Q .I‘- -I"- (Frags: LIBRARY Michigan State University This is to certify that the dissertation entitled In vivo, ultrastructural, and in Vitro studies on the pathologic effects of 2,2',4,4',5,5'-hexabromobiphenyl and 3,3',4,4',5,5'-hexachlorobiphenyl presented by Mark G. Evans has been accepted towards fulfillment of the requirements for phn mgmemPathologyz Environmental Toxicology iVK/%¢é{ Major professor Dme 2‘4‘87 MS U is an Affirmative Action/Equa! Opportunity Institution 0-12771 7‘71,» , MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. 4/75— 713/ IN VIVO, ULTRASTRUCTURAL, AND IN VITRO STUDIES ON THE PATHOLOGIC EFFECTS OF 2,2',4,4',5,5'-HEXABROMOBIPHENYL AND 3,3',4,4',5,5'-HEXACHLOROBIPHENYL BY Mark G. Evans A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Pathology and Center for Environmental Toxicology 1987 ABSTRACT IN VIVO, ULTRASTRUC'I‘URAL, AND IN VITRO STUDIES ON THE PATHOLOGIC EFFECTS OF 2,2',4,4',5,5'-HEXABROMOBIPHENYL AND 3,3',4,4',5,5'-HEXACHLOROBIPHENYL BY Mark G. Evans Female Sprague-Dawley rats were partially hepatectomized, initiated with diethylnitrosamine (DEN), and fed diets containing 2,2',4,4',5,5'—hexa- bromobiphenyl (245-HBB), or 3,3',4,4',5,5'-hexa- chlorobiphenyl (345-HCB) to determine the tumor promoting ability of these compounds in a two-stage hepatocarcinogenesis system. Tumor' promoting ability was assessed by measuring hepatic foci positive for gamma glutamyl transpeptidase (GGT) activity. Dietary concentrations of 10 or 100 mg/kg of 245-HBB caused increased numbers of GGT-positive hepatic foci. LikeWise, dietary concentrations of 0.1 or 1.0 mg/kg 345-HCB caused increased numbers of GGT—positive hepatic foci. When 245—HBB and 345-HCB were fed simultaneously, an additive effect on tumor promoting ability was observed at dietary concentrations of 10 mg/kg 245-HBB and 0.1 mg/kg 345-HCB. However, an inhibitory effect on tumor promoting ability occurred when dietary concentrations of 100 mg/kg 245-HBB and 1.0 mg/kg 345- HCB were fed simultaneously. Freeze-fracture studies revealed that less hepatocytic membrane was occupied by gap junctions in hepatic nodules from rats that were DEN-initiated and fed dietary concentrations of 10 mg/kg 245-HBB plus 0.1 mg/kg 3,3',4,4',5,5'—hexabromobiphenyl than in sur- rounding non-nodular hepatic parenchyma. However, numbers of nuclear pores were not significantly different between nodular and non-nodular areas of liver from similarly treated rats. In in Vitro studies, 245-HBB inhibited gap junction-mediated intercellular communication in WB-F344 (rat epithelial) cells in a dose-dependent manner in the metabolic cooperation assay, the fluorescence redistribution after photobleaching assay, and the scrape-loading/dye transfer assay. When the scrape- loading/dye transfer assay was combined with a technique in which fluorescence intensity was measured, quantitation of dose-responsiveness was similar to that found with the metabolic cooperation assay. In addition, Firemaster BP-6 (FM) did not inhibit intercellular communication more than its major congener, 245-HBB, in the metabolic cooperation assay. Results from these studies further characterize the carcinogenic and toxicologic properties of FM, 245-HBB, and 345-HCB. Furthermore, these results demonstrate the usefulness of the scrape-loading/dye transfer assay for in Vitro assessment of dose-dependent inhibition of intercellular communication by 245-HBB. DEDICATION To my Family ii ACKNOWLEDGEMENTS My sincerest thanks go to Dr. Stuart D. Sleight for his patient guidance during the course of my research. His assistance and advice was greatly appreciated. I wish to thank Drs. Glenn L. Waxler and Allan L. Trapp from the Department of Pathology for faithfully serving on my guidance committee. I especially thank Dr. James E. Trosko from the Department of Pediatrics and Human Development for his advice and inspiration and for serving on my guidance committee. Their assistance has been especially helpful. I am indebted to several people for their support: Drs. Robert W. Leader and Adalbert Koestner of the Department of PathOlogy for their obtainment and administration of the pathology/toxicology training grant from NIEHS; Drs. Karen Klomparens and Stanley Flegler of the Center for Electron Optics for their help with ultrastructural studies; Dr. Mohammad El-Fouly for sharing his expertise with the scrape-loading/dye transfer assay; Dr. Margaret Wade of Meridian Instruments for her help with the FRAP analysis; Irene Brett for her technical expertise; and Mrs. Cheryl Assaff for her clerical assistance. TABLE OF CONTENTS Page LIST OF TABIIESOo.o...oaI.one.Iooooooooooonooooooviii LIST OF FIGURES.o..-coo-ooooooooulnnooooooouooooooix LIST OF ABBREVIATIONS.ao.o.noon-cocoooooo'cooooonoxv CHAPTER 1: TUMOR PROMOTING EFFECTS OF 2,2', 4,4' ,5,5'-HEXABROMOBIPHENYL AND 3,3',4,4',5,5'- HEXACHLOROBIPHENYL IN AN INITIATION/PROMOTION HEPATOCARCINOGENESIS SYSTEM IN RATS............... 1 INTRODUCTIONOOOOa...so0..onto-coollloooooouoooo 1 LITERATURE REVIEW.coo.ooooooooooooooooIOIoooooo3 History and Uses of PCB's and PBB's....... 3 Environmental Distribution of PCB's and PBB's............ . .............. Toxic Effects of PCB's and PBB' s in Animals............................. 7 Acute Effects........................ 7 Dermal Effects....................... 8 Hepatic Effects...................... 9 Neurologic and Behavioral Effects....11 Immunologic Effects..................12 Reproductive Effects.................13 Porphyrinogenic Effects..............15 Metabolism of PCB's and PBB's.............17 Preneoplastic Hepatic Changes as End Points for Carcinogenicity Assays.....21 Initiation and Promotion in Carcinogenesis............. ...........28 In Vivo Carcinogenic Effects of PBB' s and PCB's.............................30 Two-Stage Models for Hepatic Tumor Induction in Rats.....................32 Carcinogen-Promoter Model............32 Partial Hepatectomy + Promotion Model...........................32 Selective Pressure Model.............33 01 iv Page MATERIALS ANDMETHODSOOOOIIIOIOI‘QOQOOII.00....34 Protocol..................................34 Test Chemicals............................35 Necropsy, Tissue Collection, and Histologic Techniques.................35 Histochemical Staining....................36 Microsomal Enzyme Assays..................37 Analysis for 245-HBB and 345-HCB in Liver and Fat.........................38 Statistical Analysis......................39 RESULTS.to.ocooooooooooo-ocoooooooo00.00-000-0041 Body Weight Gains and Organ Weights.......41 Histopathologic Evaluation of the Liver...41 GGT-positive Foci.......... ...............46 Concentraions of 245-HBB and 345-HCB in Liver and Fat.....................50 Microsomal Enzyme Assays..................52 other Findings............................54 DISCUSSION. 0 I a o o I o O o o a O O o o a n o o o u o o o o o o o o o a o o o o 055 SIIMMARY-CHAPTER 1 o o o o o u o o o a I I o o o c I a 0 I o o n o a o o a o o 59 BIBLIOGRAPHY-CHAPTER 1 o c n a o I I I o I O o 0 o 0 I I O o 0 O O I o o 61 CHAPTER 2: QUANTITATIVE ALTERATIONS OF GAP JUNCTIONS AND NUCLEAR PORES IN CHEMICALLY-INDUCED HEPATIC NODULES IN RATS...........................88 INTRODUCTIONOIOOIIOI000.00.000.00..-oooooootooosa LITERATURE REVIEWOOOuo-QOIIDolooaaooooocooooooogo Role of Gap Junctions in Metabolic Cooperation............................90 Synchronized Contraction.............91 Metabolic Coordination of Cells......92 Growth Control.......................93 Differentiation and Development......93 Regulation of Enzyme Activities......94 The Nature and Function of Gap Junctions..95 lation of Gap Junctions..........96 Gap Junctional Communication and Neoplasia..............................98 In Vivo Studies......................98 In Vitro Studies....................101 Structure and Function of Nuclear Pores..102 Page MATERIALS AND“THODSOODIOIIII'IOOOOIOOOOIOOQOI104 Rats......................................104 Electron Microscopy.......................105 Morphometric Analysis.....................106 Statistics................................107 RESULTSIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIlos Gap Junctionsttooool00.0.0.0...III-IIIIItolos Nuclear Pores.............................114 DISCUSSION. I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 118 SUMARY-CMPTER 2 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 121 BIBLIOGRAPHY-CHAPTER 2 I I I I I I I I I I I I I I I I I I I I I I I I I 122 CHAPTER 3: THE EFFECTS OF 2,2',4,4',5,5'-HEXA- BROMOBIPHENYL ON INTERCELLULAR COMMUNICATION: ASSESSMENT BY THREE IN VITRO ASSAYS...............129 INTRODUCTIONIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII129 LITERATURE REVIEWIIIIIIIIIIIIIIIIIIIIIIIIIIIIII132 In Vitro Properties of Tumor Promoters....l32 Assays for Measuring Gap Junctional Communication..........................135 Electrocoupling Assays...............135 Junctional Conductance Assays........136 Freeze-Fracture Studies..............136 Metabolic Cooperation Assays.........137 Dye Transfer Assays..................138 MATERIALS AND mTHODSIIIIIIIIIIIIIIIIIIIIIIIIII143 Metabolic Cooperation Assay...............l43 Fluorescence Redistribution After Photo- bleaching (FRAP) Assay................l46 Scrape-Loading/Dye Transfer Assay.........l48 RESULTSOOOCOOOIIOOIOIOOIOOII.OCOOOIIOOOIOOOOIOOISZ Metabolic Cooperation Assay...............152 Fluorescence Redistribution After Photo- bleaching (FRAP) Assay.................155 Scrape-Loading/Dye Transfer Assay.........155 vi Page DISCUSSION.....................................169 SUMMARY-CHAPTER 3..............................177 BIBLIOGRAPHY-CHAPTER 3.........................179 VITAIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII186 Table LIST OF TABLES Page Effect of dietary 245-HBB and 345-HCB on body weight gains, thymic weight, hepatic weight, and histologic structure of livers of ratSOIII...ICI.OIOICCOOOOOOOOCOI0.0.0....- 42 Experimental design and number of GGT-pos- itive foci per cubic centimeter of liver.... 47 Concentrations of 245-HBB and 345-HCB in liver and adipose tissue of rats fed a basal diet or diets containing phenobarbi- tal, 245—HBB, 345-HCB, or combined 245-HBB/ 345-HCB for 150 days......................... 51 Effects of 245-HBB and 345-HCB on the con- centration of cytochrome P-450 and the ac- tivity of aminopyrine demethylase and eth- oxyresorufin-o-deethylase in rat liver....... 53 Membrane surface area occupied by gap junc- tions in non-nodular sections of liver (control) and hepatic nodules in rats........lo9 Numbers of nuclear pores in hepatocytes from non-nodular sections of liver (con- trol) and from cells in hepatic nodules in rats.........................................115 Experimental design of metabolic coopera- tion assay using 245-HBB or Firemaster BP- 6 as test chemicals with resistant (HGPRT ) and sensitive (HGPRT+ ) WB- F344 cells.........l45 viii Figure/Page 1-1/ 45 1—2/ 45 1-3/ 49 2-1/ 111 LIST OF FIGURES Photomicrograph of the centrolobular and midzonal region of the liver from a rat fed a diet containing 1.0 mg/kg 345-HCB for 150 days after a partial hepatectomy and administration of 10 mg/kg diethylnitros- amine intraperitoneally. Notice diffuse hepatocyte hypertrophy, macrovesicular and microvesicular changes, loss of sinusoidal space, and mild inflammatory cell in- filtrates (H & E, 160 X). Photomicrograph of a focus of hepato— cellular alteration from a rat fed a diet containing a combination of 100 mg/kg 245— HBB plus 1.0 mg/kg 345-HCB for 150 days after a partial hepatectomy and administra- tion of 10 mg/kg diethylnitrosamine intra- peritoneally. The hepatocytes in this focus have hypertrophied, and a cell in the center of the focus is trinucleate. Notice the macrovesicular and microvesicular changes in the surrounding parenchyma (H & E stain, 160 X). Photomicrograph of‘ a histochemically stained section of liver from a rat fed a diet containing 10 mg/kg 245-HBB for 150 days after a partial hepatectomy and ad— ministration of 10 mg/kg diethylnitrosamine intraperitoneally. Notice the well-defined focus of hepatocytes that have positive staining for" gamma. glutamyl transpeptidase activity in their cytoplasms (Gamma glutamyl transpeptidase stain, 160 X). Photomicrograph of a hepatic nodule in a section of liver from a rat fed a diet containing a combination of 10 mg/kg 245- HBB plus 0.1 mg/kg of 345-HCB for 140 days af— ter partial hepatectomy and administration of diethylnitrosamine (10 mg/kg' body wt.) intraperitoneally. Notice light staining of cells within hepatic nodule and compression of hepatocytes near periphery of nodule (H & E, 90 X). ix Figure/Page 2-2/ 113 2-3/ 117 3-1/ 151 3-2/ 151 3-3/ 151 Recorded image of a freeze-fractured section of hepatocytic membrane from a section of hepatic nodule from a rat fed a diet containing a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HBB for 140 days following diethylnitrosamine administration (10 mg/kg body wt.) intraperitoneally. Notice the gap junction (star) and its close proximity to tight junctions (arrows) (platinum and carbon coating, 67,500 X). Recorded image of a freeze-fractured section of hepatocytic nuclear membrane from a section of hepatic nodule from the liver of a rat fed a combination of 10 mg/kg 245- HBB plus 0.1 mg/kg 345-HBB for 140 days after partial hepatectomy and diethyl- nitrosamine administration (10 mg/kg body wt.) intraperitoneally. Notice nuclear pores on the surface of the nuclear membrane (platinum and carbon coating, 30,000 X). Phase contrast photomicrograph of a monolayer of untreated WB-F344 cells in culture. Upper left portion of plate has been scraped with a wooden probe. Cells appear confluent and have normal conformation in unscraped portion (400 X). Photomicrograph from same field as Figure 3-1 stained with Lucifer yellow (LY) and rhodamine dextran. Notice primary LY- loaded cells along the scraped edge have the most intense fluorescence and that the fluorescence extends four to seven cell layers beyond the primary LY-loaded cells, indicating that LY has transferred via gap junctions into the secondary LY-recipient cells (Rhodamine dextran/Luci‘fer yellow, B filter, 400 X). Photomicrograph from same field as Figure 3-1 stained with Lucifer yellow and rhodamine dextran (RD). Notice intracellu- lar loading of RD in the cell layer along the scraped edge but that RD did not trans- fer to layers of cells away from scraped edge, indicating that cell membranes away from scraped edge are intact (Rhodamine dex- tran/Lucifer yellow, G filter, 400 X). Figure/Page 3-4/ 153 3-5/ 157 3-6/ 157 3~7/ 157 Effect of 245-HBB and FM on metabolic cooperation (MC) in WB-F344 cells. The top two (red and green) lines represent cytotoxicity curves for 100 HGPRT" cells/ml medium not co-cultured with HGPRT cells and exposed to various concentrations of FM and 245-HBB. The bottom two (blue and red) lines represent % of HGPRT' cells recovered in the metabolic cooperation assay when 108 HGPRT'cells are co-cultured with 4 X 10 HGPRT cells and exposed to various concentrations of FM or 2 4 5-HBB . In the metabolic cooperation assay at a concentration of 40 ug FM/ml medium, there was a four-fold increase in % recovery of 6- TG-resistant mutants (i.e., HGPRT' cells) when compared to 1 pg FM/ml medium. Similar concentrations of 245—HBB had a three-fold increase in recovery of 6-TG-resistant mutants. There was no statistical difference between ‘the ability of FM and 245-HBB to inhibit metabolic cooperation when compared at the same concentrations (i.e., l, 5, 20, and 40 ug/ml medium). Photograph of digitized pseudoimage of WB—F344 cells grown in culture, not exposed to test chemical (negative control), stained with 6—carboxyfluorescein diacetate, and analyzed for fluorescence redistribution after photobleaching with Anchored Cell Analysis and Sorting. Photobleaching of cells in boxes has not yet occurred. Notice that all boxes except one (arrow) are attached to touching cells. Cell in box (arrow) is a negative control for dye return. Photograph of above image immediately following' bleaching' with argon ion laser. Notice that cells in all boxes have been completely photobleached. Photograph of same field 15 minutes fol- lowing initial photobleaching of cells in boxes with argon ion laser. Notice return of fluorescence to prebleaching levels in all boxes except nontouching cell. Xi Figure/Page 3-8/ 159 3-9/ 159 3-10/ 159 3-11/ 161 3-12/ 161 Photograph of digitized pseudoimage of WB-F344 cells grown in culture, exposed for 24 hours to 5 ug 245-HBB/ml medium, stained with 6-carboxyfluorescein diacetate, and analyzed for fluorescence redistribution after photobleaching. Photobleaching of cells in boxes has not yet occurred. Notice that all boxes except one (arrow) are around touching cells. Cell in box (arrow) is not touching other cells and serves as a negative control for dye transfer. Photograph of above image immediately following photobleaching of the dye with argon ion laser. Notice that cells in all boxes have been completely photobleached. (Cell in box in lower left corner was not photobleached.) Photogragh of same field 15 minutes fol- lowing initial photobleaching of cells in boxes with argon ion laser. Notice slight return of fluorescence in three of the five boxes in which touching cells were located and no return of fluorescence in the other two boxes. These results suggest that gap junctional transfer of the dye was partially inhibited by 5 ug 245—HBB/ml medium. Photograph of digitized pseudoimage of WB-F344 cells grown in culture, exposed for 24 hours to 20 pg 245-HBB/ml medium, stained with 6—carboxyfluorescein diacetate, and analyzed for fluorescence resdistribution after photobleaching. Photobleaching of cells in boxes has not yet occurred. Notice that cells in all boxes, except two (arrows) are touching other cells. Cells in boxes (arrows) are negative controls for dye return. Photograph of above image immediately following photobleaching. Notice that cells in all boxes (except cell in 'upper left) have been completely photobleached. (Cell in upper left was not photobleached). Figure/Page 3-13/ 161 3-14 to 3-19/ 163 3-20 to 3-25/ 166 Photograph of same field 15 minutes following initial photobleaching of cells in boxes. Notice total lack of return of fluo- rescence in all boxes. These results suggest that gap junctional transfer of the dye was totally blocked by 20 ug 245-HBB/ml medium. Series of photomicrographs of WB-F344 cells grown in culture, exposed to various concentrations of 245-HBB, and subjected to the scrape-loading/dye transfer assay. Notice that cells not exposed to any chem- ical (Figure 3-14--negative control) have yellow dye (Lucifer yellow) in several cell layers beneath the scraped edge. This is interpreted as noninhibition of gap junc- tional communication. Cells exposed to 1 pg 245-HBB/ml medium (Figure 3-15), 5 pg 245- HBB/ml medium (Figure 3-16), 20 pg 245-HBB/ ml medium (Figure 3-17), and 40 pg 245-HBB/ ml medium (Figure 3-18) have progressively inhibited transfer of Lucifer yellow dye in- to secondary recipient cells. Control cells (DMSO only) are shown in Figure 3-19,and had similar degree of transfer of Lucifer yellow as Figure 3-14, indicating that the vehicle used (DMSO) did not interfere with transfer of Lucifer yellow (Rhodamine dextran/Lucifer yellow, B filter, 400 X). Series of photographs of digitized pseudoimages of WB-F344 cells grown in cul— ture, exposed to various concentrations of 245-HBB, subjected to the scrape-loading/dye transfer technique, and examined with An- chored Cell Analysis and Sorting to quantify fluorescence intensity. Notice that cells not exposed to any chemical (Figure 3-20—ne- gative control) have fluorescence in several cell layers beneath the scraped edge at the left of each photograph. This is interpreted as noninhibition of gap junctional communi- cation. Cells exposed to lug 245-HBB/ml me- dium (Figure 3—21), 5 ug 245-HBB/ml medium (Figure 3-22), 20 pg 245-HBB/ml medium (Fig- ure 3-23), and 40 pg 245-HBB/ml medium (Fig- ure 3-24) had progressively inhibited trans- fer of dye into secondary recipient cells. Control cells (DMSO only) are shown in Fig- ure 3-25, indicating that the vehicle used, DMSO, did not interfere with dye transfer. (Rhodamine dextran/Lucifer yellow, 400 X). xiii Figure/Page 3-26/ 168 Quantitation of fluorescence intensity relative units) in WB-F344 cells exposed to l, 5, 20 or 40 pg 245-HBB/ml medium and subjected to the scrape-loading/dye transfer assay. All treatment groups were signifi- cantly different (*) from the nontreated control group. LIST OF ABBREVIATIONS 245-HBB.....2,2',4,4',5,5'-hexabromobiphenyl 345-HBB.....3,3',4,4',5,5'-hexabromobiphenyl 345-HCB.....3,3',4,4',5,5'-hexachlorobiphenyl PBB.....polybrominated biphenyl(s) PCB.....polychlorinated biphenyl(s) FM......Firemaster BP-6 DEN.....diethylnitrosamine 2-AAF...2-acetylaminof1uorene GGT.....gamma glutamyl transpeptidase DDT.....dichlorodiphenyltrichloroethane 3-MC....3-methylcholanthrene PB......phenobarbital D-ALAS..delta—amino-levulinic acid synthetase MC......metabolic cooperation HGPRT...hypoxanthine guanine phosphoribosyl transferase SL/DT...scrape-loading/dye transfer assay FRAP....fluorescence redistribution after photobleaching ACAS....anchored cell analysis and sorting 6-CFDA..6-carboxyfluorescein diacetate 6eTG....6-thioguanine XV LIST OF ABBREVIATIONS-continued Pk—C....protein kinase C PBS.....phosphate buffered saline DMSO....dimethlysulfoxide TPA.....12-0-tetradecanoylphorbol-l3-acetate LY......lucifer yellow RD......rhodamine dextran CHAPTER 1 TUMOR PROMOTING EFFECTS OF 2,2',4,4' ,5,5'-HEXABROMOBIPHENYL AND 3,3',4,4',5,5'-HEXACHLOROBIPHENYL IN AN INITIATION/PROMOTION HEPATOCARCINOGENESIS SYSTEM IN RATS CHAPTER 1 TUMOR PROMOTING EFFECTS OF 2,2',4,4',5,5'-HEXABROMOBIPHENYL AND 3,3',4,4',5,5'-HEXACHLOROBIPHENYL IN AN INITIATION/PROMOTION HEPATOCARCINOGENESIS SYSTEM IN RATS INTRODUCTION Certain environmental contaminants resist agradation and continue to be a source of further human {posure. The polybrominated biphenyls (PBB's) and alychlorinated biphenyls (PCB's) are examples of such ampounds. The PCB's have been used extensively for irious applications because of their varied physical :operties and chemical stability and have worldwide xvironmental distribution (Mackay gt _1., 1983, Tanabe 1., 1983, Murphy gt al., 1983). The PBB's are less — Ldely distributed, but were involved in a chemical :cident in Michigan in the early 1970's in which cattle :cidentally ingested the compound, contaminating meat 1d milk products consumed by Michigan residents (Kay, D77; Jacobs g; al., 1978). Thus, residents of Michigan 1 2 have a high likelihood of carrying detectable body burdens of both PCB's and PBB's. Strong evidence that PBB's or PCB's cause cancer in people is lacking (Brown and Jones, 1981; Stross gt gl., 1981). However, experimental studies in laboratory animals suggest that these compounds have tumor promoting (i.e., epigenetic) activity (Jensen gt g1., 1982a, 1982b, 1983; Kimura. and. Baba, 1973; Kimura. gt g1., 1976; Aishizumi, 1976; Pereira gt g1., 1982; Deml and 0esterle, 1982; Hirose gt gl., 1981; Preston gt gl., 1981). It is of special concern that simultaneous exposure to combinations of these environmental toxicants may have additive, synergistic, or inhibitory effects on tumor promotion. The first objective of the following studies was to determine the tumor promoting effects of 2,2',4,4',5,5'- hexabromobiphenyl and 3,3',4,4',5,5'-hexachlorobiphenyl by using a two-stage hepatocarcinogenesis assay, the Pitot model (Pitot gt g1., 1978a). A second objective was to determine the tumor promoting ability of these compounds when fed simultaneously to rats by using the same assay. Information from this study may shed light on the additive, synergistic, or inhibitory tumor promoting effects when animals are concomitantly exposed to more than one environmental toxicant. LITERATURE REVIEW Commercial preparations of polychlorinated biphenyl (PCB) and polybrominated biphenyl (PBB) are formulations made by the chlorination and bromination, respectively, of biphenyl. Several PCB and PBB preparations have been widely utilized in most industrial countries. Most commercial producers have marketed PCB formulations with a variable chlorine content because the degree of biphenyl chlorination determines the properties of these industrial mixtures. However, only one PBB formulation, namely Firemaster BP—6, made by Michigan Chemical Company of St. Louis, Michigan, has been widely used for industrial purposes. Firemaster BP-6 was used as a flame retardant additive for polymeric resins, while the PCB's have been used for many varied applications because of their wide range of physical properties and their chemical stability with various organic compounds. The PCB's have been used extensively as hydraulic fluids, adhesives, heat transfer agents, flame retardants, plasticizers, wax extenders, lubricants, "dedusting" agents, organic diluents/extenders, and 3 4 dielectric fluids in electrical capacitors and transformers. The PCB's were detected in the en- vironment during the late 1960's and early 1970's resulting in a voluntary ban on all "open" uses of these compounds, but their use a dielectric agents ("closed" use) was permitted. until the late 1970's. It is es- timated that 14 billion pounds Of PCB's were manufactured in the United States from 1930 to 1975, and total U.S. production of PBB's from 1970 to 1976 was about 130 million pounds (Brinkman and de Kok, 1980). Currently, industrial applications of PBB and PCB have been stopped, and production of these compounds ceased during the 1970's. However, PCB's are still present as dielectric fluids in older transformers and capacitors (Pomerantz gt .g1., 1978; Brinkman and de Kok, 1980; Rappe and Buser, 1980). Commercial PBB's and PCB's are prepared with var- ious catalysts and experimental conditions. The com— mercial products are complex mixtures of isomers and congeners, and halogen substitution occurs on the phenyl ring with no apparent preference for ortho or para positions. The PBB and PCB formulations are similar with respect to their average number of bromine and chlorine atoms per biphenyl. However, there are two major dif- ferences in the composition of Firemaster BP-6 and Aroclor 1260, a commercial PCB preparation. First, 2, 5 4, 5, 2',4',5'-hexabromobiphenyl is the major PBB con- gener of Firemaster BP-6, whereas no single PCB congener predominates in Aroclor 1260. Second, there is no homology in the relative concentrations of structurally similar PCB‘s and PBB's in the commercial mixtures (Ballschmitter and Zell, 1980; Mullin gt .gl., 1983; Moore and Aust, 1978). Environmental persistence and stability of PCB's and PBB's is attributed to their resistance to breakdown by acids, bases, heat, reducing agents, and oxidizing agents. Furthermore, chemical stability of these com- pounds is partially dependent on the degree of halogenation as well as on the specific pattern of substitution (Brinkman and de Kok, 1980; Huntzinger gt gl., 1974). Analysis of PCB residues taken from the environment indicates that the more heavily chlorinated congeners are more persistent. This may be due to preferential microbial breakdown of the less chlorinated compounds (Ballschmitter gt _l., 1978). Residues of PCB's have been detected in atmospheric samples taken from both industrialized regions and remote arctic and antarctic locations (Mackay gt g;., 1983;'Harvey and Steinhauer, 1974; Tanabe gt gl., 1983; Atlas and Giam, 1981; Murphy gt gl., 1983). These 6 results imply that atmospheric transport processes contribute to the distribution of PCB's in the en- vironment. Similar residues have also been found in lake, river, and ocean sediments which provide a reservoir for the gradual release of PCB's into water, aquatic animals and plants, and eventually into the biota (Kauss gt gl., 1983; Sullivan gt gl., 1983). The PCB's have also been detected in fish (Zabik gt gl., 1982; Brunn and Manz, 1982; Wickstrom gt gl., 1981) and various wildlife species (Olsen gt gl., 1980; Barbehenn and Reichel, 1981; Passivirta and Linko, 1980). An ex- tensive study of the Great Lakes ecosystem demonstrated the preferential bioconcentration of PCB residues in the food chain. The lowest average levels were found in the water and sediments, and. highest levels were in the adipose tissue of carnivores such as the herring gull (International Joint Commission, Great Lakes Water Quality, 1977). The levels of PCB's in the environ- ment are gradually diminishing due to their limited use and regulated storage and disposal (Passivirta and Linko, 1980). The PBB's are not frequently detected in the environment because their industrial production and distribution was limited. Soils of contaminated Michigan farms and the areas adjacent to the Michigan Chemical Company, where Firemaster BP-6 was manufactured, contain detectable levels of PBB's (Kay, 1977; Carter, 1976; Jacobs, gt gl., 1978). Low PBB levels have been detected in fish taken from waters near the Michigan Chemical Company manufacturing site (Hesse and Powers, 1978; Filonow gt gl., 1976). Toxic Effects of PCB's and PBB's in Animals Acute Effects The PCB's and PBB's have similar toxic properties, and there are several generalizations that can be made about acute toxic effects. These toxic properties are dependent on the sex, strain, and age of the experimental animal, and there is wide variation in species' sensitivity to these commercial compounds. Generally, the onset of clinical signs due to the tox- icity of PCB's and PBB's occurs between one and three weeks following initial exposure to the compound. The LD50 values for commercial PCB preparations for rats, rabbits, and mice are between one and ten grams/kg. (Damstra gt gl., 1982; Matthews gt gl., 1978). In contrast, mink are exquisitely sensitive to the acute toxic effects of PBB's and PCB's (Ringer gt _l., 1981). In general, the more highly chlorinated formulations of PCB's appear to be more toxic than the less chlorinated products (Kimbrough, 1974; Fishbein, 1974). The L050 values for commercial PBB's have not been rigorously established. However, like the PCB's, PBB's 8 are relatively nontoxic to rats, mice, and cattle, but are highly toxic to mink. Typical clinical signs in acute PBB or PCB toxicosis include weight loss that is somewhat due to decreased food intake, thymic atrophy, and hepatomegaly (Gartoff gt a_1., 1977 ; Kimbrough e_t gl., 1978; Kimbrough, 1974; McConnell and Moore, 1979; Roberts e_t _a_l., 1978) . Dermal Effects The skin lesions in animals and man in PCB and PBB toxicosis are distinctive. The most common dermal le- sion is chloracne. The rabbit ear is especially sen— sitive to the toxic effects of halogenated aromatic compounds, and changes include hyperplasia and hyper- keratosis of the epidermis and hair follicle epithelium (Vos and Beems, 1971; V05 and Notenboom-Ram, 1972; Bass a a1., 1978; Needham _et _l., 1982; Patterson gt gt” 1981). Nonhuman primates also have typical dermal and ocular lesions after dietary exposure to PCB's and PBB's (McConnell and Moore, 1979; Allen gt gl., 1978; Lambrecht fl a_1., 1978; Allen gt 11., 1974; Altman _e_t 1L, 1979; McConnell gt gl., 1979; Barsotti e_t_ at” 1976; Allen e_t a_1., 1979). These clinical signs can develop with diets of less than 50 mg/kg body weight of the halogenated biphenyls. Neonatal primates suckling PCB-exposed dams also have similar dermal lesions (Allen e_t Q” 1979; Allen and Barsotti, 1976) , but this may be 9 due to exposure in utero as well as intake of PCB- contaminated breast milk. Ingestion of PBB-contaminated feed by cattle in Michigan caused hyperkeratosis (Jackson and Halbert, 1974; Kay, 1977; Carter, 1976; Fishbein, 1974). These clinical signs are similar to hyperkeratosis (i.e., "X-disease") seen in cattle following exposure to chlorinated naphthalenes (Olafson, 1947; Bell, 1953). Interestingly, rats do not have acne or' dermal lesions after exposure to PCB's or PBB's. However, hairless mice acquire dermal lesions associated with ingestion of these compounds (Inagami and. Koga, 1969; Knutson and Polland, 1982; Puhvel gt gl., 1982). Mink are especially susceptible to PBB and PCB tox- icoses but do not have dermal lesions upon exposure to these compounds. However, the ferret, a closely re- lated animal, had hyperkeratosis and excessive nail growth following ingestion of 20 mg/kg Aroclor 1242 (a commercial preparation of PCB's) for several months (Bleavins gt g;, 1982). Dermal responses to PCB's and PBB's by some animals are similar to those seen in people exposed to high levels of these compounds, but the mechanism of action responsible for this effect is not understood. Hepatic Effects The PCB's and PBB's cause toxicity to the liver in many animal species. The severity of the hepatotoxicity 10 differs according to the age and sex of the animal, the dose given, the duration of exposure, and the species tested (Matthews gt _a_l_.., 1978; Parkinson and Safe, 1981; Kimbrough 1974; Fishbein 1974; McConnell and Moore, 1979). Minimal hepatic damage has been reported in guinea pigs and monkeys, but moderate to severe liver damage has been seen in chickens, rabbits, rats, and mice. It is of interest that the guinea pig and monkey represent two species in which minimal liver damage occurs , although these two species are highly susceptible to other toxic effects of PCB's and PBB's. The most commonly observed gross lesion in PCB or PBB toxicosis in several animal species is hepatomegaly (Parkinson gt a_1., 1980; Gupta fl gl., 1983a, 1983b; Allen gt a_1., 1973; Kimbrough gt gt, 1972a; Kimbrough e_t gl., 1973; Kimbrough gt g_l_. 1972b; Jonsson, 1981; Bruckner gt {a_1., 1974). Moderate to severe liver damage was observed in rabbits after either dermal or dietary exposure to PCB's (Vos and Beems, 1971). Hepatomegaly and severe subcapsular and midzonal necrosis were evident. Hepato- megaly and necrotizing hepatitis have been seen in chickens exposed to commercial PCB and PBB preparations (Vos and Koeman, 1970; Ringer, 1978). Mink given these compounds in the diet had hepatomegaly and necrotizing hepatitis (Ringer gt _a_]_._., 1981; Aulerich gt _l., 1973). Dairy cattle given Firemaster BP—6 at a rate of 25 ll grams/day for one to two months and calves fed Firemaster FF-l for two to twelve weeks had hepatomegaly (Moorehead gt g;., 1978; Durst gt g;., 1978; Robl gt gl., 1978). Nonhuman primates had hepatic lesions after exposure to PCB's and PBB's, but gastric lesions were also observed in these species (Allen gt g;., 1978; Allen gt Q” 1974; McConnell e_t a_1., 1979; Allen gt gl., 1979; Allen and Barsotti, 1976; Allen and Norbach, 1973; Becker gt gl., 1979). Studies in fish indicated that PCB's and PBB's cause hepatotoxicity in these species as well (Klaunig gt _t., 1979). Neurologic gt; Behavioral Effects The neurotoxicity and behavioral effects of PCB's and PBB's have been studied in several animal species. Chicks had impaired and irreversible avoidance response when fed diets of 200 mg/kg of Aroclor 1254 for seven days (Kreitzer and Heinz, 1974). Coturnix quail had reduced biochemical adaptation to stress when, fed Aroclor 1254 (Deiter, 1974). Pheasant chicks whose mothers had been exposed to 50 ppm of Aroclor 1254 for 17 weeks also had impaired behavioral responses (Dahlgren and Linder, 1971). Additional studies suggest that several avian species are highly sensitive to PCB- mediated neurotoxicity (Ulfstrand gt gl., 1971; Karlsson gt g;., 1974; Peakall and Peakall, 1973), and various fish species exposed to Aroclors had alterations in 12 levels of various neurotransmitters (Fingerman 'and Shortt, 1983; Fingerman and Russell, 1980). Rats ex— posed chronically or subchronically to Firemaster BP-6 have many neurotoxic and behavioral changes including muscular impairment, irritability, decreased maze performance, and reductions in cognitive ability (Tilson and Cabe, 1979). Immunologic Effects The target for PCB and PBB toxicosis in the immune system is lymphoid tissue (Vos gt gl., 1980). Studies in poultry revealed that exposure to PCB's caused decreased splenic and bursal weights (Flick gt g;., 1965; V05 and Koeman, 1970). Results from studies by Harris gt gt. (1976) found that the offspring of chickens fed commercial PCB's had decreased splenic and bursal weights dependent on the degree of chlorination of the compounds fed. other studies indicate that ducklings fed Aroclor 1254 were more susceptible to duck hepatitis virus. This was thought to be associated with decreased immunocompetence and was not accompanied by other signs of PCB toxicosis (Friend and Trainer, 1970). Chronic dietary exposure of guinea pigs to com- mercial PCB's reduced numbers of circulating lympho- cytes and other leukocytes, suppressed delayed hyper— sensitivity reactions to tuberculin, and decreased circulating antibody titers to tetanus toxoid (Vos and 13 van Driel Grootenhuis, 1972). Results of other studies in mice and rats indicate a variety of immunotoxic effects of PCB's and PBB's, including thymic atrophy, splenic atrophy, decreased resistance to host infections, decreased antibody response, depressed T- cell responsiveness to mitogens, and diminished delayed hypersensitivity reactions (Loose gt gl., 1977; Silkworth and Loose, 1978; Loose gt g;., 1978; Smith gt gl., 1978; Thomas and Hinsdill, 1978; Luster gt g;., 1978). Results from studies in other species including monkeys, dogs, rabbits, and pigs indicate that diverse immunotoxic effects occur following exposure to halogenated biphenyls (Allen and Lambrecht, 1978; Farber gt g;., 1978; Keller and Thigpen, 1973; Thomas and Hinsdill, 1980; Howard gt gl., 1980). However, the mechanisms of immunotoxicosis by halogenated biphenyls is currently unknown. Reproductive Effects Results from several experiments have established that primates fed two to five mg/kg of Aroclor 1248, a commercial PCB preparation, had reproductive toxicosis. (Allen gt g;., 1978; Lambrecht gt g;., 1978; Allen gt g;., 1974; Barsotti gt gl.,l976; Allen gt g;., 1979; Allen and Barsotti 1976). The exposed primates had in- creased frequency of abortions, resorptions, and low birth weights. Primates fed Firemaster FF-l, a 14 commercial PBB preparation, at a rate of 0.3 ppm for seven months had prolonged menstrual cycles and diminished progesterone levels. Offspring of these animals had low birth weights (Lambrecht gt g;., 1978; Allen and Lambrecht, 1978). Rats and mice also suffer from reproductive tox- icosis when fed commercial PCB's and PBB's. Toxic ef- fects include decreased numbers of live births from PCB- fed rats, decreased survivability, and decreased suc- cessful matings in rats fed Aroclor 1254. The PBB's were fetotoxic and embryotoxic in rats in a dose- dependent manner, and commercial halogenated biphenyls were potent reproductive toxins in mink (Spencer, 1982; Hansen gt g;., 1975; Kihlstrom gt gl., 1975; Beaudoin, 1977; Aulerich and Ringer, 1979; Corbett gt gl., 1975; Ringer gt g;., 1981). Birds also suffer reproductive toxicosis from PCB's and PBB's. Hens fed a diet containing Aroclor 1254 for 39 weeks had decreased egg production (Corbett gt g;., 1975). Reduced hatchability was seen in chickens fed Aroclors 1232, 1242, or 1248 at a dose of 10 mg/kg for six weeks (Britton and Huston, 1972; Bush gt g;., 1974; Lillie gt g;., 1975; Ax and Hansen, 1975). other com- mercial PCB's and PBB's cause similar reproductive toxicoses. Peakall (1975) reviewed the role of PCB's as a cause of eggshell thinning. However, this phenomenon has been related to exposure to a ruoad spectrum of 15 organochlorine environmental pollutants, including DDT and DDE. Herring gulls of the lower Great Lakes have experienced eggshell thinning, but the role of PCB's here has not been established (Gilbertson, 1983). The mechanisms by which PCB's and PBB's cause reproductive toxicities is unknown but may be associated with altered 'levels of steroid hormones or altered steroid metabolism, which may have a deleterious effect on reproduction and on the development of sexual characteristics (Lincer and Peakall, 1970; Nowicki and Norman, 1972; Platonow and Funnell, 1972). Porphyrinogenic Effects People exposed to halogenated aromatic hydrocarbons in industrial settings have frequently been affected by hepatic porphyria, a disorder characterized by altered porphyrin metabolism (Strik .gt .gl., 1979). Porphyrin compounds are synthesized by many enzyme-catalyzed steps in. which delta-levulinic acid is converted to heme. Heme is, in turn, an important component of several enzymes, including cytochrome P-450-dependent mono- oxygenases. Many reports clearly implicate PCB's and PBB's as porphyrinogenic in many animal species and various mammalian cells (Gupta gt g;., 1983a; Nonaka gt g;., 1979; Strik gt _t., 1979; Vos and Koeman, 1970; Vos and Notenboom—Ram, 1972; Fulfs and Abraham, 1976; Goldstein l6 gt 1., 1975; Strik, 1973, 1978; Vos gt, gl., 1971). Acute administration of PCB's induced delta-amino- levulinic acid synthetase (d-ALAS) in rats and sig- nificantly increased total liver porphyrin con- centrations within one week after exposure to the com- pounds (Grote gt g;., 1975). Chronic dietary exposure of female rats to PCB's also increased levels of d—ALAS. However, increases in hepatic and urinary porphyrins were not seen until several months after initial exposure to these toxins. Commercial PBB's have similar porphyrinogenic activity in rodents, mice, and birds. The feeding of Firemaster BP-6 for six months to male and female rats and mice yielded a dose-dependent increase in hepatic porphyrins, and significant elevations were seen at doses as low as 0.3 mg/kg in male and female Fischer 344/N and male B6C3F1 mice (Gupta gt gl., 1983a). The mechanism by which these compounds cause porphyria in laboratory animals has not been elucidated. Many environmental chemicals induce d-ALAS and inhibit uroporphyrinogenic decarboxylase. These effects may be major factors in the mechanisms of action of these toxins. Porphyria induced by polyhalogenated aromatic hydrocarbons is species-specific, with effects noted in adult rats, mice, human beings, and some avian species. Interestingly, young rats, mink, and guinea pigs are less susceptible or do not have this lesion at all 17 (Strik (gt 1., 1979). There appears to be an as— sociation between species' susceptibility to hepatic lesions (such as hepatomegaly) and porphyria caused by PCB's and PBB's. Metabolism gt ngig gag Pgttg The PCB's, as well as many chlorinated organic pesticides, drugs, xenobiotics, and carcinogens, are potent inducers of hepatic and extrahepatic drug- metabolizing enzymes. Historically, microsomal enzyme inducers were exemplified by two main groups, specifically phenobarbital (PB) and 3-methylchol- anthrene (3-MC) inducers (Conney, 1967; Gillette gt gl., 1972; Parke, 1975; Lu gt g;., 1976; Snyder and Remmer, 1979; Imai and Sato, 1966). Pretreatment of laboratory animals with chemicals which induce PB-type enzyme activity causes increased levels of hepatic and extrahepatic phase I (microsomal) and phase II (cytosolic and microsomal) drug metabolizing enzymes. These induced enzymes include several cytochrome P—450- dependent microsomal mixed function oxidase (MFG, or monooxygenase) enzymes, which catalyze N- and O-de- alkylation, aromatic and aliphatic C-oxidation, glutathione S-transferases, glucuronyl transferases, and epoxide hydrolase. Additionally, 3-MC induces a similar range of enzyme activities. However, there are 18 differences in substrate and/or oxidation site specificities between PB- and 3-MC-induced. microsomal enzymes. Several studies show that there are several microsomal cytochrome P-450 isoenzymes (Lu and Levin, 1974; Guengerich, 1979; Ryan gt g;., 1982). Many individual PCB isomers and congeners have PB- or 3-MC- type inducing properties. Therefore, the mixed type induction of enzymatic activity seen with PCB's is dependent on the activity of the individual congeners present within these mixtures. Several experiments have shown that certain congeners of PCB's are metabolized by animals into hydroxylated intermediates (Hutzinger gt g;., 1972; Sundstrom gt g;., 1976; Safe, 1980). Furthermore, feeding of commercial PCB mixtures to laboratory animals has demonstrated the preferential excretion of the less chlorinated isomers (Burse gt g;., 1974; Burse, 1976). In addition, gas chromatographic and mass spectrometric analyses have demonstrated that the mono- and dihydroxylated PCB metabolites are eliminated in urine and feces (Safe gt g;., 1975). However, the pathologic effects of these metabolites has not been thoroughly examined. Some PCB and PBB congeners are not metabolized to any appreciable extent even though they are potent enzyme inducers. The congeners 2,2',4,4',5,5'- hexabromobiphenyl and 3,3',4,4',5,5'—hexachlorobiphenyl 19 are examples of such agents (Dannan gt _l., 1978; Mills gt g;., 1985). Enzyme induction, toxicity, and the rate and type of metabolism appear to be correlated with the location of bromine or chlorine of the phenyl rings. Results from in vitro studies indicate that certain PCB congeners are also metabolized by microsomal enzymes that require both oxygen and NADPH as cofactors for activity (Shimada, 1976; Shimada and Sato, 1978; Shimada gt g;., 1981). These in Vitro studies demonstrated that specific activity of the microsomal enzymes was dependent not only on PCB-pretreatment of the animal from which the cells were obtained but also on sub- strate structure. The metabolism of commercial PCB mix- tures was improved by the addition of PB and 3—MC microsomal enzymes. Shimada and co-workers (1981) have shown that PCB's are also metabolized by cytochrome P- 450 from rabbit and rat livers and that the PB-inducible enzymes from these species were the more potent metabolic catalysts. The metabolism of PCB's includes hydroxylation (i.e., Phase II metabolism), conjugation with thiols and other water soluble derivatives, and binding to macromolecules, especially proteins (Furukawa and Matsumura, 1976; Furukawa gt g;., 1979). The effects of PCB's as inducers of rat hepatic drug-metabolizing enzymes have been rigorously studied. These inductive effects occur in livers of males and females, fetal, neonatal, immature, mature, and 20 senescent rats. Moreover, PCB-induced rat microsomal enzymes readily metabolize other polycyclic aromatic hydrocarbons (Jacob gt gl., 1981; Gingell gt gl., 1981; Biggar gt g;., 1980; Jacob gt gl., 1982). The metabolism of PBB preparations, such as Firemaster BP-6, has not been fully elucidated, but compounds in the mixture are preferentially eliminated after dietary administration to rodents (Wolff and Selikoff, 1979). In addition, gas chromatographic analyses of PBB's in human tissues indicate that specific components within the Firemaster mixture are degraded (Wolff and Aubrey, 1978). Dent and co—workers first demonstrated that Fire- master BP-6 was a mixed type (i.e., PB and 3-MC) microsomal enzyme inducer in rats (Dent gt g;., 1976, 1978a). Like PB given with 3-MC, and similar to commercial PCB's, the commercial PBB's induce several MFO enzymes, including N—demethylases, O-dealkylases, PAH—hydroxylases, and steroid hydroxylases, as well as several phase II metabolic enzymes. The commercial PBB mixtures induce drug-metabolizing enzymes in many animal species and in both liver and nonliver tissues (Dent gt ‘g1., 1977a, 1977b, 1978b; Safe gt g;., 1978; Moore gt 1., 1978; Kluwe and e1., 1978a, 1978b; McCormack gt Hook, 1981). 21 Preneoplastic Hepatic Changes gg End Points for Carcinogenicity Assays Historically, the assessment of cancer risk of certain compounds has depended mainly on traditional histopathologic examination. The accepted end point in carcinogenicity testing is the histologically identified tumor. One disadvantage of this end point is the lengthy time period for these tumors to occur in the animal. Therefore, many efforts have been made to detect early biochemical or morphological markers which may be predictive of cancer lesions. During the past two decades, a number of characteristic cellular changes that regularly' precede the development of tumors has been observed, especially fix: the liver. Such changes have been designated as "preneoplastic lesions." These altered areas of preneoplastic cells usually form well- delineated foci and appear prior to the development of tumors. Hepatic preneoplasia has been exhaustively studied, especially in rats and mice, using various models (Peraino gt g;., 1983; Farber, 1984a; Ward, 1984). In rats, potentially preneoplastic hepatic foci are used as end points in carcinogenicity testing. A preneoplastic cell may be defined as a phenotypically altered cell which has no observable neoplastic nature, but which has a greater than normal 22 chance of becoming a benign or malignant tumor. Some workers contend that hyperplasia can be regarded as an early stage in tumor development (Farber and Cameron, 1980). But the use of the term "hyperplasia" in the context of carcinogenesis becomes confusing. By definition, the term hyperplasia is an increase in the number of tissue-specific cells caused by extracellular growth-stimulating factors (such as hormones), while the term "neoplasia“ implies an autonomous increase in the number of cells independent from such extracellular stimuli. A dilemma for pathologists is that early stages of neoplastic change may be characterized by a proliferation of cells which cannot be histologically distinguished from normal cells. While these lesions have the potential to become tumors without additional exposure to carcinogens or growth-stimulating factors, they are often classified as hyperplastic. It is probable that proliferating chemically-induced precursor lesions, whether classified as hyperplastic or preneoplastic, are already composed of irreversibly altered cells (Symposium of Rodent Liver Nodules, 1982). Several phenotypic patterns are seen histologically prior tx: the appearance of chemically-induced hepatic adenomas (also called "neoplastic nodules") and carcinomas in rats (Sasaki and Yoshida, 1935; Firminger, 1955; Reuber, 1965; Schauer and Kunze, 1976). Treatment of rats with nitrosamines induced focal hepatic lesions 23 characterized by excessive storage of glycogen (Bannasch, 1968). Others have shown a reduction in activity of the microsomal enzyme g1ucose-6-phosphatase (Gossner and Friedrich-Freska, 1964; Friedrich-Freska gt a_1., 1969). Results of light microscopic and ultra- structural studies have shown that rats treated. with certain diethylnitrosamines underwent a series of hepatic changes. First, clear to acidophilic glycogen- filled hepatocytes were seen. Later, basophilic cells were seen that were low in glycogen (Bannasch, 1968). These cells represent "altered foci" which persist after withdrawal of the carcinogen and may progress to adenomas and hepatocellular carcinomas (Schauer and Kunze, 1968; Scherer, 1984). The classification of "foci of altered hepatocytes" is seen as different from "neoplastic nodules" by several groups (Squire and Levitt, 1975; Stewart gt a_1., 1980; Bannasch gt _a_l., 1985). Altered hepatic foci may develop spontaneously in aged untreated rats. This may be due to small amounts of carcinogens in food or in the environment (Burek, 1978; Ward, 1984). Certain rat strains have an un- usually high incidence of spontaneous altered hepatic foci, suggesting a genetic predisposition toward their ievelopment (Ward, 1981). Many biochemical markers are used to identify rarcinogen-induced altered foci in rat hepatocytes. 24 Examples of enzymes which show a decreased activity in such foci include g1ucose-6-phosphatase, membrane-bound adenosine triphosphatase (Schauer and Kunze, 1968), acid and alkaline nucleases (Taper gt-gl., 1971; Taper gt gl., 1983), and glycogen phosphorylase (Scherer and Emmelot, 1976; Hacker gt g;., 1982). Enzymes that in- crease in activity in these foci include gamma-glutamyl- transpeptidase (GGT) (Kalengayi and Desmet, 1975; Hanigan and Pitot, 1985), glucose-6-phosphate de- hydrogenase (Hacker gt g;.,1982; Klimek gt gl., 1984), epoxide hydrolase (Enomoto gt g1., 1981; Kuhlman gt gl., 1981), uridine-diphosphate—glucuronyl transferase (Fischer gt g;., 1983; Sato gt g1., 1984), various isoenzymes of cytochrome P-450 (Schulte—Hermann gt gl., 1984; Buchmann gt gl., 1985), and glutathione transferases (Sato gt gl., 1984; Buchmann gt gl., 1985). Other alterations in preneoplastic hepatic tissue include resistance to experimental hemosiderosis (Williams gt g;., 1976; Williams and Watanabe, 1978), increased glutathione activity (Deml and 0esterle, 1980), and diminished lipid peroxidation (Benedetti gt gl., 1984). Of special interest is GGT. It has been considered a reliable indicator to assess preneoplastic changes in the liver. (Hanigan and Pitot, 1985). However, others nave found it to be lacking in some types of areneoplastic foci in rat liver (Butler gt g;., 1981; 25 Moore gt 1., 1983; Rao gt __ _l., 1984; Bannasch gt gl., 1985). Furthermore, an increase in the amount of GGT in periportal areas of the liver has been reported to occur with increasing age of the rat (Kitigawa gt gl., 1980a) or after partial hepatectomy (Bone gt gl., 1985). Some workers favor glutathione S-transferase placental form (GST-P) as a marker because it may be more accurate than GGT (Sato gt gl., 1984; Tatematsu gt gl., 1985; Thamavit gt g” 1985). Most phenotypic markers used for identifying preneoplastic cells are not stable. This represents a continuing problem in the evaluation of preneoplastic cellular changes. Therefore, given certain experimental conditions, these foci may phenotypically resemble persistent preneoplastic lesions, but may disappear after termination of the treatment. This phenomenon has been called "reversion—linked phenotypic instability" (Bannasch e_t g” 1985). Other changes that describe reversion—linked phenotypic instability of carcinogen- induced focal hepatic lesions have been described by numerous authors as "reversion," "remodeling," "neodifferentiation," or "maturation". (Kitigawa, 1971, 1976; Farber, 1976; Ito gt g;., 1976; Williams and Watanabe, 1978; Ogawa gt g;., 1979; Tatematsu gt g;., 1983). It is of interest that phenobarbital leads to a 26 more stable expression of altered hepatic foci. However, this is currently poorly understood (Moore gt gl., 1984). Farber (1984b) has stated that 95-98% of the chemically-induced nodular hepatic lesions are reversible, while only 1-3% persist and may progress to hepatocellular carcinomas. To definitively determine if such nodules will persist, some workers have recommended that the administration of the test compound should be stopped before termination of the experiment whenever foci with a disputed significance develop (Bannasch gt gl., 1982). The morphological transitions between altered hepatic foci, hepatic adenomas, and hepatocellular car- cinomas have been addressed by several workers (Farber, 1973; Williams and Watanabe, 1978; Bannasch gt .g1., 1982). These observations indicate that altered foci give rise to adenomas and that these, in turn, may progress to hepatocellular carcinomas. However, it is also probable that carcinomas can develop directly from altered foci without going through an intermediate adenomatous stage (Bannasch, 1976; Williams, 1976). Studies assessing cellular functional changes also indicate a close correlation between hepatic altered foci, nodules or adenomas, and hepatocellular carcinomas. Several workers have demonstrated a de- 'crease or increase in the activity of many enzymes using 27 enzyme histochemical methods in these lesions (Farber, 1980; Pitot and Sirica, 1980; Williams, 1980). Others have shown in both rats and mice that hepatocellular foci, nodules, and carcinomas do not accumulate iron in experimentally produced hepatic siderosis (Lipsky gt g;., 1979; Williams gt g;., 1979; Nigam gt gl., 1981). There appears to be a dose-dependent relationship for the induction of altered foci in rat livers. Several studies indicate that quantitative correlations occur between the size and number of foci and the dose or duration of treatment with the carcinogen. However, some have questioned this relationship due to large discrepancies between the number of foci appearing early during hepatocarcinogenesis and final tumor yield (Scherer and Emmelot, 1975; Emmelot and Scherer, 1980; Scherer, 1984; Kaufman gt _l., 1985). These data may suggest that only a small number of foci have the potential for progression to malignancy. An elementary interpretation of carcinogen testing usingj rat liver is generally agreed. upon.; If a test compound induces significantly more altered hepatic foci in treated animals than in untreated control animals, then the test chemical has carcinogenic potential. Because of possible reversion-linked phenotypic instability of these lesions, "stop" experiments may allow a better distinction between reversible and. irreversible preneoplastic lesions (Bannasch gt g;., 28 1982). All hepatocarcinogens tested to date have induced some level of focal hepatic lesion prior to the development of hepatic tumors. However, it remains unclear if this is always true. Therefore, the absence of hepatic foci after the administration of a test chemical does not necessarily preclude potential carcinogenicity (Bannasch gt g;., 1982). Initiation and Promotion it Carcinogenesis The process of carcinogenesis consists of multiple steps. The concepts of initiation and promotion are central to an understanding of chemically-induced carcinogenesis. Initiation is defined as an ir- reversible event involving a biochemical change in DNA. Cell proliferation is required to "fix" the biochemical lesions in DNA, and the altered DNA becomes a permanent property of the cell and its progeny (Craddock, 1976; Cairne, 1975; Ying gt gt., 1975). Nearly all initiators are in the form of procarcinogens and must be metabolically activated, usually by the cytochrome P-450 dependent monooxygenase system, to a form that has a high affinity for the genome (Czygan gt gl., 1973; Guengerich, 1977; Miller and Miller, 1969). However, some initiators are direct-acting and may cause ‘alkylation or acylation of DNA without prior metabolic activation (Miller and Miller, 1981). There is cogent 29 evidence that mutation is a major consequence' of initiation (Quintanilla gt gl., 1986). Promotion is a reversible epigenetic event that causes preferential mitogenic selection of initiated cells to become phenotypically similar to neoplastic cells upon repeated exposure to the promoter. The pro- cess of initiation may predispose a cell to the effects of promotion, but promoters generally have little tumorigenic effect on non-initiated cells. Complete carcinogens are defined as compounds that have both initiating and promoting ability. Historically, early researchers were able to distinguish between chemicals that were initiators or promoters (Rous and Kidd, 1941; Mottram, 1944; Berenblum, 1941). Classically, induction of mouse skin tumors was used to show that small doses of an initiator followed by repeated doses of a promoter caused papillomas first and carcinomas later (Berenblum and Shubik, 1949). The administration of only a single dose of initiator did not cause tumors, nor did repeated ad- ministrations of only a promoter (Boutwell, 1964). Moreover, tumors did not develop if the promoter was applied first followed by application of initiator (Williams gt _t., 1981). Currently, many initiation- promotion assays are used involving several different organ systems, including liver, thyroid gland, lung, colon, skin, mammary gland, stomach, kidney, pancreas, 30 urinary bladder, and thymus gland (Berenblum, 1979; Hicks gt g;., 1975, 1977; Leonard gt g;., 1982; Miyata e_t gt” 1985) . I Vivo Carcinogenic Effects of PBB's and PCB's The carcinogenicity of PBB's and PCB's is dependent upon several factors including the sex, strain, and species of the test animal as well as the composition of the commercial PBB or PCB formulation. Generally, these compounds are not regarded as initiators (i.e., genotoxins). However, several reports using different carcinogenicity testing systems indicate that PBB's and PCB's have promoting (i.e., epigenetic) activity (Kimura and Baba, 1973; Aishizumi, 1976; Kimura gt g;., 1976; Pereira gt gt., 1982; Deml and 0esterle, 1982; Hirose gt gl., 1981; Jensen gt gl., 1982a, 1982b; Preston gt g;., 1981;.Jensen and Sleight, 1986). Strong evidence that PBB's or PCB's cause cancer in people is lacking (Brown and Jones, 1981; Stross gt g;., 1981). Commercial PBB and PCB mixtures and specific con- geners of PBB's and PCB'c have been identified as tumor promoters. Firemaster BP-6 has been shown to enhance the development of enzyme-altered foci in DEN-initiated ‘partially hepatectomized rats (Jensen ,gt gl., 1982a, 1983). Using a similar assay, the PBB congener ,2,2',4,4',5,5‘—hexabromobiphenyl, the major congener of 31 Firemaster BP-6, caused increased numbers of enzyme- altered foci when compared to controls. (Jensen gt gt. 1982a). Another PBB congener, 3,3',4,4',5,5'—hexabromobi- phenyl was found to be hepatotoxic (Render gt gt., 1982) as well as a tumor promoter when fed at a concentration of 1.0 mg/kg to DEN-initiated partially hepatectomized rats (Jensen gt gt., 1982b; Jensen gt gt., 1983). This congener may have a different mechanism of tumor promoting action than the nonhepatotoxic congener 2,2',4,4',5,5'-hexabromobiphenyl (Jensen gt gt., 1983). Simultaneous exposure to more than one PBB congener has been shown to have a synergistic effect on tumor promotion. Initiated and partially hepatectomized rats fed a combination of 10 mg/kg 2,2',4,4',5,5'- hexabromobiphenyl plus 0.1 mg/kg 3,3'4,4',5,5'- hexabromobiphenyl had a more than additive number of enzyme-altered foci and hepatic nodules when compared to similarly treated rats fed either one congener or the other (Jensen and Sleight, 1986). Interestingly, Fire- master BP-6 had a greater ability to enhance development of enzyme—altered foci than 2,2'4,4',5,5'-hexabromo— biphenyl, the major congener of the Firemaster BP—6 mixture (Jensen gt gt., 1982b). These results suggest that additive or synergistic interactions of congeners in this commercial mixture may have been responsible for its greater tumor promoting ability. 32 Two-Stage Models for Hepatic Tumor Induction tg Rats Carcinogen-Promoter Model Peraino and co-workers (1971, 1973a, 1973b, 1977) introduced this model in which rats were given 2- acetylaminofluorene (2-AAF) for 18 days. One week later, rats were fed a diet containing 0.05% phenobarbital, a known tumor promoter in rats, for eight months. Test chemicals could be assessed by sub- stituting either the initiator or promoter. For ex- ample, in place of 2-AAF, diethylnitrosamine (Weisberger gt gt., 1975), 3'smethyl-4-dimethylamino- benzene (Kitigawa and Sugano, 1978), and 2—methyl-4-di- methylaminoazobenzene (Kitigawa gt gt., 1979) have been used as alternative initiators. This model provided a new approach to analysis of the promoting effects of chemicals. Partial Hepatectomy + Promotion Model This model was proposed as a combination of the two stages of carcinogenesis (Pitot, 1977; Pitot .gt_ gt., 1978a, 1978b). Rats are given a single dose of DEN by intubation 24 hours after partial hepatectomy. Eight weeks later, a group of the animals are given phenobarbital (0.05%) for 24 weeks as a positive control. Modifications of this model include using 33 benzo(a)pyrene as an initiator (Kitigawa gt 1., 1980b). Phenobarbital can be replaced with a choline-deficient diet for the promotional phase (Shinozuka gt _t., 1979). Another modification uses dimethylhydrazine as the initiator and orotic acid as the promoter (Laurier gt gt., 1984). Results from these studies helped to clarify the conceptual distinction between the two stages of liver carcinogenesis in rats. Selective Pressgtg Model The selective pressure model was introduced by Solt and Farber (1976, 1977). The initiator was DEN injected intraperitoneally, and two weeks later animals were placed on a basal diet containing 0.02% 2-AAF. After one week of 2—AAF feeding, the DEN—treated animals were partially hepatectomized. One week thereafter, animals were returned to the carcinogen-free basal diet for eight months. The advantage of this model is the relatively rapid and marked induction of enzyme-altered foci. Furthermore, these foci are essentially syn- chronous and, therefore, are easy to follow for tumor sequence studies. MATERIALS AND METHODS Protocol The Pitot model of two—stage (initiation/promotion) hepatocarcinogenesis was used for this study (Pitot gt gt., 1978). Outbred female Sprague—Dawley rats (Charles River Corporation, Portage, MI) weighing about 190-210 grams were acclimated for one week. Rats that were to be hepatectomized ‘ were anesthetized with ether (Mallinckrodt Inc., Paris, KY), and two-thirds of the liver' was ligated and removed (Higgins and. Anderson, 1931). Twenty-four hours later, diethylnitrosamine (DEN) (Sigma Chemical Co., St. Louis, MO) was ad- ministered intraperitoneally at a dose of 10 mg/kg body weight. Thirty days after the partial hepatectomy, rats were randomly assigned into groups (Table 1—2, page 47). Diets were prepared by adding phenobarbital (PB), 2,2‘,4,4',5,5'-hexabromobiphenyl (245-HBB), .or 3,3', 4,4',5,5'-hexachlorobiphenyl (345-HCB) in corn oil to a commercial ground diet for rats (Wayne Lab Blox, Allied Mills, Inc., Chicago, IL). Amounts of each compound in the diet are listed in Table 1-2. Water was available 34 35 ithe diet are listed in Table 1—2. Water was available free choice. Negative controls included rats not hepatectomized or given DEN but maintained either on the same basal diet or treatment diets. Rats were kept in standard clear plastic cages within laminar flow units (Contamination Control Inc., Lansdale, PA) at 220 C with a 12 hour light/dark cycle. Test Chemicals The 245-HBB used in this study was isolated from a commercial PBB mixture (Firemaster BP-6, Michigan Chemical Co., St. Louis, MI). The 345—HCB was obtained from a commercial laboratory (RFR Corporation, Hope, RI). Isolation of 245-HBB and. purification of both congeners was performed using chromatographic methods by personnel in the Department, of Biochemistry, Michigan State University (Moore and Aust, 1978). Greater than 99% purity of each congener was obtained. Necropsy, Tissue Collection, ggg Histologic Technigges Rats were maintained on diets for 150 days, after which they were anesthetized by ether (Mallinckrodt Inc., Paris, KY), killed using decapitation, and necropsied. The brain, kidneys, spleen, liver, thymus gland, and thyroid glands were removed and weighed (Mettler Instrument Corp., Highstown, NJ). Five sections .of liver, taken from the same portions of hepatic lobes from each rat, were mounted on corks (Slee International 36 Inc., London, England) and frozen by immersion into isopentane cooled with liquid nitrogen. Representative sections of liver for histological evaluation were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at a thickness of six um , and stained with hematoxylin and eosin. Other samples of liver and body fat were collected for chemical analysis, wrapped in aluminum foil, and stored at -20 0 C. Sections of liver for microsomal enzyme analysis were placed into cold 1.15% KCl containing 0.2% nicotinamide. Histochemical Staining Frozen and cork-mounted sections of liver were cut at a thickness of eight um using a cryostat (Slee International Inc., London, England) and stained for gamma glutamyl transpeptidase (GGT) activity (Rutenberg e_t _a_l_., 1969). The eight um sections of liver were placed on a cover glass, fixed in 100% acetone for 15 minutes, air-dried, and incubated for 15 minutes in a solution of one m1 of gamma-glutamyl-4-methoxy-2- naphthylamide (2.5 mg/ml) (Vega Biochemicals, Tuscon, AZ), five m1 of 0.1 M Tris buffer at pH 7.4 (sigma Chemical Co., St. Louis, MO), 10 mg glycylglycine (Sigma Chemical Co., St. Louis, MO) , 10 mg Fast Blue BBN (Sigma Chemical Co., St. Louis, MO), and 14 m1 of 0.85% saline solution. The section was then washed in 0.85% saline for two minutes and transferred to a 0.1 M cupric 37 sulfate solution (sigma Chemical Co., St. Louis, M0) for another two minutes. The section was washed a second time for two minutes with 0.85% saline, rinsed in distilled water, counterstained with hematoxylin (Gills Hematoxylin No. 3, Pblysciences Inc., Warrington,~PA) for 15 minutes, air-dried, and mounted on a glass slide. The histochemically stained slide was placed onto a Leitz Prado ‘Projector (Ernst Leitz Wetzlar GMBH, Wetzlar, West Germany), magnified 90 X, and outlines of GGT-positive foci were traced. An approximately equal area of liver was evaluated (2.5-3.5 cmz) from each rat. The area of each GGT-positive focus was determined with a planimeter (Lasico L-30, Los Angeles Scientific Co., Inc., Los Angeles, CA), and the number of GGT-positive foci/cm3 was obtained by using the formula of Scherer (1981). Microsomal Enzyme Assays Hepatic microsomes were isolated and stored by established techniques (Moore gt gt., 1978; Welton and Aust, 1974) by personnel in the Department of Biochemistry, Michigan State University. Liver in cold KCl was weighed, homogenized, and centrifuged once at 10,000 xg for 20 minutes, followed by 90 ndnutes at 105,000 xg, using the supernatant of the first cen- trifugation for the second centrifugation. Microsomes were washed and stored at —20 0 C in 0.05 M Tris-HCl at 38 pH 7.5 with 50% glycerol and 0.01% butylated hydroxytoluene. Analysis 29; 245-HBB gpg 345-HCB tg ttygt gpg Egt Concentrations of 245-HBB and 345-HCB in liver and body fat were determined using a technique by Thompson (1977) by personnel in the Department of Pathology, Michigan State University. Liver and fat samples from two rats within each group were analyzed. Samples of 0.5 grams were washed with petroleum ether, ground with washed ignited sand (Mallinckrodt Inc., Paris, KY), and dehydrated by adding 10—20 grams of granular anhydrous sodium sulfate (Mallinckrodt Inc., Paris, KY). For determining 245-HBBy concentrations, fat and liver samples had 15 ml of hexane distilled in glass (J.T. Baker Chemical Co., Phillipsburg, NJ) added, and the mixture was brought to a boil over an 80 0 C water bath. The mixture was then filtered into a flask. Hexane washes and further filtrations were repeated for a total of four extractions. Tissues for 345-HCB analysis were treated similarly except that extraction was done with toluene distilled in glass (J.T. Baker Chemical Co., Phillipsburg, NJ) instead of hexane. The sample was added to a: column filled with 1.5 grams of activated magnesium silicate (Florisil, 60—100 mesh, Fischer Scientific Co., Cleveland, OH) topped with two cm of granular anhydrous sodium sulfate. The 39 columns had been prewashed with acetone followed by hexane. After adding the sample, the column was repeatedly washed with hexane. The eluant was condensed to 0.5 ml and 2,2,4-trimethylpentane (Burdick and Jackson Laboratories, Inc., Muskegon, MI) was added to create a total volume of two ml. A volume of two ul of eluant was injected into a gas chromatograph (GC Model 3700, Varian Instrument Division, Palo Alto, CA). For 245-HBB, injector temperature was 280 0 C, column temperature was 250 0 C, and, detector temperature was 310 0 C. For 345-HCB, column temperature was 300 0 C and detector temperature was 350 0 C. The carrier gas was nitrogen at a rate of 30 ml/minute. Tracings from the gas chromatograph were recorded and compared to standards. Concentrations of 245-HBB and 345-HCB were deter- mined from lipid samples using a 20 ml aliquot of each hexane or toluene extracted sample, respectively. The solvent was evaporated and the sample was placed in a preweighed foil container and dried under vacuum. Following drying, the remaining lipid was weighed, and the percentage of lipid in the original sample was determined. Statistical Analysis Data were analyzed using the one—way analysis of _variance (Steel and Torrie, 1980a). Multiple 40 comparisons were analyzed using a Student-Newman-Keul's test (Steel and Torrie, 1980b). Differences between groups were considered significant at the P 5 0.05 level. RESULTS Body Weight Gains and Organ Weights The effects of diets containing 2,2',4,4',5,5'- hexabromobiphenyl (245-HBB), 3,3',4,4',5,5'- hexachloro- biphenyl (345—HCB), and combined 245-HBB/345-HCB on body weight gain, thymic weight, and hepatic weight are shown in Table 1-1. Rats that were DEN-initiated, partially hepatectomized, and fed diets containing 1.0 mg/kg 345— HCB or a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB had a significant decrease in total body weight gain. when compared to similarly 'treated rats fed a basal diet. Hepatic weights were significantly in- creased in DEN—initiated, partially hepatectomized rats fed diets containing 100 mg/kg 245—HBB or 1.0 mg/kg 345- HCB, as well as in those rats fed a combination of 100 mg/kg 245-HBB and 1.0 mg/kg 345-HCB. No significant changes in the weights of brain, thymus gland, kidneys, thyroid glands, or spleen were found in treated rats when compared to control rats. Histopathologic Evaluation gt the Liver Livers from rats fed diets containing 500 mg/kg of phenobarbital or 10 mg/kg 245-HBB had hypertrophy of 41 42 Table 1-1. Effects of Dietary 245-HBB and 345—HCB on body weight gains, thymic weight, hepatic weight, and histologic structure of livers of rats. Chemical Body wt. Absolute Absolute Histology mg/kg diet gain thymus wt. liver wt. of liver Basal diet 88147 .33i.10 7.5tl.0 Normal 500mg PBa 74:17 ~24i-03 8.7il.4 Hepatocyte hypertrophy in CL region 10mg 245-HBB 87116 .281.08 8.8il.0 Hepatocyte hypertrophy in CL region 100mg 245-HBB 75i21 .21i.05 10.1:2.lc Hepatocyte hypertrophy in CL region; altered foci 0.1mg 345-HCB 89i17 .24i.03 8.1+1.2 Mild macro/ microve51cu- lation; al- tered foci 1.0mg 345-HCB 73:24‘3 .3oi.o9 13.9i2.70d Moderate macro/micro- vesiculation; inflammation; altered foci 10mg 245-HBB+ 0.1mg 345-HCB 75i29 .24:.06 7.5io.8 Moderate macro/micro- vesiculation; inflammation; altered foci 100mg 245-HBB+ c 1.0mg 345-HCB 53:18c .21:.O7 11.3:4.9 Severe macro/ m1croves1cu- lation; in- flammation; altered foci Data in grams as mean i SD for 6 rats. Rats had partial hepatectomy and diethylnitrosamine 1? days before dietary treatment. a Phenobarbital. Centrolobular. c Significant difference (P50.05) from basal diet group. Significant difference (P50.05) from all groups except group fed 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB. 43 hepatocytes in centrolobular regions of hepatic lobules. Histologically altered foci and hepatocytic hypertrophy in centrolobular regions were seen in sections of liver from rats fed 100 mg/kg 245—HBB. Livers from rats fed diets containing 0.1 mg 345-HCB had mild diffuse microvesicular and macrovesicular changes (presumably due to fatty change) and occasional altered foci. Livers from rats given 1.0 mg/kg 345-HCB or a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HCB had moderate micro- and macrovesicular changes, moderate hepatocyte hypertrophy in centrolobular regions, and occasional altered foci (Figure 1-1). Hepatic changes were the most severe in rats fed a combination of 100 mg/kg 245-HBB with 1.0 mg/kg 345—HCB. Livers from these rats had severe micro- and. macrovesiculation, severe hepatocyte hypertrophy in centrolobular regions, mild to moderate inflammatory changes in periportal areas, multifocal altered foci (Figure 1-2), and mild hyperplasia of biliary epithelium. Preneoplastic changes seen in livers from these rats included foci of altered cells (Institute of Laboratory Animal Resources, National Research Council, 1980). These islands of hepatocytes had cells which were mildly basophilic and slightly enlarged with abundant cytoplasm. They contained enlarged and occasionally 44 Figure 1-1. Photomicrograph of the centro- lobular and midzonal region of the liver from a rat fed a diet containing 1.0 mg/kg 345-HCB for 150 days after a partial hepatectomy and administration of 10 mg/kg diethylnitrosamine intraperitoneally. Notice diffuse hepatocyte hypertrophy, macrovesicular and microvesicular changes, loss of sinusoidal space, and mild inflammatory cell infiltrates (H & E, 160 X). Figure 1-2. Photomicrograph of a focus of hepatocellular alteration from a rat fed a diet containing a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB for 150 days after a partial hepatectomy and administration of 10 mg/kg diethylnitrosamine intraperitoneally. The hepato- cytes in this focus have hypertrophied, and a cell in the center of the focus is trinucleate. Notice the macrovesicular and microvesicular changes in the surrounding parenchyma (H & E stain, 160 X). 46 multiple nuclei (Figure 1-2), often with multiple nucleoli. One hepatic nodule was seen in a rat given 500 mg/kg phenobarbital, but hepatic nodules were not observed in rats fed 245—HBB or 345—HCB. Hepato- cellular carcinomas were not observed in any rats in this study. GGT—positive Foci The numbers of GGT-positive foci for rats in each group are shown in Table 1-2. The typical appearance of a GGT-positive focus is seen in Figure 1—3. Rats nei- ther partially hepatectomized nor given DEN that were then fed diets containing 245—HBB, 345-HCB, or a combination of 245-HBB with 345-HCB had fewer GGT- positive foci when compared to partially hepatectomized and DEN-initiated rats fed the same diets. Rats that were partially hepatectomized, given DEN, and fed either 245-HBB, 345-HCB, or a combination of 245-HBB with 345- HCB had significantly greater GGT—positive foci/cm3 in their livers when compared to other DEN-treated partially hepatectomized rats fed only a basal diet. The DEN-treated partially hepatectomized rats with the greatest number of GGT—positive foci were fed a combination of 10 mg/kg 245-HBB and 0.1 mg/kg 345-HCB. The number of GGT-positive foci for this group was nearly a summation of the GGT-positive foci for rats fed 47 Table 1-2. Experimental Design and Number of GGTC- positive Foci per Cubic Centimeter of Liver. Group Treatment Chemical No. rats GGT+ foci/cm3 No. mg/kg diet per group mean 1 SD 1 PHa + DENb Basal diet 6 48128 2 None Basal diet 3 010 3 PH + DEN 500 PB d 6 284011297 e 4 PH + DEN 10 mg 245-HBB 6 169511800 e 5 None 10 mg 245-HBB 3 66138 6 PH + DEN 100 mg 245-HBB 6 11461536 8 7 None 100 mg 245-HBB 3 61140 8 PH + DEN 0.1 mg 345-HCB 6 2951192 e 9 None 0.1 mg 345-HCB 3 917 10 PH + DEN 1.0 mg 345-HCB 6 134311090 e 11 None 1.0 mg 345-HCB 3 76146 12 PH + DEN 10 mg 245-HBB+ 0.1 mg 345-HCB 6 18521629 elf 13 None 10 mg 245-HBB+ 0.1 mg 345-HCB 3 61135 14 PH + DEN 100 mg 245-HBB+ 1.0 mg 345-HCB 6 6121220 6'9 15 None 100 mg 245-HBB+ 1.0 mg 345-HCB 3 87169 a Partial hepatectomy. IDdDiethylnitrosamine. c Gamma glutamyl transpeptidase. Phenobarbital. Sig- nificantly different (P g 0.05) from group 1. Sig- nificantly different (P g 0.05) from group 8. g Sig- nificantly different (P 5 0.05) from group 12. 48 Figure 1-3. Photomicrograph of a histo- chemically stained section of liver from a rat fed a diet containing 10 mg/kg 245-HBB for 150 days after a partial hepatectomy and administration of 10 mg/kg diethylnitrosamine intraperitoneally. Notice the well-defined focus of hepatocytes that have positive staining for gamma glutamyl transpeptidase activity in their cytoplasms (Gamma glutamyl transpeptidase stain, 160 X). 49 Figure 1-3 50 10 mg/kg 245-HBB alone and rats fed 0.1 mg/kg 345-HCB alone. However, rats receiving a combination of 100 mg/kg 245—HBB plus 1.0 mg/kg 345-HCB had only about half as many GGT-positive foci when compared to rats receiving either 100 mg/kg 245—HBB alone or 1.0 mg/kg 345-HCB alone. Concentrations of 245-HBB and 345—HCB in liver and adipose tissue are shown in Table 1-3. Rats receiving exclusively 100 na/kg of 245-HBB in the diet had ap- proximately seven and 11 times more 245-HBB in their livers and adipose tissue, respectively, than rats receiving 10 mg/kg 245-HBB. Similarly, rats receiving exclusively 1.0 mg/kg 345-HCB in the diet had ap— proximately five and eight times more 345-HCB in their livers and adipose tissue, respectively, than rats receiving 0.1 mg/kg 345-HCB. The amount of 245-HBB was similar in livers of rats fed either 10 mg/kg 245-HBB alone or 10 mg/kg 245—HBB in combination with 0.1 mg/kg 345-HCB. Likewise, the amount of 245-HBB was similar in adipose tissue of rats fed either 10 mg/kg 245-HBB alone or 245-HBB in combination with 0.1 mg/kg 345-HCB. Generally, 245-HBB reached higher concentrations in adipose tissue than in liver, regardless of whether the 51 Table 1-3. Concentrations of 245-HBB and 345-HCB in Liver and Adipose Tissue of Rats Fed a Basal Diet or Diets Containing Phenobarbital, 245-HBB, 345-HCB, or combined 245-HBB/345-HCB for 150 Days. Tissue Concentration (mg/kg)a Chemical (mg/kg) Liver Adipose Tissue in diet % Fat HBBb HCBc % Fat HBB HCB Basal 411 010 010 8212 010 010 diet 500 mg 4:1 0:0 0:0 84:1 0:0 0:0 PB d 10 mg 612 12:1 0:0 8313 300:94 0:0 245-HBB 100 mg 3:1 88139 0:0 71:9 3117:1252 0:0 245-HBB 0.1 mg 611 010 711 8112 010 312 345-HCB 1.0 mg 11:2 0:0 38:29 77:1 0:0 25:5 345-HCB 10 mg 245-HBB plus 4:1 9:3 4:1 8114 289:92 2:1 0.1 mg 345-HCB 100 mg 245-HBB plus 1716 12831442 4214 7417 422011149 3715 1.0 mg 345-HCB a Values are expressed on lipid basis and represent the mean 1 SD for 3 rats, 2 of which received a partial hepatectomy plus 10 Eg/kg body weight diethylnitrosamine intraperitoneally. 2,2',4,4',5,5'-h§xabromobiphenyl. c 3,3',4,4',5,5'-hexachlorobiphenyl. Phenobarbital. 52 concentration of 245-HBB in the diet was 10 mg/kg or 100 mg/kg. Conversely, 345-HCB reached relatively higher concentrations in hepatic tissue than in adipose tissue regardless of the concentration of 345-HCB (0.1 mg/kg or 1.0 mg/kg) in the diet. Microsomal Enzyme Assays Concentrations of hepatic cytochrome P-450 and activities of hepatic enzymes aminopyrine demethylase and ethoxyresorufin-o-deethylase are shown in Table 1-4. Rats given 245-HBB alone or 345-HCB alone in the diet had an apparent dose-related increase in the con- centration of cytochrome P-450. Rats fed diets con- taining 345—HCB either alone or in combination with 245- HBB had a downward shift in carbon monoxide difference spectra of cytochrome P-450 when compared to rats fed either the basal diet or 245-HBB. When compared to rats fed a basal diet, aminopyrine demethylase activity was most increased in rats fed 100 mg/kg 245-HBB alone and was somewhat increased in those rats fed a combination of 100 mg/kg 245-HBB with 1.0 mg/kg 345-HCB. Ethoxyresorufin—o-deethylase activity was increased in rats fed 1.0 mg/kg 345—HCB alone or in combination with 100 mg/kg 245-HBB. The activity of ‘this enzyme was relatively low in rats fed only a basal diet, 10 mg/kg 245-HBB, or 100 mg/kg 245-HBB. 53 Table 1—4. Effects of 245-HBB and 345—HCB on the Concentration of Cytochrome P-450 and the Activity of Aminopyrine Demethylase and Ethoxyresorufin-o-deethylase in Rat Liver. Chemical Cytochrome alpgg Aminopyrine Ethoxyresorufin— (mg/kg) P-450a ma demethylasec o-deethylasec in diet Basal diet 1.0610.23 449.6 4.4210.13 0.7310.13 10 mg/kg 245-HBB 2.2810.39 449.2 6.6211.61 0.4910.03 100 mg/kg 245-HBB 2.4410.l4 449.5 12.3010.56 l.5312.30 0.1 mg/kg 345-HCB 2.32:0.25 448.7 4.97:1.24 33.90:4.45 1.0 mg/kg 345-HCB 3.52:0.09 448.5 7.29:2.24 54.00111.98 10 mg/kg 245-HBB plus 2.2110.21 448.5 5.8910.58 29.1016.06 0.1 mg/kg 345-HCB 100 mg/kg 245-HBB plus 3.8410.80 448.5 8.331l.01 57.601l.25 1.0 mg/kg 345-HCB Data are expressed as mean 1 SD for three rats from each group, analyzed as pooled samples. nmols/mg protein nanometers c nmols/mg protein/minute 54 chgt Findings Formalin-fixed tissues in their containers were examined with ultraviolet light to detect the presence of porphyrins. Rats fed a combination of 10 mg/kg 245- HBB plus 0.1 mg/kg 345-HCB tested relatively strongly for porphyrins, and rats fed a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB tested relatively weakly for porphyrins. However, porphyrins were neither fur- ther characterized run: quantified. Porphyrinogenic ac- tivity was not detected in other treatment groups or controls. DISCUSSION Compounds that enhance the development of foci positive for gamma glutamyl transpeptidase (GGT) in the livers of initiated and partially hepatectomized rats are considered tumor promoters (Pitot gt a_1., 1978a; Leonard gt gt., 1982). Dietary concentrations of 10 or 100 mg/kg of 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB), 0.1 or 1.0 mg/kg 3,3',4,4',5,5'-hexachlorobiphenyl (345- HCB) , a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345—HCB, or a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB caused significantly increased numbers of GGT-positive foci in the livers of these rats when compared to initiated and partially hepatectomized rats fed basal diets. A dose-dependent increase in the number of GGT- positive foci was seen in initiated and partially hepatectomized rats fed 345—HCB. Rats fed 1.0 mg/kg 345-HCB alone had approximately a five-fold increase in GGT-positive foci when compared to rats fed 0.1 mg/kg 345-HCB alone. Conversely, initiated and partially hepatectomized rats fed only 100 mg/kg 245-HBB had no significant difference in the number of GGT-positive foci when compared to rats fed 10 mg/kg 245-HBB. In contrast, the results of a previous study using an 55 56 identical protocol showed a significant dose-dependent increase in the number of GGT-positive foci in rats fed 100 mg/kg 245-HBB when compared to rats fed 10 mg/kg 245-HBB (Jensen gt 94., 1982b) . Small numbers of GGT-positive foci occurred in the livers of rats fed various concentrations of 245—HBB and 345-HCB that had not undergone diethylnitrosamine (DEN) administration or partial hepatectomy. These foci may arise if rats had been previously exposed to low levels of initiators from the environment (Pitot gt gt., 1980; Pitot and Sirica, 1980; Williams gt .gt., 1981). Al- ternatively, these compounds could act as both initiators and promoters and thus behave as complete carcinogens, but evidence for PBB's or PCB's acting as complete carcinogens is generally lacking (Garthoff gt gt., 1977). However, in one study a large single dose of Firemaster BP—6 caused hepatocellular carcinomas in rats not previously initiated (Kimbrough gt gt., 1981). Nevertheless, results from the current study confirm the tumor promoting ability of these compounds. Initiated and partially hepatectomized, rats fed combinations of 245-HBB plus 345-HCB are of particular interest. A combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HCB caused greatly increased numbers of GGT- positive foci. This number was nearly a summation of the GGT—positive foci in rats fed exclusively 10 mg/kg 245-HBB and exclusively 0.1 mg/kg 345-HCB. Therefore, 57 the effect of feeding this combination of compounds may best be described as an additive tumor promoting effect. This is in contrast to the results of a similar study in which 245-HBB and another polybrominated congener, 3,3',4,4',5,5'-hexabromobiphenyl (345-HBB), had a synergistic, rather than additive, effect on GGT- positive foci when fed to DEN-initiated partially hepatectomized rats at a concentration of 10 mg/kg 245- HBB plus 0.1 mg/kg 345-HBB (Jensen and Sleight, 1986). Given the structural similarity and the nearly identical toxic effects of 345-HCB and 345-HBB, it is of interest that these compounds did not have the same tumor promoting effect, whether additive or synergistic, when fed in combination with 10 mg/kg 245-HBB. Perhaps this is due to the different halogens in 345-HBB and 345-HCB. Moreover, comparison of these studies is hampered by the fact that concentrations of 345-HBB and 345-HCB were prepared in mg/kg (or ppm) concentrations rather than molar concentrations. Therefore, the number of moles of bromine in rats fed 1.0 mg/kg of 345-HBB would be different from the number of moles of chlorine in rats fed 1.0 mg/kg 345-HCB. Subtle differences in halogen concentration in these compounds may be responsible for their slightly different tumor promoting ability. However, the mechanism of action of tumor promotion for either of these compounds is unknown. 58 Jensen and Sleight (1986) found an inhibitory effect of the formation of GGT-positive foci in rats fed a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HBB compared to the number of such foci in rats fed either 100 mg/kg 245-HBB alone or 1.0 mg/kg 345-HBB alone. These results are in agreement with the results of the present study in which 345-HCB was substituted for 345- HBB. The mechanism of inhibition of tumor promotion by diets containing a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB is unknown. One possible explanation is that this combination of toxicants caused a suppression of body weight gains. Perhaps available dietary nutrients were not utilized as efficiently in these rats as in rats given only 245—HBB or 345-HCB. Such alterations of body growth may have negative effects on tumor formation, since long-term dietary restrictions have been shown to decrease the incidence of naturally-occurring tumors in rodents (Tucker, 1979). However, the reasons that one combination of poly- halogenated hydrocarbons in the diet caused an additive effect on tumor promotion while different concentrations of the same toxicants in the diet caused an inhibitory effect on tumor promotion remain to be determined. SUMMARY-CHAPTER 1 Conclusions from the previously described studies include the following: 1) The compounds 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB) and 3,3',4,4',5,5'-hexachlorobiphenyl (345- HCB) have tumor promoting ability in a two-stage hepatocarcinogenesis assay. 2) A diet containing a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HCB caused an apparent additive effect on tumor promotion as determined by measuring hepatic gamma glutamyl transpeptidase- (GGT-) positive foci in a two-stage hepatocarcinogenesis assay. 3) A diet containing a combination of 100 mg/kg 245-HBB plus 1.0 mg/kg 345-HCB caused an inhibitory effect on tumor' promotion as determined. by measuring GGT-positive hepatic foci. As determined by the preceding experiments, 245-HBB and 345-HCB behave as tumor promoters of experimental hepatocarcinogenesis in rats. However, the results of such animal studies are difficult to extrapolate to determine human health risks from exposure to such 59 60 environmental toxicants. Unfortunately, people have been exposed to relatively high doses of polybrominated and polychlorinated biphenyls. The effect of these exposures on the development of cancer in people is currently unknown. BIBLIOGRAPHY-CHAPTER l BIBLIOGRAPHY Aishizumi M: Enhancement of diethylnitrosamine hepato- carc1nogenesis in rats exposed to polychlorinated biphenyls or phenobarbital. Cancer Lett 2:11-18, 1976. Allen. JR, Norback. DH: Polychlorinated. biphenyl and triphenyl induced gastric mucosal hyperplasia in primates. Science 179:498-499, 1973. Allen JR, Carstens LA, Barsotti DA: Residual effects of short-term, low-level exposure of nonhuman primates to polychlorinated. biphenyls. Toxicol Appl Pharmacol 30:440-451, 1974. Allen JR, Barsotti DA: The effects of transplacental and mammary movement of PCBs on infant rhesus monkeys. Toxicology 6:331-340, 1976. Allen JR, Lambrecht LK: Responses of rhesus monkeys to polybrominated biphenyls. Toxicol Appl Pharmacol 45:340-341, (Abstr.), 1978. Allen JR, Lambrecht LK, Barsotti DA: Effects of polybrominated biphenyls in non-human primates. J Am Vet Med Assoc 173:1485-1489, 1978. Allen JR, Hargraves WA, Hsia MTS, Lin FSD: Comparative toxicology of chlorinated compounds on mammalian species. Pharmacol Ther 7:513-547, 1979. Altman NH, New AE, McConnell EE, Ferrell TL: A spon- taneous outbreak of polychlorinated biphenyl (PCB) toxicity in rhesus monkeys (Macaca mulatta): clinical observations. Lab Anim Sci 29:661-665, 1979. Atlas E, Giam CS: Global transport of organic pol- lutants: ambient concentrations in the remote marine atmospheres. Science 211:163-165, 1981. 61 62 lerich RJ, Ringer RK, Iwamoto 8: Reproductive failure and mortality in mink fed on Great Lakes fish. J Reprod Fertil Suppl 19:365-376, 1973. Llerich RJ, Ringer RK: Toxic effects of dietary polybrominated biphenyls on mink. Arch Environ Contam Toxicol 8:487—498, 1979. x RL, Hansen LG: Effects of purified polychlorinated biphenyl analogs on chicken reproduction. Poultr Sci 54:895-900, 1975. Sallschmitter K, Zell M: Analysis of polychlorinated biphenyls (PCB) by glass capillary gas chro- matography. Composition of technical Aroclor- and Clophen-PCB mixtures. Fresenuis Z Anal Chem 302: 20—31, 1980. Bannasch P: The cytoplasm of hepatocytes during carcinogenesis. Light and electron. microscopic investigations of the nitrosomorpholine- intoxicated rat liver. Rec Res Cancer 1921-100, 1968. Bannasch. P: Cytology and cytogenesis of neoplastic (hyperplastic) nodules. Cancer Res 36:2555-2562, 1976. Bannasdh P, Moore MA, Klimek F, Zerban H: Biological markers of preneoplastic foci and neoplastic nodules in rodent liver. Toxicol Pathol 10:19-34, 1982. Bannasch P, Benner U, Enzmann H, Hacker HJ: Tigroid cell foci and neoplastic nodules in the liver of rats treated with a single dose of aflatoxin B-l. Carcinogenesis 6:1641-1648, 1985. Barbehenn KR, Reichel WL: Organochlorine concentra- tions in bald eagles: brain/body lipid relations and hazard evaluation. J Toxicol Environ Health 8:325-330, 1981. Barsotti DA, Marlar’ RJ, Allen JR: Reproductive dys- function in rhesus monkeys exposed to low levels of polychlorinated biphenyls. Food Cosmet Toxicol 14:99-103, 1976. Beaudoin AR: Teratogenicity of polybrominated bi- phenyls in rats. Environ Res 14:81-86, 1977. 63 ter GM, McNulty WP, Bell M: Polychlorinated bi- phenyl-induced morphological changes in the gas— tric mucosa of the rhesus monkey. Lab Invest 40: 373-383, 1979. 1 WE: Relative toxicity of the chlorinated naph- thalenes in experimentally produced bovine hyper- keratosis (X-disease). Vet Med Sm Anim Clin 48: 135-140, 1953. aedetti A, Malvadi G, Fulceri R, Comporti M: Loss of lipid peroxidation as a histochemical marker for preneoplastic hepatocellular foci in rats. Cancer Res 44:5712-5717, 1984. erenblum. I: The cocarcinogenic action of croton resin. Cancer Res 1:44—48, 1941. erenblum I, Shubik P: A new quantitative approach to the study of the stages of chemical carcinogenesis in the mouse skin. Brit J Cancer 1:383—391, 1947. Biggar CAH, Tomaszewski, Andrews AW, Dipple A: Evaluation of metabolic activation of 7,12-di- methylbenz[a1anthracene in vitro by Aroclor 1254- induced rat liver S-9 fraction. Cancer Res 40: 655-661, 1980. Bleavins MR, Aulerich RJ, Ringer RK, Bell TG: Exces- sive nail growth in the European ferret induced by Aroclor 1242. Arch. Environ Contam ‘Toxicol 11: 305-312, 1982. Bone III SN, Michalopoulos G, Jirtle RL: Ability of partial hepatectomy to induce gamma—glutamyl trans—peptidase in regenerated and transplanted hepato-cytes of Fischer 344 and Wistar-Furth rats. Cancer Res 45:1222—1228, 1985. Boutwell RK: Some biological aspects of skin carcino- genesis. Prog Exp Tumor Res 4:207—250, 1964. Brixflunan 'UA, de Kok A: Production, properties and uses. In Halogenated Biphenyls, Terphenyls, Na hthalenes, Dibenzodioxins gpg Related Products, Edited by R.D. Kimbrough, Elsevier/North-Holland, Amsterdam, pp. 1-23, 1980. Britton WM, Huston TM: Yolk content and hatchability of egg from hens fed Aroclor 1242. Poultr Sci 51:1869, (Abstr.), 1972. 64 own DP, Jones M: Mortality and industrial hygiene of workers exposed to polychlorinated biphenyls. Arch Environ Health 36:120—129, 1981. ruckner JV, Khanna KL, Cornish HH: Effect of pro- longed ingestion of polychlorinated biphenyls in the rat. Food Cosmet Toxicol 12:323-330, 1974. runn H, Manz D: Contamination of native fish stock by hexachlorobenzene and polychlorinated biphenyl residue. Bull Environ Contam Toxicol 28:599—604, 1982. Buchmann A, Kuhlmann WD, Schwarz M, Kunz HW, Wolf CR, Moll E, Friedberg T, Oesch F: Regulation and ex— pression of four cytochrome P—450 isoenzymes, NADPH-cytochrome P-450 reductase, the glutathione transferase B and C and microsomal epoxide hydrolase in preneoplastic and neoplastic lesions in rat liver. Carcinogenesis 6:513—521, 1985. Burek JD: Pathology of aging rats. A morphological and experimental study of the age—associated lesions in aging BN/Bi, WA6/Rij and (WA6xBN) F1 rats. CRC Press, West Palm Beach, Florida, pp. 58-68, 1978. Burse 'VW, Kimbrough. RD, Villanueva EC, Jennings RW, Linder RE, Socovol GW: Polychlorinated biphenyls. Arch Environ Health 29:301-307, 1974. Burse VW, Moseman RF, Socovol GW, Villanueva EC: PCB metabolism in rats following prolonged exposure to Aroclor 1242 and Aroclor 1016. Bull Environ Contam Toxicol 15:122—128, 1976. Bush B, Tumasonis CF, Baker FD: Toxicity and per— sistence of PCB homologs and isomers in the avian system. Arch Environ Contam Toxicol 2:195-212, 1974. Butler WH, Hempsall V, Stewart MC: Histochemical stud- ies on the early proliferating lesions induced in the rat liver by aflatoxin. J Pathol 133:325-340, 1981. Cairns J: Mutation, selection and the natural history of cancer. Nature 255:197-200, 1975. Carter LJ: Michigan‘s PBB incident: Chemical mixup leads to disaster. Science 192:240-243, 1976. Conney AH: Pharmacological implications of microsomal enzyme induction. Pharmacol Rev 19:317-366, 1967. 65 bett TH, Beaudoin AR, Cornell RG, Anver MR, Schumacher R, Endres J, Szwambowska M: Toxicity of polybrominated biphenyls (Firemaster BP—6) in ro- dents. Environ Res 10:390-396, 1975. 1ddock VM: Cell proliferation and experimental liver cancer. In Liver Cell Cancers. Edited by H.M. Cameron, D.S. Linsell, and G.P. Warwick, Elsevier/ North—Holland, Amsterdam, pp. 153-201, 1976. ygar P, Greim H, Garro AJ, Hutterer F, Schaffner F, Popper H, Rosenthal 0, Cooper DY: Microsomal metabolism: of dimethylnitrosamine and. the cyto- chrome P-450 dependency of its activation to a mu— tagen. Cancer Res 33:2983-2986, 1973. ahlgren RB, Linder RL: Effects of PCBs on pheasant reproduction, behavior and survival. J Wildl Manag 35:313-319, 1971. Damstra T, Jurgelski W Jr, Posner WS, Vouk VB, Bernhiem NJ, Guthrie J, luster ML, Falk HL: Toxicity of polybrominated biphenyls (PBBs) in domestic and laboratory animals. Environ Health Perspect 44:175-188, 1982. Dannan GA, Moore RW, Aust SD: Studies on the micro- somal metabolism and binding of polybrominated biphenyls (PBBs). Environ Health Perspect 23:51- 61, 1978. Deml E, Oesterle D: Histochemical demonstration of en- hanced glutathione content in enzyme—altered is- lands induced by carcinogens in rat liver. Cancer Res 402490-491, 1980. Deml E, Oesterle D: Sex-dependent promoting effect of polychlorinated biphenyls on enzyme-altered is— lands induced by diethylnitrosamine in rat liver. Carcinogenesis 3:1449-1453, 1982. Dent JG, Netter KJ, Gibson JE: The induction of hepat- ic microsomal metabolism in rats following acute administration of a mixture of polybrominated bi- phenyls. Toxicol Appl Pharmacol 38:237-249, 1976. Dent JG, Roes U, Netter KJ, Gibson JE: Stimulation of hepatic microsomal metabolism in mice by a mixture of polybrominated biphenyls. J Toxicol Environ Health 8:651-661, 1977a. 66 Dent JG, Cagen SZ, McCormack KM, Rickert DE, Gibson JE: Liver and mammary aryl hydrocarbon hydroxylase and epoxide hydratase in lactating rats fed polybromi- nated biphenyls. Life Sci 20:2075-2080, 1977b. Dent JG, Elcombe CR, Netter KJ, Gibson JE: Rat hepatic microsomal cytochrome(s) P-450 induced by polybrominated biphenyls. Drug Metab Dispos 6:96- 101, 1978a. Dent JG, McCormack KM, Rickert DE, Cagen SZ, Melrose CP, Gibson JE: Mixed function oxidase activities in lactating rats and their offspring following dietary exposure to polybrominated biphenyls. Toxicol Appl Pharmacol 46:727-735, 1978b. Dieter MP: Influence of environmental contaminants on biochemical adaptation to stress in birds. Toxicol Appl Pharmacol 29:110—111, (Abstr.), 1974. Durst HI, Willett LB, Schanbacher FL, Moorhead PD: Effects of PBBs on cattle. I. Clinical evalua- tions and clinical chemistry. Environ Health Perspect 23:83-89, 1978. Emmelot P, Scherer E: The first relevant cell stage in rat liver carcinogenesis. A quantitative ap— proach. Biochim Biophys Acta 605:247-304, 1980. Enomoto K, Ying TS, Griffin MJ, Farber E: Immunohisto- chemical study of epoxide hydrolase during experi- mental liver carcinogenesis. Cancer Res 41: 3281— 3287, 1981. Farber E: Hyperplastic liver nodules. Methods Cancer Res 7:345-375, 1973. Farber E: The pathology of experimental liver cell cancer. In Liver Cell Cancer. Edited by H.M. Cameron, D.A. Linsell, and G.P. Warwick, Elsevier/ North-Holland, Amsterdam, pp. 243-277, 1976. Farber E: The sequential analysis of liver cancer induction. Biochim Biophys Acta 605:149-166, 1980. Farber E: Precancerous steps in carcinogenesis. Their physiological adaptive nature. Biochim Biophys Acta 738:171-180, 1984a. Farber E: Cellular biochemistry of the stepwise devel- opment of cancer with chemicals. Cancer Res 44: 5463-5474, 1984b. 67 Farber E, Cameron R: The sequential analysis of cancer development. Adv Cancer Res 31:125-226, 1980. Farber T, Kasza L, Giovetti A, Carter C, Earl F, Balzas T: Effect of polybrominated biphenyls (Firemaster BP—6) on the immunological system of the beagle dog. Toxicol Appl Pharmacol 45:343, (Abstr.), 1978. Filinow AB, Jacobs LW, Mortland MM: Fate of polybro- minated biphenyls (PBBs) in soils. J Agric Chem 24:1201-1204, 1976. Fingerman SW and Russell LC: Effects of the polychlo- rinated biphenyl Aroclor 1242 on locomotor ac- tivity and the neurotransmitters dopamine and norepinephrine in the brain of gulf killifish, Findulus grandis. Bull Environ Contam Toxicol 25: 682-687, 1980. Fingerman SW, Shortt EC: Change in neurotransmitter levels in channel catfish after exposure to ben- zo(a)pyrene, naphthalene and Aroclor 1254. Bull Environ Contam Toxicol 30:147-151, 1983. Firminger HJ: Histopathology of carcinogenesis and tu- mors of the liver in rats. J Natl Cancer Inst 15: 1427-1435, 1955. Fischer G, Ullrich D, Katz N, Bock WK, Schaier A: Im- munohistochemical and biochemical detection of uridine-diphosphate-glucuronyl-transferase (UDP— GT) activity in putative preneoplastic liver foci. Virchows Arch Cell Pathol 42:193-200, 1983. ?ishbein L: Toxicity of chlorinated biphenyls. Annu Rev Pharmacol Toxicol 14:139-156, 1974. ?lick DF, O'Dell RG, Childs VA: Studies of the chick disease: similarity of symptoms by feeding chlo— rinated biphenyl. Poultr Sci 44:1460-1465, 1965. Triedrich—Freska H, Papadopulu G, Gossner W: Histo- chemische Untersuchungen der Carcerogenese in der Rattenleber nach zeitlich begrenzter Verabfolgung von Diathylnitrosamin. Z Krebsforsch 72:240-253, 1969. Priend M, Trainer DO: Polychlorinated biphenyl: inter— action with duck hepatitis virus. Science 170: 1314-1316, 1970. 68 fs JC, Abraham R: Effects of mirex and chloro- quinine on PCB-induced hepatic porphyria in the rat. Toxicol Appl Pharmacol 37:119-120, (Abstr.), 1976. 'ukawa K, Matsumura F: Microbial metabolism of poly- chlorinated biphenyls. Studies on the relative degradability' of polychlorinated biphenyl compo- ents by Alcaligenes sp. J Agric Food Chem 24:251- 261, 1976. :ukawa K, Tomizuka N, Kamibayashi A: Effect of chlo- rine substitution on the bacterial metabolism of various polychlorinated biphenyls. Appl Environ Microbiol 38:301-310, 1979. :toff LH, Friedman L, Farber TM, Locke KK, Sobotka TJ, Green S, Hurley N, Peters EL, Story GE, Moreland FM, Graham CH, Keys JE, Taylor MJ, Scalera JV, Rothlein JE, Marks EM, Cerra FE, Rodi SB, Sporn EM: Biochemical and cytogenetic effects in rats caused by short—term ingestion of Aroclor 1254 or Firemaster BP-6. J Toxicol Environ Health 3:769-796, 1977. 1bertson M: Etiology of chick edema disease in her- ring gulls in the lower Great Lakes. Chemosphere 12:357-370, 1983. llette JR, Davis DC, Sasame HA: Cytochrome P-450 and its role in drug metabolism. Ann Rev Pharmacol 12:57—84, 1972. ngell R, Weber A, Ilaqua V, van de Walle C, Hertzog P: Differential effect of several enzyme inducers on hepatic and mammary benzo[a]pyrene metabolism in rat and hamster. Drug Chem Toxicol 4:101-112, 1981. ldstein JA, Hickman P, Burse V, Bergman, H: A com- parative study of two polychlorinated biphenyl mixtures (Aroclors 1242 and 1016) containing 42% chlorine on induction of hepatic porphyria and drug metabolizing enzymes. Toxicol Appl Pharmacol 32:461-473, 1975. ssner W, Friedrich-Freska H: Histochemische Unter- suchungen, uber die Glucose-6-Phosphatase in der Rattenleber wahrend der Cancerisierung durch Nitrosamine. Z Naturforsch l9b:862-864, 1964. 69 :e W, Schmoldt A, Benthe HF: Hepatic porphyrin syn- thesis in rats after pretreatment with polychlo- rinated biphenyls (PCBs). Acta Pharmacol Toxicol 36:215-224, 1975. 1gerich FP: Separation and purification of multiple forms of microsomal cytochrome P-450. J Biol Chem 252:3970-3979, 1977. igerich FP: Isolation and purification of cyto- chrome P-450, and the existence of multiple forms. Pharmacol Ther 6:99—121, 1979. :a BN, McConnell EE, Goldstein JA, Harris MW, Moore JA: Effect of a polybrominated biphenyl mixture in the rat and the mouse. I. Six-month exposure. Toxicol Appl Pharmacol 68:1—18, 1983a. ta BN, Mcconnell EE, Moore JA, Haseman JK: Effect of a polybrominated biphenyl mixture in the rat and mouse. II. Lifetime study. Toxicol Appl Pharmacol 68:19—35, 1983b. ker HJ, Moore MA, Mayer D, Bannasch P: Correlative biochemistry of some enzymes of carbohydrate metabolism in preneoplastic and neoplastic lesions in the rat liver. Carcinogenesis 3:1265-1272, 1982. igan MH, Pitot HC: Gamma-glutamyl transpeptidase- its role in hepatocarcinogenesis. Carcinogenesis 6:165-172, 1985. sen LG, Byerly CS, Metcalf RL, Bevill RF: Effect of a polychlorinated biphenyl mixture on swine reproduction and tissue residues. Am J Vet Res 36:23-26, 1975. ris SJ, Cecil HC, Bitman J, Lillie RJ: Antibody response and reproduction in bursa of Fabricius and spleen weights of progeny of chickens fed PCBS. Poultr Sci 55:1933-1940, 1976. vey GR, Steinhauer WG: Atmospheric transport of polychlorobiphenyls to the North Atlantic. Atmos Environ 8:777—782, 1974. 5 JR, McConnell EE, Harvan DJ: Chemical and toxi- cological evaluation of Firemaster BP-6. J Agric Food Chem 26:94-99, 1978. 70 esse JH, Powers RA: Polybrominated biphenyl contam- ination of the Pine River, Gratiot, and Midland Counties, Michigan. Environ Health Perspect 23: 19-25, 1978. icks RM, Wakefield J, Chowaniec J: Evaluation of a new model to detect bladder carcinogens or cocar- cinogens: results obtained with saccharin, cyclam- ate and cyclophosphamide. Chem Biol Interact 11: 225-233, 1975. icks RM, Chowaniec J: The importance of synergy between weak carcinogens in the induction bladder cancer in experimental animals and humans. Cancer Res 37:2943-2949, 1977. iggins GM, Anderson RM: Experimental pathology of the liver. I. Restoration of the liver of the white rat following partial hepatectomy. Arch Pathol 12: 186-202, 1931. irose M, Tomoyuki S, Tsuda H, Fukuchima S, Ofiso T, Ito N: Effect of phenobarbital, polychlorinated biphenyl and sodium saccharin on hepatic and renal carcinogenesis in unilaterally nephrectomized rats given N-ethyl—N-hydroxyethylnitrosamine orally. Carcinogenesis 2:1299—1302, 1981. oward SK, Werner PR, Sleight SD: Polybrominated bi- phenyl toxicosis in swine: effects on some aspects of the immune system in lactating sows and their offspring. Toxicol Appl Pharmacol 55:146—153, 1980. utzinger O, Nash Dm, Safe S, DeFreitus ASW, Norstrom RJ, Wildish DJ, Zitko V: Polychlorinated bi- phenyls: metabolic behavior of pure isomers in pigeons, rats and brook trout. Science 178:312- 314, 1972. mai Y, Sato R: Evidence for two forms of P-450 hemo- proteins in microsomal membranes. Biochim Biophys Acta 23:5-11, 1966. nagami K, Koga T: Experimental study of hairless mice following administration of rice oil used. by a "Yusho" patient. Fukuoka Acta Med 60:548-555, 1969. nstitute of Laboratory Animal Resources, National Research Council, National Academy of Sciences, Washington DC: Histologic typing of liver tumors of the rat. J Natl Cancer Inst 64:177-206, 1980. 71 ternational Joint Commission, IJC Great Lakes water quality--Appendix E, Status Report on the Per- sistent Toxic Pollutants in the Lake Ontario Basin, 1977. o N, Hananouchi M, Sugihara S, Shirai T, Tsuda H, Fukushima S, Nagasaki H: Reversibility and ir- reversibility of liver tumors in mice induced by the alpha isomer of 1,2,3,4,5,6-hexachlorocyclo- hexane. Cancer Res 36:2227—2234, 1976. cob J, Schmoldt A, Grimmer G: Time course of oxida- tive benz[a]anthracene metabolism by liver microsomes of normal and PCB-treated rats. Carcinogenesis 2:395-401, 1981. cob J, Grimmer G, Raab G, Schmoldt A: The metabolism of pyrene by rat liver microsomes and the influence of various monooxygenase inducers. Xenobiotica 12:45—53, 1982. cobs LW, Chou SF, Tiedje JM: Field concentrations and persistence of polybrominated biphenyls in soils and solubility of PBB in natural waters. Environ Health Perspect 23:1—8, 1978. ckson TF, Halbert FL: A toxic syndrome associated with the feeding of polybrominated biphenyl con- taminated protein concentrate to cattle. J Am Vet Med Assoc 165:437-439, 1974. nsen RK, Sleight SD, Goodman JI, Millis CD, Aust SD, Trosko JE: Assessment of the capacity of 3,3',4,4',5,5'-hexabromobiphenyl to serve as a promoter of hepatocarcinogenesis. Toxicologist 2: 531, 1982a. nsen RK, Sleight SD, Goodman SD, Aust SD, Trosko JE: Polybrominated biphenyls as promoters in experimental hepatocarcinogenesis in rats. Carcinogenesis 3:1183—1186, 1982b. nsen RK, Sleight SD, Aust SD, Goodman JI, Trosko JE: Hepatic tumor promoting ability of 3,3',4,4',5,5'- hexabromobiphenyl: The interrelationship between toxicity, induction of hepatic microsomal drug- metabolizing enzymes and tumor promoting ability. Toxicol Appl Pharmacol, 71:163—176, 1983. isen RK, Sleight SD: Sequential study on the synergistic effects of 2,2',4,4',5,5'-hexabromobi- phenyl and 3,3',4,4',5,5'-hexabromobiphenyl on hepatic tumor promotion. Carcinogenesis 7:1771- 1774, 1986. 72 sson HT, Walker EM, Greene WB, Hughson MD, Hennigar GR: Effects of prolonged exposure to dietary DDT and PCB on rat liver morphology. Arch Environ Contam Toxicol 10:171-183, 1981. angayi MMR, Desmet VJ: Sequential histological and histochemical study of the rat liver during af- latoxin B-l-induced carcinogenesis. Cancer Res 35:2845-2852, 1975. .sson B, Persson B, Sodergren S, Ulfstrand S: Locomotory and dehydrogenase activities of red starts, Phoenicurus phoenicurus given PCB and DDT. Environ Pollut 7:53-56, 1974. fmann WK, Mackenzie SA, Kaufman DG: Quantitative relationship between hepatocyte neoplasms and islands of cellular alterations during hepato- carcinogenesis in the male F344 rat. Am J Pathol 119:171-174, 1985. :5 PP, Suns K, Buckley EH: Monitoring of PCB's in water, sediments and biota of the Great Lakes-- some recent examples. In Physical Behavior' gt PCB's tg tgg Great Lakes. Edited by D. Mackey, S. Paterson, S.J. Eisenreich, and M.S. Simmons, Ann Arbor Science, Ann Arbor, Michigan, pp. 367—381, 1983. K: Polybrominated biphenyls (PBB)—-environmental contamination in Michigan, 1973-1976. Environ Res 13:74-93, 1977. .strom JE, Lindberg C, Orberg J, Danielsson PO, Sydhoff J: Sexual functions of mice neonatally exposed to DDT and PCB. Environ Physiol Biochim 5:54—57, 1975. trough RD: The toxicity of polychlorinated poly- cyclic compounds and related compounds. Crit Rev Toxicol 2:445-498, 1974. trough RD, Linder RE, Gaines TB: Morphological changes in the livers of rats fed polychlorinated biphenyls. Arch Environ Health 25:354-364, 1972. rrough RD, Linder RE, Burse VW, Jennings RW: Adeno- fibrosis in the rat liver. Arch Environ Health 27:390-395, 1973. rough R, Buckley J, Fishbein L, Flamm G, Kasza L, Marcus W, Shibko S, Teske R: Animal toxicology. Environ Health Perspect 24:173—184, 1978. 73 .mbrough RD, Groce DF, Korver MP, Burse VW: Induction of liver tumors in female Sherman strain rats by polybrominated biphenyls. J Natl Cancer Inst 66: 535-542, 1981. .mura NT, Baba T: Neoplastic changes in the rat liver induced by polychlorinated biphenyl. Gann 64:105, 1973. .mura NT, Kanematsu T, Baba T: Polychlorinated biphenyl(s) as a promoter of experimental hepatocarcinogenesis in rats. Z Krebsforsch 266: 257-266, 1976. .tigawa T: Histochemical analysis of hyperplastic lesions and hepatomas of the liver of rats fed 2- fluorenylacetamide. Gann 62:207-216, 1971. .tigawa T: Sequential phenotypic changes in hyper- plastic areas during carcinogenesis in the rat. Cancer Res 36:2534-2539, 1976. ,tigawa T, Sugano H: Enhancing effects of pheno— barbital on the development of enzyme-altered islands and hepatocellular carcinomas initiated by 3'-methyl-4-dimethylaminoazobenzene (n: diethylni- trosamine. Gann 69:679-687, 1978. tigawa T, Pitot HC, Miller EC, Miller JA: Promotion by dietary' phenobarbital of' hepatocarcinogenesis by 2-methy1—N,N-dimethyl—4—aminoazobenzene in the rat. Cancer Res 39:112—115, 1979. tigawa T, Imai F, Sato K: Re-evaluation of gamma- glutamyl transpeptidase activity in periportal hepatocytes of rats with age. Gann 71:362-366, 1980a. tigawa T, Hirakawa T, Ishikawa T, Nemoto N, Takayama S: Induction of hepatocellular carcinoma in rat liver by initial treatment with benzo(a)pyrene after partial hepatectomy and promotion by phenobarbital. Toxicol Lett 6:167-171, 1980b. aunig JE, Lipsky MM, Trump BF, Hinton DE: Biochemical and ultrastructural changes in teleost liver following acute exposure to PCB. J Environ Pathol Toxicol 2:953-963, 1979. imek F, Mayer' D, Bannasch P: Biochemical micro- analysis of glycogen content and glucose-6-phos- phate dehydrogenase activity in focal lesions of rat liver induced by N-nitrosomorpholine. Carcinogenesis 5:265-268, 1984. 74 :luwe WM, Hook JB: Comparative induction of xenobiotic metabolism in rodent kidney, testis and liver by commercial mixtures of polybrominated biphenyls, polychlorinated biphenyls, phenobarbital and 3- methylcholanthrene: absolute and temporal effects. Toxicology 20:259-273, 1981. :nutson JC, Poland A: Response of murine epidermis to 2, 3, 7, 8-tetrachlorodibenzo-p—dioxin: inter— action of the Ah and hr loci. Cell 30:225-232, 1982. :oller LD, Thigpen JE: Reduction of antibody to pseudorabies virus in polychlorinated biphenyl— exposed rabbits. Am J Vet Res 34:1605-1606, 1973. Ireitzer JF, Heinz GH: The effect of sublethal dosages of five pesticides and a polychlorinated biphenyl on the avoidance response of coturnix quail chicks. Environ Pollut 6:21-28, 1974. Zuhlmann WD, Krishan R, Kunz W, Guenthner TM, Oesch F: Focal elevation of liver microsomal epoxide hydrolase ix: early preneoplastic stages and its behavior in the further course of hepatocarcino- genesis. Biochim Biophys Res Commun 98:417-423, 1981. .ambrecht LK, Barsotti DA, Allen JR: Responses of non- human primates to a polybrominated biphenyl mixture. Environ Health Perspect 23:139—145, 1978. aurier C, Tatematsu M, Rao PM, Rajalakshmi S, Sarma DSR: Promotion by orotic acid of liver carcino- genesis in rats initiated by 1,2-dimethylhy- drazine. Cancer Res 44:2186—2191, 1984. .eonard. TB, Dent JG, Graichen. E, Lyght O, Popp JA: Comparison of hepatic carcinogen initiation-promo- tion systems. Carcinogenesis 3:851-856, 1982. illie RJ, Cecil HC, Bitman J, Fries GF, Verrett J: Toxicity of certain polychlorinated and poly- brominated biphenyls on reproductive efficiency in caged chickens. Poultr Sci 54:1550-1555, 1975. incer JL, Peakall DB: Metabolic effects of polychlo- rinated biphenyls in the American kestral. Nature 228:783-784, 1970. inder RE, Gaines TB, Kimbrough RD: The effect of polychlorinated biphenyls on rat reproduction. Food Cosmet Toxicol 12:63-77, 1974. 75 psky MM, Hinton DE, Goldblatt PJ, Klaunig JE, Trump BF: Iron negative foci and nodules in safrole- exposed mouse liver made siderotic by iron—dextran injection. Pathol Res Pract 164:178-185, 1979. ase LD, Pittman KA, Benitz KF, Silkworth JB: Poly- chlorinated biphenyl and hexachlorobenzene induced humoral immunosuppression. J Retic Soc 22:253- 271, 1977. ase LD, Silkworth JB, Pittman KA, Benitz KF, Mueller W: Impaired host resistance to endotoxin and malaria in polychlorinated biphenyl-and hexachlo— robenzene-treated mice. Infect Immun 20: 30-36, 1978. AYH, Levin W: The resolution and reconstitution of the liver microsomal hydroxylation system. Biochim Biophys Acta 344:205-218, 1974. AYH, Kuntzman R, Conney AH: The liver microsomal hydroxylation enzyme system, induction and properties of the functional components. Front Gastrointest Res 2:1-31, 1976. :ter MI, Faith RE, Moore JA: Effects of polybromi- nated biphenyls (PBB) on immune response in ro- dents. Environ Health Perspect 23:227-232, 1978. :kay D, Shui WY, Billington J, Huang GL: Physical- chemical properties and behavior of PCBs. In Physical Behavior o_f PCB's _i_n 123 Great Lakes. Edited by S. Peterson, S.J. Eisenreich, and M.S. Simmons, Ann Arbor Science, Ann Arbor, Michigan, pp. 59-69, 1983. :thews A, Fries G, Gardner A, Gartoff L, Goldstein J, Ku Y, Moore J: Metabolism and biochemical toxi- city of PCBs and PBBs. Environ Health Perspect 24:173-184, 1978. :onnell EE, Moore JA: Toxicopathology characteris- tics of the halogenated aromatics. Ann NY Acad Sci 320:138-150, 1979. :onnell EE, Hass JR, Altman N, Moore JA: A spontane- ous outbreak of polychlorinated biphenyl (PCB) toxicity in rhesus monkeys (Macaca mulatta): toxi- copathology. Lab Anim Sci 29:666-673, 1979. 76 :ormack KM, Kluwe WM, Rickert DE, Sanger VL, Hook JB: Renal and hepatic microsomal enzyme stimulation and renal function following three-month dietary exposure to polybrominated biphenyls. Toxicol Appl Pharmacol 44:539-553, 1978. Ller JA, Miller EC: The metabolic activation of carcinogenic aromatic amines and amides. Prog Exp Tumor Res 11:273-301, 1969. Ller JA, Miller EC: Mechanisms of chemical carcino- genesis. Cancer 47:1055-1064, 1981. Lls RA, Millis CD, Dannan GA, Guengerich FP, Aust SD: Studies on the structure-activity relationships for the metabolism of polybrominated biphenyls by rat liver microsomes. Toxicol Appl Pharmacol 78: 96-104, 1985. Iata Y, Fukushima s, Hirose M, Masui T, Ito N: Modifying potentials of various environmental chemicals on N-butyl-N-(4-hydroxybutyl)-nitros- amine-initiated urinary bladder carcinogenesis in rats with ureteric ligation. Jpn J Cancer Res 76: 828-834, 1985. >re MA, Hacker HJ, Kunz HW, Bannasch P: Enhancement of NNM-induced carcinogenesis in the rat liver by phenobarbital: a combined morphological and enzyme histochemical approach. Carcinogenesis 4:473-479, 1983. >re MA, Tsuda H, Ogiso T, Mera Y, Ito N: Enhancement of phenotypic instability by alpha-difluoromethyl- ornithine and butylated hydroxyanisole in rapidly induced rat liver lesion. Cancer Lett 25:145-151, 1984. ire RW, Aust SD: Purification and structural char- acterization of polybrominated biphenyl con- geners. Biochim Biophys Res Commun 84:936-942, 1978. >re RW, Dannan GA, Aust SD: Induction of drug— metabolizing enzymes in polybrominated biphenyl- fed lactating rats and their pups. Environ Health Perspect 23:159-165, 1978a. vre RW, Sleight SD, Aust SD: Induction of liver mi- crosomal drug-metabolizing enzymes by 2, 2', 4, 4', 5, 5'- hexabromobiphenyl. Toxicol Appl Pharmacol 44:309-321, 1978b. 77 orhead PD, Willett LB, Schanbacher FL: Effects of PBBs on cattle. II. Gross pathology and histopathology. Environ Health Perspect 23:111- 118, 1978. ttram JC: A developing factor in experimental blas- togenesis. J Pathol Bacteriol 56:181-187, 1944. llin MD, Pochini CM, Safe SH, Safe LM: Analysis of PCBs using high resolution capillary gas chromatography. In PCBs: Human gpg Environmental Hazards. Edited by F.M. D'Itri and M.A. Kamrin, Ann Arbor Science, Ann Arbor, Michigan, pp. 165- 176, 1983. rphy TJ, Pokojowczyk JC, Mullin MD: Vapor exchange of PCB's with Lake Michigan: the atmosphere as a sink for PCB's. In Physical Behavior gt PCB's tg thg Great Lakes. Edited by D. Mackey, S. Paterson, S.J. Eisenreich, and M.S. Simmons, Ann Arbor Science, Ann Arbor, Michigan, pp. 49-58, 1983. edham LL, Hill RH, Orti D1, Patterson DG, Kimbrough RD, Groce DF, Liddle JA: Identification of poly- brominated biphenyls in Firemaster FF-l that pos- sess hyperkeratotic activity. J Toxicol Environ Health 9:877-887, 1982. yam SK, Aravinda Babu K, Bhatt DK, Karnik AB, Thakore KN, Lakkad BC, Kashyap SK, Chatterjee SK: Pattern of glycogen and iron accumulation in early appearing BHC induced liver lesions and liver tumors. Indian J Med Res 74:289-296, 1981. [aka S, Shimoyama T, Honda T, Yoshida H: Analysis of urinary porphyrins in polychlorinated biphenyl poisoning (Yusho) patients. In Chemical Porphyria tg Mgg. Edited by J.J.T.W.A. Strik and J.H.Koeman, Elsevier/North—Holland, Amsterdam, pp.69-73, 1979. Vicki HG, Norman AW: Enhanced hepatic metabolism of testosterone, 4-androstene-3, l7-dione and estra- diol-17 in chickens pretreated with DDT or PCB. Steroids 19:85-91, 1982. .wa K, Medline A, Farber E: Sequential analysis of hepatic carcinogenesis. .A comparative study of the ultrastructure of preneoplastic, malignant, prenatal, postnatal, and regenerating liver. Lab Invest 41:22-35, 1979. 78 Dlafson P: Hyperkeratosis (X-disease) of cattle. Cor- nell Vet 37:279-291, 1947. Jlsen P, Settle H, Swift R: Organochlorine residues in wings of ducks in southeastern Australia. Aust Wildl Res 7:139-143, 1980. Parke DV: Induction of the drug-metabolizing enzymes. In Enzyme Induction. Edited by D.V. Park, Plenum Press, London, pp. 207—228, 1975. Parkinson A, Robertson LW, Safe S: Reconstituted breast milk PCBs as potent inducers of aryl hydro- carbon hydroxylase. Biochim Biophys Res Commun 96: 882-803, 1980. Parkinson A, Safe S: Aryl hydrocarbon hydroxylase induction and its relationship to the toxicity of halogenated aryl hydrocarbons. Toxicol Environ Chem 4:1-46, 1981. Passivirta J, Linko R: Environmental toxins in Finnish wildlife. A study of trends of residue contents in fish during 1973-1978. Chemosphere 9:643—661, 1980. Patterson DG, Hill RH, Needham LL, Orti DL, Kimbrough RD, Liddle JS: Hyperkeratosis induced by sunlight degradation products of the major polybrominated biphenyl in Firemaster. Science 213:901-902, 1981. >eakall DB: p,p'-DDT: effect of calcium metabolism and concentration of estradiol on the blood. Science 168:592-594, 1970. ’eakall DB, Peakall ML: Effect of a polychlorinated biphenyl on the reproduction of artificially and naturally incubated dove eggs. J Appl Ecol 10: 863-868, 1973. ’eakall DB: PCBs and their environmental effects. CRC Crit Rev Environ Contam pp. 469-488, 1975. 'eraino C, Fry RJM, Staffeldt E: Reduction and en- hancement by phenobarbital of hepatocarcinogenesis induced in the rat by 2-acetylaminofluorene. Cancer Res 31:1506-1512, 1971. ’eraino C, Fry RJM, Staffeldt E, Kisieleski WE: Effects of varying the exposure to phenobarbital on its enhancement of 2—acetylaminofluorene-in- duced hepatic tumorigenesis. Cancer Res 33:2701- 2705, 1973a. 79 :raino C, Fry RJM, Staffeldt E: Enhancement of spon- taneous hepatic tumorigenesis in L3H mice by dietary' phenobarbital. J Natl Cancer Inst 51: 1349-1350, 1973b. :raino C, Fry RJM, Staffeldt E, Christopher JP: Comparative enhancing effects of phenobarbital, amobarbital, diphenylhydantoin, and dichlorodi- phenyltrichloroethane on 2-acetylaminofluorene-in— duced hepatic tumorigenesis in the rat. Cancer Res 35:2884-2890, 1975. :raino C, Fry RJM, Staffeldt E, Christopher JP: Enhancing effects of phenobarbitone and butylated hydroxytoluene on 2-acetylaminofluorene-induced hepatic tumorigenesis in the rat. Food Cosmet Toxicol 15:93-96, 1977. raino C, Richards WL, Stevens FJ: Multistage hepato- carcinogenesis. In Mechanisms gt Tumor Promotion. Edited by T.J. Slaga, Vol 1, CRC Press, Boca Raton, Florida, pp. 1-53, 1983. reira MA, Herren SL, Britt AL, Khoury MM: Promotion by polychlorinated biphenyls of enzyme-altered foci in rat liver. Cancer Lett 15:185-190, 1982. tot HC: The natural history of neoplasia. Am J Pathol 89:402-411, 1977. tot HC, Barsness L, Goldsworthy T, Kitigawa T: Biochemical characterization of stages of hepatocarcinogenesis after a single dose of di- ethylnitrosamine. Nature 271:456-458, 1978a. tot HC, Barsness L, Kitigawa T: Stages in the process of hepatocarcinogenesis in rat liver. In Carcinogenesis: A Comprehensive Survey. Edited by T.J. Slaga, A. Sivak, and R.K. Boutwell, Vol 2, Raven Press, New York, pp. 433—442, 1978b. tot HC, Sirica AB: The stages of initiation and pro— motion in hepatocarcinogenesis. Biochim Biophys Acta 605:191-215, 1980. tot HC, Goldsworthy T, Campbell HA, Poland A: Quantitative evaluation of the promotion by 2,3,7,8- tetrachlorodibenzo—p-dioxin of hepatocar- cinogenesis from diethylnitrosamine. Cancer Res 40:3616-3620, 1980. 80 Platonow NS, Funnell HS: The distribution and some ef- fects of PCBs (Aroclor 1254) in cockerals during prolonged feeding trial. Can J Comp Med 36:89—93, 1972. Pomerantz I, Durke J, Firestone D, McKinney J, Roach J, Trotter J: Chemistry of PCB's and PBB's. Environ Health Perspect 24:133—146, 1978. Preston BD, VanMiller RW, Moore RW, Allen JR: Promoting effects of polychlorinated biphenyls (Aroclor 1254) and polychlorinated dibenzofuran- free Aroclor 1254 on diethylnitrosamine-induced tumorigenesis in the rat. J' Natl Cancer Inst 66:509-515, 1981. Puhvel SM, Sakamoto M, Ertl DC, Reisner RM: Hairless mice as models for chloracne: a study of cutaneous changes induced by topical applications of es- tablished chloracnegens. Toxicol Appl Pharmacol 64:492-503, 1982. Quintanilla M, Brown K, Ramsden M, Balmain A: Car- cinogen-specific mutation and amplification of Ha- ras during mouse skin carcinogenesis. Nature 322: 78- 79, 1986. Rao .MS, Lalwani ND, Reddy "JK: Sequential histologic study of rat liver during peroxisome proliferator [4-chloro—6—(2,3-xylidin0)-2-pyrimidinylthio]-ace- tic acid (Wy-l4,643)—induced carcinogenesis. J Natl Cancer Inst 73:983—990, 1984. Rappe C, Buser HR: Chemical properties and analytical methods. In Halogenated Bi hen ls, Ter hen ls, Naphthalenes, Dibenzodioxins gpg Related Products. Edited by R.D. Kimbrough, Elsevier/North-Holland, Amsterdam, pp. 41—66, 1980. Render JA, Aust SD, Sleight SD: Acute pathologic effects of 3,3',4,4',5,5-hexabromobiphenyl in rats: comparison of its effects ‘with ‘Firemaster BP-6 and 2,2',4,4’,5,5'-hexabromobiphenyl. Toxicol Appl Pharmacol 71:163-176. Reuber MD: Development of preneoplastic and neoplastic lesions of the liver in 'male rats given 0.025 percent N-2—fluorenyldiacetamide. J Natl Cancer Inst 34:697-724, 1965. Ringer RK: PBB fed to immature chickens: its effect on organs weights and function and on the cardiovas- cular system. Environ Health Perspect 23:247-255, 1978. 81 inger RK, Aulerich RJ, Bleavins MR: Biological ef- fects of PCBs and PBBs on mink and ferrets--a re- view. In Toxicology gt Halogenated Hydrocarbons-— Health ggg Ecological Effects. Edited by M.A.Q. Khan and R.H. Stanton, Pergamon Press, New York, pp. 329-343, 1981. aberts JR, Rodgers DW, Bailey JR, Rorke MA: Polychlo- rinated biphenyls: biological criteria for an assessment of their effects on environmental qual- ity. National Research Council of Canada, NRCC No. 16077, Ottawa, Ontario, 1978. Dbl MG, Jenkins DH, Wingender RJ, Gordon DE, Keplinger ML: Toxicity and residue studies in dairy animals with Firemaster FF-l (polybrominated biphenyls). Environ Health Perspect 23:91-97, 1978. bus P, Kidd JG: Conditional neoplasms and subthreshold neoplastic states: A study of the tar tumor of rabbits. Cell Diff 6:25-39, 1941. ltenberg AM, Kim H, Fischbein JW, Hanker JS, Wasserkrug HL, Seligman AM: Histochemical and ultrastructural demonstration of gamma-glutamyl transpeptidase activity. J Histochem Cytochem 17: 517-526, 1969. y'an DE, Thomas PE, Reik LM, Levin W: Purification, characterization and regulation of five hepatic cytochrome P—450 isoenzymes. Xenobiotica 12:727- 744, 1982. afe S, Platonow N, Hutzinger O, Jamieson WD: Analysis of organochlorine metabolites in crude extracts by high-resolution photoplate mass spectrometry. Biomed Mass Spectrom 2:201-208, 1975. afe S, Kohil J, Crawford. A: Firemaster' BP—6: frac- tionation, metabolic and enzyme induction studies. Environ Health Perspect 23:147-152, 1978. ife S: Metabolism, uptake, storage and bioaccumula- tion. In Halogenated Bi hen ls, Terphenyls, Naphthalenes, Dibenzodioxins and Related Products. Edited by R.D. Kimbrough, Elsevier/North-Holland, Amsterdam, pp. 77-107, 1980. :saki T, Yoshida T: Experimentalle Erzeugung des Leber-carcinoms durch Fuuterung mit o-Amidoazo- tolu-ol. Virchows Arch 295:175-200, 1935. 82 [to K, Kitahara A, Satoh K, Ishikawa T, Tatematsu M, Ito N: The placental form of glutathione s-trans- ferase as a new marker protein for preneoplasia in rat chemical carcinogenesis. Gann 75:199-202, 1984. :hauer A, Kunze E: Enzymhistochemische und autoradio- graphische Untersuchungen wahrend der Kanzerisie- rung der Rattenleber durch Diathylnitrosamin. Z Krebsforsch 70:252-266, 1968. :hauer A, Kunze E: Liver tumors of the rat. In Pathology gt Laboratory Animals. Edited by V.S. Turosov, Vol 1, International Agency for Research on Cancer, Lyon, pp. 41-72, 1976. :herer E: Use of a programmable pocket calculator for the quantitation of precancerous foci. Carcinogenesis 2:805-807, 1981. :herer E: Neoplastic progression in experimental hepatocarcinogenesis. Biochim Biophys Acta 738: 219-236, 1984. :herer' E, Emmelot P: Foci of altered liver cells induced by a single dose of diethylnitrosamine and partial hepatectomy: their contribution to hepato- carcinogenesis in the rat. Europ J Cancer 11:145- 154, 1975. :herer E, Emmelot P: Kinetics of induction and growth of enzyme-deficient islands in hepatocarcinogene- sis. Cancer Res 36:2544-2554, 1976. :hulte-Hermann R, Roome N, Timmermann-Trosiener I, Schuppler J: Immunocytochemical demonstration of a phenobarbital-inducible cytochrome P-450 in pu- tative preneoplastic foci in rat liver. Carcinogenesis 5:143-153, 1984. limada T: Metabolic activation of [14-C] polychlo- rinated biphenyl mixtures by rat liver microsomes. Bull Environ Contam Toxicol 16:25-32, 1976. 1imada T, Sato R: Covalent binding in vitro of polychlorinated biphenyls to microsomal macromole- cules. Biochim Pharmacol 27:585-590, 1978. :imada T, Imai Y, Sato R: Covalent binding of poly- chlorinated biphenyls to proteins by reconstituted monooxygenase system-containing cytochrome P-450. Chem Biol Interact 38:29—34, 1981. 83 Shinozuka H, Sell MA, Katyal SL, Sell S, Lombardi B: Effects of choline-devoid diet on the emergence of gamma-glutamyltranspeptidase-positive foci in the liver of carcinogen-treated rats. Cancer Res 39: 2515-2521, 1979. Silkworth JB, Loose LD: Cell-mediated immunity in mice fed either Aroclor 1016 or hexachlorobenzene. Toxicol Appl Pharmacol 45:326-327, (Abstr.), 1978. Smith SH, Sanders VM, Barret BA, Borzellera JF, Munson AE: Immunotoxicological evaluation on mice ex- posed to polychlorinated biphenyls. Toxicol Appl Pharmacol 45:330, (Abstr.), 1978. Snyder R, Remmer H: Classes of hepatic microsomal mixed function oxidase inducer. Pharmacol Ther 7: 203- 211, 1979. Solt DB, Farber E: A new principle for the analysis of chemical carcinogenesis. Nature 263:702—703, 1976. Solt DB, Medline A, Farber E: Rapid emergence of car- cinogen-induced hyperplastic lesions in a new model for the sequential analysis of liver carci- nogenesis. Am J Pathol 88:595—618, 1977. Spencer F: An assessment of the reproductive toxic potential of Aroclor 1254 in female Sprague-Dawley rats. Bull Environ Contam Toxicol 28:290-297, 1982. Squire RA, Levitt MH: Report of a workshop on classi- fication of specific hepatocellular lesions in rats. Cancer Res 35:3214-3223, 1975. Steel RGD, Torrie JH: Analysis of variance I: The one- way classification. In Principles ggg Procedures gt Statistics. A Biometrical Approach. Edited by C. Napier and J.W. Maisel, McGraw-Hill, New York, pp. 137-167, 1980a. Steel RGD, Torrie JH: Multiple Comparisons. In Principles and Procedures gt Statistics. A Biometrical Approach. Edited by C. Napier and J.W Maisel, McGraw-Hill, New York, pp. 172—191, 1980b. Stewart HL, Williams G, Keysser CH, Lombard LS, Montali RJ: Histologic typing of liver tumors of the rat. J Natl Cancer Inst 65:179-206, 1980. Strik JJTWA: Species differences in experimental por- phyria caused by polyhalogenated aromatic com- pounds. Enzyme 16:224—230, 1973. 84 Strik JJTWA: Porphyrinogenic action of polyhalogenated aromatic hydrocarbons with special reference to porphyria and environmental impact. In Diagnosis ggg Therapy gt Potphyrias ggg Lead Intoxication. Edited by 1L Doss, Springer-Verlag, Berlin, pp. 151-164, 1978. Strik JJTWA, Kip H, Yoshimura T, Masuda Y, Harmsen EGM: Porphyrins in urine of Yusho patients. In Chemical Porphyria tg Mgp. Edited by J.J.T.W.A. Strik. and J.H. Koeman, Elsevier/North-Holland, Amsterdam, pp.63—68, 1979. Stross JK, Smokler IA, Isbister' J, Wilcox KR: The human health effects of exposure to polybrominated biphenyls. Toxicol Appl Pharmacol 58:145-150, 1981. Sullivan JR, Delfino J, Buelow CR, Sheffy TB: Poly- chlorinated biphenyls in the fish and sediment of the Lower Fox River. Wisconsin Bull Environ Contam Toxicol 30:58-63, 1983. Sundstrom G, Hutzinger 0, Safe S: The metabolism of chlorobiphenyls-—a review. Chemosphere 5:267-287, 1976. Symposium on Rodent Liver Nodules: Significance to Human Cancer Risk. Toxicol Pathol 10:1-227, 1982. Tanabe S, Hidaka H, Tatsukawa R: PCB's and chlorinated hydrocarbon pesticides in Antarctic atmosphere and hydrosphere. Chemosphere 12:277-288, 1983. Taper HS, Fort L, Brucher JM: Histochemical activity of alkaline and acid nucleases in rat liver parenchyma during N-nitrosomorpholine carcinogene- sis. Cancer Res 31:913-9l6, 1971. Taper HS, Lans M, de Gerlache J, Fort L, Roberfroid M: Morphological alterations and DNase deficiency in phenobarbital promotion of N—nitrosomopholine—ini- tiated rat hepatocarcinogenesis. Carcinogenesis 4:231-234, 1983. Tatematsu M, Nagamine Y, Farber E: Redifferentiation as a basis for remodeling of carcinogen-induced hepatocyte nodules to normal appearing liver. Cancer Res 43:5049-5058, 1983. 85 Tatematsu M, Mera Y, Ito N, Satoh K, Sato K: Relative merits of immunohistochemical demonstration of placental A, B, and C forms of glutathione S- transferase and histochemical demonstration of gamma-glutamyltranspeptidase as markers of altered foci during liver carcinogenesis in rats. Carcinogenesis 6:1621—1626, 1985. Thamavit W, Tatematsu M, Ogiso T, Mera Y, Tsuda H, Ito N: Dose-dependent effects of butylated hydroxy- anisole, butylated hydroxytoluene and ethoxyquin in induction of foci of rats liver cells con- taining the placental form of glutathione S- transferase. Cancer Lett 27:295—303, 1985. Tilson HA, Cabe PA: Studies on the neurobehavioural effects of polybrominated biphenyls in rats. Ann NY Acad Sci 320:325-336, 1979. Thomas PT, Hinsdill RD: Effect of polychlorinated biphenyls on the immune responses of rhesus monkeys and mice. Toxicol Appl Pharmacol 44:41- 51, 1978. Thomas PT, Hinsdill RD: Perinatal PCB exposure and its effects on the immune system of' young rabbits. Drug Chem Toxicol 3:173-184, 1980. Thompson JS: Analysis of Pesticide Residue in Human and Environmental Samples. U.S. and Environmental Protection Agency, Health Effects Research Labora- tory, Environmental Toxicology Division, Research Triangle Park, North Carolina, 1977. Tucker MJ: The effect of long-term food restriction on tumors in rodents. Int J Cancer 23:803—807, 1979. Ulfstrand S, Sodergren S, Rabol J: Effect of PCB on nocturnal activity in caged robins, Eruthacus rubecula t. Nature 231:467—468, 1971. Vos JG, Koeman JH: Comparative toxicologic study with polychlorinated biphenyls in chickens with special reference to porphyria, edema formation, liver necrosis and tissue residues. Toxicol Appl Pharmacol 17:656-668, 1970. Vos JG, Beems RB: Dermal toxicity studies of technical polychlorinated biphenyls and fractions thereof in rabbits. Toxicol Appl Pharmacol 19:617—633, 1971. 86 Vos JG, Strik JJTWA, van Holsteyn CWM, Pennings JH: Polychlorinated biphenyls as inducers of hepatic porphyria in Japanese quail, with special refer- ence tx: delta-aminolevulinic acid synthetase ac- tivity, fluorescence and residues in the liver. Toxicol Appl Pharmacol 20:232-240, 1971. Vos JG, van Driel Grootenhuis L: PCB-induced sup- pression of of the humoral and cell-mediated im- munity in guinea pigs. Sci Total Environ 1:289- 297, 1972. Vos JG, Notenboom-Ram E: Comparative toxicity of 2, 2', 4, 4', 5, 5'—hexachlorobiphenyl and a poly— chlorinated biphenyl mixture in rabbits. Toxicol Appl Pharmacol 23:563-578, 1972. Vos JG, Faith RE, luster MI: Immune alterations. In Halogenated Bi hen ls, Terphenyls, Na hthalenes, Dibenzodioxins ggg Related Products. Edited by R.D. Kimbrough, Elsevier/North-Holland, Amsterdam, pp. 241-258, 1980. Ward JM: Morphology of foci of altered hepatocytes and naturally-occurring hepatocellular tumours in F344 rats. Virchows Arch Pathol Anat 390:339—345, 1981. Ward JM: Morphology of potential preneoplastic hepato- cyte lesions and liver tumors in mice and a com- parison with other species. In Mouse Liver Neoplasia. Edited by J.A. Popp, Hemisphere Pub- lishing, New York, pp. 1-26, 1984. Weisburger JH, Madison RM, Ward RJM, Vignera C, Weisburger EK: Modification of diethylnitrosamine liver carcinogenesis with phenobarbital but not with immunosuppression. J Natl Cancer Inst 54: 1185-1188, 1975. Welton AF, Aust SD: The effects of 3-methyl-cholan- threne and phenobarbital induction of the struc- ture of the rat liver endoplasmic reticulum. Biochim Biophys Acta 373:197-210, 1974. Wickstron: K, Pyysalo H, Perttila M: Organochlorine compounds in the liver of cod in the northern Bal- tic. Chemosphere 10:999-1004, 1981. Williams GM: Functional markers and growth behavior of preneoplastic ‘hepatocytes. Cancer' Res 36:2540- 2543, 1976. 87 Williams GM: The .pathogenesis of rat liver cancer caused by chemical carcinogens. Biochim Biophys Acta 605:167-189, 1980. Williams GM, Watanabe K: Quantitative kinetics of de- velopment of N-2-fluorenylacetamide—induced, al- tered hyperplastic hepatocellular foci resistant to iron accumulation and of their reversion of persistence following removal of carcinogen. J Natl Cancer Inst 61:113-121, 1978. Williams GM, Klaiber M, Parker SE, Farber E: Nature of early appearing, carcinogen-induced liver lesions resistant to iron accumulation. J Natl Cancer Inst 57:157-165, 1976. Williams GM, Hirota N, Rice JM: The resistance of spontaneous mouse hepatocellular neoplasms to iron accumulation during rapid iron loading by paren- teral administration and their transplantability. Am J Pathol 65-74, 1979. Williams GM, Katayama S, Ohmori T: Enhancement of hepatocarcinogenesis by sequential administration of chemicals: Summation versus promotion effects. Carcinogenesis 2:1111-1117, 1981 Wolff MS, Aubrey B: PBB homologs in sera of Michigan dairy farmers and Michigan chemical workers. Environ Health Perspect 23:211-215, 1978. Wolff MS, Selikoff IJ: Variation of polybrominated bi- phenyl homolog peaks in blood of rats following treatment with Firemaster BP-6. Bull Environ Contam Toxicol 21:771-774, 1979. Ying TS, Sarma DSR, Farber E: Role of acute hepatic necrosis in the induction of early steps in liver carcinogenesis by diethylnitrosamine. Cancer Res 41:2096-2101, 1981. Zabik ME, Merrill C, Zabik MJ: PCB's and other xeno— biotics in raw and cooked carp. Bull Environ Contam Toxicol 28:710-715, 1982. CHAPTER 2 QUANTITATIVE ALTERATIONS OF GAP JUNCTIONS AND NUCLEAR PORES IN CHEMICALLY-INDUCED HEPATIC NODULES IN RATS CHAPTER 2 QUANTITATIVE ALTERATIONS OF GAP JUNCTIONS AND NUCLEAR PORES IN CHEMICALLY-INDUCED HEPATIC NODULES IN RATS INTRODUCTION Gap junctions are ultrastructural channels found in the plasma membrane of most cells that permit the intercellular sharing of metabolites. They are re- sponsible for "cell-cell communication" and are quantitatively altered in certain pathologic conditions, including malignant neoplasia (Schindler _t _t., 1982; Alroy, 1979; Inoue and Skoryna, 1979; McNutt and Weinstein, 1971; Martinez—Palomo, 1975; Swift gt gt., 1983) and hyperplasia (Yancey gt 1., 1981; Yee and Revel, 1978). Hepatic neoplasia has several identifiable stages, and precursor (i.e., "preneoplastic") lesions have been described (Farber, 1984). The hepatic nodule is thought to be a precursor to the hepatocellular carcinoma. Gap junctions are decreased in number in hepatocellular carcinomas (Swift gt gt., 1983), but it is unknown if they are decreased in number in hepatic nodules. 88 89 Another ultrastructural channel, the nuclear pore, permits the sharing of low molecular weight metabolites between the nuclear and cytoplasmic compartments within a cell (deRobertis, 1983; Paine gt gt., 1975). Quantitative alterations in nuclear pores in neoplastic cells have not been rigorously studied, but some reports suggest that they are diminished in number in neoplastic cells (Codd gt gt., 1981; Czerniak gt gt., 1984). The first objective of the following study was to determine if the surface area occupied by gap junctions differs between cells from normal liver and cells comprising hepatic nodules. A second objective was to compare the numbers of nuclear pores within nuclear membranes of cells in normal liver with cells in hepatic nodules. LITERATURE REVIEW Role gt Gap Junctions tp Metabolic Cooperation Gap junctions are integral protein structures of the plasma membrane found ix: all metazoan animals and in nearly all cells comprising organized tissues and organs (Hertzberg gt gt., 1981; Hooper and Subak-Sharpe, 1981; Pitts, 1980; Loewenstein, 1981). They form pore—like openings, called connexons, connecting the cytoplasms of two adjacent cells. A group of coupled cells will therefore form a compartment within which ions and small molecules are easily exchanged. The junctions have a characteristic appearance in freeze-fractured pre- parations and appear as an array of hexagonally packed connexons. Small molecules of less than 1000 daltons may pass through these connexons (Flagg-Newton gt gt., 1979; Spray gt gt., 1977). Examples of such molecules. include cAMP (Hertzberg gt gt., 1981) calcium (Loewenstein, 1981), nucleotides (Hertzberg gt gt., 1981; Hooper and Subak-Sharpe, 1981; Pitts, 1980), and amino acids (Pitts, 1980). These molecules can pass between cells via gap junctions without entering the interstitial space. 90 91 Transfer of small molecules between cells via gap junctions has been termed "metabolic cooperation" and allows adjacent cells to communicate with one another (Subak-Sharpe gt ,gt., 1969; Hooper and Subak-Sharpe, 1981). Intercellular communication may be required for several fundamental biological events, including: (a) synchronized contraction of cells within a tissue, (b) metabolic coordination of cells within a tissue, (c) growth control, (d) differentiation and development, and (e) enzymatic regulation. Synchronized Contraction In tissues such as the heart, cardiac muscle cells are interfaced with each other by gap junctions allowing for electrical synchronization of cardiac tissue (De Mello, 1982). Synchronized contraction is also a feature of the gravid uterus. Approximately two days before the onset of parturition there is a 100-fold increase in the number of gap junctions in the endometrium (Garfield gt gt., 1978) which is apparently in response to increasing levels of estrogen. Two days after parturition, the number of gap junctions decreases and returns to pre-pregnancy levels (Garfield gt gt., 1980). 92 Metabolic Coordination gt Cells Intercellular sharing of metabolites via gap junctions may help cells in some tissues respond to hormones or growth factors. For example, the rat pancreas contains four different subsets of cells: the A (or alpha) cells which produce glucagon, the B (or beta) cells of the islets of Langerhans which produce insulin, the D (or delta) which produce somatostatin, and the PP cells which make pancreatic polypeptide (Micheals and Sheridan, 1981; Meda gt gt., 1981). Groups of beta cells are connected with each other via gap junctions but are connected, to surrounding non—beta cells with fewer gap junctions, thus forming discrete functional domains of cells within the pancreas. With. prolactin stimulation, the beta cells increase their numbers of gap junctions two-fold and increase the numbers of gap junctions with their neighboring A, D, and PP cells by 10 to 20-fold (Micheals, 1982). Rats made hyperglycemic by chemically-induced block- age of insulin secretion had a two-fold increase in gap junctions in beta cells, while the ability of these cells to share metabolites increased nine-fold (Meda gt gt., 1983). Conversely, rats made hypoglycemic with a chemical that depletes beta cell insulin content had a two-fold increase in the number of gap junctions between beta cells while intercellular communication with neighboring cells increased three-fold. This experiment 93 is an in vivo example of how an effect of hormones or blood-borne factors can spread from the target cells to nontarget cells by the intercellular exchange of metabolites via gap junctions. Growth Control Certain organs depend on intact communication with surrounding cells for normal growth. An example of to- tal metabolic dependence of an organ upon neighboring cells is the lens of the eye. The cells of the lens are not in direct contact with the blood vasculature but are nourished totally by intercellular communication via gap junctions with neighboring epithelial cells which are in intimate contact with the blood supply. Another similar example is the mammalian oocyte. Its surrounding granulosa cells appear to be required for maintenance of the oocyte in meiotic arrest (Wassarman and Letourneau, 1976). Furthermore, results of studies on. metabolic cooperation indicate that the uridine used for RNA synthesis by the oocyte is obtained from neighboring cumulus cells via gap junctions (Gilula gt gt., 1978). Differentiation ggg Development Gap junctions occur in the Xenopus embryo as early as the four—cell stage (Hertzberg gt gt., 1981). Cells of the "grey crescent" are known to develop into the Xenopus eye. If a polyvalent antibody against gap 94 junctional protein is injected into such cells at an early stage of embryogenesis, then intercellular communication of the injected cells with surrounding cells is prevented. This may lead to abnormal dif- ferentiation and development of the injected cells (Warner gt gt., 1984). Regglation gt Enzyme Activities Metabolic cooperation regulates the activities of several enzyme systems, including HGPRTase (hypoxanthine guanine phosphoribosyl transferase) (Vitkauskas and Canellakis, 1984; Sheridan gt gt., 1979; Vitkauskas gt gt., 1983), sodium and potassium ATPase (Ledbetter and Lubin, 1979; Ledbetter and Young, 1983), and some protein kinases (Fletcher gt gt., 1983; Murray and Fletcher, 1982). For example, the enzyme HGPRTase produces the product inosine monophosphate from its substrates hypoxanthine and phosphoribosyl phosphate (PRPP). Lesch—Nyhan cells lack HGPRTase and die when grown in hypoxanthine-aminopterine-thymidine (HAT) medium. Normally, Lesch—Nyhan cells have higher PRPP levels than other cells and increase their PRPP content three—to-four-fold when grown in HAT medium. If Lesch— Nyhan cells lacking HGPRT activity (HGPRT— cells) are co-cultured in HAT medium with normal fibroblasts (HGPRT+ cells), the HGPRTase activity in the HGPRT+ cells is increased three to four fold (Benke and 95 Dittman, 1977). By modifying the PRPP content of the HGPRT" Lesch-Nyhan cells, the HGPRTase activity in such co-cultures varies depending‘ upon the amount of PRPP available from Lesch-Nyhan cells (Vitkauskas and Canellakis, 1984). Therefore, the increase in HGPRTase activity is associated with the equilibration of excess PRPP of the Iesch—Nyhan cells with the HGPRT+ cells. However, it is unclear if the increase in HGPRTase activity is due to induction of additional enzymes or to increased activity of existing enzymes in response to higher levels of substrate. Nevertheless, metabolic cooperation appears to be central to the regulation of the activity of this enzyme. The Nature and Structure gt Gap Junctions The physical structure of gap junctions was first described by Revel and Karnovsky (1967) when they found that gap junctions appear as a pair of apposed plasma membranes separated by a 2-3 nm gap. Studies suggest that gap junctions are assembled from protein units that have a molecular weight of about 27,000 daltons (Hertzberg gt gt., 1982). Cholesterol and phospholipids are the other main constituents of gap junction pro- teins (Hertzberg and Gilula, 1979). The inner core of the gap junction is considered to be hydrophilic (Hirokawa and Heuser, 1982; Loewenstein, 1981). 96 Gap junctional proteins have a high turnover rate, and different half-lives have been reported depending on the technique used. Yancey gt gt. (1981) reported a 19 hour half-life. However, Fallon and Goodenough (1981) reported a shorter half-life of 5.5 hours using a different technique. Cells apparently contain ap- preciable levels of gap junctional proteins which can be rapidly inserted into the plasma membrane when junctional contact between adjacent cells has been established (Gilula, 1984). Metabolic cooperation does not appear to be dependent upon ongoing gap junctional protein synthesis (Epstein gt gt., 1977). The current structural model of gap junctions was proposed by Unwin and Zampighi (1980). Their model is composed of six closely associated junctional proteins that extend individually through the full thickness of a cell membrane to form a hemichannel that can open and close by lateral and circular movement of the proteins. The complete junctional channel is the bipartite structure formed by suitable alignment of hemichannels of two adjacent cells. Regulation gt ggp Junctions Several substances are known to alter' the perme- ability of gap junctions. Increased levels of intra- cellular calcium can inhibit intercellular electrical coupling ‘within. seconds (Rose and Loewenstein, 1975). 97 Several other cations, including sodium, barium, and cobalt, have similar functions when injected into individual cells (De Mello, 1984). Increased acidity, causing a corresponding increase in intracellular calcium concentrations, has also been shown to decrease gap junctional coupling (Rose and Rick, 1978) . Calcium is thought to elicit its inhibitory electrical coupling effect via calmodulin or a calmodulin-like protein (Peracchia e_t gt., 1981). It has been hypothesized that calmodulin binds to gap junctional proteins causing conformational changes in them, resulting in partial or complete occlusion of the hemichannels within connexons. (Peracchia, 1984). Damage to tissue may modulate gap junctions. Myo- cardial infarction (Nealy e_t a_1., 1976) and other heart lesions (De Mello, 1972) lead to ischemia and lowering of the intracellular pH in the cells adjacent to the afflicted area. Normal cells uncouple from the damaged cells in a phenomenon known as "healing over." This phenomenon appears to be a homeostatic mechanism by which normal cells are protected from the deleterious effects of ischemia and cell death. By stopping intercellular conununication with their surrounding damaged cells, the nondamaged cells may help restore function to remaining tissue. Another example of the effect of tissue damage on gap junctions is seen with partial hepatectomy. When 98 two-thirds of the liver was removed in rats there was progressive loss in the number of gap junctions in the remaining lobes of the liver undergoing hyperplasia (Yee and Revel, 1978). The number of gap junctions decreased and became minimal at 29-35 hours after partial hepatectomy. It then increased and returned to normal by 48 hours after partial hepatectomy (Meyer gt a_1., 1981). Gap Junctional Communication and Neoplasia In Vivo Studies Chemically-induced Cancers. There is limited ex- perimental evidence to suggest that neoplastic cells have decreased numbers of gap junctions when compared to their normal tissue counterparts. Wistar rats given N- 4-(5-nitro—2-furyl)-2-thiazolyl formamide (FANFT) had decreased numbers of gap junctions in urothelial cell tumors compared with normal tissue. Furthermore, the decrease in numbers of gap junctions was associated with progression of the tumors to a more malignant stage, and gap junctions were absent in the most malignant tumor cells. Additionally, in FANFT-induced urinary bladder carcinomas, there was a selective loss of larger gap junction plaques but a preservation of the numbers of smaller gap junctional plaques (Pauli and. Weinstein, 1981). Rats with methylcholanthrene-induced dermal 99 tumors, including squamous cell carcinomas, had detectable gap junctions in all primary tumors but had no gap junctions in metastatic squamous cell carcinomas in lymph nodes and lung (Horak gt gt., 1984). Mice treated with tumor-promoting doses of phorbol esters had epidermal tumors with decreased numbers of gap junctions (Kalimi and Sirsat, 1984). Janssen-Timmen gt gt. (1986) and Willecke gt gt. (1985) have described the usefulness of monoclonal antibodies to demonstrate diminished numbers of gap junctions in diethylnitrosamine-induced hepatomas and hepatocellular carcinomas in rats. Janssen—Timmen gt gt. (1986) found that the numbers of gap junctions in hepatocellular carcinomas were reduced by 71% when compared to normal control livers. However, they found no decrease in gap junctional numbers in most, but not all, of small ATPase-deficient preneoplastic cell populations. Perhaps those few enzyme-altered pre- neoplastic foci which have decreased numbers of gap junctions have the greatest potential for autonomous proliferation and may, therefore, be more likely to develop into hepatocellular carcinomas. Decreased numbers of gap junctions may contribute to inhibited cell-cell communication, a mechanism by which tumor promotion may occur (Trosko gt gt., 1983). Although the molecules which regulate tissue homeostasis are not known, one would predict that cells which have lost 100 their gap junctions are more likely to escape growth control. Spontaneous Neoplasia. Gap junctions in progres- sive human cervical neoplasia have been studied, and were found to progress from normal in number to nearly zero ix: more invasive cervical nonsquamous epithelial tumors (Schindler gt gt., 1982). Gap junctions were also few in number in invasive squamous cell carcinomas of the cervix (Schindler gt gt., 1982). Numbers of gap junctions were decreased in urinary bladder adeno- carcinomas of dogs (Alroy, 1979). In human and murine mammary adenocarcinomas, gap junctions were decreased in number, and gap junctional plaques were considered decreased in size when compared to normal mammary epithelial cells. Furthermore, the number of gap junctions was significantly decreased in non-neoplastic parenchymal cells immediately peripheral to the tumor mass (Inoue and Skoryna, 1979). No gap junctions could be demonstrated in cells comprising malignant human glioblastoma multiforme (McNutt and Weinstein, 1971), and few gap junctions could be found in human hepatocellular carcinomas (Swift gt gt., 1983). These studies suggest that decreased numbers of gap junctions are associated with the progression of cells toward a malignant state, and that this trait is not merely a random phenotype present in malignant cells. 101 There is contrary evidence that suggests that not all tumors have decreased numbers of gap junctions. For example, virally-transformed cells, such as Rous sarcoma-transformed cells, have numbers of gap junctions similar to that found in normal, non-virally-transformed cells (Pinto de Silva and Gilula, 1972). Furthermore, in spontaneous pulmonary metastases of virus-induced murine mammary adenocarcinomas, gap junctions appeared to be similar in number to that found in normal cells (Shamsuddin, 1984). In another study, spontaneous pul- monary metastases of mammary adenocarcinomas in virus- infected mice had numbers of gap junctions similar to that seen in cells of the normal mammary gland (Pitelka gt ,gt., 1980). Human benign meningiomas and astro— cytomas had similar numbers of gap junctions when compared to control tissues (McNutt and Weinstein, 1971). However, in none of these studies was the functional status of gap junctions determined. In Vitro Studies A relationship between intercellular communication and tumor promotion was made when it was observed that tumor-promoting chemicals, used at noncytotoxic and nongenotoxic concentrations, inhibited gap junction— mediated metabolic cooperation (Yotti gt gt., 1979; Murray and Fitzgerald, 1979) and electrocoupling activity between cells (Enomoto et al., 1981). Chinese 102 hamster V79 cells treated with a tumor promoter were shown, using freeze-fracture techniques, to have fewer numbers of gap junctions on their plasma membranes than nontreated control cells (Yancey gt gt., 1982). Somatic cell hybrids had a good correlation between decreased junctional communication and neoplastic growth (Azarnia and Loewenstein, 1977). However, none of these studies determined if the decrease in intercellular com- munication was due exclusively to qualitative or quantitative changes in gap junctions. Structure gmg Function gt Nuclear tgtgg The nuclear pore complex provides a channel of communication between the nuclear and cytoplasmic compartments of cells (deRobertis, 1983; Feldherr, 1965; Kessel, 1973; Paine gt gt., 1975). The complex spans the two nuclear membranes and the perinuclear space and is an octagonally symmetrical structure (Gall, 1967; Unwin and Milligan, 1982). Ultrastructural studies have revealed the nuclear pore complex to be composed of a central channel, or annulus, the diameter of which may vary from 0-400 angstroms (Gall, 1967). The central channel is flexible and may expand or contract in a manner similar to a muscle sphincter. Limited evidence indicates that the pore diameter may be influenced by 103 alterations in the activity of ATPase within the cell (Jiang and Schindler, 1986). Functional studies involving transport of molecules through nuclear pores have been performed in normal and neoplastic tissues. Some findings indicate that functional transport via nuclear pores is decreased in neoplastic tissue when compared to its normal counterpart (Drews gt gt., 1968; Garret gt _t., 1973a, 1973b). Few studies have tried to assess quantitative morphological changes of nuclear pore complexes in neoplastic tissue. Czerniak gt gt. (1984), using freeze-fracture techniques, found fewer nuclear pores in human urinary bladder carcinomas than in the normal tissue counterpart. Similarly, Codd gt, gt. (1981), found fewer nuclear pore complexes in an experimentally- induced oral neoplasm than in normal tissue. These findings suggest that decreases in the number of nuclear pores per area of nuclear membrane may be associated with the transition of a cell toward neoplasia, but such changes have yet to be rigorously studied, especially in hepatocarcinogenesis systems. MATERIALS AND METHODS Rats Female Sprague-Dawley rats used for tumor promotion studies (described previously in Chapter 1, Materials and Methods) were fed a combination of 10 mg/kg 2,2',4,4',5,5'—hexabromobiphenyl (245-HBB) plus 0.1 mg/kg 3,3',4,4',5,5'-hexabromobiphenyl (345—HBB) for 140 days following partial hepatectomy and diethylnitrosamine (DEN) administration intra- peritoneally (10 ng/kg body weight). Rats were then maintained on diets free of HBB until day 480 at which time they were killed with ether anesthesia and decapitation, and liver sections were taken during necropsy. Sections of six grossly visible hepatic nodules were taken from six rats, and six sections of liver were taken from the same rats' livers from non— nodular areas. The tissue section was bisected, and one half was fixed in 10% neutral buffered formalin, processed for light microscopy, and stained with hematoxylin and eosin. The other half of each liver 104 105 section was placed in 4% gluteraldehyde for two hours and processed for freeze-fracturing and electron microscopy. Electron Microscopv For assessment of gap junctions, samples fixed in 4% gluteraldehyde ‘were prepared for freeze-fraCturing' by gradual infiltration over three hours with 25% glycerol in phosphate buffered saline (pH 7.2). Sections were cut using a vibratome (Lanar vibratome, Series 1000, Brunswick Co., St. Louis, MO) to a thickness of two mm and "glued" (using a: 1:2 solution of glycerol to 30% polyvinyl alcohol in double distilled water), to a gold replica holder (Electron Microscopy Sciences, Fort Washington, PA) followed by immersion into Freon 22 in its liquid state (- 1500 C) for 10 seconds. Tissues were ‘then stored on their replica holders in liquid nitrogen (- 1900 C). Tissue replicas were freeze- fractured 'using a Balzer's BA-360 M freeze—fracture apparatus (Balzers, Hudson, NH), coated with platinum and carbon, cleaned for 30 minutes in 5.24% sodium hypochlorite (CloroxR Bleach, The Clorox Co., Oakland, CA), placed onto 270—mesh honeycomb copper grids (Ted Pella, Inc., Tustin, CA), and examined using a JEOL 100— CX II scanning/transmission electron microscope (Japanese Electron Optics Laboratory, Tokyo, Japan) at an accelerating electron beam voltage of 100 kilovolts. 106 The magnification of the microscope was calibrated regularly using a diffraction grating replica of 2160 lines/mm. For assessment of nuclear pores, samples were slightly overfixed (eight hours) in 4% gluteraldehyde to increase chances of transmembrane fracture. Sections were then gradually infiltrated with 25% glycerol as before and freeze-fractured as described above. Morphometric Analysis ggp Junctiong. Total membrane surface area was estimated by calculating the amount of grid space occupied by hepatocyte cell membranes during electron microscopic examination. Selected images from the "P" faces of hepatocyte cell membranes from control sections and from hepatic nodules were recorded at a magnification of 30,000 X. The smooth portion of the hepatocyte membrane extending from the bile canaliculus toward the periphery where the cell surface is thrown into numerous irregular projections as it interfaces with another hepatocyte was sought. This has been shown to be the most likely area for the occurrence of gap junctions (Meyer gt gt., 1981; Yancey gt gt., 1981). The recorded images were analyzed, using a technique described by Yancey‘ gt gt.‘ (1981), with an AppleR computer (Apple Computer Corporation, Cupertine, CA), and a digitizing tablet with which outlines of gap 107 junctions were traced and quantified. The total gap junctional area was measured and expressed as a percentage of total measured membrane occupied by gap junctions. A total of 6,000 umz (1,000 umz/sample) of hepatocyte membranes was surveyed from control tissue and 6,000 umz was surveyed from hepatic nodules. Nuclear Pores. Freeze-fractured sections of con- trol liver and hepatic nodules were examined for transmembrane fracture, and images of hepatocytic nuclear membranes were recorded at a magnification of 30,000 X. Numbers of nuclear pores per 9 cm2 of recorded image were determined by averaging six measurements in a single electron micrograph. A total of 25-30 cells per tissue sample was used in this determination. Statistics A rank-sum test was used to compare the area occupied by gap junctions in sections of control livers with that from sections of hepatic nodules (Steel and Torrie, 1980) . Similarly, a rank-sum test was used to determine statistical significance between the number of nuclear pores in hepatocytic nuclei from control liver sections with the number in cell nuclei of hepatic nodules. Significance was defined as P 5 0.05. RESULTS ggp Junctions The total hepatocyte membrane surface area examined and the percent ratio of gap junctional area to total measured membrane area in sections of control liver and hepatic nodules are listed in Table 2-1. The typical histologic appearance of a: hepatic nodule is seen in Figure 2—1. There was less area occupied by gap junctions in sections of hepatic nodules (1.13%) when compared to control sections (2.81%) from the same liver in which hepatic nodules occurred. The typical appearance of gap junctions from freeze- fracture preparation is seen in Figure 2—2. The morphology of gap junctions in control sections of liver was similar to that found in hepatic nodules. Packing, spacing, density, and shape of individual connexons were similar in gap junctions from control livers and hepatic nodules, but resolution of freeze-fracture replicas was not great enough to permit morphological assessment of the size of hemichannels within individual connexons. 108 109 Table. 2-1. Membrane Surface Area Occupied ”by Gap Junctions 1n Non-nodular Sections of Liver (Control) and in Hepatic Nodules in Rats. Ratio of gap junctional area to Rat total membrane area (%) 1 2.36 0.93 2 2.97 1.52 3 3.15 1.03 4 2.71 0.98 5 2.80 1.30 6 2.87 1.02 Mean : SD 2.81 : 0.30 1.13 : 0.23 a a Significantly different from controls (P g 0.05). Sections of control liver and hepatic nodules were taken from rats receiving a partial hepatectomy and diethylnitrosamine (10 mg/kg body wt.) followed by 140 days dietary treatment with a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HBB. Rats were then fed diets free of HBB until day 480. Sections of control liver were from non-nodular areas of livers in which hepatic nodules occurred. The tota membrane area examined was approximately 6,000 pm for control liver and 6,000 pm for hepatic nodules. 110 Figure 2-1. Photomicrograph of a hepatic nodule in a section of liver from a rat fed a diet containing a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg of 345-HBB for 140 days after partial hepatectomy and administration of diethylnitrosamine (10 mg/kg body wt.) intraperitoneally. Notice light staining of cells within hepatic nodule and compression of hepatocytes near periphery of nodule (H & E, 90 X). qumrro 111 Figure 2-1 112 Figure 2-2. Recorded image of a freeze- fractured section of hepatocytic membrane from a section of hepatic nodule from a rat fed a diet containing a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HBB for 140 days following di- ethylnitrosamine administration (10 mg/kg body wt.) intraperitoneally. Notice the gap junction (star) and its close proximity to tight junctions (arrows) (platinum and carbon coating, 67,500 X). 113 . w :38“ gfigt: Figure 2-2 114 Nuclear Pores The numbers of nuclear pores in control sections of liver and in sections of hepatic nodules are shown in Table 2-2. The number of nuclear pores was not significantly different when nuclei from sections of control liver were compared to nuclei from. hepatic nodules. Surface area of individual nuclear pores was not quantified, but there appeared to be little variation in the size of nuclear pores from one nuclear membrane surface to another. Resolution of freeze- fractured replicas was not great enough to permit a detailed morphological assessment of individual nuclear pores or their annuli, but morphological variation appeared minimal between nuclear pores from sections of control hepatocytes and those from hepatic nodules (Figure 2-3). 115 Table 2-2. Numbers of Nuclear Pores in Hepatocytes from Non-nodular Sections of Liver (Control) and from Cells in Hepatic Nodules in Rats. Rat Number of Nuclear Pores per 9 cm2 a number ----------------------------------- Control Sections b Hepatic Nodules b l 18 20 2 25 17 3 17 22 4 19 25 5 16 19 6 21 23 Mean + SD 19.3 + 3.3 21.0 + 2.9 C a On recorded image at magnification of 30,000 X. b A total of 25-30 nuclei per tissue section was examined. c Not significantly different from controls (P g 0.05). 116 Figure 2—3. Recorded image of a freeze—fractured section of hepatocytic nuclear membrane from a section of hepatic nodule from the liver of a rat fed a combination of 10 mg/kg 245-HBB plus 0.1 mg/kg 345-HBB for 140 days after partial hepatectomy and diethylnitrosamine administration (10 mg/kg body wt.) intraperitoneally. Notice nuclear pores on the surface of the nuclear membrane (platinum and carbon coating, 30,000 X). 117 ‘°"=°-\Qrv~m _- Figure 2-3 DISCUSSION Freeze-fracture studies provide useful information regarding morphological and quantitative alterations in ultrastructural cellular organelles such as gap junctions and nuclear pores. However, freeze-fracture studies are limited because they do not provide information about the functional status of these structures. In addition, some workers have estimated that for quantitative studies of gap junctions in liver tissue, freeze-fracture allows examination of only approximately 7% of the the total contact area of a hepatocyte (Meyer gt gt., 1981). Therefore, freeze- fracture studies such as these provide useful but limited information about such organelles. The results of this study indicate that there is less membrane surface area occupied by gap junctions in hepatic nodules than in control sections from the same livers in which hepatic nodules occurred in DEN- initiated and partially hepatectomized rats given known hepatic tumor promoters. Hepatic nodules have been proposed as precursor lesions to hepatocellular carcinomas in rats (Farber, 1984; Scherer, 1984; Schulte-Hermann, 1985; Williams, 1982). Hepatocellular carcinomas are known to have fewer gap junctions than 118 119 either normal or cirrhotic liver (Swift gt 1., 1983). A reduction in the number of gap junctions on cell surface membranes has been associated with nonhepatic neoplasia (Schindler gt gt., 1982; Alroy, 1979; Inoue and Skoryna, 1979; McNutt and Weinstein, 1971; Martinez- Palomo, 1975), hepatic neoplasia (Swift gt gt., 1983), and hepatic regeneration (i.e., hyperplasia) (Yancey gt gt., 1981; Yee and Revel, 1978). The amount of hepatocytic membrane area occupied by gap junctions in sections of control liver was similar to that reported for normal rat liver by others (Meyer gt gt., 1981). It has been postulated that decreases in gap junctional surface area may alter the intercellular sharing of metabolites, decrease electrocoupling between cells, and inhibit cell-cell communication (Swift gt gt., 1983; Meyer gt gt., 1981). Diminished gap junctional function has been observed specifically during tumor promotion (Enomoto gt gt., 1981; Yancey gt gt., 1982). Blockage of intercellular communication may contribute to the autonomous proliferative behavior of neoplastic cells and has been proposed as a mechanism of tumor promotion (Trosko gt gt., 1982). However, the role of gap junctions in in Vivo intercellular communication in cells undergoing preneoplastic or hyperplastic changes is largely unknown. Furthermore, it is unknown if there is a critical number of gap junctions that must still be functioning in a cell in 120 order for it not to be metabolically "blocked" from other adjacent cells. Studies in which anti-gap junction antibodies are used could allow rigorous quantitation of gap junctions under various conditions of cellular dysfunction. Little is known about morphological or quantitative changes in nuclear pores of neoplastic or preneoplastic cells. Results from the present study indicate that there was no difference in numbers of nuclear pores in individual hepatocytes when sections of control liver were compared with hepatic nodules. These results dif— fer from those in another study in which a decrease in numbers of nuclear pores was found in experimentally- induced oral epithelial cancer (Codd gt_ gt., 1981). Functional studies indicate that transport via nuclear pores is decreased in neoplastic tissue when compared to normal tissue (Drews gt gt., 1968; Garret gt gt., 1973a, 1973b). No reports on quantitative alterations in nuclear pores in hepatocarcinogenesis systems were found. SUMMARY-CHAPTER 2 Conclusions from the preceding experiments include the following: 1) The numbers of gap junctions in freeze-fractured preparations of hepatic nodules induced by a two-stage hepatocarcinogenesis assay were significantly decreased when compared to non-nodular areas of liver. 2) The numbers of nuclear pores in freeze-fractured preparations of cells from hepatic nodules induced by a two-stage hepatocarcinogenesis assay were not significantly different when compared to hepatocytes from sections of non-nodular liver. The results from these studies suggest that decreased numbers of gap junctions are associated with the development of hepatic nodules. Such a phenomenon may cause decreased intercellular communication and may be an in vivo mechanism of tumor promotion. However, the role of nuclear pores in the development of hepatic nodules is less clear. 121 BI BLIOGRAPHY-CHAPTER 2 BIBLIOGRAPHY Alroy J: . Ultrastructure of canine urinary bladder carc1noma. Vet Pathol 16:693-701, 1979. Azarnia R, Loewenstein WR: Intercellular communication and tissue growth. VIII. A genetic analysis of junctional communication and cancerous growth. J Membrane Biol 34:1-28, 1977. Benke PJ, Dittman B: Phosphoribosylpyrophosphate synthe51s in cultured human cells. Science 197:1171-1172, 1977. Codd RM, White FH, Goharti K: Quantitative alterations in nuclear pores during experimental oral carcinogenesis. Br J Cancer 44:303 (Abstr.), 1981. Czerniak B, Kuss LG, Sherman A: Nuclear pores and DNA ploidy in human bladder carcinomas. Cancer Res 44:3752-3756, 1984. De Mello WC: The healing-over process in cardiac and other muscle fibers. In Electrical Phenomena in the Heart. Edited by W.C. De Mello, AcademiE Press, New York, pp.323-351, 1972. 1 De Mello WC: Cell-cell communication in heart and other tissues. Prog Biophys Mol Biol 39:147-182, 1982. De Mello WC: Modulation of junctional permeability. Proc Fed Am Soc Exp Biol 43:2090-2092, 1984. deRobertis EM: Nucleocytoplasmic segregation of proteins and RNA's. Cell 32:1021-1025, 1983. Drews J, Brawerman G, Morris HP: Nucleotide sequence homologies in nuclear and cytoplasmic ribonucleic acid from rat liver and hepatomas. Europ J Biochem 3:284-292, 1968. 122 123 Enomoto T, Sasaki Y, Kanno Y, Yamasaki H: Tumor promoters cause a rapid and reversible inhibition of the formation and maintenance of electrical cell coupling in culture. Proc Natl Acad Sci 78:5628-5632, 1981. Epstein ML, Sheridan JD, Johnson RG: Formation of low- resistance in vitro in the absence of protein synthesis and ATP production. Exp Cell Res 104:24-30, 1977. Fallon RF, Goodenough DA: Five-hour half-life of mouse liver gap junction protein. J Cell Biol 90:521- 526, 1981. Farber, E: Pre-cancerous steps in carcinogenesis-— their physiological adaptive nature. Biochem Biophys Acta 738:171-180, 1984. Feldherr CM: The effect of the electron-opaque pore material on exchanges through the nuclear annuli. J Cell Biol 25:43-53, 1965. Flagg-Newton J, Simpson I, Loewenstein WR: Permeability of the cell—to—cell membrane channels in mammalian cell junction. Science 205:405—407, 1979. Fletcher WH, Tusa JD, Greenman JRT: Gap junction mediation of hormone action that causes cAMP- dependent protein kinase dissociation in ovarian granulosa cells. J Cell Biol 97:80a (Abstr.), 1983. Gall JG: Octagonal nuclear pores. J Cell Biol 32:391— 399, 1967. Garfield RE, Sims SM, Kannan MS, Daniel EF: Possible role of gap junctions in activation of myometrium during parturition. Am J' Physiol 253:168-176, 1978. Garfield RE, Kannan MS, Daniel EF: Gap junction formation i3: myometrium: control kn! estrogens, progesterones, and prostaglandins. Am J Phy51ol 238:81-88, 1980. Garret CT, Katz C, Moore RE, Pitot HC: Competitive DNA-RNA. hybridization of microsomal and nuclear RNA in normal tissues of the rat. Cancer Res 33:1662-1669, 1973a. 124 Garret CT, Moore RE, Katz C, Pitot HC: Competitive - DNA-RNA. hybridization. of nuclear and microsomal RNA in normal, neoplastic and neonatal liver tissues. Cancer Res 33:2464-2468, 1973b. Gilula NB: The biosynthesis of gap junctions. Proc Fed Am Soc Exp Biol 43:2678—2680, 1984. Gilula NB, Epstein ML, Beers WH: Cell-cell communication and ovulation. J Cell Biol 78:58- 75, 1978. Hertzberg EL, Gilula NB: Isolation and characteriza- tion of gap junctions from rat livers. J Biol Chem 254:2138-2147, 1979. Hertzberg EL, Lawrence TS, Gilula NB: Gap junctional communication. Ann Rev Physiol 43:479—499, 1981. Hertzberg EL, Anderson DJ, Freidlander M, Gilula NB: Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions. J Cell Biol 92:52-59, 1982. Hirokawa N, Heuser J: The inside and outside of gap junction membranes visualized by deep etching. Cell 30:395-406, 1982. Hooper ML, Subak-Sharpe H: Metabolic cooperation between cells. Int Rev Cytol 69:45—104, 1981. Horak E, Lelkes G, Sugar J: Intercellular junctions of methylcholanthrene-induced rat skin basocellular and squamous carcinomas. Br J Cancer 49:637—644, 1984. Inoue S, Skoryna SC: Intercellular communication in human breast cancer. Proc Am Assoc Cancer Res 20:29 (Abstr.), 1979. Janssen-Timmen U, Traub O, Dermietzel R, Rabes HM, Willecke K: Reduced number of gap junctions in rat hepatocarcinomas detected by monoclonal antibody. Carcinogenesis 7:1475-1482, 1986. Jiang LW, Schindler M: Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study. J Cell Biol 102:853-858, 1986. Kalimi GH, Sirsat SM: Phorbol ester tumor promoter affects the mouse epidermal gap junctions. Cancer Lett 22:343-350, 1984. 125 Kessel RG: Structure and function of the nuclear envelope and related cytomembranes. Prog Surf Membr Sci 6:243-329, 1973. Ledbetter MLS, Imbin M: Transfer of potassium: a new measure of cell-cell coupling. J Cell Biol 80:150-159, 1979. Ledbetter MLS, Young GL: Use of a potassium transfer assay to demonstrate communication among cultured epithelial cells. J Cell Biol 97:82a (Abstr.), 1983. Loewensteix: WR: Junctional intercellular communica- tion: The cell-cell membrane channel. Physiol Res 61:829-913, 1981. Martinez-Palomo, A: Ultrastructural modifications of tight junctions in epithelia with different permeability. Proc Natl Acad Sci 72:4487-4491, 1975. McNutt NS, Weinstein RS: Further observations on the occurrence of nexuses in astrocytomas and glioblastoma multiforme. J Cell Biol 51:805—825, 1971. Meda P, Kohen E, Kohen C, Rabinovitch A, Orci L: Direct communication of homologous and .heterologous endocrine islet cells in culture. J Cell Biol 92:221-226, 1981. Meda P, Micheals RL, Halban PA, Orci L, Sheridan JD: In vivo modulation of gap junctions and dye coupling between B-cells of the intact pancreatic islet. Diabetes 32:858-868, 1983. Meyer DJ, Yancey B, Revel JP: Intercellular communication in normal and regenerating rat liver: a quantitative analysis. J Cell Biol 91:505-523, 1981. Micheals RL: Increased dye coupling among cells in prolactin-stimulated. pancreatic islets. J Cell Biol (Suppl) 95:94a (Abstr.), 1982. Micheals RL, Sheridan JD: Islets of Langerhans: dye coupling among immunohistochemically distinct cell types. Science 214:801-803, 1981. Murray SA, Fitzgerald DJ: Tumor promoters inhibit metabolic cooperation in co-cultures of epidermal and 3T3 cells. Biochem Biophys Res Commun 91:395— 401, 1979. 126 Murray SA, Fletcher WH: Contact dependent signal transfer. that leads to cAMP-dependent protein kinase dissoc1ation. J Cell Biol 95:99a (Abstr.), 982. Nealy JR, Whitmer JT, Rovetto MJ: Effect of coronary blood flow on glycolytic flux and intracellular pH in isolated rat hearts. Circ Res 37:733-741, 1976. Paine PL, Moore LC, Horowitz SB: Nuclear envelope permeability. Nature 254:109-114, 1975. Pauli BU, Weinstein RS: Structure of gap junctions in cuTtures of normal and neoplastic bladder epithelial cells. Experientia 37:248—250, 1981. Peracchia C, Bernardini G, Peracchia LL: A calmodulin inhibitor prevents gap junctional crystallization and electrical uncoupling. J Cell Biol 91:124a (Abstr.), 1981. Peracchia C: Communicating junctions and calmodulin: inhibition of electrical uncoupling in Xenopus embryo by calmidazolium. J Membrane Biol 81:49- 58, 1984. Pinto de Silva P, Gilula NB: Gap junctions in normal and transformed fibroblasts in culture. Exp Cell Res 71:393-401, 1972. Pitelka DR, Hamamoto ST, Taggart BN: Epithelial cell junctions in primary and metastatic mammary tumors of mice. Cancer Res 40:1588—1599, 1980. Pitts JD: The role of junctional communication in animal tissues. In Vitro 16:1049—1055, 1980. Revel JP, Karnovsky MJ: Hexagonal array of subunits in intercellular junctions of the mouse heart and liver. J Cell Biol 33:C7-C12, 1967. Rose B, Loewenstein WR: Permeability of cell junction depends on local cytoplasmic calcium activity. Nature 254:250-252, 1975. Rose B, Rick R: Intracellular pH, intracellular free Ca, and junctional cell—cell coupling. J Membrane Biol 44:377-415, 1978. Neoplastic progression in experimental Scherer E: _ Biophys Acta hepatocarcinogeneSis. Biochem 738:219—236, 1984. 127 Schindler AM, Amaudruz MA, Kocker O, Riotton G, Gabbiani G: Desmosomes and gap junctions in various epidermoid preneoplastic and and neoplastic lesions of the cervix uteri. Acta Cytol 26:466-470, 1982. Schulte-Herman R: Tumor promotion in the liver. Arch Toxicol 57:147-158, 1985. Shamsuddin AM: Comparative studies of primary, metastatic and transplanted colon adenocarcinomas in Fischer 344 rats. J Submicrosc Cytol 16:327- 339, 1984. Sheridan JD, Finbow ME, Pitts JD: Metabolic interactions between animal cells through permeable intercellular junctions. Exp Cell Res 123:111-117, 1979. Spray DC, Harris AL, Bennett MVL: Voltage dependence of junctional conductance in early amphibian embryos. Science 204. 432- 434, 1977. Steel RGD, Torrie .JH: Nonparametric Statistics. In Principles and Procedures of Statistics. A Biomedical Approach. Edited by C. Napier and J. W. Maisel, McGraw—Hill Book Co., New York, pp. 533- 554, 1980. Subak-Sharpe H, Burk RR, Pitts JD: Metabolic co- operation between biochemically marked mammalian cells in culture. J Cell Sci 4:353-367, 1969. Swift JG, Mukherjee TM, Rowland R: Intercellular junctions in hepatocellular carcinoma. J Submicrosc Cytol 15:799-810, 1983. Trosko JE, Yotti LP, Warren ST, Tsushimoto G, Chang CC: Inhibition of cell-cell communication by tumor promoters. In Carcinogenesis: A Comprehensive Survey. Edited In? E. Hecker, N.E. Fusenig, W. Kunz, F. Marks, and H.W. Thielmann, Vol 7, Raven Press, New York, pp. 565—584, 1982. Trosko JE, Chang CC, Medcalf A: Mechanism of tumor promotion: potential role of intercellular communication. Cancer Invest 45:3742—3749, 1983. Unwin PNT, Zampighi G: Structure of the junction between communicating cells. Nature 283:545-549, 1980. 128 Unwin PNT, Milligan RA: A large particle associated With the perimeter of the nuclear pore complex. J Cell Biol 93:3-75, 1982. Vitkauskas G, Kole J, Canellakis ES: Biochemical assay of inhibitors of metabolic cooperation. Exp Cell Res 145:15-30, 1983. Vitkauskas G, Canellakis ES: The regulation of hypoxanthine guanine phosphoribosyl transferase activity through transfer of PRPP by metabolic cooperation. Exp Cell Res 152:541—551, 1984. Warner AE, Guthrie SC, Gilula NB: Antibodies to gap junction protein selectively disrupt junctional communication in the early amphibian embryo. Nature 311:127-131, 1984. Wassarman PM, Letourneau GE: RNA synthesis in fully grown mouse oocytes. Nature 261:73-74, 1976. Williams GM: Phenotypic properties of preneoplastic rat liver lesions and applications to detection of carcinogens and tumor promoters. Toxicol Pathol 10:3-11, 1982. Willecke K, Traub O, Janssen—Timmen U, Frixen U, Dermietzel R, Leibstein A, Paul D, Rabes H: Immunochemical investigations of gap junction protein in different mammalian tissues. In: Gap Junctions. Edited by M.V.L. Bennett and D.C. Spray, Cold Spring Harbor Laboratory, New York, pp. 67—76, 1985. Yancey SB, Nicholson BJ, Revel JB: The dynamic state of liver gap junctions. J Supramol Struct Cell Biochem 16:221-232, 1981. Yancey SB, Edens JE, Trosko JE, Chang CC, Revel JP: Decreased incidence of gap junctions between Chinese hamster V79 cells upon exposure to the tumor promoter l2-0-tetradecanoyl-phorbol-13— acetate. Exp Cell Res 139:329-340, 1982. Yee A, Revel JP: Loss and reappearance of gap junctions in regenerating liver. J Cell Biol 78:554-564, 1978. Yotti LP, Chang CC, Trosko JE: Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206:1089—1091, 1979. CHAPTER 3 THE EFFECTS OF 2,2',4,4',5,5'-HEXABROMOBIPHENYL 0N INTERCELLULAR COMMUNICATION: ASSESSMENT BY THREE IN VITRO ASSAYS CHAPTER 3 THE EFFECTS OF 2,2',4,4',5,5'-HEXABROMOBIPHENYL 0N INTERCELLULAR COMMUNICATION: ASSESSMENT BY THREE IN VITRO ASSAYS INTRODUCTION Intercellular communication is the phenomenon of sharing metabolites, ions, and other small molecules between cells. The cellular organelles responsible for distribution of these molecules between cells are gap junctions. Thus, functional gap junctions are important for tissue homeostasis (Trosko gt gt., 1982; DeMello, 1982; Loewenstein, 1979). Certain environmental toxicants behave in vivo as tumor promoters. Such compounds apparently have little genotoxicity and therefore are not categorized as "mutagens." Rather, tumor promoters seemingly have "epigenetic" activity and affect the target cell in some way other than by genomic alteration. However, the mechanisms of tumor promotion have yet to be fully elucidated. 129 130 One proposed mechanism by which tumor promotion may occur is inhibition of cell-cell communication (Saxen gt gt., 1976; Loewenstein, 1979; Trosko gt gt., 1983a). Tumor promoters may disrupt the function of gap junctions such that previously initiated cells may be liberated. from 'the control of normal cells. Perhaps shared metabolites responsible for control of cellular growth are no longer able to enter neighboring cells and prevent expansion of initiated clones. Cell-cell communication is measured by several different in vitro techniques. The first objective of the following studies was to assess the ability of 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB), the major congener of Firemaster BP-6 (FM), to inhibit intercellular communication in vitro using a rat liver epithelial cell line (WB-F344) in the metabolic cooperation assay at various concentrations of PM or 245-HBB. An ancillary objective of this study was to determine if 245-HBB had the same ability to inhibit cell—cell communication as PM at similar concentrations. A second objective was to assess the ability of 245-HBB to inhibit cell-cell communication using a novel technique of "Fluorescence Redistribution After Photobleaching" ("FRAP") from a technique known as Anchored Cell Analysis and Sorting (ACAS). A third objective was to determine the usefulness of a new in vitro assay, termed "scrape-loading/dye transfer," for 131 assessing inhibition of cell-cell communication in a dose-dependent manner. A final objective was to combine the new techniques provided by ACAS with results from the scrape-loading/dye transfer assay to rigorously quantify dose-responsiveness of inhibited intercellular communication at various concentrations of 245-HBB. LITERATURE REVIEW t_ Vitro Properties gt Tumor Promoters One property of known tumor promoters that has been recently elucidated is their ability to inhibit metabolic cooperation between cells in vitro by cell membrane interactions (Trosko gt, gt., 1981; Yotti gt gt., 1979). One manner in which tumor promoters may interact with the cell membrane involves alteration of cell-cell communication via gap junctions (Loewenstein, 1979; Peracchia, 1980; Larsen, 1983). Cell—cell com- munication is an important determinant in the control of cellular growth, differentiation, and development, as well as tissue function and homeostasis (Bertram, 1979; Trosko gt gt., 1982; Andrew gt gt., 1981; DeMello, 1982; Gilula, 1980; Lawrence gt gt., 1978; Loewenstein, 1979). -Contact inhibition between cells may be dependent upon functional gap junctions (Levine gt gt., 1965). Tumor promoters appear to disrupt gap junctional intercellular communication such that previously initiated cells may be freed from the control of normal cells. Many tumorigenic cell lines have been shown to have modified gap junctional characteristics. Therefore, inhibition 132 133 of intercellular communication may be associated with the tumorigenic process (Saxen gt gt., 1976: Loewenstein, 1979; Trosko gt gt., 1983a) Tumor promoters may exert their cell membrane effects by two general means. One involves direct interaction with the plasma membrane of the cell (Weinstein gt gt., 1979). Another mechanism may be the altering of gene expression (Yamasaki, 1984) without altering' the genetic ‘material of the cell. This is consistent with the observation that most tumor promoters have little or no mutagenic potential (Trosko g a_1., 1983b). While tumor promoters are not generally mutagens, sudh compounds have been conceptualized as "mitogens" (Trosko gt gt., 1983a). Two major hypotheses have emerged to explain how certain tumor' promoters cause’ clonal expansion of previously initiated cells. First, tumor promoters may activate protein kinase C (Pk-C), a calcium-dependent phospholipid enzyme (Nishizuka, 1986). This sets off a chain reaction to phosphorylate a sequence of cellular structures. An end point of Pk-C activation has been postulated to be loss of gap 3 £1 nctional permeability (Castagna gt gt., 1982; Fujiki |‘.': _t., 1984). It is not known if the activated Pk-C directly phosphorylates gap junctional proteins, rendering gap junctions impermeant, or indirectly inactivates gap junctional permeability via 134 phosphorylation of other membrane-bound enzymes or proteins. A second possible mechanism for the cellular effects of tumor promoters is that they may induce a "prooxidative" state in the cell. According to this hypothesis, oxygen radical species are generated by the action of certain tumor promoters (Cerutti, 1985). The target for cellular damage by such oxygen radicals is DNA, implying that tumor promoters are somehow mutagenic. This leaves open the possibility that oxygen radicals might directly effect gap junctions or membrane components regulating gap junctional function. However, this hypothesis fails to account for the very different biological responses between tumor initiators and tumor promoters. Many different cell types have been used in studying chemicals which could inhibit intercellular com- munication. Chinese hamster V79 cells (Yotti gt gt., 1979), various human cell types (Davidson gt gt., 1985; Enomoto gt gt., 1981; Friedman and Steinberg, 1982; Mosser and Bols, 1982), rat cells of hepatic origin (Telang gt gt., 1982; Walder and Lutzelschwab, 1984), and murine cell lines (Fitzgerald gt gt., 1983; Murray and Fitzgerald, 1979) have been used. It is important Vto use various cell lines because chemicals which inhibit intercellular communication may show organism or organ specificity. Therefore, employing a wide variety 135 of cell lines improves the chances of detecting inhibition of cell—cell communication by a wide variety of chemicals. Assays for Measuring Gap Junctional Communication Electrocoupling Assays The sharing of passive electrical potential between touching cells is termed electrocoupling. These assays involve placing‘ microelectrodes into the cytoplasm of two contiguous cells (Yamasaki gt _t., 1983). Pulses of current are then passed into one of the cells. The electrical potentials of the two cells are determined concurrently with respect to the external environment. The ratio of voltage change of the second cell to that of the injected cell is the coupling coefficient or coupling ratio. When cells are joined by functional gap junctional channels, the coupling coefficient is relatively high (Socolar and Loewenstein, 1978). This assay is a sensitive test for the presence of functional gap junctions. However, one limitation is that the assay only detects the passage of the very smallest molecules, namely inorganic ions, that are responsible for the intercellular spread of the electrical current. In addition, it is unknown what the effects of trauma of microinjection are to the cell membrane or to the integrity of gap junctions. 136 Junctional Conductance Assays Quantitative determinations of gap junctional permeability can be measured by junctional electrical conductance. This technique requires the insertion of multiple microelectrodes with simultaneous measurements of several end points and is technically more difficult than electrocoupling. Normal values for junctional conductance for various tissues have yet to be established, making such measurements limited in their usefulness. It is further restricted for use with cells in pairs or short chains (Socolar and Loewenstein, 1978). Freeze-Fracture Studies Freeze-fracturing of intercellular membranes allows visualization of gap junctions. The technique requires electron microscopy, and relatively large areas of membrane must be scanned to obtain accurate estimates of the amount of area of the membrane occupied by gap junctions, making this method time-consuming. Furthermore, decreases in gap junction numbers are not positively associated with reduced electrical coupling (Meyer gt gt., 1981). In fact, electrical coupling appears possible with only a small amount of the membrane occupied by' gap junctional channels. These channels may be difficult to locate in freeze-fractured 137 preparations. Another limitation of freeze-fracture studies is that they do not assess gap junctional function. In spite of these limitations, such studies have been used for qualitative and quantitative information about gap junctions. Metabolic Cooperation Assays Subak-Sharpe gt gt. (1969) first used the term "metabolic cooperation" to describe the intercellular exchange of metabolites by direct cell contact. One method for the detection of metabolic cooperation involves measurement of survival of mutant cells in the presence of a toxic precursor compound. Wild-type cells capable of metabolizing the compound to its toxic product died when cultured in the presence of the compound. However, mutant cells that lack the ability to metabolize the compound survived during exposure to the toxicant. When wild-type and mutant cells were co- cultured the mutant cells received, via gap junctions, the toxic metabolite from the wild-type cells. Gap junctional communication was thus measured as decreased survival of mutant cells in co-cultures compared to mutant cells cultured without wild-type cells (Fujimoto gt gt., 1971; Davidson gt gt., 1985; Mosser and Bols, 1982; Yotti gt gt., 1979; Jone gt gt., in press, 1987). Further evidence that metabolic cooperation was positively correlated with electrocoupling and with the 138 presence of gap junctions was demonstrated by Gilula gt gt. (1972), who measured the transfer of radiolabeled nucleotides between cells. Radioactive metabolite transfer between cells has been used by others as an assessment of intercellular communication (Mosser and Bols, 1982; Newbold gt gt., 1981; Davidson gt gt., 1985). Several modifications and cell types have been successfully incorporated into the metabolic cooperation assay. Williams gt gt. (1981) have modified the metabolic cooperation assay to include primary rat hepatocytes that provide a metabolizing system for activation of certain. toxicants in order for ‘them “to behave as tumor promoters. Kavanagh _t .gt. (1986) characterized ea human cancer cell line useful in the metabolic cooperation assay. Jone g g. (in press, 1987) have described the development of a rat hepatic nonparenchymal epithelial cell line, termed WB—F344, for measuring metabolic cooperation. ng Transfer Assays Functional gap junctional communication can be assessed using low molecular weight fluorescent dyes as tracers (Enomoto and Hamasaki, 1984; Fitzgerald and Murray, 1980; Friedman and Steinberg, 1982). These dyes may be injected into cells or may be introduced into cells by transmembrane diffusion if the dye is of the 139 appropriate nonpolar ester (Rotman and Papermaster, 1966; Goodall and Johnson, 1982). Once the esterified dye is inside the cell, the dye is rapidly hydrolyzed by esterases to a fluorescent compound. These dyes are typically hydrophilic and do not readily cross into other cells by other than gap junctional transfer. Scrape—LoadinggDye Transfer Agggy. El-Fouly gt gt. (i3: press, 1987) have introduced a rapid and simple technique for' measuring gap junctional communication. This assay, termed "scrape-loading/dye transfer," introduces dyes into cells in culture by creating a tear in the cell membrane without affecting cell viability or colony-forming capacity (McNiel gt gt., 1984). The tracer dye, Lucifer yellow, has a molecular weight of 457.2 and is a brightly fluorescent 4-aminoaphthalimide compound with a high quantum yield of 0.25 (Stewart, 1978, 1981). The quantum yield is stable from pH one to 10 and is easily detectable with epifluorescence microscopyx Lucifer' yellow does not diffuse through intact cell membranes but its low molecular weight permits its transfer from one cell to another via patent gap junctions (Stewart, 1978, 1981; Lo and Gilula, 1979). Another dye, rhodamine dextran, has a high molecular weight (10,000) and is administered con- currently with Imcifer yellow. Rhodamine dextran can neither diffuse through intact plasma membranes nor pass though gap junctional channels. Rhodamine dextran emits 140 red fluorescence whereas Lucifer yellow emits yellow-to— apple green fluorescence. The simultaneous introduction of both dyes into cells allows the identification of primary dye-recipient cells, whose cell membranes have been torn by scraping with a wooden probe, and verifies that transfer of Lucifer yellow into contiguous cells (i.e., secondary dye—recipient cells) has occurred via gap junctions. Major advantages of the scrape- loading/dye transfer assay include low cost, rapidity, and direct visualization of results. Its greatest potential is for a quick screening assay to determine inhibition of cell-cell communication by various chemicals. Furthermore, it requires minimal metabolic and biochemical integrity of the cells. Other advantages include minimization of physiological alterations or artifacts that may be induced by other lengthy or_complicated procedures. It has application for both quantitative and qualitative evaluation of cell-cell communication. However, the sensitivity of this technique is unknown. Fluorescence Redistribution After Photobleaching. Another dye transfer technique, termed "fluorescence , redistribution after photobleaching" ("FRAP"), involves labeling of cells in tissue culture with 6- carboxyfluorescein diacetate (Wade gt gt., 1986). All cells in the culture are internally labeled by this stain. Upon contact with the cell cytoplasm the dye is 141 hydrolyzed and a hydrophilic fluorescein derivative is maintained in the cell (Rotman and Papermaster, 1966). Any labeled cell may be photobleached by a laser beam ' whose width is approximately equal to the diameter of the cell. 'Alternatively, the dye may be photobleached by a series of laser pulses with each pulse having a diameter of about one pm. Following photobleaching, the bleached dye molecules from one cell and the nonbleached dye molecules from an adjacent contacting cell may be redistributed via gap junctions. The dye and labeling conditions do not affect cell viability and all measurements can be performed at room temperature. However, leaching of the dye may occur through the cell membrane after a certain period of time, depending on the cell type used and the culture conditions employed. The FRAP technique requires sophisticated and expensive equipment. In the original assay, a tissue culture plate of labeled cells was placed on a rdgh speed computer-controlled two dimensional stage of an instrument known as ACAS 470 (Anchored Cell Analysis and Sorting, Meridian Instruments, Okemos, MI). The stage moves the culture plate in a defined manner above the objective lens of an inverted epifluorescence microscope. The objective lens of the microscope focuses an argon ion laser beam (excitation wavelength of 488 nm) to a one um spot that excites fluorescence in individual cells. Digitized pseudoimages record the 142 fluorescence intensity of the cells, and this information can be stored in a computer. Several compounds that inhibit cell-cell com- munication in the metabolic coOperation assay have been found to inhibit dye transfer using FRAP. Many of the compounds are tumor promoters in vivo such as dieldrin and 12—0-tetradecanoylphorbol-13-acetate (TPA). Results from FRAP studies using these compounds correlate with dye microinjection techniques (Fitzgerald gt gt., 1983). A major advantage of the FRAP assay is that it has the capacity to make multiple measurements within the same cell without inducing traumatic alterations to the plasma membrane. Another advantage is its ability to measure various end points of all anchored cells types and cell configurations. MATERIALS AND METHODS Metabolic Cooperation A_ss_a_y C_el_lg m Culture Methods. Cells used for this as- say were WB-F344 (rat epithelial) cells, previously shown to metabolically cooperate (Jone gt Q” in press, 1987). These cells had been previously isolated from the livers of adult male Fischer 344 rats (Tsao gt gt., 1984). The WB—F344 cells (courtesy of Dr. J. W. Grisham, University of North Carolina) were maintained in vials and were frozen in liquid nitrogen. Cells had been previously biochemically characterized as either lacking activity for the enzyme hypoxanthine guanine ribosyl transferase (HGPRT‘) or having such activity (HGPRT+) . Thawed cell suspensions were entered into sterile 25 cm2 culture flasks (Corning Glass Works, Corning, NY) and were grown in modified Eagle‘s medium (Gibco Inc., Grand Island, NY) with Earle's balanced salt solution (Gibco Inc., Grand Island, NY). This medium was supplemented with a 50% increase in vitamins and essential amino acids (except glutamine), a 100% increase in nonessential amino acids, 5% fetal calf serum (Gibco Inc., Grand Island, NY), insulin (10'6 143 144 M/liter) (Sigma Chemical Co., St. Louis, MO), and gentomycin (12 mg/L of medium) (Quality Biologicals, Gathersburg, MD). Phenol red (10 mg/L of medium) (Sigma Chemical Co., St. Louis, MO) was used as an indicator of pH. Cells were grown in humidified incubators (Heinicke Instrument Co., Hollywood, FL) at 370 C with 5% C02. Cultures were tested routinely for contamination with Mycoplasma spp. using the Hoechst 33258 (Hoechst Inc., Philadelphia, PA) staining technique (Chen, 1977). Three days prior to the experiment, cells in flasks were rinsed twice with 10 ml sterile phosphate buffered saline (PBS), detached with a solution of trypsin (Worthingham Diagnostic Systems, Freehold, NJ) with 2% EDTA (Sigma Chemical Co., St. Louis, MO), placed onto a warming plate (370 C) for five minutes, and 1/10 of the cells were placed into a new flask (i.e., 1:10 split). On the day of the experiment, the same procedure was followed and cells were split 1:2. Cell density in each flask was calculated by using a hemocytometer (American Optical Co., Buffalo, NY), and cells were dispensed into 60 mm plates (Corning Glass Works, Corning, NY) each with five m1 of medium (Table 3-1). Cells were incubated as before for two to four hours, then the desired concentrations of FM or 245-HBB (Table 3—1) were added. Each treatment group consisted of ten plates. One hour later, 50 pl of 6-thioguanine 145 Table 3-1. Experimental Design of Metabolic Cooperation Assay using 2, 2', 4, 4', 5, 5'-hexabromobiphenyl (245-HBB) or Firemaster BP-6 (FM) as Test Chemicals with Resistant (HGPRT ) and Sensitive (HGPRT+ ) WB- F344 cells. Group Volume Resistant Sensitive Volume of Final [ ] name . of cells/m1 cells/ml chemical per ml of medium medium medium per plate medium (m1) PE 4 100 ---— 25 pl DMSO 5 pl N 3 100 4 x 105 25 U1 DMSO 5 “1 AL 3 100 4 x 105 10 ul Aldrin 10 ul TA 4 100 ---- 25 “1 a 1 ug a TB 4 100 ~—-- 25 ul a 5 ug a TC 4 100 ---- 25 pl a 20 ug a TD 4 100 -—-— 25 p1 a 40 ug a MA 3 100 4 x 105 25 ul a 1 ug a MB 3 100 4 x 10 25 ul a 5 pg a MC 3 100 4 x 105 25 ul a 20 ug a MD 3 100 4 x 105 25 p1 a 40 pg a a- — Firemaster BP-6 or 2,2',4,4',5,5'-hexabromobiphenyl n = 10 plates per group Plating efficiency group Abbreviations: PE N Negative control group y [2" II II II II II Aldrin (positive control) group T* Cytotoxicity assay groups M* Metabolic cooperation assay groups DMSO = Dimethylsulfoxide (vehicle) [ ] = Concentration 146 (6-TG) (Sigma Chemical Co., St. Louis, M0) were added to each plate. Four days after the beginning of the experiment, the medhnn in the flasks was discarded and replaced with fresh medium, and 50 pl of 6-TG were again added to each plate. Eight days after the beginning of the experiment the medium was discarded, cell colonies were rinsed twice with PBS, fixed and stained with a solution of 10% ethanol and 1.0% crystal violet (Sigma Chemical Co., St. Louis, MO), dried, and counted (Colony Counter, American Optical Co., Buffalo, NY). Statistics. A one-way analysis of variance was used to determine significance between treatment and control groups (Steel and. Torrie, 1980a). A Student—Newman- Keul's test was used for multiple comparisons (Steel and Torrie, 1980b). Significance was defined as P g 0.05. Fluorescence Redistribution After Photobleaching (FRAP) Aéfiéx- The WB-F344 cells were plated at low density (not confluent) on 35 mm plates (Corning Glass Works, Corning, NY) in two ml of medium as described above (but without phenol red) in a humidified incubator at 370 C and 5% CO2. Cells were allowed to settle and attach to the plate for one hour. After that time, each plate received 1, 5, 20, or 40 pg 245-HBB/ml of medium. The 245-HBB was dissolved in DMSO as a vehicle. Cells were allowed to incubate for 24 hours, after which the medium was decanted from the plates. Cells were rinsed twice with calcium/magnesium saline solution (made by adding one gram CaC12 and one gram MgCl2 to 10 liters PBS), and two ml of calcium/magnesium saline solution were added back to the plates. Fourteen ul (seven ul/ml of medium) of 0.1 mg/ml 6-carboxyfluorescein diacetate (6—CFDA) (Molecular Probes, Inc., Eugene, OR) were slowly added to the plates, mixed with gentle agitation, and allowed. to stain the cells for a period of 20 minutes in incubator conditions. After 20 minutes, the calcium/magnesium saline solution and 6-CFDA were decanted, two nu. of medium (without phenol red) were added, and cells were examined by using Anchored Cell Analysis and Sorting (ACAS 470, Meridian Intruments, Okemos, MI). Cells appearing in touching pairs and as single cells were evaluated at a magnification of 400 X using phase contrast microscopy. One cell in a touching pair was selected for photobleaching, and single nontouching cells were selected as negative controls for fluorescence redistribution. The same number of photobleaching points was selected for each photobleached cell. The digitized pseudoimages were examined, and selected cells were then marked with boxes and photobleached at photomultiplier tube voltage of 25- 30%, argon ion laser power of 200 milliwatts, blast 148 strength of 30%, scan strength of 7%, blast time of 250 milliseconds, and a stage speed of 40.0 megahertz. Cells were then analyzed for fluorescence recovery following photobleaching. Recovery of fluorescence was monitored for a period of 15 minutes after initial photobleaching, with one post—bleaching scan every five minutes for the duration of the 15 minute period. Digitized pseudoimages were recorded and saved in the computer's memory (Model XT, International Business Machines, Boca Raton, FL), and previously selected cells were compared for return of fluorescence in 245- HBB-treated and nontreated (DMSO only) cells. Scrape-Loadingsze Transfer Assay Cell and Culture Conditions. The WB-F344 cells were grown overnight to confluency in 35 mm plates in incubator conditions and using medium (without phenol red) as previously described. Final concentrations of 1, 5, 20, and 40 pg 245—HBB/m1 medium (in DMSO as the vehicle) were added. After 24 hours, cells were rinsed twice with room temperature PBS. There were six plates in each treatment group. .A dye mixture of 0.05% Lucifer yellow and 0.05% rhodamine dextran (Molecular Probes, Inc., Eugene, OR) was dissolved in PBS and added to the cell culture. Care was taken to not expose the dye mixture to excessive room light. A wooden probe was used to scrape several rows of cells, and the dye 149 solution was left on the cells for two minutes after scraping. The dye was decanted and cells were rinsed twice with PBS at 250 C to remove background fluorescence and any detached cells. Two milliliters of medium were added to each plate. Plates were examined for the distribution of yellow-green fluorescence (from Lucifer yellow) and red fluorescence (from rhodamine dextran) from scraped edges using' a phase 'microscope with epifluorescence capacity and appropriate filters for fluorescence detection of the two different fluorochromes (Figures 3—1 through 3-3). Quantitation 9; Fluorescence Intensity. Plates from each treatment group were examined on an Anchored Cell Analysis Sorter (ACAS 470, Meridian Intruments, Okemos, MI). Digitized pseudoimages of scraped edges were recorded at a magnification of 400 X using a photomultiplier tube voltage of 25-30% and a scan strength of 7%. Digitized pseudoimages were stored in the computer, and fluorescence' intensity values were obtained for each field examined. A total of ten fields from each plate was quantified for fluorescence intensity. Statistics. Data were analyzed using a one-way analysis of variance (Steel and Torrie, 1980a). Significance was defined at a level of P 5 0.05. 150 Figure 3-1. Phase contrast photomicrograph of a monolayer of untreated WB-F344 cells in culture. Upper left portion of plate has been scraped with a wooden probe. Cells appear confluent and have normal conformation in unscraped portion (400 X). Figure 3-2. Photomicrograph from same field as Figure 3-1 stained with Lucifer yellow (LY) and rhodamine dextran. Notice primary LY-loaded cells along the scraped edge have the most intense fluorescence and that the fluorescence extends four to seven cell layers beyond the primary LY-lOaded cells, indicating that LY has transferred via gap junctions into the secondary LY-recipient cells (Rhodamine dextran/Lucifer yellow, B filter, 400 X). ‘ . Figure 3-3. Photomicrograph from same field as Figure. 3—1 stained with Lucifer yellow and rhodamine dextran (RD). Notice intracellular load1ng of RD in the cell layer along the scraped edge but that RD did not transfer to layers of cells away from scraped edge, indicating that cell membranes away from scraped edge are intact ‘(llglgogamine dextran/ Lucifer yellow , G filter , M 4 ve m-o 3:..Hm m ‘D-in 151 Figure 3-1 Figure 3-2 Figure 3-3 RESULTS Metabolic Cooperation Assay The results from the metabolic cooperation assay are depicted in Figure 3-4. The chemical 2,2',4,4',5,5'- hexabromobiphenyl (245-HBB) inhibited metabolic cooperation at the noncytotoxic concentrations used and in a dose—dependent manner such that the highest concentration of 245—HBB (40 ug/ml medium) had a three- fold greater inhibition of nmtabolic cooperation than those cells receiving a concentration of l ug/ml medium. .A similar pattern was seen when the chemical Firemaster BP-6 (FM) was tested at identical concentrations of 245—HBB, as FM caused a nearly four- fold increase in recovery of mutant (i.e., HGPRT‘) cells. However, the degree of inhibition of metabolic cooperation with FM was not significantly different from 245—HBB when tested at the same concentrations as FM. These results indicate that FM and 245-HBB inhibit metabolic cooperation in WB—F344 cells in a dose— dependent manner, but that one chemical does not inhibit metabolic cooperation more than the other in this in vitro testing system. 152 153 Figure 3-4. Effect of 245-HBB and FM on metabolic cooperation (MC) in WB-F344 cells. The top two (red and green) lines represent cytotoxicity curves for 100 HGPRT” cells/ml medium not co- cultured with HGPRT+ cells and exposed to various concentrations of FM and 245-HBB. The bottom two (blue and red) lines represent % of HGPRT' cells recovered in the metabolic cooperation as ay when 100 HGPRT cells are co-cultured with 4 X 10 HGPRT+ cells and exposed to various concentrations of FM or 245-HBB. In the metabolic cooperation assay at a concentration of 40 ug FM/ml medium, there was a four-fold increase in % recovery of 6-TG-resistant mutants (i.e., HGPRT" cells) when compared to 1 ug FM/ml medium. Similar concentrations of 245-HBB had a three-fold increase in recovery of 6-TG-resistant mutants. There was no statistical difference between the ability of FM and 245-HBB to inhibit metabolic cooperation when compared at the same concentrations (i. e., 1, 5, 20, and 40 ug/ml medium). Vim musmflm 25:52 _s\a3 cozutwcmucoo 9. Mn mm MN ow mm my m a a wow 38V 4 5 l V . mm Scions: Stu: Ill, mmznmvm 5.: o: .IXI. :0m 2m 5:: o: I? mmzlmvm +0 3.23935 IE!5 z... are 1. 3.42336 LT I Uam: ozmwwq paJaAoan 53419an lu815!531-91-9 % 155 Fluorescence Redistribution After Photobleaching (FRAP) Assay Redistribution of the f1uoreScent dye 6- carboxyfluorescein diacetate (6-CFDA) into touching photobleached WB-F344 cells treated. with 245-HBB was most inhibited in those cells treated with 40 pg 245- HBB/ml medium (Figures 3-5 through 3-13). Touching cells treated with lower concentrations of 245-HBB had a greater degree of return of the 6-CFDA into photobleached cells such that the lowest concentration, 1.0 ug 245-HBB/m1 medium, had only slightly less return of 6-CFDA than non-treated controls. These results were similar to those obtained with cells treated with DMSO only (control group). Scrape—Loadinngye Transfer Assay In those plates treated with 40 pg 245-HBB/ml medium, transfer of Incifer yellow into confluent WB— F344 cells was totally blocked and did not spread from the primary dye-loaded cells into secondary recipient cells. (Figures 3-14 through 3-19). Lucifer yellow spread into one secondary dye—recipient layer of cells in those plates treated with 20 11g 245-HBB/ml medium. Redistribution of Lucifer yellow into secondary 156 Figure 3-5. Photograph of digitized pseudo- image of WB-F344 cells grown in culture, not exposed to test chemical (negative control), stained with 6-carboxyfluorescein diacetate, and analyzed for fluorescence redistribution after photobleaching with Anchored Cell Analysis and Sorting. Photobleaching of cells in boxes has not yet occurred. Notice that cells in all boxes except one (arrow) are attached to touching cells. Cell in box (arrow) is not touching other cells and is a negative control for dye return. Figure 3- 6. Photograph of above image im- mediately following bleaching with argon ion laser. Notice that cells in all boxes have been completely photobleached. Figure 3— —7. Photograph of same field 15 minutes following initial photobleaching of cells in boxes with argon ion laser. Notice return of fluorescence to prebleaching levels in all boxes except nontouching cell. 157 . - unmnmmmmmmsuuoa nammnmmmwmmfiaann nlllllli .- -!§IIII Figure 3-5 (mull. ofimflflmmm mull-III. .- Figure 3-6 a I flmlmmmwmmsnuln N . . . . . . . . . . . . . . . amulmmmwmmsnann leg I '2' Figure 3-7 158 Figure 3-8. Photograph of digitized pseudo- image of WB-F344 cells grown 511 culture, exposai for 24 hours to 5 pg 245—HBB/ml medium, stained with 6—carboxyfluorescein diacetate, and analyzai for fluorescence redistribution after photo- bleaching. Photobleaching of cells in boxes has not yet occurred. Notice that all boxes except one (arrow) are around touching cells. Cell in box (arrow) is not touching other cells and serves as a negative control for dye transfer. Figure 3-9. Photograph of above image im- mediately following photobleaching of the dye with argon ion laser. Notice that cells in all boxes have been completely photobleached. (Cell in box 1n lower left corner was not photobleached.) . Figure 3—10. Photogragh of same field 15 minutes following initial photobleaching of cells in boxes with argon ion laser. Notice slight return of fluorescence in three of the five boxes 1n wh1ch touching cells were located and no return of fluorescence in the other two boxes. These re- sults SUQQESt that gap junctional transfer of the 311431112515 partially inhibited by 5 pg 245-HBB/ml mu JM:. .5 ‘ "Ev . H.W. o , ooooooooooooooo mammammmwmmsnoca cumulmmwmmmfiaaon A .ulllllai ll;i!slllllln _m Ami. r a? l '1. Figure 3-8 Figure 3-9 Flgure 3-10 5.2 pm: 160 Figure 3-11. Photograph of digitized pseudo- image of WB-F344 cells grown in culture, exposed for 24 hours to 20 119 245-HBB/ml medium, stained with 6—carboxyfluorescein diacetate, and analyzai for fluorescence redistribution after photo- bleaching. Photobleaching of cells in boxes has not yet occurred. Notice that cells in all boxes except two (arrows) are touching other cells. Cells in boxes (arrows) are negative controls for dye return. Figure 3—12. Photograph of above image ink mediately following photobleaching of the dye with argon ion laser. Notice that cells in all boxes (except cell in upper left) have been completeLy photobleached. (Cell in upper left was not photobleached). _ Figure 3-13. Photograph of same field 15 minutes following initial photobleaching of cells 1n boxes. Notice total lack of return of fluorescence in all boxes. These results suggest that gap junctional transfer of the dye was totally blocked by 20 pg 245-HBB/ml medium. IIIIIIIIIIIII ~— -uassafiisfiafiifii '- 4 Bail!!! I I I I I I I , i. -m .m .’ s I. In “I ”a a- . a. 3" . ‘0 IIIIIIIIIIIIII h 1 .uasaafifieififilli Figure 3—13 162 Figures 3-14 through 3-19 -14 3-15 3-16 3-17 _3-18 3-19_ Figures 3-14 through. 3-19. Series of photo- micrographs of WB-F344 cells grown in culture, exposed to various concentrations of 245-HBB, and subjected to the scrape-loading/dye transfer assay. Notice that cells not exposed to any chemical (Figure 3-14-negative control) have yellow dye (Lucifer yellow) in several cell layers beneath the scraped edge. This is interpreted as noninhibition of gap junctional communication. Cells exposed to l p g 245—HBB/ml medium (Figure 3- 15), 5 pg 245- HBB/ml medium (Figure 3- 16), 20 ug 245-HBB/ml medium (Figure 3-17) and 40 11g 245— HBB/ml medium (Figure 3- 18) have progressively inhibited transfer of Lucifer yellow dye into secondary recipient cells. Control cells (DMSO only) are shown in Flgure 3-19, and had a similar degree of transfer of lucifer yellow as Figure 3-14, indicating that the vehicle used (DMSO) did not interfere with transfer of Lucifer yellow (Rhodamine dextran/Lucifer yellow, B filter, 400 X.) Figure 3—14 Figure 3-16 Figure 3—18 163 Figure 3-15 Figure 3-17 Figure 3—19 164 recipient cells was more noticeable in cells treated with 5 pg 245—HBB/ml medium. Cells treated with 1.0 ug 245-HBB/ml medium appeared to have only slightly less redistribution of Lucifer yellow into secondary recipient cells than cells receiving DMSO only. In all plates, the larger molecular weight dye, rhodamine dextran, was confined to the primary loaded cell layer, and in no instance did this dye redistribute into secondary' recipient cells. This indicates that cell membranes remained functionally intact during the experiment, and that the concentrations of 245-HBB used were probably not high enough to cause membrane damage to the WB-F344 cells. Results from quantitation of fluorescence from scrape-loading/dye transfer experiments are seen in Figures 3-20 through 3-26. These results confirm the visual observation of an inverse correlation between the concentration of 245-HBB and the amount of dye transfer into secondary recipient cells. 165 Figures 3-20 through 3-25 __3-20 3-21 3-22 3-23 3-24 3-25 Figures 3-20 through 3-25. Series of photographs of digitized pseudoimages of WB-F344 cells grown in culture, exposed to various concentrations of 245-HBB, subjected to the scrape- 1oading/dye transfer technique, and examined wifll Anchored Cell Analysis and Sorting to quantify fluorescence intensity. Notice that cells not exposed to any chemical (Figure 3—20—negative control) have fluorescence in several cell layers beneath the scraped edge at the left of each photograph. This is interpreted as noninhibition of gap junctional communication. Cells exposed to 1 ug 245—HBB/m1. medium (Figure 3-21), 5 pg 245—HBB/m1 medium (Figure 3-22), 20 pg 245-HBB/ml medium (Figure 3-23), and 40 ug 245-HBB/ml medium (Figure ?'24) had progressively inhibited transfer of dye 1nto secondary recipient cells. Control cells (DMSO only) are shown in Figure 3-25, indicating that the vehicle used, DMSO, did not interfere with dye transfer (Rhodamine dextran/Lucifer yellow, 400 X). Figure 3-24 166 Figure 3-25 167 _ Figure 3-26. Quantitation of fluorescence lnten51ty (relative units) in WB-F344 cells exposed to '1, 5, 20, or 40 11g 245-HBB/ml medium and subjected to the scrape-loading/dye transfer assay. All treatment groups were significantly different (*) from the nontreated control group. _.//-m M 417/-.. 1 ////////// DISCUSSION The results of the metabolic cooperation assay, the fluorescence redistribution after photobleaching assay (FRAP), and the scrape-loading/dye transfer assay indicate that 2,2',4,4',5,5'-hexabromobiphenyl (245- HBB), at noncytolethal concentrations, blocks in vitro gap junction-mediated intercellular communication in a dose-dependent manner. These results agree with the results from previous in vitro studies in which 245-HBB, the major congener in Firemaster BP-6 (FM), was shown to inhibit metabolic cooperation in Chinese hamster V-79 cells (Trosko gt a_1., 1981). Results from the current study are also in agreement with those from another experiment in which 245-HBB was shown to inhibit metabolic cooperation in a dose-dependent manner in Chinese hamster V79 cells (Tsushimoto g; g” 1983). However, results from the present study when compared with those of Tsushimoto fl a_l. (1983) indicate that there are differences in the slopes of the dose-response curves between WB-F344 cells and Chinese hamster V79 cells when each cell line was exposed to similar concentrations of 245—HBB. This may be due to inherent biochemical and metabolic differences between these two 169 170 cultured cells lines. However, the precise differences between these cell lines responsible for these results are unknown. Results from this study indicate that the cytotoxic concentration for WB-F344 cells exposed to FM is probably between 20 and 40 pg/ml medium. At a concentration of 40 119 FM/ml medium, there is recovery of about 80% of 6-thioguanine-resistant (i.e., HGPRT- cells) (Figure 3-4). This concentration is in the range of cytotoxicity for these cells as indicated by the cytotoxicity curves. However, it is unusual that the number of mutant cells recovered at this concentration in the metabolic cooperation plates was greater than the number recovered from cytotoxicity plates. One explanation for this may be that at cytotoxic levels of a chemical, killing of wild type (i.e., HGPRT+) cells may allow some mutant (i.e., HGPRT“) cells to survive by not receiving the lethal biochemical product from a dead neighboring cell. This phenomenon could result in an increased survival of mutant cells at cytotoxic concentrations of the chemical. Another explanation is that the cell density in cytotoxicity plates is lower than 131 metabolic cooperation plates. Therefore, the effective concentration per cell is greater in cytotoxicity plates. Firemaster BP-6 has been shown to be a more potent tumor promoter than 245-HBB at the same doses when both 171 chemicals were tested with in vivo tumor promotion studies. However, PM was not a stronger inhibitor of metabolic cooperation than 245-HBB when given at the same concentrations in the present in vitro study. Findings from previous studies with various tumor promoting agents have shown that results from metabolic cooperation assays are reasonably predictive of the tumor promoting activity of these compounds in certain in vivo initiation/promotion hepatocarcinogenesis sys- tems (Ito gt gt., 1980; Tennekes gt _l., 1982; Tatematsu gt _l., 1983; Trosko gt gt., in press, 1987). The rea— son for the discrepancy between the results of previous in vivo studies and those of the present in vitro studies with FM and 245-HBB is not readily apparent. The characterization and successful isolation of the WB—F344 cell line has been described by Tsao e_t gig. (1984). It is an epithelial cell, isolated from the livers of adult male Fischer 344 rats, that expresses the oval cell phenotype. Oval cells are a population of nonparenchymal cells in the liver which have the capacity to proliferate and undergo phenotypic and karyotypic changes in response to hepatocarcinogens (Tsao gt gt., 1985). Histologically, WB-F344 cells resemble biliary epithelial cells. The WB—F344 cells are useful because they can be transformed in vitro, yet are diploid. Many other cell lines that continuously grow in culture conditions are not diploid. 172 The WB-F344 cells in culture may more closely resemble in vivo metabolic characteristics than many other cultured cell lines. Therefore, culture systems using this cell line may be of more value than other cell lines for predicting the in vivo effects of certain environmental toxicants. The usefulness of the WB-F344 cell line in the metabolic cooperation assay has been recently established (Jone gt gt., in press, 1987). Since metabolic cooperation is dependent upon functioning gap junctions, this cell line could be capable of detecting certain chemicals which inhibit cell-cell communication in rat epithelial cells. Previous studies in which two organochlorine pesticides, dieldrin and aldrin, were used 131 the metabolic cooperation assay indicate that cell-cell communication was inhibited in several different cell lines when exposed to these environmental toxicants at noncytolethal concentrations (Jone gt gt., 1985; Kurata gt g_l_., 1982; Trosko gt _1., in press, 1987; Lin gt gt., 1986). In vivo studies have shown that these compounds are tumor promoters in rat initiation/promotion hepatocarcinogenesis systems (Ito gt gt., 1980; Tatematsu gt gt., 1983; Tennekes gt gl., 1982). Therefore, results from these in vitro studies have been predictive of the in vivo behavior of certain environmental toxicants and suggest that one possible mechanism of tumor promotion may be the inhibition of 173 gap junction-mediated intercellular communication. While in vitro studies are not a replacement for more conventional animal studies, such in vitro experiments provide another means by which to detect nongenotoxic hepatocarcinogens and hepatic tumor promoters which may be missed by traditional in vitro short term genotoxic assays. One limitation of the metabolic cooperation assay, as with several in vitro cell systems, is that it does not totally reflect in vivo biological complexity. This limitation has been recognized by Williams and co- workers (1981) who have used cultures of primary rat hepatocytes co—cultured with other cells to enhance the metabolic capabilities of their in vitro system. The WB-F344 cells probably have their greatest potential in co-cultured cell systems with normal rat hepatocytes. Such a co—culture system would more closely resemble in vivo metabolic characteristics. The three assays used in the present study have different strengths and weaknesses. The metabolic cooperation assay is well-established and measures gap junctional intercellular communication over a period of approximately 3-4 days. However, cell-cell com- munication is not. measured at any intermediate times during the course of a metabolic cooperation assay. Therefore, if a test chemical blocks intercellular communication for only a short time then this transient 174 phenomenon would not be detected. Reversibility of inhibited cell-cell communication would not be detected in our metabolic cooperation assays. The FRAP assays have some advantages over the metabolic cooperation assay. With FRAP, there is direct visualization of the return of dye via gap junctions into ‘touching cells. With the metabolic cooperation assay, other factors, such as contamination of culture plates by low numbers of bacterial or fungal organisms, may influence cell survival. If not detected, even slight amounts of contamination could lead to erroneous results. The FRAP analysis is performed over a shorter time period, somewhat reducing the chances of microbial contamination. The FRAP assay is not dependent on consistent biochemical or metabolic factors within the cell. With the metabolic cooperation assay it is assumed that certain biochemical pathways are not only present in the cells but that they are consistently functioning as well. These assumptions are not made with FRAP analysis. However, FRAP analysis is done over a relatively short duration when compared to the metabolic cooperation assay. It is possible that test chemicals which at first inhibit gap junctional communication may reverse this inhibition after only a short time. Analysis with FRAP may be of a short enough duration to allow detection of only temporary blockage of cell-cell 175 communication, whereas results from the metabolic cooperation assay may indicate no inhibition of communication. Perhaps testing chemicals with both assays would permit better characterization of transient inhibition of cell—cell communication. The scrape—loading/dye transfer assay has several advantages over the other assays used in this study. It is relatively simple when compared to the FRAP and metabolic cooperation assays and gives a rapid and clear visual assessment of spread of a dye into cell layers. It is of low cost and probably has its greatest potential as a rapid screening assay to quickly assess the ability of test chemicals to inhibit gap junctional communication (El—Fouly gt gt., in press, 1987). It is similar to the FRAP assay in that detailed characterization of metabolic and biochemical pathways in the cells used is not needed to perform the assay. Both the FRAP assay and the scrape-loading/dye transfer technique depend on transfer of an exogenous dye from one cell to another. The toxicity of dyes to cultured cells is unknown, but these biological tracers are assumed to be physiologically inert. However, it is possible that certain cell lines used with these dyes may be exquisitely sensitive to deleterious effects from such exogenous compounds. Nevertheless, the scrape- loading/dye transfer technique, as used in the present 176 study, appears to be sensitive enough to be used for the determination of dose-response curves with certain test chemicals. The nature of the loss of gap junctional function is generally unknown. However, there are several con- ceivable mechanisms by' which this may occur. Perhaps patency of gap junctional hemichannels is lost, as hypothesized by Loewenstein (1979). other possible mechanisms include inhibition of assembly of gap junctional precursor proteins into the plasma membrane, inhibition of mRNA coded for production of gap junctional precursor proteins, or increased degradation of assembled gap junctional plaques. It is currently unknown which of these phenomena is most responsible for chemically-induced communication incompetence between these cultured cells. SUMMARY-CHAPTER 3 Conclusions from the preceding experiments include the following: 1) The compound 2,2',4,4',5,5'-hexabromobiphenyl (245-HBB) inhibits gap junctional intercellular communication in a dose-dependent manner in the metabolic cooperation assay, the fluorescence re- distribution after photobleaching ("FRAP") assay, and the scrape-loading/dye transfer assay. 2) Both Firemaster BP-6 (FM) and its major congener, 245-HBB, inhibit gap junctional intercellular com- munication as determined by the metabolic cooperation assay. The difference between the ability of these two agents to do so was not significantly different. 3) The scrape—loading/dye transfer assay, a new technique for assessing gap junctional intercellular communication, was useful for measuring the dose- responsiveness of inhibition of cell-cell communication by 245-HBB. This technique has potential as a rapid and simple method for assessing the ability of many 177 178 environmental toxicants to inhibit intercellular communication. Results from these studies suggest that the ability of 245—HBB to behave as a tumor promoter in vivo may be associated with the ability of this compound to inhibit intercellular communication in vitro. In addition, these results imply that the metabolic cooperation assay, the FRAP assay, and, the SL/DT assay are each sensitive enough to be used for dose/response studies to measure the ability of certain environmental toxicants to inhibit gap junctional intercellular communication. BI BLIOGRAPHY-CHAPTER 3 BIBLIOGRAPHY Andrew RD, MacVicar BA, Dudek FE, Hatton GI: Dye transfer through gap junctions between neuroendocrine cells of rat hypothalamus. Science 211:1187-1189, 1981. Bertram JS: Modulation of cell-cell interactions in vitro by agents that modify cAMP metabolism. Proc Am Assoc Cancer Res 20:212, (Abstr.), 1979. Castagna M, Takai Y, Kaibuchi K, Sano K, Kikkawa U, Nishizuka Y: ' Direct activation of calcium- activated phospholipid-dependent protein kinase by tumor promoting phorbol esters. J Biol Chem 257:7847-7851, 1982. Cerutti PA: Prooxidant states and ‘tumor' promotion. Science 227:375-381, 1985. Chen TR: In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp Cell Res 104:255-262, 1977. Davidson JS, Baumgarten I, Harley EH: use of a new citrulline incorporation assay to investigate inhibition. of intercellular" communication by l,1,1-trichloro-2,2-bis (p-chlorophenyl)-ethane in human fibroblasts. Cancer Res 45:515—519, 1985. DeMello WC: Cell-cell communication in heart and other tissues. Prog Biophys Mol Biol 39:147-182, 1982. El-Fouly MH, Trosko JE, Chang CC: Scrape-loading and dye transfer: .A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res, in press, 1987. Enomoto T, Sasaki Y, Shiba Y, Kanno Y, Yamasaki H: Inhibition of the formation of electrical cell coupling of FL cells by tumor promoters. Gann 72:631-634, 1981. Enomoto T, Yamasaki H: Lack of intercellular communication between chemically-transformed and surrounding nontransformed BALB/c3T3 cells. Cancer Res 44:5200-5203, 1984. ' 179 180 Fitzgerald DJ, Murray AW: Inhibition of intercellular communication by tumor-promoting phorbol esters. Cancer Res 40:2935—2937, 1980. Fitzgerald DJ, Knowles SE, Ballard FJ, Murray AW: Rapid and reversible inhibition of junctional communication by tumor promoters in a mouse cell line. Cancer Res 43:3614-3618, 1983. Friedman EA, Steinberg M: Disrupted communication between late-stage premalignant human colon epithelial cells by 12—O-tetradecanoylphorbol~13- acetate. Cancer Res 42:5096—5105, 1982. Fujiki M, Tanaka Y, Miyake R, Kikkawa U, Nishizuka Y, Sugimura T: Activation of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) by new classes of tumor promoters: teleocidin and dibromoaplysiatoxin. Biochem Biophys Res Commun 120:339-343, 1984. Fujimoto WY, Subak-Sharpe JH, Seegmiller JE: Hypoxanthine—guanine phosphoribosyl—transferase deficiency: chemical agents selective for mutant or normal cultured fibroblasts in mixed and heterozygote cultures. Proc Natl Acad Sci 68:1516-1519, 1971 Gilula NB: Cell to cell communication and development. In Cell Surface: Mediator pf Developmental Processes. Edited by s. Subtelny and N.K. Wessells, New York Academic Press, New York, pp. 23-42, 1980. Gilula NB, Reeves OR, Steinbach A: Metabolic coupling, ionic coupling and cell contacts. Nature 235:262- 265, 1972. Goodall H, Johnson MH: Use of carboxyfluorescein diacetate to study formation of permeable channels between mouse blastomeres. Nature 295:524-526, 1982. Ito N, Tatematsu M, Nakanishi K, Hasagawa R, Takano T, Imaida K, Ogiso T: The effects of various chemicals on the development of hyperplastic liver nodules in hepatectomized rats treated. with. N- nitrosodiethylamine of N-z-fluorenylacetamide. Gann 71:832-842, 1980. 181 Jone C, Trosko JE, Aylsworth CF, Parker L, Chang CC: Further characterization of the in vitro assay for inhibitors of metabolic cooperation in the Chinese hamster V79 cell line. Carcinogenesis 6:361—366, 1985. Jone C, Trosko JE, Chang CC: Characterization of a rat epithelial cell line to detect inhibitors of metabolic cooperation. In Vitro, in press, 1987. Kavanagh TJ, Chang CC, Trosko JE: Characterization of a human teratocarcinoma cell assay for inhibitors of metabolic cooperation. Cancer Res 46:1359- 1366, 1986. Kurata M, Hirose K, Umeda M: Inhibition of metabolic cooperation 1n Chinese hamster cells by organochlorine pesticides. Gann 73:217-221, 1982. Larsen.‘WJ: Biological implications of gap junction structure, distribution, and composition: a review. Tissue Cell 15:645-671, 1983. Lawrence TS, Beers NH, Gilula NB: Transmission of hormonal stimulation by cell-to-cell communica- tion. Nature 272:501-506, 1978. Levine EM, Beck Y, Boone CW, Eagle H: Contact inhibition, macromolecular synthesis . and polyribosomes in cultured human diploid fibroblasts. Proc Natl Acad Sci 53:350-356, 1965. Lin ZX, Kavanagh T, Trosko JE, Chang CC: Inhibition of gap junctional intercellular communication in human teratocarcinoma cells by organochlorine pesticides. Toxicol Appl Pharmacol 83:10-19, 1986. - Lo CW, Gilula NB: Gap junctional communication in the post-implantation mouse embryo. Cell 18:411-422, 1979. Loewenstein WR: Junctional intercellular communication and the control of growth. Biochim Biophys Acta 560:1-65, 1979. McNeil PL, Murphy RF, Lanni F, Taylor DL: A method of incorporating macromolecules into adherent cells. J Cell Biol 98:1556-1564, 1984 Meyer DJ, Yancey SB, Revel JP: Intercellular com— munication and normal and regenerating rat liver: a quantitative analysis. J Cell Biol 91:505-523, 1981. V. 182 Mosser DD, Bols NC: The effects of phorbols on metabolic cooperation between human fibroblasts. Carcinogenesis 3:1207-1212, 1982. Murray AW, Fitzgerald DT: Tumor promoters inhibit metabolic cooperation in cocultures of epidermal and 3T3 cells. Biochem Biophys Res Commun 91:395- 401, 1979. Newbold RF, Amos J: Inhibition of metabolic cooperation between mammalian cells in culture by tumor promoters. Carcinogenesis 2:243-249, 1981. Nishizuka Y: Studies and perspectives of protein kinase C. Science 233:305—312, 1986. Peracchia C: Structural correlates of gap junction permeation. Int Rev Cytol 66:81-146, 1980. Rotman B, Papermaster BW: Membrane properties of living' mammalian cells as studied by enzymatic hydrolysis of fluorigenic esters. Proc Natl Acad Sci 55:134-141, 1966. Saxen L, Karkinen-Jaaskelainen M, Lehtonen E, Nordling S, Wartiovaara J: Inductive tissue interactions. In Egg Cell Surface tp Animal Embtyogenesis gpg Development. Edited by G. Poste and G. L. Nicolson, Elsevier/North—Holland, Amsterdam, pp. 331-407, 1976. Socolar SJ, Loewenstein WR: Methods for studying transmission through permeable cell—to-cell junctions. In Methods it Membrane Biology. Edited by E. Korn, Vol 10, Plenum Press, New York, pp.123-l79, 1978. Steel RGD, Torrie JH: Analysis of variance I: The one- way classification. In Principles gpg Procedures gt Statistics. A Biometrical Approach. Edited by C. Napier and J.W. Maisel, McGraw-Hill, New York, pp. 137-167, 1980a. Steel RGD, Torrie JH: Multiple comparisons. In Principles and Procedures pt Statistics. A Biometrical Approach. Edited by C. Napier and J.W. Maisel, McGraw-Hill, New York, pp. 172-191, 1980b. Stewart WW: Functional connections between cells as revealed by dye-coupling with a highly fluorescent aminoaphthalimide tracer. Cell 14:741-759, 1978. 183 Stewart WW: Lucifer dyes- highly fluorescent dyes for biological tracing. Nature 292:17-21, 1981. Subak-Sharpe H, Burk RR, Pitts JD: Metabolic co- operation between biochemically marked mammalian cells in culture. J Cell Sci 4:353-367, 1969. Tatematsu M, Hadehawa R, Imaida K, Tsuda H, Ito W: Survey of various chemicals for initiating and promoting activities in a short term in vivo system based on generation of hyperplastic liver nodules in rats. Carcinogenesis 4:381-386, 1983. Telang S , Tong C , Williams GM: Epigenetic membrane effects of a possible tumor promoting type on cultured liver cells by non—genotoxic organo— chlorine pesticides chlordane and heptachlor. Carcinogenesis 3:1175—1178, 1982. Tennekes HA, Edler L, Kunz HW: Dose—response analysis of the enhancement of the liver tumor formation in CF—l mice by dieldrin. Carcinogenesis 3:941-945, 1982. Trosko JE, Yotti LP, Dawson B, Chang CC: In vitro assays for tumor promoters. In Short Term Tests fpt Chemical Carcinogens. Edited by H. Stitch and R.H.C. San, Springer-Verlag, New York, pp. 420- 427, 1981. Trosko JE, Yotti LP, Warren ST, Tsushimoto G, Chang CC: Inhibition of cell-cell communication by tumor promoters. Carcinogenesis 3:181-186, 1982. Trosko JE, Jone C, Chang CC: The role of tumor promoters on phenotypic alterations affecting intercellular communication and tumorigenesis. In Cellular Systems for Toxicity Testing. Edited by G.M. Williams, V.C. Dunkel, and V.A. Ray, New York Academy of Sciences, New York, pp. 316-327, 1983a. Trosko JE, Chang CC, Medcalf A: Mechanisms of tumor promotion: potential role of intercellular communication. Cancer Invest 1:511-526, 1983b. Trosko JE, Jone C, Chang CC: Inhibition of gap junctional-mediated intercellular communication, in vitro, by aldrin, dieldrin and toxaphene: a possible cellular mechanism for their tumor promoting and neurotoxic effects. Molec Toxicol, in press, 1987. 184 Tsao MS, Smith JD, Nelson KG, Grisham JW: A diploid epithelial cell line from normal adult rat liver with phenotypic properties of "oval" cells. Exp Cell Res 154:38-52, 1984. Tsao MS, Grisham JW, Nelson KG, Smith JD: Phenotypic and karyotypic changes induced in cultured rat . hepatic epithelial cells that express the "oval" cell phenotype by exposure to N-methyl-N‘-nitro-N— nitrosoguanidine. Am J Pathol 118:306—315, 1985. Tsushimoto G, Asano S, Trosko JE, Chang CC: Inhibition of intercellular communication by various congeners of polybrominated biphenyl and polychlorinated biphenyl. In PCBs: Human gpg Environmental Hazards. Edited by F.M. D'Itri and M.A. Kamrin, Butterworth Publishers, Woburn, Massachusetts, pp. 241-252, 1983. Wade MH, Trosko JE, Schindler M: A fluorescence photobleaching assay of gap junction—mediated communication between human cells. Science 232:525-528, 1986. Walder L, Lutzelschwab R: Effects of lZ-O-tetra- decanoylphorbol-l3-acetate (TPA), retinoic acid and diazepam on intercellular communication in a monolayer of rat liver epithelial cells. Exp Cell Res 152:66-76, 1984. Weinstein IB, Lee LS, Fisher BB, Mufson A, Yamasaki H: Action of phorbol esters in cell culture: mimicry of transformation altered differentiation and effects on cell membranes. J Supramol Struct 12: 195- 208, 1979. Williams GM, Telang S, Tong C: Inhibition of intercellular communication between liver cells by the liver tumor promoter 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane. Cancer Lett 11:339-344, 1981. Yamasaki H: Modulation of cell differentiation by tumor promoters. In Mechanisms pt Tumor Promotion. Edited by T.J. Slaga, Vol 4, CRC Press, Boca Raton, Florida, pp. 1-26, 1984. Yamasaki H, Enomoto T, Martel N, Shiba Y, Kanno Y: Tumor promoter—mediated reversible inhibition of cell-cell communication (electrical coupling). Exp Cell Res 146:297-308, 1983. 185 Yotti LP, Chang CC, Trosko JE: Elimination of metabolic cooperation in Chinese hamster cells by a tumor promoter. Science 206:1089-1091, 1979. VITA Mark G. Evans was born in Berrien Springs, Michigan, and completed primary and secondary education there. He earned. his Doctor of Veterinary' Medicine degree from Michigan State University in 1978 and returned to Berrien Springs to engage in private veterinary practice. In 1981, he entered the Department of Pathology at Michigan State University and completed a Master’s Degree in 1983 with Dr. Glenn L. Waxler. He was then the recipient of a training award from the National Institutes of Health and completed his PhD in pathology/environmental toxicology in 1987 with Dr. Stuart D. Sleight at Michigan State University. The author was recently awarded a postdoctoral fellowship in immunotoxicology at Harvard Medical School. He is a member of Phi Zeta, Sigma Xi, and Phi Kappa Phi. 186 “"'WWW/(11311[iiiujfigfil’yifljfimES