- Oibromobiphenyl was obtained from K and K Rare and Fine Chemicals,_ Plainview, New York. Deuterobenzene, 99.5 atom % D, was from ICN, Irvine, California. The Firemaster PBB mixture was manufactured by the Michigan Chemical Corp., St. Louis, Michigan. The sample was obtained from a feed mixing plant shortly after the accidental contamination of the Michigan food chain was discovered, and is most probably Firemaster FF-l, lot 7042. For the purification of several brominated biphenyls, Firemaster BP-6, lot 6244A, was used as the starting material. The 32 column packing for gas chromatography, 3% OV-l on Gas Chrom Q, was purchased from Supelco, Bellefonte, Pennsylvania. The carrier and purge gas, 95% argon, 5% methane, was from Matheson Gas Products, Joliet, Illinois. Gas Chromatography PBBS were assayed with a Hewlett Packard Model 402 gas chromato- graph equipped with a pulsed 63Ni electron capture detector. The col- umn, 3% OV-l on Gas Chrom 0, 100-120 mesh, was maintained at 270°, using 95% argon-5% methane for both carrier and purge gas. The normal settings used were range = 10 and attenuation = 8. Gas Chromatography-Mass Spectrometry An LKB Type 9000 gas chromatograph-mass spectrometer was used to determine the molecular weights of the P88 components. Source tempera- ture was 290°, a 3500 volt accelerating voltage was used, and the col- umn was 3% OV-l. I thank Joelle André of the Michigan State University Mass Spectrometry Facility for these analyses. Determination of Spectra 180 MHz 'H-NMR spectra were obtained on a Bruker WP 180 spectro- meter at ambient temperature. Dilute solutions (less than 3% w/v) were prepared in CDC13, and chemical shifts are reported in ppm relative to the internal standard tetramethylsilane. I thank the Michigan State University Chemistry Department and Frank J. Bennis for these spectra. 15.08 MHz 13C-NMR spectra were determined at ambient temperature on a Bruker WP 60 spectrometer, equipped with quadrature detection. Both CDC13 (for peaks 6, 8, and 12) and deuterobenzene (for peak 12) 33 were used as solvents; all solutions were saturated. (See Figure l for the numbering system.) Chemical shifts are reported in ppm rela- tive to internal tetramethylsilane. I am grateful to John O'Conner and John Pierce for determining these spectra. Infrared spectra were taken as KBr pellets on a Perkin-Elmer 167 grating infrared spectrophotometer. Other Determinations Melting points were determined with a HOover Unimelt capillary melting point apparatus. Estimates of the percent purity of congeners were made assuming that the electron capture detector on the gas chromatograph responded with equal intensity to each congener. Values obtained by this method were in good agreement with those estimated by the 'H-NMR spectra. The percentage compositions of peaks 4 and 8 in Firemaster and OBB were determined by gas chromatographic analyses of standard (w/v) solutions. Fractionation of Firemaster and 088 Mixtures - General Procedures Chromatography on alumina in the presence of hexane was the only chromatographic method found to fractionate the PBB congeners. A large number of other column packings and solvents were tried, without success. These included: silicic acid with hexane, Florisil with hexane, silanized silica gel with acetone, hexane, acetonitrile and 95% ethanol, Sephadex LH-20 with ethyl acetateImethanol (4:1), aceto- nitrile, and hexane, activity grade IV (hydrated) alumina with hexane, AgN03 impregnated alumina with hexane, paraffin oil-saturated alumina with paraffin oil-saturated methanol, Bio-Beads S-X2 (BioRad, neutral 34 porous styrene-divinylbenzene copolymer beads) with hexane, acetoni- trile, and methanol, Celite with hexane, and phenyl derivatized silica gel with methanol. In all cases, all congeners eluted with or very close to the void volume, with no evidence of separation of congeners. Vacuum distillation met with only limited success as a fractionation procedure. Recrystallization of partially purified congeners was at times a very selective purification technique. Exact procedures for preparing the individual bromobiphenyls will be given below, following a general discussion of the protocol used. Most separations were performed in a 2.2x35 cm glass column fitted with a fritted glass bottom and a Teflon stopcock. The column was filled with hexane, then typically 110-115 g of alumina powder was added while the column was drained to prevent overflowing and to mini- mize sticking of the alumina to the top of the column. Excess hexane was drained, then the sample was applied in a minimum volume of hexane (about 40 m1/g of Firemaster, and 400 ml/g of OBB). The samples were washed into the column with several small volumes of hexane before the reservoir was filled. Basic, neutral, and acidic alumina all gave comparable results, the only difference being in the retention volumes, which decreased with decreasing pH. Neutral alumina was used for all purifications described in this Chapter. Hydrated alumina did not re- tain PBBs. The order of elution of Firemaster congeners was as follows (see Figure 1 for nomenclature): trace quantities of relatively volatile components including (probably) 2,2'-dibromobiphenyl (see Chapter 3), nearly all the peak 4, then peaks 1, 6, 7, and 8, 6a immediately fol- ‘ lowed by 2, then 5 and 3, then 10, 11, and 12. Peak 9 and a number of 35 additional unidentified components, plus substantial quantities of peaks 10, 11 and 12, could then be eluted with a more polar solvent, such as ethyl acetate, chloroform, or acetonitrile. Mixtures of com- pounds eluted from alumina columns with these solvents are referred to as the polar fractions. When 088 was chromatographed on alumina, the order of elution was peaks 4, 8, 6a, 7, ll, 12, 10, 13, and 14. Com-' ponents of these mixtures having identical gas chromatographic reten- tion times also had indistinguishable column chromatographic properties and were therefore assumed to be identical. . All peaks visible by gas chromatographic analyses of Firemaster or 068 behaved as single components during column chromatography. A large number of trace components were revealed in the course of various purifications which had retention times similar but not identical to those of the major congeners. One property encountered during column chromatography was that retention volumes were dependent upon the quantity of sample applied. For example, a small number of fractions containing only peaks 1 and 4 can be obtained once about 90% of the peak 4 has eluted from alumina, although the peak 1 is only about 20% pure. But when the partially purified peak 1 preparation is rechromatographed, the peak 4 displays a much longer elution volume, and only a small portion of it elutes be- fore peak 1. This change in relative elution volumes was encountered a number of times with other partially purified fractions, and it se- verely limited the possible purifications of a number of the congeners. Column fractions were assayed by gas chromatography. Except when very small amounts of bromobiphenyls were being chromatographed, or when more than about 500 fractions (about 20 ml each) were collected, 36 one pl injections were usually sufficient to monitor the progress of the column chromatographic separations. Fractions were pooled and concentrated using a rotary evaporator. While the solvents used did not bump, bromobiphenyls could usually be found in the trap, indicating that a small amount of co-distillation could take place. Because a dibromobiphenyl could be found in the distilled solvent, solvents were not reused. Samples for recrystallization were dissolved in hexane with con- tinuous swirling in round bottom flasks, using an electric heating mantle as the source of heat. Samples were always filtered through a Pasteur pipet tightly plugged with glass wool before beginning re- crystallizations in order to remove particulate matter. Unless the volume of solvent was very small, recrystallizations were allowed to proceed overnight at room temperature. Flasks were then placed in an ice bath for several hours, followed by fifteen minutes in'a dry ice- acetone bath. Mother liquors were then removed with a Pasteur pipet. Purification of Individual Bromobiphenyl Congeners Peak 1 was purified 20-30 fold, by two different routes. One preparation was the first mother liquor from the peak 6 purification (see below), a mixture which contained approximately 35-40% peak 1, as shown in the insert in Figure 2. This is designated peaks 1 plus 6. while the second is designated as peaks 1 plus 4. The second prepara- tion contained approximately 60% peak 1, with peak 4 as the predominant 'contaminant, as shown in the insert in Figure 3, and was prepared as follows. One gram of Firemaster was applied to each of two alumina columns. Fractions containing peaks 1, 4, and 6 only were pooled and 37 reapplied to a third column of alumina. The final preparation con- sisted of the peak 1-containing fractions which eluted before peak 6 began to emerge from the column. Peak 2 was purified approximately ten-fold. The starting material was one gram of Firemaster from which most of peaks 4, 1, and 6 had been removed by alumina chromatography. This was further purified by three additional columns until a final purity of 35-40% was achieved (see Figure 4, insert). Peak 4 could easily be isolated in large quantities. One gram of Firemaster was applied to an alumina column. Trace quantities of material with short gas chromatographic retention times eluted, fol- lowed by most of the peak 4. The peak 4 fractions were pooled and re- crystallized twice from hexane. More than 450 mg of 99.9% pure crys- tals could be obtained from one gram of Firemaster. Peak 5 was obtained in approximately 80% purity. Fractions con- taining the highest concentrations of peak 5 (about 10% by weight of the starting material) were pooled from the first two columns used to purify the peak 1 plus 4 preparation, then rechromatographed on alumina. While this third column had relatively little effect on the purity, it did remove all the contaminating peak 3, and increased the purity of peak 5 by about 10%. This peak 5 preparation was the only one which behaved as an oil, and attempts to recrystallize it from several sol- vents were unsuccessful. 7 Peak 6 was prepared from Firemaster to about 95% purity. After the fractions from the last column used for the final peak 1 plus 4 preparation eluted, the column was washed with chloroform and aceto- nitrile. The remaining fractions, which contained all the peak 6, were 38 then applied to another alumina column. Peaks 1 and 4 eluted first, then the column was washed with chloroform and acetonitrile and all fractions containing peak 6 were combined. This preparation was then recrystallized twice, to remove peak 4 and most of the peak 1 (the first mother liquor was the peak 1 plus 6 preparation). Peak 8 and all subsequent peaks were purified from 088 rather than from Firemaster. One gram of 088 was dissolved in hexane and applied to an alumina column. The most concentrated fractions of peak 8, which were also contaminated with peaks 4, 11, and 12, were pooled and reap- plied to a second column. Peak 8-containing fractions which eluted be— fore peaks 11 and 12 did were pooled and recrystallized from hexane. About 150 mg of 98% pure crystals were obtained per gram of 088. The first column used to purify peak 8 was also used to purify peak 11. Fractions containing the greatest concentration of peak 11, but with only small amounts of peak 13, were pooled and applied to another alumina column. Once most of the peak 8 eluted, all fractions before peak 10 began to emerge were pooled and recrystallized. The first mother liquor was enriched in peak 11, and was in turn recrystal- lized. The second mother liquor contained peak 11 in about 80% purity. Peak 12 was purified by a series of columns and recrystallizations. The starting material was two grams of 088 from which most of the peak 8 (and 4) had been removed by chromatography on two alumina columns. This was applied to a third column, and fractions containing peaks 10, 11, and 12 (and some 8) were pooled and recrystallized. The mother liquor contained most of the peaks 10 and 11, while the crystals were mostly peaks 8 and 12. The crystals were dissolved in hexane, then applied to a fourth column. Peak 8 eluted first, then chloroform was 39 applied, and all fractions containing peak 12 were pooled. A fifth column identical to the fourth was then run, resulting in peak 12 at a purity of about 90%. Peaks 10 and 11 were easily removed by recrys- tallization, but peak 8 tended to co-crystallize with peak 12, and three recrystallizations were required to raise the purity of peak 12 to 98%. The preparation of peak 13 began with the third column used to purify peak 12. After most of the peaks 8, 10, 11, and 12 had eluted ' from this column, chloroform was applied to elute all remaining con- geners. The chloroform fractions contained much of the original peak 13 (about 60% pure), and all of the peak 14, plus other contaminants. They were reapplied to a fourth column, and all fractions containing peak 13 but not 14 were pooled and recrystallized four times. A final purity of 95% was achieved. . Peak 14 was obtained from the final column used to purify peak 13. After peak 14 began to elute, a number of fractions containing most of the remaining peaks 10, 11, 12, and 13 were eluted. Peak 14 was then obtained at 30% purity by washing the column with chloroform. Nomenclature Rules for naming halogenated biphenyls are as presented by Hutzinger e1_a1, (1974). 1. An unprimed locant is of lower order (i.e., preferred) than its corresponding primed locant. For example, 2 is lower than 2', but 2' is lower than 3. 2. Locants as low as possible should be given to all substituents, ignoring primes at this stage. 4O 3. As few primed locants as possible Should be used.’ A 4. When the number of halogen substituents in each ring is the same, the ring with the lower numbered substituents receives the un- primed numbers. If everything else is equal, the locant cited first is unprimed. RESULTS The gas chromatographic profiles of Firemaster and 088 are shown in Figure 1. The isomeric compositions of these mixtures had previ- ously been partially determined by gas chromatography-mass spectrometry in three laboratories. Fries and Marrow (1975) identified Firemaster peak 4 as a hexabromobiphenyl, and stated that peak 8 was a mixture of hepta- and octabromobiphenyl. Jacobs e1_a1, (1976) identified Fire- master peaks 1 and 2 as pentabromobiphenyls, peaks 3, 4, 5, and 6 as hexabromobiphenyls, and peaks 7 and 8 as heptabromobiphenyls. Zitko (1977) identified 088 peak 8 as a heptabromobiphenyl, the 10 and 11 region and peak 12 as octabromobiphenyls, and peak 13 as a nonabromo- biphenyl. I The results of gas chromatographic-mass spectrometric analyses of Firemaster, partially purified Firemaster fractions, and 088 are pre- sented in Table 1. Resolution improved considerably with each run and was excellent for the last several samples, but the initial poor resolu- tion limited the number of positive assignments which could be made with the Firemaster and 088 mixtures. Partially purified samples al- lowed the unambiguous assignment of molecular weights to several 41 .mc cow umcwmucou mFanm epxcmzawnosognmuuo: mzu mpmgz .mme m: 00¢ umcwmucou Lmummsmgwd do mpaEmm ugh mmmakxmz 4>zmxaum omh40a do mpzmzoazou mzh mom Ewhm>m wszmmzzz oz< mmgumoma u~xmzmxa_mozommfioo. N. C m 1M. 3 a 3+ a. Pmm 0.: mm a 1 _ mu m _ r , _ m m m 5542me _ r BSNOdSBH 43 Table l DETERMINATION OF THE NUMBER OF BROMINES PRESENT IN POLYBROMINATED BIPHENYL CONGENERS Peak Number Sample 1 2 3 4 5 6 7 8 9 10 ll 12 13 14 Firemaster T 6 6 7 8 088 6 7 8 9 Partially purified 9 10 peak 14 Partially purified 5 6 6 7 peak 3* Firemaster polar 7 7 8 8 fraction *This preparation contained no peak 4 and therefore allowed the molecular weights of peaks 3 and 5 to be unambiguously determined. 44 congeners with very similar gas chromatographic retention times or which were present in only small concentrations in the mixtures. ‘ The only disagreement with the literature was the finding that peak 8 contained no octabromobiphenyl. Peak 6 was shown to contain six bromines by taking the molecular weight scan at the leading edge of the peak. The molecular weight of peak 7, and of the shoulder be- tween peaks 6 and 7, could not be unambiguously determined. Hepta- bromobiphenyl was clearly present in one or both of these peaks, but a hexabromobiphenyl may also be present. These analyses have resulted in four new molecular weight assign- ments. While Zitko (1977) had previously shown the peak 10 and 11 re- gion to contain an octabromobiphenyl, it was shown here that peak 10 is a heptabromobiphenyl and peak 11 is an octabromobiphenyl. Peak 9 was shown to be a heptabromobiphenyl, and peak 14 was found to contain ten bromines. Since the maximum number of bromines a biphenyl can con- tain is ten, these analyses identify the structure of peak 14 to be 2,2',3,3',4,4',5,5',6,6'-decabromobiphenyl. Other structural analyses cannot be made based on the fragmentation patterns of halogenated bi- phenyls because of rearrangement during fragmentation (Sundstrfim et_ a1,, 1976a). Characterization of Peak 1 Peak 1 is a pentabromobiphenyl, of which forty-six possible iso- mers can exist. Although it could not be completely purified, peak 1 was obtained in highly enriched form in two different mixtures, whose gas chromatographic profiles are shown in the inserts in Figures 2 and 3. Fortunately, since the NMR spectra of the two major contaminants, 45 peaks 6 and 4 respectively, are known, it was possible to clearly assign NMR signals to peak 1, and in this way the structure of the molecule was determined. As shown in Figure 2, a complicated 'H-NMR spectrum was obtained from the peak 1 plus 6 mixture. Peak 6 gives 'H-NMR signals at 6 = 7.93 (1H), 7.59 (2H) and 7.54 (1H) ppm, however, in this spectrum, the integrals for these areas are nearly 2:2:1 rather than 1:2:1, so there must be one proton at a = 7.93 ppm from peak 1. The other signals must all arise from peak 1. In addition to the signal at 6 = 7.93 ppm, there is another unsplit signal at 7.49 ppm of equal magnitude. While the remaining signals were not electron- ically integrated, the 1:1 ratio of peak areas appears to hold. Unsplit signals on a halogenated biphenyl can only result from two types of structures. The proton can be the lone proton on one ring (at any position), or two protons can exist para_to each other on one ring (necessarily in the positions grthg_and mg1a_to the bridge carbon). Since there are five protons in this molecule it is impossible for these signals to arise from lone protons on each ring, therefore, one ring must contain two protons para_to each other. This ring must be brominated at the 2, 4, and 5 positions. The bromination of benzene deshields the grthg_protons by 0.19 ppm (Williams and Fleming, 1973), and since the proton at the 3 position is adjacent to two bromines, it must give rise to the singlet at 7.93 ppm. The proton at the 6 position is less deshielded, being adjacent to only one bromine, and causes the singlet at 7.49 ppm. The quartet centered at 6 = 7.53 ppm shows Qrthg_and para_splitting, the quartet at 7.41 is split g:thg_and mega, and the quartet centered at 7.36 ppm is split E959 and para, Based on these splitting patterns, only three structures for the second 46 .cmpo: mew p xemq op xppewuema to xpmpom mac mpecowm mg» 40 mm:_m> as» apco .ucmmcw any cw czozm we cowumemgmea tawdrgaa xppmwueeq mpg» do mPvdoLa umzamemoumsocgu mam ugh AF ¥zmmemozomm¢hzmau.m.m.e..N.N mo mashuamhm oz< Ezmhummm «2211“ .N mgzmwd 47 N «Lamp; 83 00d 006 00.0 006 CON 00d «duJfid—lfidH—44_.1_q_«4qq ut‘ .1131 Ill-3 11"-.. II.I..(I I I... .ll II .. . ; 4 2 JP \ . _ — rt. 11..) ) 15‘. I 4. .. u c 5.. 3 . D J L. ‘3‘. I; . ‘. I 2'... 4 o . J. c J. r7 (2. J3.... T . r 4...] . 1. 4... . 1...?;..\ (in... 2:41.... J.)\ (elf. . I? .\_.'>\f.1\7(.r? c )4 “\44“ . . 4 .. ..~.. 23.2..) .. . . . I. 315 ., .3. r 4... m... 2.. .11.. c . c .fJ .L. .. .4; _ ”i... 4...... 4 .,.: 4.4.1.14. .. i _.Z L 44:“ __: at... .1; : um 4... 4m 4. a _. . r. _._ _ . I _u m . f 4 _. T m . w . w _.w _ 4 fl _ 45.5 ms: 20:239.. _ .. ... .. . _ N. m o _ _ —-——~_—.—-—-—-—- -- ___— ..——’ _._...__—— -~ 4—__.._...______.. ._.___. ——" ' ‘ __......_..__ . . _ __.._..- _._-_.._.._....- .4 --‘-r' . I . . .4 . m _ _ W m m .44.. m m M one mes _ .3 a M _ fl _. mmN hm hm moxo 48 ring are poSsible. If the bromines are at the 2 and 4 positions, the signal split para and g§1h9_would have to occupy the 6 position, but 6 = 7.53 ppm is far too downfield for an grghg_proton adjacent to another unsubstituted carbon. If the bromines are at the 3 and 4 posi- tions, the signal split g;1hg_and mega_would have to occupy the 6 posi- tion and be adjacent to an unsubstituted carbon. The shift of 7.41 ppm is again too far downfield for such a proton between a bridge carbon and an unsubstituted carbon. As will be shown, this is actually the structure of peak 2. The third posssibility is for bromines to occupy the 2 and 5 positions, which results in very reasonable chemical shift values for the three protons on this ring. The protons at the 3, 4, and 6 positions have 'H-NMR signals centered at O = 7.53, 7.41, and 7.36 ppm, respectively. The ortho, meta, and para_coupling constants are 8.4, 2.4, and 0.4 Hz, respectively, in agreement with the coupling constants of a wide variety of halogenated aromatic compounds. Analysis of the 'H-NMR spectrum of the peak 1 plus 4 preparation (Figure 3) permits the same conclusions to be drawn. The signals at a = 7.59 and 7.54 ppm are absent because peak 6 is absent. The peak 4 signals are at 7.47 and 7.93 ppm (Sundstrbm _1__1,, 1976a), and several other smaller signals which are absent from the peak 1 plus 6 NMR spec- trum can be seen to arise from one or more compounds obscured by peak 4 in the gas chromatographic analysis. When the peak 4 contribution is subtracted from the integral of the signal at 7.93 ppm, an excellent ratio of unity is obtained for the integrated areas of the signals from all five of the peak 1 protons. From both the peak 1 plus 4 and 1 plus 6 preparations, the structure of peak 1 is determined to be 2,2',4,5,5' pentabromobiphenyl, as shown in Figures 2 and 3. 49 .umuoc men P xmma op xFmeuLmq Lo 44mpom use mpccmvm wcu mo mma—m> mzu spec .pcmmc4 exp :4 exocm m4 co4ueemamea nmwmwcza XPPMmutma was» mo upwwoca owgaecmoumeoczu mam mgh AP xqmav 4>zmzmHmozomm/ “.53... ~ ..5 1. _ 4 _._ . “a :. , ..E , : MTK _ m. _ .m. , ..._ ,1. k __ ; .M . _ f} r... :4, i w E . ...4 .\ . a). a..¢.\ \- ”I". .1. run a»: . -..-a" - .W w v w 3.5 we: zofizmfim ’ f. __ _ , a N. o o . w ‘ u q . d F E .M v 41:4 . 4 e .. ,_ a _ u 4 _ H \ _ and. . . m8. .3. am am © © .. .. A 2 4.046 _ 5] Characterization of Peak 2 Like peak 1, peak 2 was not completely purified, as shown by the gas chromatographic analysis presented in the insert of Figure 4. Peak 6 gives rise to singlets at 7.93, 7.59 (2H), and 7.54, while the other major contaminant (peak 8) gives rise to singlets at 7.93, 7.47 and 7.45 ppm. When these signals are disregarded, and the splitting pat- terns shown in Figure 4 are assigned, the five protons from this penta- Vbromobiphenyl are found to cause NMR signals of nearly equal intensity at the shift values shown in Figure 4. Two singlets are observed, and by the same reasoning as was used during the assignment of the structure of peak 1, one ring must be brominated at the 2, 4, and 5 positions. The proton at the 3 position causes the signal at 6 = 7.93 ppm, while the proton at the 6 position causes the 7.54 ppm signal. Egra_splitting is not observed in the second ring. Based on the splitting pattern observed, only three possible structures can be drawn for the second ring. If the bromines are at the 2 and 5 posi- tions, the proton at the 4 position, which is adjacent to one bromine and one proton, would cause the signal at 7.18 ppm, which is not nearly downfield enough. Indeed, this is the structure of peak l. If the bromines are at the 2 and 4 positions, the proton at the 5 position would cause the signal at 7.18 ppm, which again isn't nearly far enough downfield for a proton adjacent to one bromine. However, if the bro- mines are at the 3 and 4 positions, the 2, 5, and 6 protons would cause the reasonable shifts of 7.63, 7.68, and 7.l8 ppm, respectively. The structure of peak 2 is therefore 2,3',4,4',5-pentabromobiphenyl (Figure 4). Ortho, meta and para coupling constants are 8.4, 2.2, and <0.5 Hz, respectively. 52 .umuo: mgm N xmma op x..w.pgma go me.om man m.mcm.m mg. .0 mm:.m> as. xPco .ugmmc. mg“ :. czogm m. co.umgmamga ww...g=n a..m.ugma mwnu we mpwwoga owgamgmoumeognu mam ugh AN ¥zmzmHmozomm x . \ .._\ ./ _. J g t f .. : ... , _ .. ._ ____ .: f. : ‘ . , . _. _ _ ._ T . _ . __ _ . .m _ . m em» mm~ . . m 8. _. mi. __., 2.5 us: 20:23”... . u o. N. o o h m was ..m .m . .m © o .m . ._ . £010 .. m .m x 54 Characterization of Peak 4 Peak 4 is the only PBB congener for which the structure had been elucidated. Sundstrbm _t_al, (1976a) concluded that the structure was 2,2',4,4',5,5'-hexabromobiphenyl, based on the 'H-NMR spectrum which showed two singlets (J < 1 Hz) of equal intensity at 7.47 and 7.93 ppm. Jacobs gt_g1, (1976) similarly obtained the structure by 13C-NMR spectroscopy. Only six signals were observed, indicating that the molecule is symmetrical. Structural assignments were as follows: bridge carbons, 140.3; C-2 (Br), 122.4; C-3 (H), 136.7; C-4 (Br), 125.9; C-5 (Br), 123.8; and C-6 (H), 134.8 ppm. Peak 4 was obtained in a virtually homogeneous form, and was found to have a melting point ' of 159-160°, in agreement with the value of 159-161° reported by Norstrom gt_al, (1976), and far higher than the values of 128-130° re- ported by Sundstrbm £3.21: (1976a) for an impure preparation. The infrared spectrum has not yet been published, and is presented in Figure 14. By gas chromatographic analysis of standard solutions, peak 4 was found to comprise 56% by weight of the Firemaster lot (#7042) which contaminated much of the Michigan food chain. The Structure of peak 4, which is 2,2',4,4',5,5'-hexabromobiphenyl, is shown in Figures 15 and 16. Characterization of Peak 5 The sample of peak 5 was approximately 80% pure, being contaminated with peak 8 (6 = 7.93, 7.47, and 7.45 ppm) and a small amount of peak 2. When the contributions from the peak 8 signals were subtracted, an excellent ratio of unity was found between the signals arising from peak 5. Peak 5 is a hexabromobiphenyl (forty-two possible structures) 55 and contains four protons. The doublets centered at a = 7.68 and 7.02 ppm (J = 7.8 Hz) arise from protons orthg_to each other, and since these are not also meta_split, they must be the only two protons on one ring. There are only two possible substitution patterns for such a ring. If bromines occupy the 2, 3, and 6 positions, each proton will be orthg_to one proton and one bromine. However, the doublet centered at 7.02 ppm is far too upfield to be compatible with this structure. The second possibility is for the bromines to occupy the 2, 3, and 4 positions, which is what the spectrum shows to be the case. The sig: nals centered at 7.68 ppm are from the proton at the 5 position, and the doublet centered at 7.02 ppm represents the 6 proton, which is ad- jacent to a bridge carbon and to an unsubstituted carbon. The second ring must contain the remaining two protons. Since neither orthg nor mg;a_splitting is observed, these protons must be para_to each other, and so the bromines occupy the 2, 4, and 5 posi- tions on this ring. The downfield signal (6 = 7.93 ppm) must be from the proton at the 3 position, while the upfield one at 7.46 ppm must be from the proton at the 6 position. The structure of peak 5 is therefore 2,2',3,4,4',5'—hexabromobiphenyl, as shown in Figure 5. Characterization of Peak 6 Peak 6 is a hexabromobiphenyl, for which forty-two isomeric struc- tures may exist. The 'H-NMR spectrum (Figure 6) is very simple, with a = 7.93 (lH), 7.59 (2H), and 7.54 (1H) ppm. Since no Splitting is observed, and since threedifferent protons are present, two of the protons must be pg§g_to each other on one ring, and the other two pro- tons must occupy equivalent positions on the second ring. The first 56 0110:, Br Br Br 3. 0 0 8. Br 1 i I ll .. 1 I l «H. l l“ - ,. L l l l J l l l l L l l l l L l l 8.00 7.00 6.00 5.00 ppm Figure 5. 1H-NMR SPECTRUM AND STRUCTURE OF 2,2',3,4,4',5'-HEXA- BROMOBIPHENYL (PEAK 5). 57 CHCI35 Br Br BrHBr Br Br l i I L l l ll HWJ-NQLWJL-MWWMH-----_ m-----m--.q-m-_ i 1 4 1 IL] I 1 J l J l l l l l 8.00 7.00 6.00 5.00 ppm Figure 6. 1H-NMR SPECTRUM AND STRUCTURE OF 2,3',4,4',5,5'-HEXA- BROMOBIPHENYL (PEAK 6) 58 ring must therefore be brominated at the 2, 4, and 5 positions. The signal at 7.93 ppm is assigned to the 3 proton, and the 7.54 ppm sig- nal is assigned to the 6 proton. Two possible structures can be postulated for the second ring. If the bromines occupy the 2, 4, and 6 positions, the protons would be expected to cause NMR signals at about 7.8 or 7.9 ppm, because each proton would be adjacent to two bromines. However, if the bromines occupy the 3, 4, and 5 positions, the two proton singlet at 7.59 ppm can be easily rationalized. As will become evident when the structures and 'H-NMR spectra of several additional congeners are presented, the number of bromines in biphenyls grthg_to the bridge carbons has a char- acteristic effect on the shifts of the protons orthg_to the bridge car- bons. All other things being equal, as bromines are added to the grthg_ positions, the signals of the orthg_protons are shifted upfield by about 0.08 ppm. If the bromines on the second ring were at 2, 4, and 6 positions, the proton at the 6 position on the first ring would be expected to have a shift of about 7.37 ppm, not 7.54 ppm as was ob- served. However, with these bromines at the 3, 4, and 5 positions, only one bromine in the molecule occupies an grthg_position, and the observed signals are where they would be expected to be. The structure of peak 6 is 2,3',4,4',5,5'-hexabromobiphenyl, as shown in Figure 6. The protons at the 2' and 6' positions are equivalent and are respon- sible for the singlet at 6 = 7.59 ppm. The quantity of peak 6 was not sufficient to determine the entire 13C-NMR spectrum, however, the shifts of the protonated carbons were determined in chloroform solution (Figure 7). The signals at 137.9, 134.9, and 133.0 ppm are assigned to the 3, 4, and the equivalent 2' 59 .uwNPPmam.> on u.:ou mcoagmu vmumcouogn sou. m.m:m.m as» zpco was» seam 0.03 mco.u.vcou .mucmewgmaxm Am xzuzmHm020mmzm=aHmozommzmza~m omh40a no m4~uomm u~xazwzm~mozommzmxaum omhaom no mgmmomm o~=mdoa zH mhmuzm 4wuumowuag mo magnoEm ms» ecu .mmuwpoamuws use mmumgumnam ucaoaca m>osmc op pocmzum gum: vauumeuxm xpm>mumzmcxo ecu umumpomm see» was <29 .«zo emeeeee new we me am new :aowp «we we as op guy: emumasuc+ appouwnocmm mew: mcmLABHuHo~cmnuxm mo AmmpoE: oomv on com go mmmauu:H mo Ammpos: owv a: zpmvm <2: G» mwkugomamquNzwmuzn oz< mmauu:~ mo wZHQZHm kzm4<>ou bzmozwamouzmo.mw mzk zo onbuaozu wz>sz A<20momu~z no mkuwmum v mpamh 95 metabolite binding, while P88 and especially MC induction greatly in- creased the microsome-catalyzed covalent binding of benzo[a]pyrene metabolites to DNA (Table 4). The ethanol-soluble components from the incubations of 1°C-PBBs with microsomes (in the absence of DNA) were examined for the presence of metabolites. The TLC system used was one with which the hydroxy- lated products of chlorinated biphenyls were shown to have markedly smaller Rfs than did the unmetabolized parent compounds (Ghiasuddinlgt 31,, 1976). As shown in Figure 18, no radioactivity could be detected between the origin and the unmetabolized PBBS. The profile shown is from PBB-induced microsomes; identical results were obtained with the other three types of microsomes. An average of 99% of the applied radioactivity was recovered from each TLC plate. DISCUSSION PBBs are a complex mixture of chemicals whose biological effects and metabolic fates are not well understood. Technical mixtures of PCBs have been shown to be carcinogenic to mice (Ito g£_gl,, 1973; Kimbrough and Linder, 1974) and rats (Kimbrough et_al,, 1975). One likely mechanism for the expression of carcinogenicity would be the metabolic activation of PCB components by one or more of the microsomal cytochrome P450 hemoproteins into epoxides. Epoxides are usually reactive electrophiles, which can covalently bind to protein, RNA and DNA. The binding of chemicals to DNA can lead to mutations and cancer. Because of the demonstrated carcinogenicity of PCB mixtures, and because 4-chlorobiphenyl and PCBs can covalently bind to .uwcwsmxw ems» wng mwumpa wcu cw xpv>wuumowumc mo mcowmanwgumwu wgh .Apuwpv mumpwuw szuwnwcw~cwn cw vwaopw>wv new mwum—a ugh o me wwwpwm co wwpuoam mew: mwmwga mwcmmgo wee .cowuapom upmm m.:upom cup: vwmumcuxw new AFUNV pocmzpwe-scomocopgu cw uw>Pommmu mew: mcvawp mg» .Focmcpw saw; uwuumguxw >Pw>wmmzmcxw MW cwzu mew: «weauxme cowuumwm .IwowF pm; we as o_ cup: owmwaamcw ap—muPnogwm mew: mmmaiu:d we on xumwm mmmauu:H IF“: mzomkzm<4 szh uzmzoqdou >FH>Hbu_ PBBs .068 .1 l l l l l 1 l 0 2 4 6 8 10 12 14 TIME AFTER INJECTION (daySI 0 2 4 6 810121416182022 TIME AFTER INJECTION (days) Figure 21 1— I S2 .060 LIJ ; .050 HBB, E C O m .040 g DBB \ I— .030 ~ I (D E .020 *- 3 a: .0I0 — LU MC .050 Pb .057 PBBS .068 _>_: I I I I I I I I I I I I .J 124 fourteen days after the injection. Except for a slight increase on day 1, DBB had no effect. HB87 caused a steady increase of up to 43% by day 10, and livers were still 28% heavier than controls at day 22. The effects of HBB6 and HBB7 were comparable to that caused by Pb, but were far less than the 60% increase seen in response to PBBs. MC was not as effective at inducing liver weight as were the other agents. The effects of bromobiphenyl congeners on liver microsomal protein are shown in Figure 22. The induction by HBBE5was apparent within one day of injection, and continued to increase sharply for several days thereafter, until the amount of protein was nearly triple the control value by day 10. The content of microsomal protein in response to HBstwas only slightly greater than that caused by Pb. HBB7 caused a persistent induction of up to 2.8-fold, a level somewhat greater. than that caused by Pb. Both H886 and H887 were less effective inducers than the mixture from which they were derived. MC induced microsomal protein far less than did these four agents, and DBB had no effect on the quantity of microsomal protein. The data in Figure 22 have been placed on a body weight basis in order to compensate for the small variations in the body weight of the rats on a given day and for the growth of the rats over the course of the experiment. The data in the figures which follow are all expressed as units per mg of microsomal protein. Thus, for example, while cyto- chrome P450 was induced 2.6-fold in microsomes by HBB6 (Figure 24), because the microsomes were also induced (Figure 22), the treated rats actually contained 5-8 times the amount of hepatic cytochrome P450 as did the controls. Figure 22. 125 EFFECTS OF 90 mg 088, H886, AND HBB7/kg ON MICROSOMAL PROTEIN Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means t SE (N=3). Responses to maximally effective doses of MC, Pb, and PBBs are shown for comparison. 126 2.0“ 2 1 E; I 53 >.-.—---.--—-‘]://///1\\\\\\\1 8 '0; . H886 CL 6‘ -__I E; 25 1C) 2 o c» m \ 0 o. 1.4 . 5 5’05» I’z/ N3} 0 .3 - 1 MC 0.76 Pb 1.32 PBBS 1.90 I I I I J I I I O 2 4 6 8 1012 14 TIME AFTER INJECTION (days) 1.5 - 1.0 - MICROSOMAL PROTEIN (mg/g body wt) MC 0.64 Pb 108 PBBS 1.57 I J J I I L I I J I I I O 2 4 6 810121416182022 TIME AFTER INJECTION (daySI Figure 22 127 The microsomal mixed-function oxidase system consists of NADPH- cytochrome P450 reductase plus the cytochrome P450 hemoproteins. Figure 23 shows that HBB6 and HBB7 induced the reductase to about the same extent as was caused by Pb or PBBs, and that MC had little effect on this activity. The only effect of DBB was to increase the reduc- tase at day 1. The induction of cytochrome P450 content by H886 reached a maximum by day 2, and stayed essentially the same through the end of the study (Figure 24). The magnitude of the induction by HBB6 was nearly identical to that caused by Pb, but markedly less than that caused by the PBB mixture. The extent of induction in response to H887 was nearly the same as that caused by HBBS, however, the differ- ences in magnitude between the effects of Pb, H887 and PBBs were not as pronounced. Induction by MC was smaller, and it shifted Amax in the spectral assay to 448 nm. PBBs also shifted the spectral maximum, to 449.5 nm. Neither H886, H887, 088, nor Pb caused any shift from 450 nm, and DBB had no effect on the specific content of the cytochrome P450 hemoproteins. Pb and MC are the two classically distinct inducers of microsomal drug metabolizing enzymes, and aminopyrine demethylation can be used as a measure of the extent of Pb-type induction (Conney, 1967). Benzo- [a]pyrene hydroxylation (arylhydrocarbon hydroxylase) is similarly a measure of the MC-type induction (Parke, 1975). Figure 25 shows that H886 strongly induced aminopyrine demethylation, but to a level only two-thirds of that produced by Pb or PBBS. HBB7 also strongly induced this activity, but to a level comparable to that produced by Pb and PBBs. The levels attained in response to both HBB5 and HBB7 showed no signs of diminishing when the studies were concluded. Neither MC nor Figure 23. 128 EFFECTS OF 90 mg 088, HBBG, AND HBB7/kg 0N NADPH-CYTOCHROME P450 REDUCTASE - Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means t SE (N=3). Responses to maximally effective doses of MC, Pb, and PBBs are shown for comparison. UJ (I) g 400 0 £3 LIJ 93::- 02300 100. 2... we 85200 m\ 12 82 '— $5100 I (L C) <12 2 LIJ 22 500 F. ‘92 w-400 ‘r'e 0Q @3300 O. 5 Lee g_._,_o,200 1: 5 0v 8 100 >- ‘3 I (L C) <1 2 Figure 23 129 H88, 0 ' MC 232 Pb 333 PBBs 349 l I l I I I I o 22 4 es 8 I0 E214 TIME AFTER INJECTION (days) I— . . 0 - IR - HBB, I ' I“ I . ...—£3...“ . C Y ““4 088 MC 265 Pb 434 PBBS 478 I. J l I l l J l I l l A O 2 4 6 8 IO 12 I4 16 18 20 22 TIME AFTER INJECTION (dayS) Figure 24. 130 EFFECTS OF 90 mg DBB, H886, AND HBB7/kg 0N CYTOCHROME P450 Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means t SE (N=3). Responses to maximally effective doses of MC, Pb, and PBBS are shown for comparison. 131 2.4 r- 20 o “’ =3 E 2 O 2 E I .5 I2 0 E E 5 o .8 .4 _ MC L49 Pb 2.|4 PBBS 2.62 I I I I J I I O 2 4 6 8 IO l2 I4 TIME AFTER INJECTION (doyS) HBB7 DBB CYTOCHROME P450 (nmole / mg proi) MC I72 Pb 2.68 P885 2.96 I I I I I I I I I I I o :2 4. 6 £3 KDIZ I4 KSIB 20 22 TIME AFTER INJECTION (days) Figure 24 Figure 25. 132 EFFECTS OF 90 mg DBB, H886. AND HBB7/kg 0N AMINOPYRINE DEMETHYLATION Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means 2 SE (N=3). Responses to maximally effective doses of MC, Pb, and P885 are shown for comparison. (nmole /min. mg prof.) — ... m o 01 o AMINOPYRINE DEMETHYLATION 0| 2 9. '— 85 (LC. 0V g 2 (I Figure 25 133 I W C 7 MC 670 Pb 24.7 PBBS 24.I I I I I A I J I HBB6 O 2 4 6 8 IO I2 I4 TIME AFTER INJECTION (dayS) - HBB, I I I I I I MC 6.3 Pb I75 PBBs l6.8 I I I 02468IOI2I4I6I82022 TIME AFTER INJECTION (day8) T34 DBB had any effect on aminopyrine demethylation. Benzo[a]pyrene hydrox- ylation was induced ten-fold by MC, as shown in Figure 26, and while HBBG. H887 and Pb all caused a moderate increase in this activity, the level was only one-fifth of the maximum possible induction. PBBS, in contrast, caused a strong induction in benzo[a]pyrene hydroxylation, to a level half of that induced by MC. DBB had little if any effect on the specific activity of this enzyme. SDS-polyacrylamide gel electrophoresis has proven to be a useful. tool in elucidating the structure and function of microsomal proteins (Alvares and Siekevitz, 1973; Welton and Aust, 1974a,b; Haugen et 21,, l976). Figure 27 shows the effects of all agents tested on the pro- files of microsomal proteins (top) and cytochrome P450 hemoproteins (bottom). The gels stained for protein fall into four categories. Microsomes from DBB-pretreated rats were identical to control micro- somes. Pb, H886 and H887 all caused the same pattern, while MC caused a unique induction pattern. The proteins induced by PBBs were those induced by the Pb-type inducers. plus those induced by MC. When heme staining was performed, similar results were seen (bottom). except that the difference between control and MC microsomes was very subtle and could not be seen inthis gel. The differences can be observed when the proteins smaller than 40,000 daltons are electrophoresed off such a gel (results not shown). A number of substrates are metabolized by the cytochrome P450 family into epoxides. These reactive products can bind covalently to a variety of nucleophiles including protein, RNA and DNA and thereby cause great cellular damage (Jerina and Daly, l974). Epoxide hydratase converts epoxides into much less reactive dihydrodiols, and H885 tripled 135 Figure 26. EFFECTS OF 90 mg DBB, H886, AND HBB7/kg ON BENZO[a]PYRENE HYDROXYLATION Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means t SE (N=3). Responses to maximally effective doses of MC. Pb. and P885 are shown for comparison. J> CD 9” CD BENZO[a]PYRENE HYDROXYLATION (nmole/mun mg prof) 6 8 .N .04 .«b .0' o o o o BENZOIalPYRENE HYDROXYLATION (nmole/mm mg prot) ‘5 Figure 26 I36 I * MC Pb PBBS I 20. I 4.00 9.30 I I I k I HBB6 O 2 4 6 8 IO I2 l4 TIME AFTER INJECTION (days) " . HBB7 - a 5 DBB . . - "”/,/’ *-—T (3 ...T..'....,,fim ‘ MC 22.8 Pb 4.44 PBBS I57 I I I I J I I I I I I I O 2 4 6 8 IO I2 I4 I6 l8 20 22 TIME AFTER INJECTION (days) Figure 27. I37 EFFECTS OF POLYBROMINATED BIPHENYL CONGENERS AND OTHER XENOBIOTICS ON THE PROTEIN AND HEME PROFILES OF MICROSOMES SUBJECTED TO SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS From left to right, the microsomes are from control rats. or from rats pretreated with MC, Pb, PBBs, HBBG, HBB , and DBB. Rats were injected i.p. with 90 mg/kg of the brominated agents seven days before sacrifice. MC and Pb pretreatments were likewise maximally effective. Pro- tein staining (t0p) was performed on gels containing 20 ug of microsomal protein, while l20 ug of microsomal protein was applied to gels stained for heme. The broad. rapidly migrating band in the heme-stained gel represents dissoci- ated heme. I38 E t E ‘ G D . ' u 4 «W ' 1 f I a V J v dd 7H ’ l "-_-4' unnuu ...-II ‘I'II. lllll' ...-t, .-'-.’ ‘ ‘ Figure 27 I39 this activity, while HBB7 quadrupled it. Pb and especially PBBS were also good inducers, while MC and DBB had no effect (Figure 28). Bresnick et_al, (l977) have previously shoWn Pb to be a good inducer, and MC to have little effect on this enzyme. Another pathway for the further disposition of compounds metabo— lized by the cytochrome P450 hemoproteins involves the conjugation of a hydroxylated metabolite with glucuronic acid. Neither HBBG, HB87 nor DBB had much effect on UDP-glucuronyltransferase, while PBBS were a good inducer, as shown in Figure 29. When assayed with p-nitrophenol as the acceptor, MC is known to induce, while Pb has no effect (Bock .et l., I973); these observations were verified by the data presented here. DISCUSSION PBBS have been shown by several laboratories to induce a variety of microsomal enzymes in several different tissues and species (Farber and Baker, 1974; Cecil gt_ 1., I975; Corbett gt_ 1., I975; Troisi, 1975; Babish 3:91., 1975, l976; Farber e 1., l976; Sleight and Sanger, l976; Moore et al. ,l976, I978; Dent g_a al. ,l976a, b, l977a,b, c,d, I978; Roes et al. ,l977; McCormack gt_ 1., T977, l978). In rat liver, this induction is of a mixed type in that it includes the proper- ties of microsomes induced both by MC and by Pb (Troisi, l975; Moore et. l., l976, I978; Dent g__ l. ,l976a, b, l977a,b,d, T978; McCormack g__ l. ,l977, l978). PBBs, however, are a complex mixture of chemicals, and it had not previously been known which components of the mixture were able to induce microsomal drug metabolizing enzymes, nor what Figure 28. I40 EFFECTS OF 90 mg DBB, H886, AND HBB7/kg 0N EPOXIDE HYDRATASE Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means t SE (N=3). Responses to maximally effective doses of MC, Pb, and PBBs are shown for comparison. I2 I4I H886 C _ MC 4.68 Pb I23 PBBS I5.4 O 2 4 6 8 IO I2 I4 IO 3% "TB 53 <1 :1 Eu. >86 Is; m 322 O 85 ME 30 “In 23' L— t; CL $320 >- I: ME 9.92 lo) x0. 85:. m Figure 28 TIME AFTER INJECTION (days) "nan...“ Pb I927 H887 M3 088 MC 7.6 P885 I I I I I I I I I I 024 6 8 IO I2 I4 I6 I82022 TIME AFTER INJECTION (day5) Figure 29. I42 EFFECTS OF 90 mg DBB, H886, AND HBB7/kg ON UDP-GLUCURONYL- TRANSFERASE Rats were injected on day 0 and sacrificed at intervals up to twenty-two days later. Values shown are means t SE (N=3). Responses to maximally effective doses of MC, Pb, and P885 are shown for comparison. N 01 O O (nmole/min. mg proI.) UDP-GLUCURONYLTRANSFERASE 5 UDP - GLUCURONYLTRANSFERASE (nmole/mm mg pro’I) Figure 29 I43 I HBBd I c I ' MC 87.3 Pb 29.6 PBBS 99.2 o 2 4 6 8 I0 I2 I4 TIME AFTER INJECTION (days) MC 50.5 I I I I J Pb 25.7 I I I I I PBBS 5L3 I I 02468 TIME AFTER IO I2 I4 I6 l8 2O 22 INJECTION (days) T44 pattern of induction would result from treating animals with these puri- fied components. The results presented in this Chapter demonstrate which enzymes can be induced by a rapidly metabolized suspected trace component of P335, and by the two most prominent congeners, which to- gether comprise 83% by weight of the Firemaster mixture of PBBS. As with any study of this sort, it is important to know that the chemicals under investigation are indeed pure, or that any contaminants present would have only minimal effects on the test animals. The re- sults of gas chromatographic analyses of DBB, HBBG and H387 are pre- sented in Figure 19, and show that by this criterion they were 98, 99.9 and 98% pure, respectively. The contaminant in HBB7 was HBBG, but the amount present was shown by preliminary experiments to have no detecta- ble effects on microsomal enzymes. Since PBBS might contain brominated dibenzofurans, it was important to exclude the possibility that even trace quantities of them could be present in the final preparations of H885 and HBB7. Also, the DBB (purchased commercially) was probably specifically synthesized: if the Ullman reaction was used, brominated dibenzofurans were probably present (based on the work of Moron gt g1: (l973) and Morita gt_al, (1977) with chlorinated compounds). For these reasons, the presence of oxygenated contaminants was of concern. How- ever, since the bromobiphenyl congeners were all eluted from alumina with pure hexane before use, no such contaminants should have been present in the preparations. (While PCBs are eluted from alumina with 1% methylene chloride in hexane, 20% methylene chloride was required to elute chlorinated dibenzo-p-dioxins and dibenzofurans (Porter and Burke. l97l; Zitko, l972)). The purified compounds were also recrystallized, I45 to minimize the possibility of contamination by brominated naphthalenes or other components. DBB had little if any effect on rat liver microsomal drug metabo- lizing enzymes. Results of the pathological analysis are not yet avail- able. DBB caused slight increases in several parameters one day after treatment, and an occasional value after that marginally differed from the corresponding control values, but it was clearly without major ef- fect. DBB has been shown to be very rapidly metabolized jfl_yitrg_ (Dannan, I978), but whether the metabolites or the parent compound caused these apparent minor effects is unknown. It can be concluded,. because the rats in this study were treated with approximately five thousand times the amount of DBB as is found in PBBS, that whatever DBB is present in the Firemaster mixture is completely without effect. The effects of the chlorinated analog of DBB, 2,2'-dichlorobi- phenyl (DCB) have been investigated by other researchers. DCB had lit- tle effect on microsomal drug metabolizing enzymes in rats, although it 'doubled the cytoplasmic glutathione-S-epoxide transferase (Johnstone gt al., l974; Ecobichon and Comeau, I975), presumably as a consequence of its metabolism. Another study with DBB showed it to have no effect on rat liver microsomal drug metabolizing enzymes, either as a Pb- or as an MC-type inducer (Goldstein gt__l., 1977). When injected into rats, HBstwas found to have structural effects similar to PBBs. Results of H887 treatment are not yet available. Microscopic evaluation of tissues revealed that hepatic cells were swollen and vacuolated in a majority of rats given H386, Special stain- ing did not reveal an appreciable increase in fat. A previous study with PBBs (Sleight and Sanger, I976) revealed similar lesions, although . 146 at extremely high levels fat deposits and necrosis were also observed. As was the case with P885, H386 did not cause microscopically visible lesions in any other organ examined. Kimbrough gt_al, (l977) have also stated that the principal organ affected by PBBs in the rat is the liver, and a variety of light microscopic changes were noted. McCormack _t_al, (l977), however, have observed renal in addition to hepatic changes as a consequence of exposure to PBBs. The biochemical results show that HBB6 and H887 are Pb-type in- ducers of rat liver microsomal drug metabolizing enzymes. Pb, H885 and H887 had comparable effects on every parameter examined, although HBB7 was a slightly better inducer of microsomal protein and epoxide hydrae tase than was Pb, and HBBG did not cause as great an induction of amino- pyrine demethylation as did Pb, though it was still a good inducer. Neither congener appears to be metabolized in_yitr9_(Chapter 2, and Dannan, I978), and while it is conceivable that metabolites of these congeners could have been formed jn_vivo during the course of these experiments, they did not, if present, cause any detectable shift towards an MC-type induction. It is clear that neither H836 nor HB37 are MC-type inducers. HB86 and H887 had far greater effects on liver weight, microsomal protein, and cytochrome P450 than did MC, and they strongly induced NADPH-cyto- chrome P450 reductase, aminopyrine demethylation, and epoxide hydratase, which MC did not affect. MC, unlike H886 or H887, strongly induced benzo[a]pyrene hydroxylation and UDP-glucuronyltransferase. MC also shifted the cytochrome P450 spectral maximum to 448 nm, while these congeners caused no shift, and it caused patterns of microsomal T47 proteins and hemoproteins on SDS gels which were distinct from those caused by H386 and HBB7. PBBs induced liver weight and microsomal protein to a greater ex- tent than did either H886 or H887, and the effects of these three agents on NADPH-cytochrome P450 reductase were comparable. PBBs were somewhat more effective inducers of Cytochrome P450 and epoxide hydratase than were H387 or especially H886. Aminopyrine demethylation was induced to comparable levels by H887 and PBBs, while H886 was not as effective an inducer as PBBS. These were all major effects, however, and are indica- tive of a Pb-type induction. The differences between PBBS and theSe congeners were most pronounced as revealed by the following assays. PBBS also strongly induced benzo[a]pyrene hydroxylation and UDP- glucuronyltransferase, and decreased the cytochrome P450 spectral maxi- mum by 0.5 nm, responses which are indicative of an MC-type induction. PBBs also caused an SDS gel electrophoretic pattern of proteins and hemoproteins that was the summation of the effects of not only Pb but also MC. The strong induction of benzo[a]pyrene hydroxylation by PBBs was only half as great as that caused by MC, an observation consistent with the results of Dent gt_al, (l976b) fbr both P835 and for MC plus Pb. Because of its effects on all parameters, PBBS are a mixed-type inducer. But because neither H886 nor HBB7 caused the latter category of MC-type changes, they are only Pb-type inducers and are therefore in a different class of inducing agents (Pb-type) than are PBBS (mixed- type). PBBs typically caused a greater induction than did H886 or H887; the components of P835 responsible for the differences have yet to be identified. I48 Several laboratories have investigated the effects of 2,2',4,4',5,5'- hexachlorobiphenyl (HCBe), the chlorinated analog of HBBG, on microsomal drug metabolizing enzymes. Johnstone gt_gl, (1974) and Ecobichon and Comeau (T975) demonstrated that HCB had effects consistent with a Pb- type induction. Stonard and Grieg (1976) showed that HCB, unlike two other hexachlorobiphenyls, was strictly a Pb-type inducer in rats. Goldstein §t_al, (T976, 1977) have also shown HCB to cause only a Pb- type induction in both rats and chicks, and while two other hexachloro— biphenyls were also Pb-type inducers, another was inactive, and the 3,3',4,4',5,5'-isomer was a strong MC-type inducer. Poland and Glover (l977) have also demonstrated that HCBe is a Pb-type inducer in rat liver. The results obtained with HBBESare in agreement with these studies. While it is not possible from the literature to compare the relative potencies of the two molecules, it appears that the substitu- tion of one halogen for the other has little effect on the nature of the microsomal induction. No studies have been reported on the effects of the analogous fluorinated or iodinated compounds. There are no reports in the literature on the effects of 2,2',3,- 4,4',5,5'-heptachlorobiphenyl, the H387 analog. The only congener with seven chlorines which has been examined is 2,3,3',4,4',5,5'-heptachloro- biphenyl, which surprisingly had no effect on either MC- or Pb-inducible drug metabolizing enzymes (Goldstein §t_al,, T977). DBB was investigated in part because it appears to be a trace com- ponent of PBBS, but mainly because of its physico-chemical properties, as will be discussed later. When HBB6 is isolated from Firemaster by column chromatography, very small quantities of several compounds with very short (less than one minute) gas chromatographic retention times I49 are eluted before HBBB. The major one of these has a gas chromatographic retention time identical to that of DBB, but different from the retention times of biphenyl, 2-, 3-, or 4-bromobiphenyl, or 4,4'-dibromobiphenyl. Firemaster contains only about 200 ppm of this component, and gas chromatographic-mass spectrometric analysis of these early eluting peaks unfortunately was insufficiently sensitive to confirm the pres- ence of a dibromobiphenyl. While biphenyl appears to not be a planar molecule in solution (d'Annibale §t_al,, l973; Niederberger gt_al,, l973; Lunazzi and Macciantelli, I975), the existence of a large energy barrier to free rotation about the axis of the molecule appears improbable, and the molecule should be able to attain a planar state relatively easily. H886 and HBB7 are both Pb-type inducers, and each undoubtedly has a twisted structure because of the steric effect of the bromines at the 2 and 2' positions (adjacent to the bridge carbon) in each molecule. In fact, the energy barrier to rotation caused by having this substi- tution pattern may be sufficient fbr the 2,2'-dibrominated compounds to exist as enantiomers, as is clearly the case for other orth9_substi- tuted biphenyls (Eliel, 1962). (Whether the enantiomers differ in their biological effects is unknown). DBB was studied to detenmine whether bromination of the 2 and 2' positions, and the consequent twisting of the molecule, was alone suf- ficient to induce drug metabolism. DBB also offered the possibility of studying a bromobiphenyl with numerous (eight) unsubstituted posi- tions, a property which may be expected to facilitate metabolism. Dannan (I978) has since showed that DBB is indeed rapidly metabolized in vitro by rat liver microsomes, although it should be pointed out I50 that a large number of unsubstituted positions is alone not sufficient to guarantee metabolism, since 4,4'-dibromobiphenyl was not metabolized under the same conditions. Poland and Glover (l977) have very recently examined several as- pects of the induction caused by two additional brominated congeners. 3,3',5,5'-Tetrabromobiphenyl was incapable of inducing benzo[a]pyrene hydroxylation in chick embryo liver, but it was not examined for its ability to induce Pb-responsive enzymes. 3,3',4,4',5,5'-Hexabromobi- phenyl was an excellent inducer of benzo[a]pyrene hydroxylation in chick embryo, mouse and rat liver, and it caused only a weak induction of aminopyrine demethylation in rat liver. It can thus be classified as an MC-type inducer. Because of the absence of bromines adjacent to the bridge carbons, both 3,3',5,5'-tetrabromobiphenyl and 3,3',4,4',5,5'- hexabromobiphenyl would be expected to maintain the same solution con- formation as biphenyl. Based on the studies presented in this Chapter, and on the more limited investigations by Poland and Glover, several conclusions can now be drawn about the relationships between the structures of bromina- ted biphenyl congeners and their effects on microsomal drug metabolizing enzymes. Biphenyl itself is not an inducer (Ecobichon and Comeau, T975; Goldstein gt al., T977). As evidenced by the lack of effects of DBB, bromination at the 2 and 2' positions, and the concomitant twisting of the molecule, is alone insufficient to cause induction. HBBG is a Pb- type inducer, and so bromination at some or all of the 4,4',5, and 5' positions, alone or in combination with the 2 and 2' bromines, must be necessary for this molecule to exert its effects on drug metabolizing enzymes. HBB7 differs from HB86 in that it has one additional bromine. I5I at the three position. Because both molecules cause the same responses in microsomes, it is obvious that this extra bromine is unnecessary for the induction and does not affect the type of induction. A planar con- figuration, or at least the lack of a major energy barrier to planarity (i.e., the absence of 2,2',6, and 6' bromines), is alone insufficient to cause an MC-type induction, since 3,3',5,5'-tetrabromobiphenyl is not an MC-type inducer although 3,3',4,4',5,5'-hexabromobiphenyl is. Bromination at the 4 or at both the 4 and 4' positions must be neces- sary for 3,3',4,4',5,5'-hexabromobiphenyl to be an MC-like inducer. All three congeners which have been established as microsomal in- ducers contain bromines at the 4,4',5, and 5' positions, although it is not known whether all four are necessary. Nor is it known whether these four alone are sufficient to cause induction. It is clear, how— ever, that the presence of these four bromines in more highly bromina— ted biphenyls does not by itself determine the nature of the induction, since the 2,2'-derivative of this core structure (HBBG) is a Pb-type inducer, and the 3,3'-derivative is an MC-type inducer. The effects of these five pure bromobiphenyl congeners on microsomal drug metabo- lizing enzymes are summarized in Table 5. The effects of a large number of chlorinated biphenyls and struc- turally related compounds on microsomal drug metabolizing enzymes have been examined. As a result of testing twenty-two chlorinated biphenyls, Goldstein _t al. (T977) concluded that biphenyls chlorinated symmetri? cally in both the meta_and para_positions (with respect to the bridge carbons) are MC-type inducers. Biphenyl congeners chlorinated in both the parg_and orthg_positions are Pb-type inducers, regardless of the chlorination in the meta poSition. Congeners which are chlorinated in I52 Table 5 SUMMARY OF THE EFFECTS OF PURE POLYBROMINATED BIPHENYL CONGENERS ON THE TYPE OF INDUCTION OF MICROSOMAL DRUG METABOLIZING ENZYMES COMPOUND Q Q Q Q 9 TYPE OF INDUCTION Phenobarbital 3-Meihylcholonihrene T53 only one ring, or are chlorinated in both rings but not in the Qaaa position, have very little activity as inducers of liver microsomal enzymes. Results to date with brominated biphenyls are consistent with these conclusions, although it cannot yet be concluded that these rules fully apply to brominated biphenyls. Poland and Glover (1977) studied the effects of sixteen halobi- phenyls and a number of isostereomers on the induction of benzo[a]pyrene hydroxylation, and concluded that the presence of at least two adjacent halogens in the mata and Eaaa positions of each benzene ring, as well as the absence of halogens at the QEEEQ positions, were required for MC-type induction. The requirement for a lack of aataa_substitution was attributed to the concomitant loss of planarity. The most potent MC-type inducers are the most planar ones, particularly those with two bonds between benzene rings. Again, results with brominated biphenyls are consistent with these observations. The metabolism of drugs, other xenobiotics, and some steroids would be expected to be altered as a consequence of the inductions such as have been observed with HBB6, H837, and PBBs (Conney and Burns, T972), however, the biological consequences of these inductions, either alone or in combination with other environmental chemicals, are unclear. Of particular interest are increases in the enzyme or enzymes which catalyze the hydroxylation of benzo[a]pyrene, enzymes which also cata- lyze the metabolic activation of a wide variety of other polycyclic aromatic hydrocarbons into carcinogenic derivatives (Jerina and Daly, T974; Grover and Sims, T974). One might expect that the induction of benzo[a]pyrene hydroxylation by PBBs could enhance the chemical carcino- genicity of other agents. Indeed, treatment of mice with polychlorinated l54 biphenyls was found to enhance the carcinogenicity of the a and 8 iso- mers of l,2,3,4,5,6-hexachlorocyclohexane (Ito _t_al,, l973). However, polychlorinated biphenyls were found to decrease the carcinogenicity of 3'-methyl-4-dimethylaminoazobenzene, N—Z-fluorenylacetamide, and diethyl- nitrosamine in rats (Makiura at al., T974). Further investigations will be required to elucidate the mechanisms underlying such interactions. Similar experiments concerning the interactions between P885 or their components with other chemical carcinogens have not been reported, although several related experiments have been performed. Crawford and Safe (l977) found that when liver microsomes induced by PBBS were in- cubated 1a yitaa_with 4-chlorobiphenyl, the rate of NADPH-dependent co- valent binding to microsomal macromolecules was twelve times greater than when control microsomes were used. The binding of 4-chlorobiphenyl metabolites to DNA was not examined. Results presented in Chapter 2 demonstrate that rat liver microsomes induced by PBBS catalyzed the co- valent binding of benzo[a]pyrene metabolites to DNA at a rate six-fold greater than was observed with control or Pb-induced microsomes. While neither experiment, particularly the first, allows any direct conclu- sions to be drawn concerning the effects of PBBs on chemical carcino- genesis, both support the idea that induction of microsomal drug metab- olizing enzymes may enhance the carcinogenic potential of certain other chemicals. The experiments described in this Chapter have determined the effects of three pure brominated biphenyls on rat liver microsomal drug metabolizing enzymes. DBB, a suspected trace component of P885, is at best a marginal, transient inducer, and in no way accounts for the induction caused by PBBs. HBBG and HBB7 account for 56 and 27%; I55 respectively, of P835 (Firemaster). Both are Pb-type inducers and contribute heavily to the'Pb-like aspects of the mixed-type induction caused by PBBs. It is probable that other PBB components also contri- bute to the Pb-like aspects of the induction caused by PBBs. Both HBB6 and HBB7 cause long lasting effects. However, it is clear that neither compound is responsible for all the biological effects caused by an identical dose of PBBs. In several cases, the effects of PBBs were far greater than those resulting from the purified congeners, despite the fact that the rats injected with PBBs received only 56 and 27%, respectively, of the amount of HBB6 and HBB7 received by the rats in- jected with the pure chemicals. The remaining biological effects caused by PBBs but not by HBB6 and HBB7 must result from one or more of the components in the l7% by weight of Firemaster which has not yet been fully characterized. PBB congeners which have been identified in this fraction include 2,2',4,5,5'-pentabromobiphenyl, 2,3',4,4',5-pentabromobiphenyl, 2,2',3,4,4',5'-hexabromobiphenyl, 2,3',4,4',5,5'-hexabromobiphenyl, and 2,2',3,3',4,4',5,5'-octabromobiphenyl (Chapter l). However, the effects of these compounds on microsomal drug metabolizing enzymes are not known. Of particular interest are 2,3',4,4',5-pentabromobiphenyl and 2,3',4,4',5,5'-hexabromobiphenyl, because they contain a 3,4- dibromo and a 3,4,5-tribromo substitution pattern, respectively, and because 3,3',4,4'-tetrachlorobiphenyl, 3,3',4,4',5,5'-hexachlorobi- phenyl, and 3,3',4,4',5,5'-hexabromobiphenyl are all MC-type inducers of microsomal enzymes (Goldstein at_al,, T977; Poland and Glover, l977). These two components of the Firemaster mixture may well be bifunctional molecules, with the 2,4.5-tribrominated ring acting as a Pb-type T56 inducer (as it does in HBBG), and the 3,4-dibrominated or 3,4,5-tri- brominated ring acting as an MC-type inducer. In this way, these com- pounds might account for the MC-like aspects of the mixed-type induc- tion caused by PBBs. Also a candidate for the MC-type inducer in PBBs is 3,3',4,4',5,5'- hexabromobiphenyl. It is not known whether this congener is present in PBBs, or if it is, whether the quantity present is sufficient to ac- count for the MC-like aspects of the mixed-type induction caused by PBBs. Dr. John A. Liddle of the Center for Disease Control has found that 3,3',4,4',5,5'-hexabromobiphenyl constitutes, at most, one part per million of Firemaster (personal communication). In a preliminary experiment, an MC-type inducer was shown to exist in a polar PBB fraction. PBBS were applied to a column of alumina, and most were-eluted with hexane. A polar fraction was then obtained by washing the column with acetonitrile. This fraction induced benzo[a]- pyrene hydroxylation, and shifted the cytochrome P450 spectral maximum to 449 nm. It is possible, however, that MC-type or mixed—type inducers were also present in the other fractions, but that their activity was masked by the presence of much larger quantities of Pb-type inducers. The results presented in this Chapter raise an important question regarding the routine assay used to monitor PBB contamination of the environment, which only measures H886 concentrations. If low molecular weight brominated biphenyls, naphthalenes, dibenzofurans, terphenyls, or other such compounds turn out to have significant biological effects distinct from those of HBBB (or H887), then this assay may prove to be an invalid measure of contamination, because the absorption, distribu- tion, and excretion rates of these classes of compounds would likely be l57 different from those of the major congeners. It is important to iden- tify which of the less prominent components cause the MC-like aspects of the mixed-type induction caused by the PBB mixture. These studies are now in progress in this laboratory. REFERENCES REFERENCES Alvares, A.P., and Siekevitz, P. (T973). Biochem. Biophys. Res. Comm. .Efl; 923-929. Ames, B.N., Durston, W.E., Yamasaki, E., and Lee, F.D. (T973). Proc. Nat. Acad. Sci. USA m, 228T-2285. d'AnnibaTe, A., Lunazzi, L., Biocelli, A.C., and Macciantelli, D. (T973). J. Chem. Soc. Perkin II T396-l400. Aust, 5.0., and Stevens, J.B. (l97l). Biochem. Pharmacol. 29, l06l-l069. Babish, J.G., Gutenmann, W.H., and Stoewsand, G.S. (T975). J. Agric. Food Chem. ga, 879-882. BabiSh, J.G., Stoewsand, G.S., and Lisk, D.J. (T976). Fed. Proc._§§, 376. Bahn, A.K., Rosenwaike, I., Herrmann, N., Grover, P., Stellman, J., and O'Leary, K. (T976). New England J. Med. 295, 450. Bahn, A.K., Grover, P., Rosenwaike, 1., O'Leary, K., and Stellman, J. (T977). New England J. Med. 296, T08. Bartle, K.D. (T972). J. Assoc. Offic. Anal. Chem. aa, llOT-TT03. Birnbaum, L.S., Baird, M.B., and Massie, H.R. (T976). Res. Comm. Chem. Pathol. Pharmacol. 15, 553-562. Bock, K.W., Frbhling, W., Remmer, H., and Rexner, B. (T973). Biochem. Biophys. Acta 321, 46-56. Bresnick, E., Mukhtar, H., Stoming, T.A., Dansette, P.M., and Jerina, D.M. (l977), Biochem. Pharmacol. 25, 89l-892. Breckenridge, A. (T975). In Enzyme Induction (D.V. Parke, ed), pp. 273-30l, Plenum Press, New York. Brodie, B.B., Gillette, J.R., and LaDu, B.N. (T958). Ann. Rev. Biochem. 2_7_. 427-454. Burke, M.D., Prough, R.A., and Mayer, R.T. (T977) Drug Metab. Disposit. a, T-B. I58 I59 Carter, L.J. (T976). Science 132, 240-243. Cecil, H.C., Harris, S J., and Bitman, J. (T975). Arch. Environ. Contam. Toxicol. 2, l83—T92. Claude, A. (T969). In Microsomes and Drug Oxidations (J.R. Gillette, A.H. Conney, G.J. Cosmides, R.W. Estabrook, J.R. Fouts, and G.J. Mannering, eds.), pp. 3—39, Academic Press, New York. Conney, A.H. (T967). Pharmacol. Rev. 12, 3T7-366. Conney, A.H., and Burns, J.J. (T972). Science 112, 576-586. Corbett, T.H., Beaudoin, A.R., Cornell, R.G., Anver, M.R., Schumacher, R., Endres, J., and Szwabowska, M. (T975). Environ. Res. 19, 390- 396. Crawford, A., and Safe, S. (T977). Res. Comm. Chem. Pathol. Pharmacol. ,LB. 59-66. Dannan, G.A. (l978). M.S. Thesis, Michigan State University, East Lansing, Michigan. Dent, J.G., Netter, K.J., and Gibson, J.E. (l976a). Res. Comm. Chem. Pathol. Pharmacol._12, 75-82. Dent, J.G., Netter, K.J., and Gibson, J.E. (l976b). Toxicol. Appl. Pharmacol. 22, 237-249. Dent, J.G., Cagen, S.Z., McCormack, K.M., Rickert, D.E., and Gibson, J.E. (l977a). Life Sciences 29, 2075-2080. Dent, J.G., Cagen, S.Z., McCormack, K.M., Rickert, D.E., and Gibson, J.E. (l977b). Fed. Proc. 26, l009. Dent, J.G., Roes, U., Netter, K.J., and Gibson, J.E. (l977c). J. Toxicol. Environ. Health, in press. Dent, J.G., Elcombe, C.R., Netter, K.J., and Gibson, J.E. (l977d). Drug Metabol. Disposit., in press. Dent, J.G. (l978). Environ. Health Perspect., in press. Dutton, G.J. (T975). Biochem. Pharmacol. 24, T835-T84T. Ecobichon, D J., and Comeau, A.M. (T975). Toxicol. Appl. Pharmacol. 33, 94—105. Eliel, E.L. (T962). Stereochemistry aj_Carbon Compounds, McGraw-Hill Book Co., Inc., New York. Fairbanks, G., Steck, T.L., and Wallach, D.F.H. (l97l). Biochemistry 19, 2606—26l7. l60 Farber, T.M., and Baker, A. (T974). Toxicol. Appl. Pharmacol. 22, l02. Farber, T.M., Balazs, T., Marks, E., and Cerra, F. (T976). Fed. Proc. aa, 376. Fishbein, L. (T974). Ann. Rev. Pharmacol. 14, T39-T56. Folch, J., Lees, M., and Stanley, G.H.S. (T957). J. Biol. Chem. 222, 497-509. Fries, G.F., and Marrow, G.S. (T975). J. Dairy Sci. §§, 947-95I- Ghiasuddin, S.M., Menzer, R.E., and Nelson, J.O. (T976). Toxicol. Appl. Pharmacol. 22, T87-l94. Gielen, J.E., Goujon, F.M , and Nebert, D.W. (1972). J. Biol. Chem. .241. 1125-1137. Gillette, J.R., Davis, D.C., and Sasame, H.A. (T972). Ann. Rev. Pharmacol. 12, 57-84. Gillette, J.R., Mitchell, J.R., and Brodie, B.B. (T974). Ann. Rev. Pharmacol. 13, 27l-288. Gnosspelius, Y., Thor, H., and Orrenius, S. (l969/70). Chem.-Biol. Interact. l, T25-T37. . Goldstein, J.A., McKinney, J.D., Lucier, G.W., Hickman, P., Bergman, H., and Moore, J.A. (T976). Toxicol. Appl. Pharmacol. 22, 8T-92. Goldstein, J.A., Hickman, P., Bergman, H., McKinney, J.D., and Walker, M.P. (T977). Chem.-Biol. Interact. 11, 69-87. Grote, W., Schmoldt, A., and Dammann, H.G. (T975). Biochem. Pharmacol. 23, 1121-1125. Grover, P.L., and Sims, P. (T968). Biochem. J. 112, T59-l60. Guengerich, F.P. (I977). J. BIOI. Chem. 222, 3970-3979. Gunsalus, I.C., Pederson, T.C., and Sligar, S.G. (I975). Ann. Rev. Biochem. 33, 377-407. Gurtoo, H.L., and Bejba, N. (T974). Biochem. Biophys. Res. Comm. 21, 685-692. Haugen, D.A., Coon, M.J., and Nebert, D.W. (T976). J. Biol. Chem. 221, I8I7-I827. Hutzinger, 0., Safe, 5., and Zitko, V. (T974). The Chemistry 9: PCB'S; CRC Press, Cleveland, Ohio. I6I Ito, N., Nagasaki, H., Arai, M., Makiura, S., Sugihara, S., and Hirao, K. (T973). J. Natl. Cancer Inst. 21, l637-T646. Jacobs, L.W., Chou, S.-F., and Tiedje, J.M. (T976). J. Agric. Food Chem. 23, ll98-T20l. Jerina, D.M. and Daly, J.W. (l974). Science, 122, 573-582. Johnstone, G.J., Ecobichon, D.J., and Hutzinger, O. (T974). Toxicol. Appl. Pharmacol. 22, 66-8T. Kay, K. (1977). Environ. Res. 12, 74-93. Kimbrough, R.D., Linder, R.E., and Gaines, T.B. (T972). Arch. Environ. Health 25, 354-364. Kimbrough, R.D. (T973). J. Natl. Cancer Inst. 21, 679-68l. Kimbrough, R.D. (l974). CRC Crit. Rev. Toxicol. 2, 445-498. Kimbrough, R.D., and Linder, R.E. (T974). J. Natl. Cancer Inst. 22, 547-552. Kimbrough, R.D., Squire, R.A., Linder, R.E., Strandberg, J.D., Montali, R.J., and Burse, V.W. (T975). J. Natl. Cancer Inst. 22, T453-T459. Kimbrough, R.D., Burse, V.W., Liddle, J.A., and Fries, G.F. (T977). The Lancet II, 602-603. Kimura, N.T., and Baba, T. (I973). Gann 22, I05—TO8. King, H.W.S., Thompson, M.H., and Brookes, P. (T975). Cancer Res. 23, I263-I269. Kohli, J., and Safe, S. (T976). Chemosphere 433-437. Kuntzman, R. (T969). Ann. Rev. Pharmacol. 2, 2T-36. Lawrence, C. (T977). New England J. Med. 222, T08. Levy, G.C., Cargioli, J.D., and Anet, F.A.L. (T973). J. Am. Chem. Soc.' gg, 1527-1535. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (T95T). J. Biol. Chem. 122. 265-275. Lu, A.Y.H., Somogyi, A., West, 5., Kuntzman, R., and Conney, A.H. (T972). Arch. Biochem. Biophys. 122, 457-462. Lucier, G.W., Sonawane, B.R., and McDaniel, 0.8. (T977). Drug Metabol. Disposit. 2, 279-287. I62 Lunazzi, L., and Macciantelli, D. (T975). Gazz. Chim. Ital. 122, 657- 664. Makiura, S., Aoe, H., Sugihara, S., Hirao, K., Arai, M., and Ito, N. (T974). J. Natl. Cancer Inst. 22, T253-T257. Malik, N., and Berrie, A. (T972). Anal. Biochem. 22, T73-T76. McCormack, K.M., Kluwe, W.M., Rickert, D.E., Sanger, V.L., and Hook, J.B. (l977). Toxicol. Appl. Pharmacol., in press. McCormack, K.M., Cagen, S.Z., Rickert, D.E., Gibson, J.E., and Dent, J.G. (T978). Drug Metab. Disposit., in press. Moore, R.W., Dannan, G., and Aust, S.D. (T976). Fed. Proc. 22, 708. Moore, R.W., Dannan, G.A., and Aust, S.D. (l978). Environ. Health Perspect., in press. Morita, M., Nakagawa, J., and Akiyama, K. (T977). Bull. Environ. Contam. Toxicol. 12, 200—204. Moron, M., Sundstrbm, G., and Wachtmeister, C.A. (T973). Acta Chem. Scand. 22, 312T-3122. Nash, T. (T953). Biochem. J. 22, 4T6-42l. Niederberger, W., DiehT, P., and Lunazzi, L. (T973). Molec. Physics 22, 57l-576. O Norstrbm, A., Andersson, K., and Rappe, C. (T976). Chemosphere 255-26T. Oesch, F., Jerina, D.M., and Daly, J. (T97T). Biochim. Biophys. Acta 221. 685-691. Omura, T., and Sato, R. (T964). J. Biol. Chem. 222, 2370-2378. Parke, D.V. (T975). In Enzyme Induction (D.V. Parke, ed.), pp. 207- 27T, Plenum Press, New York. Pederson, T.C., and Aust, S.D. (T970). Biochem. Pharmacol. 12, 222T-2230. Pederson, T.C. (T973). Ph.D. Thesis, Michigan State University, East Lansing, Michigan. Pederson, T.C., Buege, J.A., and Aust, S.D. (T973). J. Biol. Chem. .248; 7T34-7T4T. Poland, A., and Glover, E. (T977). Molec. Pharmacol. 12, 924-938. Porter, M.L., and Burke, J.A. (T97T). J. Assoc. Off. Anal. Chem._22, I426-I43I. I63 Recknagel, R.D., and Glende, Jr., E.A. (T973). CRC Crit. Rev. Toxicol. _2_. 263-297. Rickert, D.E., and Fouts, J.R. (T970). Biochem. Pharmacol. 12, 38T-390. Roes, U., Dent, J.G., Netter, K.J., and Gibson, J.E. (l977). J. Toxicol. Environ. Health, in press. Rutter, W.J. (T967). In Methods 12_Developmental Biology (F.H. Wilt and N.K. Wessells, eds.), pp. 67l-683, Thomas Y. Crowell Co., New York. Ruzo, L.0., Safe, 8., and Hutzinger, 0. (l976a). J. Agric. Food Chem. 2_4_, 291-293. Ruzo, L., Jones, 0., Safe, 8., and Hutzinger, O. (l976b). J. Agric. Food Chem. 22, 58T-583. Sadtler Research Laboratories (T976). Sadtler Standard Carbon-l3 NMR Spectr , Sadtler Research Laboratories, Philadelphia, Pennsylvania. Safe, 5., Jones, D., and Hutzinger, 0. (T976). J. Chem. Soc. Perkin I, 357-359. Shimada, T. (T976). Bull. Environ. Contam. Toxicol. 12, 25-32. Sims, P., Grover, P.L., Swaisland, A., Pal, K., and Hewer, A. (l974). Nature 252, 326-328. Sims, P., and Grover, P.L. (T974). Adv. Cancer Res. 22, T65-274. Sissons, D., and Welti, D. (T97T). J. Chromatog. 22, l5-32. Sleight, 8.0., and Sanger, V.L. (T976). J. Am. Vet. Med. Assoc. 122, l23T-l235. Stonard, M.D., and Nenov, P.Z. (l974). Biochem. Pharmacol. 22, 2l75-2T83. Stonard, M.D. (T975). Biochem. Pharmacol. 22, T959-l963. Stonard, M.D., and Grieg, J.B. (T976). Chem.-Biol. Interact. 12, 365-379. Studier, F.W. (T973). J. Mol. Biol. 22, 237-248. Sundstrbm, G., Hutzinger, 0., and Safe, S. (T976a). Chemosphere ll-T4. Sundstrbm, G., Hutzinger, 0., Safe, 5., and Zitko, V. (T976). Sci. Total Environ. 2, 15-29. Tas, A.C., and deVos, R.H. (T97T). Environ. Sci. Technol. 5, l2T6-T2T8. Tas, A.C., and Kleipool, R.J.C. (T972). Bull. Environ. Contam. Toxicol. 5..- 32-37. I64 Thomas, P.E., Lu, A.Y.H., Ryan, 0., West, 5.8., Kawalek, J., and Levin, W. (T976). Molec. Pharmacol. 12, 746-758. Troisi, C.L. (l975). M.S. Thesis, Michigan State University, East Lansing, Michigan. Uchiyama, M., Chiba, T., and Noda, K. (T974). Bull. Environ. Contam. Toxicol. 12, 687-693. Welti, 0., and Sissons, D. (T972). Organic Magnetic Resonance g, 309- 3T9. Welton, A.F., and Aust, S.D. (l974a). Biochim. Biophys. Acta 373, T97- 2IO. Welton, A.F., and Aust, S.D. (l974b). Biochem. Biophys. Res. Comm. 22, 898-906. ‘ Williams, D.H., and Fleming, 1. (T973). Spectroscopic Methods 12 Organic Chemistry, McGraw-Hill Book Co., New York. WiIson, N.K. (I975). J. Am. Chem. SOC._21, 3573-3578. Wyndham, C., Devenish, J., and Safe, S. (1976). Res. Comm. Chem. Pathol. Pharmacol._l2, 563—570. Vessey, D.A., and Zakim, D. (T973). Biochim. Biophys. Acta 212, 43-48. Zitko, V. (T972). Bull. Environ. Toxicol. Z, lOS-TTO. Zitko, V. (T977). Bull. Environ. Contam. Toxicol. 11, 285-292. APPENDIX APPENDIX LIST OF PUBLICATIONS In Press Robert W. Moore, Ann F. Welton, and Steven 0. Aust. Detection of Hemoproteins in SDS-Polyacrylamide Gels, in Methods ia_Enzymology, 221, 21_- Biomembranes 2 - Biological Oxidations IS. Fleischer and L. Packer, eds.), Academic Press, New York. . Robert W. Moore, John V. O'Connor, and Steven 0. Aust. Identification of a Major Component of Polybrominated Biphenyls as 2,2',3,4,4',5,5'- Heptabromobiphenyl. Bull. Environ. Contam. Toxicol. Robert W. Moore, Stuart 0. Sleight, and Steven 0. Aust. Induction of Liver Microsomal Drug Metabolizing Enzymes by 2,2',4,4',5,5'- Hexabromobiphenyl. Toxicol. Appl. Pharmacol. Robert W. Moore, Ghazi A. Dannan, and Steven 0. Aust. Induction of Drug Metabolizing Enzymes in Polybrominated Biphenyl-Fed Lactating Rats and their Pups. Environ. Health Perspect. Ghazi A. Dannan, Robert W. Moore, and Steven 0. Aust. Studies on the Microsomal Metabolism and Binding of Polybrominated Biphenyls (PBBS). Environ. Health Perspect. _22 Preparation Robert W. Moore, Stuart 0. Sleight, and Steven 0. Aust. Effects of 2,2'- Dibromobiphenyl and 2,2',3,4,4',5,5'-Heptabromobiphenyl on Liver Microsomal Drug Metabolizing Enzymes. To be submitted to Toxicol. Appl. Pharmacol. Robert W. Moore, and Steven 0. Aust. Purification and Characterization of Polybrominated Biphenyl Congeners. To be submitted to J. Am. Chem. Soc. I65 I66 Abstracts R.W. Moore, F.D. O'NeaT, L.C. Chaney, and 5.0. Aust. Specificity of Antibody to the Cytochrome P-450 Hemoprotein Induced by Pheno- barbital. Fed. Proc. 22, 623 (T975). Robert W. Moore, Ghazi Dannan, and Steven 0. Aust. Induction of Drug Metabolizing Enzymes in Rats Nursing from Mothers Fed Polybrominated Biphenyls. Fed. Proc. 22, 708 (T976). Robert W. Moore and Steven 0. Aust. Induction of Drug Metabolizing Enzymes by 2,2',4,4',5,5'-Hexabromobiphenyl. The Pharmacologist 12, T62 (T977). Robert W. Moore, Ghazi A. Dannan, and Steven 0. Aust. Effects of Firemaster and Selected Pure Brominated Biphenyls on Microsomal Drug Metabolizing Enzymes. Presented at the Workshop on Scien- tific Aspects of Polybrominated Biphenyls, East Lansing, Michigan, October 24 and 25, l977. Ghazi A. Dannan, Robert W. Moore, and Steven 0. Aust. Studies on the Microsomal Metabolism and Binding of PBBS. Presented at the Work- shop on Scientific Aspects of Polybrominated Biphenyls, East Lansing, Michigan, October 24 and 25, l977. Robert W. Moore and Steven 0. Aust. Effects of 2,2'—Dibromobiphenyl (DBB) and 2,2',3,4,4',5,5'-Heptabromobiphenyl (HBB7) on Microsomal Drug Metabolizing Enzymes. Toxicol. Appl. Pharmacol., in press, to be presented at the Seventeenth Annual Meeting of the Society of Toxicology, San Francisco, California, March T3-T6, T978. MICHIGAN sTnTE UNIV. LIBRARIES IIIIIIIIIII III III IIIIIIIIII II IIIIIIIIIIIII III III“ I II 31293103155259