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We I" x" ’M « LIBRARY Michigan State University This is to certify that the dissertation entitled Biochemical Mechanisms of Toxicity of 2,3,7,8-Tetrachlorodibenzo-p—dioxin in the Rat, Guinea Pig, Hamster, Rabbit, and Mouse- presented by David William" Brewster has been accepted towards fulfillment of the requirements for Ph.D. degree in Env1ronmental Tox1cology an ntomo ogy r \ /- X It W \ Ct \ Major professor Date November 12, 1985 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 ’1‘.- llllll ”l 3 1293 00659 MSU LIBRARIES “ lllllll lllllllfllllflll ‘ 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 betow. AWL .5925153 *3 285 BIOCHEMICAL MECHANISMS OF TOXICITY OF 2,3,7,8- TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN THE RAT, GUINEA PIG, HAMSTER, RABBIT, AND MOUSE BY David William Brewster A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Center for Environmental Toxicology Department of Entomology 1985 ABSTRACT BIOCHEMICAL MECHANISMS OF TOXICITY OF 2,3,7,8- TETRACHLORODIBENZO-P-DIOXIN IN THE RAT, GUINEA PIG, HAMSTER, RABBIT, AND MOUSE BY David William Brewster TCDD (2,3,7,8-tetrachlorodibenzo-p—dioxin) caused a gross alteration in isolated rat hepatic plasma membrane protein constituency. Changes occurred as soon as two days after administration and showed a time and dose dependent response over a 20 day observation period. Na-K ATPase, Mg .ATPase, Ca ATPase, and gamma-glutamyltranspeptidase activi- ‘ties along with insulin, epidermal growth factor, and Concanavalin A binding were all reduced by single i.p. achninistration of this dioxin. Protein kinase activity was gyignificantly increased relative to control levels. These changes were not seen in the hepatic plasma membrane of guinea pigs or hamsters, species in which TCDD produced lijrtle liver pathology. These results indicate impaired icniic transport, depressed binding of critical growth factors, and increased cellular phosphorylation in rat lixnar- If membrane alterations occur at vital physiological sites in other organs and tissues, specific biochemical manifestations of TCDD's toxicity may be explained. David William Brewster Hypertriglyceridemia in guinea pigs was found to be caused by a dose and time dependent reduction of adipose lipoprotein lipase (LPL) activity. Rabbits and hamsters showed similar effects to guinea pigs. LPL was not affected in rats, nor did hypertriglyceridemia develop. Increased serum triglycerides segregated with the Ah locus in several responsive and non-responsive mouse strains. Mink had lowered concentrations of serum triglycerides but no change in LPL. The cause for the decreased LPL activity seemed to be the result of TCDD acting at 2 different organs: the pancreas had an abnormal response to glucose administration in its insulin synthetic ability, and the adipocyte was unable to produce active LPL. Altered epidermal growth factor receptor synthesis and enhanced tyrosine kinase activity is postulated to be the underlying biochemical mechanism for TCDD's toxicity. For Mom and Dad with love ii ACKNOWLEDGMENTS I would like first to thank God for His wonderful creation and for giving me the opportunity, the ability, and the wisdom to study and enjoy it. 7 I would like to express my sincere appreciation to Dr. Fumio Matsumura for his unending guidance and scientific insights and for the most part for keeping me out of the swamp. Thanks also to Drs. James Miller, John Giesy, Robert Roth, and George Ayers for their input and suggestions concerning this research and my entire Ph.D. program. To B.V. Madhukar and Dave Bombick thanks for the "tremendous" discussions and intellectual pursuits; remember we've done it before! To Louise Partridge, for the hours of typing, retyping, and retyping, I give you a big hug and giant thank you. Finally to my loving wife Janet, for your unending support, understanding, love, and countless trips to the PRC, I wish to express my deepest gratitude. Thank you Princess, and thank you for giving me a happy, healthy, and beautiful little boy. To you Jeffrey David, thank you for your love and trust and the joy you've brought into our lives. iii "Whatever is true, whatever is noble, whatever is right, whatever is pure, whatever is lovely, whatever is admirable -- if anything is excellent or praiseworthy -- think about such things." Philippians 4:8 iv TABLE OF CONTENTS List Of Tables 00.00.000.00......OOOO.........OOOOOOOOOO x List of Figures ..... . ............. . .................... xiv [—3 I. IntrOduction 00.0.00.........OCOOOOOOOOOOOCOOO... Brief History of TCDD ....... ..... ......... ...... .. Environmental Significance ........................ Symptoms of Toxicity ................ ....... . ...... Objectives ..... ............................... .... Significance ...................................... NH OKD\l\li-‘ II. Influence of 2,3,7,8-tetrach1orodibenzo-p-dioxin on Protein Composition of Rat Hepatic Plasma Mem- brane and Overall Body Weight ...................... 22 Introduction ......... ............ ... ........ ...... 22 Materials and Methods ............................. 23 Plasma Membrane Preparation ........... .......... 23 Concanavalin A Binding .......................... 24 Results ................ ............. .. ........ .... 25 Plasma Membrane Protein Composition ............. 25 conABinding ......OOOOOOOOOOOOOOOOOO0.0.0.0.... 30 Wasting ......................... ........ ........ 3O TOXiCity .........OOOOOOOOOOOO00...........OOOOOOO 40 Discussion ............................. ...... . ..... 40 conClUSionS .........OOOOOOOOOOOOO...0.00.00.00.00 46 III. Alteration of Rat Hepatic Plasma Membrane Func- tions by 2,3,7,8-tetrachlorodibenzo-p-dioxin ........ 47 Introduction ........... ....... ... ............. ..... 47 Materials and MethOds O O O C O O O O O O O O O O O O C O O O I O 0 O O O O O O O 48 ATPase Enzymatic Activity ......... ....... ........ 49 Protein Kinase Assay ................... ...... .... 49 Gamma-glutamyl Transpeptidase Assay .............. 50 Page Insulin and Epidermal Growth Factor Binding ...... 51 Concanavalin A Binding ........................... 51 Results ......OOIOOOOOOOOOOOOOOO.....OOOOOOOOQOOOOOO 52 Time Course of Plasma Membrane Protein Altera- tions ......OOOOOOOOOOOOOOOO......OOOOOOOOOOOOO. 52 Alterations of Enzymatic Activity and Receptor Binding .....OOOOOOOOOO00.000.00.000...00......O 53 Effect of TCDD on Hepatic Enzymatic Activity in the Guinea Pig and Hamster ..................... 62 DiscuSSion ... ........ .....OOOOOOOOOOOOO......OOO... 62 Conclusions ................ ...... ..... ....... .... 72 IV. Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the Thymus .............OOOOOOOOOOOOOOOO0.0.0.0000... 73 IntrOduction O O O O O O O O O O O O O O O O 0 O O O O O O O O O O O O O 0 Q 0 O O O O O O 73 Materials and Methods ...... .......... ....... . ...... 75 Thymocyte Isolation and Plasma Membrane Prepara- tion 0.00.........0.0.0..........OOOOOOOOOOOOOOO 75 BiOChemical Assays O O O O O O O O O O O O O O O C O O O O O O O O O O O O O O O 75 Results 0.00.00.00.00.......OOOOOOOOOOOOOOOOO0...... 76 Electron Microscopy .............................. 76 BiOChemj-Stry .OOOOOOOOOOOOOO.......OOOOOOOOOOOOOOO 76 DiscuSSion 0.0.0000....0.........OOOOOOOOOOOOOOO.... 83 V. 2,3,7,8-Tetrachlorodibenzo-p-dioxin Inhibition of Guinea Pig Adipose Lipoprotein Lipase Activity as Cause for Serum Hypertriglyceridemia ............. 86 IntrOduCtion ......OOOOOOOOOO.........OOOOOOOOOO.... 86 Materials and Methods .............................. 88 Preparation of Acetone/Ether Fat Powder .......... 88 LPL Assay OOOOOOOOOOOOOOOOOOOO......OOOOOOOOOOOOOO 89 Resu1ts ....0.00.0000...O.........OOOOOOOOOOOOOOOOO. 89 Wasting and "Slobbering" ......................... 89 Time and Dose Dependent Changes of LPL Activity and Serum Triglyceride Concentration ........... 92 vi Page Glucose Effect on LPL Activity ........ ........... 103 Serum Apoprotein Effect on LPL ................... 104 DiSCUSSion 0............OOOOOOOOOOOOOOO00......0.... 106 Cause for Loss of Adipose Tissue ... ............ .. 109 LPL and Serum Triglycerides .. ................... . 110 Conclusions ...................................... 114 VI. Differential Species Response of Adipose Lipopro- tein Lipase to 2,3,7,8-tetrachlorodibenzo-p-dioxin .. 116 IntrOduction 0.00.00... ........... O ..... 0.00000... 116 Materials and Methods ............ ...... .......... 117 Results . ..... ... ........... ......... ..... ........ 118 Rabbits ........................................ 118 Hamsters ....................................... 120 Rats 0............OOOOOOOOOOOOOOOO......OOOOOOOO 120 Mice .............. ...... ........... .......... .. 122 Discussion ....... ..... ........................... 122 conCIUSion 0.00.0000.............OOOOOOOOOOOO... 128 VII. Effects of 2,3,7,8-tetrachlorodibenzo-p—dioxin upon Guinea Pig Heart 0.00............OOOOOOOOOOOOOOO 131 Introduction ..................................... 131 Known cardiotoxicity of dioxin ................. 132 Materials and Methods ............................ 134 cardiac LPL Assay ......OOOOOOOOOOOOOOOOC0...... 134 Atrial Isolation and Heart Function Tests ...... 134 Results ......OOOOOOOO OOOOOOOOOOOOOOOOO 0.0.0.0.... 136 LPL ......OOOOOOOOOOOOOOO0.........OOOOOOOOOOOO. 136 Atrial Function 0.0.0.000.........OOOOOOOOO0.... 136 Isoproterenol Dose Response .................... 140 DiscuSSion 00.000.00.000...........OOOOOOOOOOOOCOO 151 vii Page conCluSions ..OOOOOOOOOOOOOOOOOOO.... 0000000 O... 154 VIII. Studies on the Cellular Mechanism of LPL Inhibi- tion Caused by in vivo Administration of 2,3,7,8- tetrachlorodibefiEo-p-dioxin ...... ................ ... 156 Introduction ..... ...... .......................... 156 Lipogenesis .................................... 161 LipOIYSis O .SRC. O O O O O O O O O O O O O O O O O O O O ......... O O O 163 Adipose pp60 . ................... . ........... 164 Materials and Methods ............. . ......... ..... 164 Adipose Plasma Membrane Isolation ......... ..... 165 Intestinal Plasma Membrane Isolation ........... 165 Epinephrine Binding ............................ 166 Phosphodiesterase Assay ........ ...... . ......... 167 Hormone Sensitive Lipase Assay ..... ............ 168 Serum Analyses .... 0 ......... . ...... . .......... 168 SRC Gene Product pp Assay .... ...... .. ..... ... 169 Results ... ............ . .................. . ....... 171 Glucose Reversal of LPL ........................ 171 Insuéfie and Epinephrine Binding ..... ........... 181 pp60 Estimation ..... ........ . ............... 182 DiscuSSion ............OOOOOOOOOOOOOOO ...... .00... 184 Conclusions ...... ..... . ...... .................. 188 IX. Summary and Final Conclusions ......... ........... 192 Proposed Unifying Theory of TCDD's Biochemical Mechanism of Action ............................ 197 Significance ..... .. .............................. 201 X. Appendices ............ . .......................... 202 Appendix A. The Influence of 2,3,7,8-tetrachlorodi- benzo-p-dioxin on Epidermal Growth Factor Binding in the Rat, Guinea Pig, Mouse, and Hamster ........ 202 Appendix B. Characterization of the Serum Hyperli- pedemia Produced in the Rabbit Upon Administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin ............ 215 Appendix C. The Effect of TCDD on Adipose LPL and Serum Lipid Parameters in the Mink .. ...... ........ 227 viii Page Appendix D. Additional Data Concerning the Biochem- ical Mechanism for LPL Inhibition ................. 231 List of References ............................. ...... ... 233 ix LIST OF TABLES Table Page 1 2,3,7,8 TCDD LD50 values for several animal species........................................... 3 2 Major pathological lesions of the rat hepatic system-00.0.0.0...0..........OOCOOOOIO0.00.0000... 1.0 3 Liver weight, liver/body w ight ratio, liver plasma membrane yield and H—concanavalin A binding between control rats and TCDD treated rats 10 days after dosing.................... ..... 26 4 Effect of in vivo treatment of TCDD on the protein composition of hepatic plasma membrane.... 29 5 Organ weightsa of rats 25 days after dosing with either acetonezcorn oil or TCDD.............. 42 6 Difference in enzyme activities between hepatic plasma membrane preparation from control and in Vivo TCDD-treated rats.........OOOOOOOOOOOOOOOO... 54 7 Effect of in vivo TCDD treatment on ligand binding to cell-surface membrane receptors in the rat liverOOOOOOOOO...O0.00......OOOOOOOOOOOOOOOOOO 55 8 Activity of liver plasma membrane bound enzymes and receptors from rats maintained on limited diets or fed ad libitum for 10 days after dosing with acetonezcorn oil.. ..... .... ..... ............. 61 9 Enzyme activity of control guinea pig, rat, or hamster hepatic plasma membrane, 10 days after treatment with 1, 25, or 6000 ug/kg TCDD respec- tively............................................ 63 10 Effects of 25 ug/Kg TCDD on thymus weight at 2 and 10 days after administration..... .......... ... 81 Table Page 11 The effect of TCDD on various biochemical parameters of thymocytes or thymocyte plasma mem- brane isolated 10 days after treatment with acetone:corn oil or 25 ug/Kg TCDD................. 82 12 Adipose LPL activity, serum triglyceride concen- tration, body weight, and adipose weight in ad lib control, TCDD treated, and pair-fed control guinea pigs 10 days after treatment with either acetone:corn oil or 1 ug/kg TCDD.................. 93 13 Dose response relationship of LPL activity, serum triglyceride concentration, body weight, and adipose weight 10 days after TCDD administra- tion....................... ........ ...... ......... 98 14 Effects of serum factors from ad lib control, TCDD treated, and pair-fed control guinea pigs and rats on triglyceride hydrolyzing capability of control, TCDD treated, and pair-fed control guinea pig LPL.................................... 105 15 Adipose LPL activity and serum triglyceride con- centration (TG) of the guinea pig, rat, rabbit, and hamster 10 days after the in- dicated dose of TCDD........................................... 119 16 Serum triglyceride, cholesterol, and protein con- centrations 10 days after administration of 1 or 50 ug/kg TCDD or acetone:corn oil and pair-fed to treated rabbits................................ 121 17 Serum triglyceride and cholesterol concentrations (mg/d1) in responsive and nonresponsive mice strains 2 days after dosing with either 30 ug/kg TCDD or acetone:corn oil.......................... 123 18 Reported cardiac lesions after treatment with TCDD-....00.00.0000.........OOOOOOOOOOOOOOOOOI.... 133 19 Guinea pig absolute heart weight or heart weight as a function of body weight (mean: S.D., n), at 2, 10, or 20 days after a single i.p. ad- ministration of 1 ug/kg TCDD or acetone:corn OiIOOOOOOC............OOOOOOOOOOOOOOOO0.0.0.0....O 137 20 Cardiac LPL activity 2 or 10 days after single i.p. treatment with 1 or 4 ug/kg TCDD or vehicle alone (mean i S.D., n).. ..... ................ ..... 138 xi Table Page 21 Force and rate of contraction of guinea pig atria 20 days after exposure to 1 ug/kg TCDD or corn oil only and fed ad libitum or pair-fed to the TCDD treated animals (mean i S.D., n animals)................................. ......... 139 22 Effective concentration of isoproterenol to pro- duce 50% of maximal contractile force in isolated guinea pig atria 10 or 20 days after i.p. admin- istration of l ug/kg or acetone:corn oil and pair-fed or fed ad libitum ........ .... ..... . ...... 150 23 Twenty day "dioxin" factor for atrial contraction ED50 as a function of treatment................... 152 24 Serum glucose concentrations (mg/d1) 2 or 10 days after treatment with acetone:corn oil or TCDD, with and without oral glucose supplement (mean i S.D., n) ........ ................................ 175 25 Na-K, Mg, and Ca ATPase activity (nM P. hy- drolyzed/mg protein/hr) in isolated guinea pig intestinal plasma membrane 10 days after treat- ment with either 1 ug/kg TCDD or vehicle alone and pair-fed to the exposed animals ............... 176 26 Serum insulin (uU/ml) concentrations for 8 pairs of TCDD treated (1 ug/kg) or pair-fed control guinea pigs, 2 days after exposure ....... . ..... ... 179 27 Effect of TCDD on various lipogenic and lipolytic parameters in blood serum and fat tissue of guinea pigs 2 days after treatment with either lug/kg TCDD or vehicle alone (mean 1 SD for 3-8 pairs of animals).... ........ ..... ......... ... 180 28 Adipose pp6O activity of pair-fed control, TCDD treated guinea pigs (1 ug/kg), or T treated (105 ug/kg) guinea pigs 2 days poét- treatment.................................. ...... . 183 29 Changes in 125I-labeled EGF binding to hepa- tic plasma membrane, body weight, and thymus weight in mouse strains 10 days after TCDD treatment......... ..... ... ...... .................. 209 30 Effect of TCDD on eyelid opening, incisor erup- tion, and hair growth on neonatal BALB/c mice..... 210 xii Table Page 31 Various physiological parameters of rabbits 10 days after administering 1 or 50 ug/kg TCDD and pair-fed or fed ad libitum ........................ 219 32 Adipose LPL activity (nM 3H oleic acid/mg acetdggzether powder/hr) and hepatic LDL binding (ng I-LDL bound/200,000 cells/hr) in rabbits 10 days after ad lib feeding or pair-fed to those dosed with 50 or 1 ug/kg TCDD ............... 220 33 Effects of orally administered TCDD on mink adipose LPL activity and various serum lipid components at 5, 28, and 43 days after adminis- tration......................... ....... .. ...... ... 228 34 Adipose LPL activity and serum triglyceride con- centration of ad lib, 4 ug/kg TCDD or pair- fed mink after acute exposure..................... 230 xiii LIST OF FIGURES Figure Page 1 One possible mechanism for the spontaneous for- mation of 2,3,7,8 TCDD during the production of the herbicide 2,4,5 T (adapted from Esposito et al. 1980)........................................ 2 2 Normal appearance and curiosity of a control rat 10 days after the administration of acetone:corn oil ........ ................ ......... 5 3 Normal posture of a control animal with the paws under the body.............................. 5 4 Typical appearance of a TCDD treated rat (10 days after administration) displaying piloerec- tion, hair loss, and lowered carriage............ 5 5 Cage huddling and hunched posture of a TCDD treated animal 10 days after administration of 25 ug/kg TCDD.... .................. ...... ........ 5 6 SDS-polyacrylamide slab gel electrophoresis pat- terns of standard protein mixture (S) and hepa- tic plasma membranes from control (C) or TCDD treated (T) rats................................. 27 7 SDS-polyacrylamide slab gel electrophoretogram of hepatic plasma membrane from rats 40, 20, 10, 2, or 0 (untreated control) days after TCDD treatment........................................ 31 8 Body weight as % initial body weight over the 25 day observation period for control (0) and TCDD treated (.) ratSOO......OOOOOIOOOOOOOOOOO0.0 33 9 Food consumption of 5 control (0) and 5 TCDD treated (0) rats over a 25 day observation periOdOOOOOOOOO0................OOIOOOOOOOOOOOOOO 36 10 Water consumption of 5 control (0) and S TCDD treated (0) rats over a 25 day observation periOdOOOOOOOO0.0.0...OOOOOOOOOOOOOOOOOO:00...... 38 xiv Figure , Page 11 Fecal output (mean i S.D., n=5) of control (0) and TCDD (0) rats as a function of time after dOSingOOOOOOOOOOOOOOO......OOOOOOOOO ....... 41 12 Time course of body weight changes and changes in specific binding of insulin, Con-A, or EGF to liver plasma membranes from TCDD-rats (0) or control rats (0) .............................. 57 13 Time course of changes in plasma membrane associated enzyme activities in the rat liver as a result of in vivo TCDD exposure.............. 58 14 Time course of c-AMP independent (A) and c-AMP dependent (3) protein kinase activity for control (0) and TCDD treated (0) rats. ........... . ........ 59 15 Dose response of rat hepatic plasma membrane Na-K (A), Mg (I), and Ca ATPase (.)...... ...... 64 16 Dose response of guinea pig hepatic plasma mem- brane Na-K (0), Mg (4;), and Ca ATPase (o)...... 66 17 Dose response of hamster hepatic plasma membrane Na-K (0), Mg (.), and ca ATPase (.)000000000000 67 18 Scanning electron micrographs of rat thymocytes isolated 10 days after administration of either acetone:corn oil (A) or 25 ug/kg TCDD (B)......... 77 19 Transmission electron micrographs of rat thymo- cytes 10 days after administration of either acetone:corn oil (A) or TCDD (B) .......... ........ 79 20 A. Typical appearance of a control and (B) a 1 ug/kg TCDD treated guinea pig 10 days after ad- ministration.0.00.00.00.00. OOOOOOOOOOOOOOO 0.00.... 90 21 Abdominal dissection of (A) pair-fed control and (B) TCDD treated guinea pig, (20 days after 1 ug/kg single i.p. dose)........................... 94 22 Do,se response relationship of LPL activity (0) and body weight (A) (as % control) 10 days after a single i.p. dose of TCDD...... ...... ............ 96 23 Adipose LPL activity (nM 3H oleic acid re- leased/mg extracted powder/hr) at 0,1,2, and 10 days in TCDD treated (. ) , ad lib. control ( o), or pair-fed control (A) guinea pigs ......... 99 XV Figure Page 24 Time course of changes in body weight (as % ini- tial) TCDD treated (o ) , or pair-fed control (0) guinea pigs, after ANOVA and Dunnett's test....... 100 25 Time course of changes in serum triglyceride con- centration (mg/d1) of ad lab ((3), TCDD treated (O ) , or pair-fed control (A) guinea pigs....................................... ....... 102 26 Relationship of body weight and adipose LPL actiVity... ..... 0.0.0.0.... 0000000000 00...... ..... 107 27 Chronotropic (A) and inotropic (B) responses of isolated guinea pig heart atria to isoproterenol.. 141 28 Chronotropic (A) and inotropic (B) responses of isolated guinea pig atria, to isoproterenol, 20 days after treatment with either 1 ug/kg (i.p.) . TCDD (0) or acetone:corn oil (0) and pair-fed..... 143 29 Inotropic response (as a percent of the maximum developed force) of isolated guinea pig atria (from young animals 250-300 g) 20 days after treatment with 1.0 ug/kg TCDD (0) or acetone:corn oil and pair-fed (0), or fed ad libitum (0).. ..... 145 30 Chronotropic response (as a percentage of maximum developed rate) of isolated guinea pig atria 20 days after administration of 1 ug/kg TCDD (o) and pair-fed (0) or fed ad libitum (A) ................ 147 31 Isolated guinea pig atrial inotropic dose response ~to isoproterenol 10 or 20 days after treatment.... 148 32 Simplistic schematic of lipogenesis and lipolysis in adipose tissue; TG-triglyceride, LPL-lipopro- tein lipase, FFA-free fatty acids, HSL-hormone sensitive lipase.................. ................ 157 33 Three possible mechanisms for the production and cellular regulation of lipoprotein lipase......... 159 34 Cellular control and kinase regulation of adipose HSL and the reciprocal regulation of LPL ....... ... 160 35 Time course effect of orally administered glucose on adipose LPL activity of TCDD treated (o), pair-fed control (A) or ad lib control (0) guinea pigs........ ............ . .................. 173 xvi Figure Page 36 Adipose LPL activity as a function of serum insu- lin concentrationOOOQO......OOOOOOOOOOO0.00.0.0... 177 37 Proposed unifying hypothesis for TCDD's mode of actionOOOOO......OOOOOOOOOOOO......OOOOOOOOOOOOOO. 198 xvii CHAPTER I INTRODUCTION An unwanted by-product in the synthesis of the herbicides 2,4- dichlorophenoxy acetic acid (2,4-D) and 2,4,5—trichloro-phenoxy acetic acid (2,4,5-T) is 2,3,7,8 tetrachlorodibenzo-p-dioxin (Figure 1). Many scientists are fascinated with TCDD because of its high toxicity to certain animal species and its wide species variation of toxicity manifested by acute oral LD50 determinations ranging from 0.6 ug/kg to > 3000 ug/kg (Table 1). Hepatocellular damage has been described in rats, mice, and rabbits; an edematous syndrome observed in chickens and certain mice strains; chloracne and hyperkeratosis noted in humans, rabbits, monkeys, and nude mice; hepatic porphyria witnessed in humans; and bone marrow depression seen in monkeys (Poland and Glover 1980). Recently, evidence of TCDD has been found in industrial high combustion effluent smoke stacks, and it is thought to be environmentally ubiquitous (Bumb et a1. 1980). Brief History of TCDD Two major events are responsible for the vast amount of research concerning this compound. First, a mixture of n-butyl esters of 2,4-D and 2,4,5-T, coded as Agent Orange, and used extensively for defoliation in Vietnam between 1962 l MCI-l . HEAT. PRESSURE HEAT AMYL ALCG-K]. H20 FFESSJE CHLOROACEToc ACID EXCESS Nam -Nao I C | m km: i 2,4,5 T 2,3,7,8 TCDD Figure 1. One possible mechanism for the spontaneous formation of 2,3,7,8 TCDD during the production of the herbicide 2,4,5 T (adapted from Esposito et a1. 1980). m hc< m .z cmesm omma .Hm um COmHo ooomA Hmmcouflummmnuca cmxflz kumemm smaumm cocaow owma .Hm um comfio mmaa Homo cmxaz umumemm ameuwm cocfioo mhma .Hm um muummm oooa Hmmcouanmmmuch coxflz moumflaom whoa .Hm um mo> oma Hmuo 2 mmdoz mmma .Hm um Num3com mHH ammo cmxflz pannmm mmsmfi .Hm um Hmccoooz on Hmuo m mmxaoz mnma .Hm um mflmuo omnmm Hmuo cm>flo poz :mxoflno mmma .Hm um Num3nom mw ammo m pom mhma .Hm um Nuw3£0m mm Homo 2 pom mama .Hm um Numznum H.m Hmuo m mam mmcazo mmma .Hm um Num3com v.0 Homo z mam mocflsw mocmumwmm Amm\msvomaq whomomxm xwm wmflomdm owmom .moflommm Hmeflcm Hmuw>om no“ mosfim> omoq onus m.n.m.~ .H manna 4 and 1969 was found to contain TCDD in concentrations from an average of 0.05 ppm to a maximum of 47 ppm (Hay 1978). The toxic manifestations of this mixture on American soldiers and Vietnamese civilians are just now beginning to be examined. The second major environmental contamination episode occurred in June 1976, in Sevesso, Italy where an industrial explosion caused the release of several hundred grams of TCDD into the atmosphere. Within weeks of the explosion thousands of animals died and soil samples revealed up to 5,477 ug TCDD/m2 (Fanelli et al. 1980). Animals treated with TCDD typically exhibit depressed behavior such as cage huddling, failure of grooming, and a general disinterest in their surroundings within 2-3 weeks post. dosing (personal observation). This behavioral modification may be accompanied by a ruffled hair appearance, piloerection, and occasional subcutaneous hemorrhages seen in the tail, paws, and under the nails (Figures 2-5). Ventral hair loss and an icteric appearance of the ears, paws, tail, subcutaneous tissue, and visceral organs may follow (Gupta et a1. 1973). This jaundiced appearance may result from increased levels (HE porphyrins since TCDD can be a powerful inducer of delta-aminolevulinic acid synthetase in some species (Poland and Glover 1973a), or from depressed uroporphyrinogen decarboxylase activity (Poland and Knutson 1982). Figure 2. Normal appearance and curiosity of a control rat 10 days after the administration of acetone:corn oil. Figure 3. Normal posture of a control animal with the paws under the body. Figure 4. Typical appearance of a TCDD treated rat (10 days after administration) displaying piloerection, hair loss, and lowered carriage. Note the positioning of the fore and hind limbs. Figure 5. Cage huddling and hunched posture of a TCDD treated animal 10 days after administration of 25 ug/kg TCDD. Environmental Significance The importance of TCDD as an environmental contaminant is unquestionable (see Poland and Kende 1976, Bumb et al. 1980, Holmstedt 1980, Kociba and Schwetz 1981). It is an extremely toxic compound with a wide diversity of toxic manifestations, a wide species susceptibility, and a biologically long halflife. Furthermore, Pitot et al. (1980) have shown it to be a tumor promoter. Most studies indicate the biodegradation of TCDD to be a very slow process, appearing (up be mediated through. microbial organisms in the soil (Esposito et al. 1980). However, Ward and Matsumura (1978) concluded the limited degradation of TCDD in aquatic systems to be favored by the presence of sediment, organic matter, and or microbial activity in the aqueous phase since the half life was longer in water without sediments than with sediments. Although biological degradation is slow, photo degradation. of this chemical occurs quite readily in the presence of artificial or natural sunlight whether the compound is in solution or soil bound (Esposito et al. 1980). Symptoms of Toxicity The most consistent symptom of TCDD intoxication in laboratory animals is body weight loss and the consequent "wasting" syndrome (Greig et al. 1973, Harris et al. 1973, Kociba et al. 1976, V08 et a1. 1973, Faith and Moore 1977, Kociba et al. 1979, Neal et a1. 1979). Oral doses of 50 8 ug/kg to rats, resulted in an average weight loss of 38% from the time of dosing (Allen et al. 1975). They concluded that this loss was not from an anorectic effect since the dead animals had significant amounts of food in the stomach and gut. Furthermore, Gasiewicz et a1. (1980) noticed this same body weight loss in their treated animals but not in the pair-fed controls. The question arises as 13) whether TCDD causes a decreased absorption of nutrients from the intestine cm: a decreased biochemical utilization of nutrients at the cellular level, since animals lose weight although food consumption seems normal and the gut apparently full. After the infusion of a: "total parental nutritjrnfl' diet, containing ‘vital Ininerals, vitamins, and amino acids, the body weight changes (HE rats administered TCDD were not found to be significantly different from controls (Gasiewicz et and .1980). Additional evidence for malabsorption caused by TCDD stems from the work of Ball and Chhabra (1981). Doses of TCDD as low as 5 ug/kg inhibited intestinal glucose and leucine absorption. The greatest and most consistent pathological change caused by dioxin in many species, is thymic atrophy or involution as revealed by gross necropsy (Gupta et al. 1973, Harris et al. 1973, V05 et al. 1973, Kociba et al. 1976, Neal et al. 1979, Van Logten et al. 1980, Poland and Knutson 1982). Although severely affected by TCDD, few observable pathological lesions are noticed. Involution appears to result from increased thymocytic migration to the medulla from the cortex and consequent secretion of cortical thymocytes into the cardiovascular system (Kociba et al. 1976, Albro et al. 1978). The pyknosis noted by Buu-Hui et al. (1972) is not routinely seen except at very high doses. The pituitary gland does not cause involution since blood vessel dilatation and hemorrhages are observed in both hypophysectomized and normal rats administered 20 ug/kg TCDD (Van Logten et al. 1980). Increased serum glucocorticoid levels can result in thymic atrophy, however, adrenal hyperfunction is not responsible since adrenalectomized animals still underwent thymic involution when treated with TCDD (Neal et al. 1979). This dioxin also was not found to bind to glucocorticoid receptors which would result in a stimulation of plasma glucocorticoids. Neal et al. (1979) theorized that thymic atrophy could occur from decreased catabolism of steroid hormones, loss of feedback control of ACTH production, or hypersensitization of tissues (other than adrenals) to stimulate glucocorticoid production. The tremendous differential species response to TCDD administration is demonstrated with liver pathology. Little hepatotoxicity is noticed in guinea pigs which are the most sensitive laboratory animal to TCDD, but in rats the liver is the organ most severely affected and exhibits the greatest number of histopathological lesions (Table 2). Briefly, the most common effects observed include lO .mwmocxmm “mmusmflw ofiuouHE o: cufl3 Afimaos: omnvv mHHmo Hoe Iwnocmumm mummaoscauasa «mmoc umom om mop um cam> Hmubcwo mo mcflcwxoace “moumoocoe cam mcHOmscHw onwccsouusm mo coflu .mamcflwv Impmaflc “mflmouom: Hmfionoaauu mhma .Hm um mflouo m m.z 0 com Icmo “ucmpcoo Hmumz ommmmuocH .onMEuoc o» :Hsumu con» mam ommmmuomc an omzoaaom mmm commouocfl hm Uo3oHH0u mmummmum 1mm mmmummm sh nmzoaaou nose Amamcflmv mafia ou unmUMncm mummHUCH mnmfl .Hm um umflzom DO 2 O mm\m mmmllmocmsvmm mmusoo mafia .mmflme low Ca mum>mm muos muoommm .mHHmo ummmmsu mo MHmMHmHmdmc Hmoom «:oflmSHocfl mcflamwc 0cm mmaosom> ownocmoeouno Amamcflmv «coflumwaoscfln «manomumx mnma .Hm um Hozsssm z m.z mH ooo.oa nowacm “mammum umflsbofiauucmo mocmuommm ucflmuum mem mmusom Amx\m5. :oammq COwumuu wmoo IchHE©< nova .Ewumxm ofipmmwn you 0:» Mo mcoflmma HmufimoHosumm Mono: .N QHDMB ll .mmum Hmuuom aw mHHmo whoa IMEEmHmcfi meow mo coflumuufifiw use «pcmucoo caged commmuoca «coflummmummm Howflmcuoccmomso Amaflmcv (Hymn «mamouom: «maflmo mummfio mead .Hm hm abaoom om m.z o H (seepage N$253 omuuoumao .mmaamcmmno Honuo can mumdmouc oaaaa .mauocoaoouae maaao nuaocm mmmuum ammunems Daub Icmocoo mcHEAOM mam CH mummuo .mamcflmv new “umamouc named commmuocfl mnma .Hm um cmaflc mm 2 O om “bones: muhooummmz oommmuocH .mHHoo mummaoocflufise «coflmmu HugoNOHE Ugo cam> Hmuucmo on mcflmmmuo Amamcflmv use HmcuOQ Hoasoflamamo mcu vbma mocon m 2 0 com macaw >ua>auom mmmma< mo mmoq .coflu IoNHHosom> “QOADMHHHHMQH muumw “cofiummmummm cam Emmflmouho Hmflscmum “mafiaaozm «maamo Hoe uwnocoumd mo mflmoufle commmuoca «mHHoo mummaoscfiufine «mflmou swooammme “monoo cmNflcmmuoch mnma .Hm Do madam DO m.z O om\ooa “mmu%00pmmwn cmmHchm «mflmonomz mocmuowmm ocfimuum xom mmusom Amx\msv coammq Q coflumuu mmoo Imflcaeca nova ...o.u:oo. m manna 12 OHEmmHmouho mucuoo mo amouum can cumuumm MMHSQOH mo cofiuuou Imflc “mammmsouau “mmmcmnu o>fia Awaflmcv IMMOMHHOHQ can .OHuouomc whoa mean .Hm um abaoom om m.z swam H.o (assmfimca “coanmuwcmmmo oaummm: .ucoucou tamed CH momcmco “mmum Hmwnom Ifinom ou meflu sufl3 mcficcmmxm wua>fluom ommme< umasnoflfluu Icmo mo mmon “mmm cam mmeom mmma Amflmcflmv nowha commmHUCfl “mmumooummm: ocMOQmO cam macho m m 0 com mummaoscflufise “wasnomflupcou .mcflom mupmm mowm cam .mumumm Houmummm loco .cflmfia commmuocfl «mmHUOQ UHoHome on HMHHEHm mauflzw ocmuQEmE mamcflm an coccsou Imam mmfiooum> ummHo “mm mo AmHmCHmv mumwma >2 coccsouusm muflmomwc mead .Hm #6 ounaa a a o ooa\om\oa camaH “magnoflmouoas cmmmmuocH .mHHwo Hmflflmcuoccmofisoflumu Hoe uhcocwumm commmuocfi «monoN umH ISQOHHuucmO can Hmuuomfluom :H Assumes coaumuusauca Hamo snowmeemfimca hhma .Ho um pmouu z z 0 mm\0H «mflmouomc «mcflaamzw ou»ooumamm mocmummwm ocflmuum nxmm mwusom Amx\msv cowqu coflumnu wmoa amacaso< oboe ...o.u:ou. m manna l3 ommH .Hm um NoHsmHmmu I I mH mhaH .Hm HO :Omumuwm : E O onmcmousm nuHB mHucco:OOH (HE commuHm «mHHmo cmummHoscHn “coHumuowHHoum bozo mHHQ «hHmm IOEOHmo «mHmmmeoummm: «mHHnmc umHoHHmo SHHS mmommm UHDmmo «monoum cmmoomHm commmuomc “EmmHmouwo cmmcwccoo «mHuccono IouHE Hmauo: nuHs mmusuosupm HmHsoHHo cH mum mDOSCHucooch “COHHMEEmHmcH «:onmu HmcoNcHE Aonchv cH mHmouooc «coHumNHHozom> OOH “manhooummmn cmmHchm :mHHOBm .3on mHHQ cam :oHuouoxm mumHHHQ :Hmn loco commmuowc mommm9< m: can mm\oH x\mz wcmunEmE MEmmHm cmmmmuomo .mHmemw map cH mmHscoc oHummHmHmdmn umHsHHmooummm: cam meocHonmo HmHsHHmooummmc “mHmonnHm «:oHu IMEEmecH HmuuomHme «wmummmummm acmemHQ «:oHumnmuHm HmHsHHwo Ioummmn mo wmoum Ho HUOM “mmumo noummwn coummHoscHuHDE “coHu IMEEmHmcH can mHmouow: 0Huwmon “mmHunmoum HmHHouocHu comm» IHm “mmcmno muumm «GOHumHosom> mocmumumm ochuum bxmm mmgsom oHnmuu uchHEc< Amx\ms. aonmq meD DOOR .A.U.UGOUV N GNOME l4 .CMENDHOEI: .meamolwsmmummuam .HmmcouHHmmmuucHlmH .HMHOIOIIODDOH coHumuuchHEc< .HonomHmlm .couuomsm .oosoo .umumHznz:u:Hmuum .mHMEOMIm .OHMEIZIIxmm .00 M .mcoN HmHsn IoHHHucmo mo momma Hmoow mo ome AmHmchv coHumumcommc cam .mcHHHmBm .Hm um coumoq cm> m m 0 cm .mHmouom: HHwo mHmch soccmm .conmH HmcoNcHE cam HmHsnoH IHHucmo cH momemc umoe «:oHumuu IHchH Hmeouc UHQHH “mmHosow> mocmummmm ocHMHbm xmm modom Amx\msv :onOH 3 moHumuu omoa :chHso< oboe ...C.UCOUV N manna 15 hyperplasia, hypertrophy from increased smooth endoplasmic reticulum production, parenchymal necrosis, inflammatory cell infiltration, lipid accumulation, multinucleate cells, and single cell necrosis. These changes are seen most often in the centrilobular and periportal zones of the liver, and coincide with loss of some hepatic biochemical functions such as ATPase activity noted by Jones (1974) and Peterson et al. (1979b). A survey of the literature shows the effect of TCDD on other organs of the rat to range from hemorrhages, hyperplasia, necrosis, and degeneration to significant reductions in the incidences of spontaneous lesions normally observed in chronic long term studies. Different animal species show different susceptibilities tx> TCDD and emphasize different manifestations of its toxic actions. The variety of lesions caused by this chemical demonstrate the difficulty in proposing a unifying theory for its biochemical mechanism of toxicity. The inductive effects of apolychlorinated dioxins on microsomal systems had been suspected as early as 1967 (see Firestone 1973), but microsomal induction as a result of TCDD administration was first shown by Greig in 1972. Subsequently, Lucier et al. (1973) demonstrated the pattern of induction by this compound to be somewhat different from that observed by the classical inducer phenobarbital. Induction by TCDD was instead similar to that seen with l6 3-methyl cholanthrene (3-mc) which earlier had been shown to correlate with increased aryl hydrocarbon hydroxylase (AHH) activity. AHH activity is inherited in an autosomal dominant fashion controlled by 1 or more loci designated as the Ah locus (Nebert et al. 1972). Poland and Glover (1973b) quickly realized that TCDD also induces AHH activity. Furthermore, they demonstrated that both compounds are recognized turaa common cytosolic receptor in the liver which somehow stimulates the induction of AHH and other' detoxifying' enzymes (Poland. and (Glover 1976). 5 daltons Characterized as a heat labile protein of 2 x 10 this receptor is specifbc for P-448 inducers since phenobarbital, pregnenolone-16 alpha carbonitrile, and steroid hormones do not compete against it. In addition, the receptor affinity for TCDD congeners is positively correlated with their induction capability as well as their biological potency. That is, those congeners having the greatest affinity for the receptor show the greatest amount of microsomal induction and time greatest amount of biological toxic manifestations. Carlstead-Duke (1979) noted the highest concentration of these cytosolic receptors to be in the thymus gland -- the organ typically exhibiting the greatest deleterious effect from TCDD. Upon binding of the polychlorinated aromatic compound to the cytosolic receptor, the receptor-substrate complex is translocated to the nucleus where a presumable interaction with DNA is 17 thought to occur (Greenlee and Poland 1979). The nuclear binding capability of this complex has been confirmed by Mason and Okey (1982), amd the nuclear receptor substrate complex has been isolated by Poellinger et al. (1982). Instead of examining the effect of TCDD on microsomal and other detoxification systems preliminary research will investigate alterations occurring on the plasma membrane. It is at this site where many physiological functions are performed which may be modified in such a way as to implicate toxic manifestations. Previous reports have indicated that TCDD does influence membrane constituency. After a: single oral dose of 200 ug/kg TCDD, electron microscopy revealed the formation of giant multinucleate cells which probably occurred as a result of parenchymal cell fusion (Jones and Butler 1973). Greig and Osborne (1978) further observed a loss of membrane continuity between parenchymal cells, wide intercellular spaces, and loss of ATPase activity in the centrilobular portion of the liver. A single oral dose of 10 or 25 ug/kg TCDD inhibited biliary excretion of ouabain as soon as 2 days post treatment (Yang et al. 1977). The transport of neutral substrates across the plasma membrane is proposed to be regulated by various membrane bound ATPase's, and Peterson et al. (1979b) have demonstrated lower plasma membrane Na-K and Mg ATPase activity within 2 days of TCDD treatment. 18 Pair-fed control rats were not affected. TCDD is not thought to directly bind to the plasma membrane and influence enzyme activity, since only 0.01357 pmole TCDD/ug protein (equivalent to 10-10 to 10.9 14 dioxin) -- accumulated in the membrane after a dose of 25 ug/kg. Plasma membrane incubated £2 XEEES with up to 10..5 M dioxin showed no inhibition of activity. Ivanovic and Weinstein (1982) noted the suppression of EGF (epidermal growth factor) binding to the cellular surface of cultured fibroblast cells after treatment with different 3-MC type microsomal inducers. They did not assay TCDD but.noted the potency of the inducers paralleled their ability to alter the cellular surface and decrease binding. This potency is thought to correlate with their ability to bind with the cytosolic receptor, therefore they concluded this decreased binding phenomenon to be a result of the pleiotypic changes caused by these compounds. Pitot et al. (1980) used canalicular ATPase as a parameter to detect TCDD-promoted development of foci in the rat liver and found them to be almost devoid of this membrane bound enzyme. Cell surface changes induced by TCDD may resemble some of those seen with precancerous cells as described by Hynes (1979). Transformed cells typically display reduced numbers of surface glycoproteins and a reduction in gap junctions. Since TCDD has been implicated as ea tumor promoter by the work of Pitot et al. (1980) this dioxin may result in 19 decreased intercellular recognition and attachment or inhibited cell-cell communication; both are mediated by the cellular surface. Objectives Previous reports implicate cell membrane alterations resulting from the administration of TCDD. The objective of this research will be to elucidate some of those _i__n_ vivo biochemical transformations. Rat liver will be used in the preliminary studies for several reasons: It is a large, easily accessible organ which displays a number of prominent pathological lesions; the size of this organ enables a high yield of relatively pure plasma membrane from a single animal; finally, many’ biochemical and metabolic pathways have been clearly defined in the liver. The guinea pig is the most sensitive species to this dioxin and demonstrates little liver pathology, therefore to ascertain whether these membrane alterations are related to a specific toxic manifestation associated with TCDD, other organ systems will be investigated in this species. TCDD represents a class of chemicals (including PCB's, PBB's, and other dioxins present in the environment as pollutants) the toxicological significance of which is unclear. Previous investigations of TCDD's action have provided much information in the area of microsomal induction but no satisfactory explanation has been given for its toxic actions. The hypothesis to be tested by this 20 research is that manifestations of TCDD's toxicity occur as a result of organ membrane alterations. These pertubations which alter the binding capability and the enzymatic capacity of vital physiological substrates, are responsible for the toxicological implications generally associated with this dioxin. The rationale of this research is to examine toxicity by investigating TCDD's effects upon the plasma membrane. Since many vital physiological functions are dependent upon the cellular membrane, our results could yield valuable information as to the mechanism of toxicity and insight for TCDD's biochemical actions. Significance The importance of TCDD in environmental toxicology is unquestionable. It has a very long biological half life, is extremely toxic, seems 1x) be ubiquitous, and represents compounds such as PCB's, PBB's, other halogenated dioxins, dibenzofurans, and polynucleated aromatics which are also present as pollutants. This research will examine TCDD's toxicity by investigating its effects on the plasma membrane. Possibly one or more of these membrane alterations result in one of TCDD's toxic manifestations such as thymic atrophy, body weight loss, decreased immunological competence, tumorigenesis, or alterations in lipid metabolism. The significance of this project would be the elucidation of 21 biochemical causes of TCDD toxicity. With this research one could gain a better understanding of the mechanisms by which other hazardous environmental pollutants may cause deleterious effects. This knowledge would enable the design of effective treatments for or antidotes against these materials. CHAPTER II INFLUENCE OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN ON PROTEIN COMPOSITION OF RAT HEPATIC PLASMA MEMBRANE AND ON OVERALL BODY WEIGHT INTRODUCTION TCDD has been considered to be a very serious 50 15 estimated to be 0.6 ug/Kg making it the most toxic small environmental pollutant (Moore 1973). The guinea pig LD size man-made chemical known to exist (Schwetz et al. 1973), and it has been shown to be teratogenic, acengenic, and carcinogenic (Kociba et al. 1978). Toxicity is unusual because a single administration to laboratory animals produces few obvious external symptoms except body weight loss, and death usually occurs 15-30 days post-treatment depending on species. TCDD is a potent enzyme inducer in the rat liver resulting in massive increases of microsomal protein (Poland and Glover 1973a, 1973b). Poland and Glover (1976) found a cytosolic receptor which specifically binds with TCDD and apparently leads to various biochemical changes which occur as a result of the dioxin administration. However, the induction of hepatic microsomal proteins does not adequately explain why TCDD is toxic. The animal most sensitive to TCDD (guinea pig) shows little hepatic induction while more resistant species, such as the hamster, are highly induced 22 23 (Neal 1981). There are many microsomal inducers which do not show high toxicities or cause body weight loss, i.e. phenobarbital or 3-methylcholanthrene. In view of the importance of TCDD to environmental toxicology and the lack of an explanation for its toxic action, the hepatic plasma membrane as a potential site of TCDD action has been examined. MATERIALS AND METHODS Male Sprague-Dawley rats (175-200 g) were obtained from Spartan Research Animals Inc. Haslett, Michigan. Food (Wayne Lab Blocks, Chicago, IL) and water were provided ad libidum and the animals were maintained on a 12 hour light, 12 hour dark cycle. Plasma Membrane Preparation Animals were dosed intraperitoneally’ with either 25 ug/kg TCDD (Dow Chemical Co. Midland, MI), dissolved in a 1:9 solution of acetone:corn oil or an equal volume of vehicle alone. At 10 days post-treatment the animals were sacrificed by decapitation, and the hepatocyte plasma membranes (PM) were isolated according to the procedure of Yunghans and Morre (1973). Examination by electron microscopy (via procedures set forth by Hooper et al. 1979) and marker enzyme assays (Na-K ATPase and 3H-Concanavalin A binding) verified the presence of PM vesicles and absence of significant ndtochondrial and/or ndcrosomal contamination. 24 Electron microscopy was a service of the Center for Electron Optics at MSU. Gel electrophoresis was performed with a Bio-Rad 221 Dual Slab Gel system using the method of Laemmli (1970) as modified by Hoefer Scientific (1980). Gels were subsequently dried using a Bio-Rad Gel Slab Dryer (Model 224) for densitometric scanning. The bands resulting from Coomassie blue staining were scanned. for their relative intensity utilizing an ACD-18 Gelman Densitometer, and the areas under the peaks integrated for comparison between plasma membrane preparations. Concanavalin A Binding Concanavalin A binding was determined by mixing 25 ug 3H-Concanavalin A membrane (in 0.25 M sucrose) with 0.21 ug (New England Nuclear, Specific activity 25 Ci/mmole) to a total reaction volume of 0.2 ml. Each reaction tube was subsequently incubated for 10 min at 370C after which the reaction was terminated with the addition of 8 ml cold 0.1 M Tris-HCl. The mixture was then quickly passed through 0.45 u HA filter (Millipore Corp., Bedford, MA), washed with an additional aliquot of 8 ml Tris-HCl, allowed to air dry, and the remaining radioactivity assayed with liquid scintilla- tion counting. Specific binding was determined in the presence of .01 M alpha methylmannoside (Sigma Chemical Co., St. Louis, MO). Parallel tubes were incubated for 5 to 10 25 min. in the presence and absence of alpha-methylmannoside before the addition of 3H-Concanavalin A. Specific binding was calculated by subtracting the radioactivity remaining in the tube without alpha-methylmannoside and was on the order of 3-6% of total radioactivity added. Protein concentration was measured by the method of Lowry et al. (1951). RESULTS A slight difference was seen in liver weights between control and TCDD-treated animals 10 days after dosing (Table 3). Although a significant difference was noticed in the liver/body weight ratio, the difference in the yield of plasma membrane per gram of hepatic tissue between treated and control rats was indistinguishable. The PM fractions, as examined with electron microscopy, were free from, contaminating mitochondria and microsomal vesicles. Absence of microsomal contamination in these preparations was further indicated by electrophoresis and the lack of many densely stained protein bands between 52,000 and 60,000 daltons (Figure 6). Plasma Membrane Protein Composition Analysis of the membrane preparations via SDS- polyacrylamide gel electrophoresis clearly showed qualitative differences in the protein composition (Figure 6). Band intensity was measured with a densitometric technique and the results (Table 4) indicated the levels of 26 .chuoum m: mm\c::on mc wmmum>< .wsmmHu Hm>HH usmHOB H03 Emum\m:mubeme mammHm mo UHme mo. v ms .ummu =u= mucmcsum cmHHmu oBu mch: cmmmecm mama .mHmmcucmHmm cH mHMEHcm mo Hones: 02H mo mm + some mnu mm commmumxm mHHsmmmw U n 41m. m.m H o.o Am. o.m + o.HH umchcHn a coosmm oHuHommm Ami m.m H m.m Ami m.4 H o.mH omaHocHn a :ooumm fiance Ame ms H omm Ase moH H cam aims. nHmHs mamunamz mammfim .AGH. m.o H s.o LVHV m.o H m.m Aw. oHumu uanmz Hcom\nw>Hq «ASHE mm.m H em.mH mAHHV mG.H H oo.HH Amsmumi uzmHmz Hm>Hq Amx\m: mmv nave Honucoo .mchoc Houmm mhmc oH mumu cmummub 0009 can mumu Houucoo :mo3uon mchch < :HHm>w:mo:oolm can chHw ocmubewe mammHm Hm>HH .pomu ucmez moon\um>HH .uanmz Hm>Hm .m mnm) and TCDD treated (0) rats. Each point represents the average 1 SD of 5 animals. Data was tested for normality then subjected to random factorial ANOVA which indicated a significant difference between the two groups of animals at P<.01. 34 300‘" 200 ‘— IOO PIG-u; >OOD ..(F—LZ. i DAYS POST DOSE 35 ug/kg TCDD or acetone:corn oil, body weight as a % of initial body weight was 186% and 260%, respectively. Results from subsequent experiments indicated that this loss of body weight produced by TCDD administration is variable. Some animals demonstrated a considerable loss of body weight while others neither gained nor lost weight while still others actually showed a gain in body weight. Those rats which did gain weight never did reach that attained by the control animals. This variability in response was thought to be due to inherent variability in the population. That is, some individuals were tolerant to dioxin at this dose while others were seemingly more sensitive to TCDD. The majority of TCDD treated animals neither gained nor lost weight as would be expected from a normal population. It is anticipated that a higher dose of dioxin would produce the characteristic loss of body weight in a greater proportion of animals while the reverse would occur at lower doses. To ascertain whether the decreased weight gain was due to depressed food consumption, food and water intake and fecal output were measured over a 25 day observation period. Results indicate an initial brief drop in intake 2-3 days after“ dosing in kxnjl control and. TCDD treated animals (Figures 9 anui 10). However after that, both food and. ‘water‘ consumption 1J1 the treated. animals were significantly' reduced. from! that. of the controls, and as 36 Figure 9. Food consumption of 5 control (0) and 5 TCDD treated (0) rats over a 25 day observation period. Each point represents the average 1 SD of the % of food or water consumed daily in relation to that provided each ani— mal. Analysis by random factorial ANOVA after normalization by angular transformation indicated the control and TCDD populations to be significantly different in both experi- ments at P<.05. 37 l 4 7 DAYS POST DOSE omiauZOO DO “— 3 26 20 38 Figure 10. Water consumption of 5 control (0) and 5 TCDD treated (0) rats over a 25 day observation period. Each point represents the average 1 SD of the % of food or water consumed daily in relation to that provided each animal. Analysis by random factorial ANOVA after normalization by angular transformation indicated the control and TCDD popu- lations to be significantly different in both experiments at P<.05. 39 % WATER CONSUMED N 4:- o o I l° ' o—I H H I-—-o o—I o—I o——I H o—I o-I o—I o—I I-——o I—o o—-I D—l g I—————o c4 I—————o O 2 8 l4 DAY POST DOSE 40 would be expected fecal output from the control animals was higher than that from the treated (Figure 11). Toxicity All animals dosed with TCDD showed noticeable hair loss beginning approximately 12 day after treatment. Some of those animals near death in the latter part of the experiment. exhibited. ocular hemorrhaging’ and lacrimation, pilo erection, minor salivation, obvious capillary dilation in the ears, and penile erection. They were considered to be abnormally aggressive and to have some difficulty in breathing. Fecal pellets were abnormally small and drier than usual and some contained a green fibrous material reminiscent of undigested food. Observation. upon. dissection revealed many fatty deposits among the organs. The liver looked very granulated with enlarged cells and obvious fatty deposits. Kidneys, lungs, trachea, spleen, pancreas, heart, and thymus all appeared normal except for large fat deposits surrounding, and in some instances infiltrating, the organ. The thymus showed signs of involution and was barely detectable in some specimens. Only liver and thymus weight were significantly different from control animals (Table 5). DISCUSSION Although TCDD caused a 10% decrease in liver weight after 10 days, the liver to body weight ratio was actually 41 FECES WEIGHT (9; 14— I o—- I - . . ° ° ° ° . . ’ ' 9 L- 6 I it JL. ‘I At it 2 I “L 0 J J 1 1 l o 2 8 14 20 26 DAYS POST DOSE Figure 11. Fecal output (mean i S.D., n=5) of control (0) and TCDD (CI) rats as a function of time after dosing. Random factorial ANOVA indicated these populations to be significantly different at P < .01. 42 TABLE 5. Organ weightsa of rats 25 days after dosing with either acetone:corn oil or TCDD. Organ Control TCDD (25 ug/kg) Liver 18.65 1 3.23a 13.71 1 3.14* Kidney - Left 1.32 1 0.26 1.52 1 0.23 Right 1.30 1 0.18 1.48 1 0.09 Lungs (and trachea) 2.22 + 0.15 2.27 1 0.13 Heart 1.21 1 0.12 1.29 1 0.22 Spleen 0.93 1 0.26 0.76 0.13 Thymus 1.11 1 0.16 0.27 1 0.10* Testis & epididymis - Left 3.41 1 0.78 3.62 1 0.70 Right 3.26 1 0 79 3 73 1 0.50 Urinary Bladder 1.43 1 0.61 1.33 1 0.48 Adrenal Gland - Left 0.04 1 0.01 0.03 1 0.01 Right 0 04 1 0 01 0 03 1 0.01 Brain 2.14 + 0.21 2.33 1 0.11 aResults expressed as of 5 animals. Data test. *P (0.05 mean organ weight in grams 1 SD analyzed with two tailed students "t" 43 increased since there was a large difference in final body weights. Therefore in proportion to the rest of the body the liver' actually' became larger. Because the yield of plasma membrane did not change, this increase was probably not due to hyperplasia but rather from an increase of intracellular material. In view of the fact that TCDD is a powerful microsomal inducing agent (Poland and Glover 1973a,b) this is not surprising. Little contamination of these preparations with other organelles was noted upon examination with electron microscopy' and Ielectrophoresis. As mentioned. previously, the absence of an increase of band 54 to 56K in the treated preparation indicated minimal microsomal contamination. The 48K band is consistently found in all plasma membrane studied (Neville and Glossman 1971a,b), is probably a structural protein, and therefore was used as an internal standard. It typically made up 8-13% of the total protein in the membrane. While no effort was made to establish the identity of the specific bands that were altered, it is important to note that if these changes occur at critical sites in critical organs, symptoms of TCDD's toxicity may be explained. Therefore changes of tflua plasma membrane shown here may be important in understanding TCDD's toxic action. The activity of 2 plasma membrane bound ATPases, Na-K and Mg-ATPase, in the rat liver have been previously shown to be reduced by 1a vivo treatment of TCDD (Peterson et al. 44 1979a, 1979b). Biliary transport of ouabain, a model neutral substrate, was also reduced. These events can be traced to the function of the plasma membrane, and it is therefore concluded that they are caused by TCDD's action 1a 1119 on the plasma membrane. These pertubations are not due to 51 direct effect of the dioxin on the membrane since Peterson et al. (1979a) showed that TCDD added to membranes 13 11119 failed to produce the toxic effect. The protein changes may be due to membrane fluidity changes or altered synthetic and/or turnover rates CHE membrane components. Whether the same type of action is responsible for TCDD's toxic manifestations in other species remains to be determined. However, the importance of plasma membrane surface proteins in normal cellular functions is unquestionable, therefore it is imperative to examine these alterations in the future. Concanavalin A is a plant lectin which binds specificalLy to membrane glycoproteins containing the mannose moity. The decreased Con-A binding caused by TCDD suggests altered hepatic cell surface glycoproteins in these animals. This may lead to a loss of cell-cell communication which in turn can decrease contact inhibition and result in tumor formation. Many precancerous or transformed cells are known to have reduced surface glycoproteins (Walborg et al. 1979), and TCDD has been shown to be a very good tumor promoter in the rat liver (Pitot et al. 1980). 4S Treated animals consumed significantly less amounts of food and water. This could account for the severe wasting syndrome experienced by these animals, however an examination of intestinal absorption along with pair-feeding needs to be evaluated. The high variability in body weight gain by the treated animals in Figure 8 and in food and water consumption (Figures 9 and 10) was due to some animals drastically cutting food intake and beginning to die at about day 14. These data confirm that of Seefeld and Peterson (1984) in that hypophagia plays a major role in the loss of body weight after TCDD treatment. These authors also measured fecal energy loss (as a percentage of feed intake) and digestible energy (percentage of feed energy absorbed by the intestine) and they found no significant differences between control and dioxin treated animals. They concluded that TCDD treated rats reduced feed intake until a new weight level abnormal for the age of the animal was obtained. Although fecal output decreased (Figure 11) there was no change in potential energy lost with the feces since feed intake was also reduced. Symptoms of toxicity other than weight loss included: minor ocular hemorrhaging, lacrimation, pilo erection, minor salivation, and penile erection. Hair loss, a common characteristic of other polychlorinated aromatics, was also observed. The major pathological change observed grossly was the infiltration of many of the major organs with fatty 46 deposits. Only the liver and thymus showed significantly lower weights than control animals. Conclusions In conclusion, TCDD caused decreased food and water intake followed by significant loss of body weight gain and typical behavioral symptoms of starvation. Fatty infiltra- tion of many' major organ systems was noted as ‘well as significant decreases in both liver and thymic weight. The major significance of this study is the hepatic membrane alterations. If these changes occur at critical physiological sites in the liver or other organs, some of TCDD's toxic symptoms may be explained. CHAPTER III ALTERATION OF RAT HEPATIC PLASMA MEMBRANE FUNCTIONS BY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN INTRODUCTION Low level residues of TCDD have been detected in soil, fish, industrial and municipal fly ashes in various locations throughout the United States (Bumb et al. 1980). Because of the many diverse effects of TCDD, the search for the Jbiochemical mechanisms (n3 its toxic action. has been difficult. Investigations to find interspecific differences among guinea pigs, rats, hamsters, etc. in the metabolism, disposition, and cytosolic receptor binding of TCDD have produced modest differences, but they probably do not explain the enormous species difference in susceptibility to this toxicant (Gasiewicz et al. 1983). Peterson (Peterson 6H: al. 1979a) has found that a dose-dependent reduction in plasma membrane ATPase accompanies the depression cu? ouabain biliary excretion in rats treated with TCDD. Although this relationship was later shown to be causally unrelated (Peterson et al. 1979b), the phenomenon that these two plasma membrane mediated functions are simultaneously reduced by dioxin treatment warrants further investigation. It was found that protein profiles of hepatic plasma membrane from TCDD—treated rats were 47 48 different from those of untreated controls (Brewster et al. 1982). Evidence is here presented that la 1119 exposure of TCDD affects A a number of physiologically important components of the rat liver plasma membrane. MATERIALS AND METHODS Young (125-1509)“ male Sprague-Dawley rats were fed Purina Laboratory Chow, ad libitum. TCDD was dissolved in corn oil with acetone (9:1 ratio) and used for intraperitoneal (i.p.) injection. Control rats received the same volume of corn oil-acetone. After specified time periods they were sacrificed, and the hepatocyte plasma membrane was isolated according to the method of Yunghans and Morre (1973). In an effort to compensate for the decreased food intake by the TCDD treated rats, a third group of rats underwent severe food restriction for the 10 day observation period after which liver plasma membrane was isolated. Young (200-225g) male albino English Shorthair Guinea Pigs (Strain MDH:SR(A)) were purchased from the Michigan Department of Health. Sprague Dawley Rats and Golden Syrian Hamsters were obtained from Harlan Animals, Haslett, Michigan. Housing, plasma membrane fractionation, enzyme assays, and electrophoresis were as before. 49 ATPase Enzymatic Activity ATPases were assayed by the method described by Matsumura and Clark (1980). Modifications were: 25 ug plasma membrane protein in 0.1 ml sucrose (0.25 M) added to 0.9 ml reaction buffer containing 10 ul 2,4 dinitrophenol (10 mM) was preincubated at 370C (10 min), (gamma-32P) ATP was added (final concentration 10 mM), and the mixture incubated at 37°C for an additional 10 min. The reaction was stopped with the addition of 300 1L1 ice cold trichloroacetic acid (10%) followed by 100 ul H20 containing 1 mg bovine serum PO . The solution was centrifuged (5 2 4 min, 1000 x g) and the clear supernatant decanted and mixed albumin and 1.36 mg KH with 200 ug activated charcoal. The sides of the tube were rinsed with 200 ul ethanol, and after a second 5 min spin, 0.5 ml of the supernatant was mixed with aqueous scintillation cocktail for liquid scintillation counting. Buffers for determining various ATPase activities contained 30 mM imidazole (pH 7.1) with the following ion combinations: Na-K ATPase, 120 mM NaCl, 20 mM KCl, and 5 mM basal medium for MgCl Mg-ATPase, 120 mM NaCl, 5 mM MgCl 2’ 2’ Ca-ATPase , 120 mM NaCl , 2 0 mM KCl , 0 . 5 mM EGTA; and for Ca-ATPase , 120 mM NaCl , 20 mM KCl , 0 . 5 mM EGTA and 2 mM CaClz. Protein Kinase Assay Protein kinase activity was determined by the method of Corbin and Reiman (1974) with the following modifications: 50 Plasma membrane (50 ug protein) in 50 ul of 0.25 M sucrose were added to 50 ul reaction buffer containing 1.1nl 50 mM potassium phosphate (pH 6.8), 1 ml 30 mg/ml histone (Sigma 32P)-ATP in 18 Chemical Co., II A-S), and 1 ml 1 mM (gamma- mM magnesium acetate. For the determination of c-AMP dependent protein kinase activity, 6 nmoles c-AMP were added to the reaction buffer. After incubating for 10 min at 30°C, the reaction was stopped with 3 ml cold trichloroacetic acid (10%). Then 100 ul water, containing 1 was added. The mg bovine serum albumin and 1.36 mg KH PO 2 4 tubes were allowed to stand for 5 min to complete protein precipitation, spun for 55 min (1000 )c g), the supernatant decanted, and the pellet redissolved with 0.5 ml NaOH (0.2N). Reprecipitation with trichloroacetic acid and a second centrifugation followed. After repeating this washing procedure once more (total 2x), the final pellet was resuspended with 0.3 ml formic acid, of which 0.1 ml was used for liquid scintillation counting. Gamma-glutamyl Transpeptidase Assay Gamma-glutamyl transpeptidase activity was assessed by the method. of Tate: and. Meister (1974). Additional information on this technique is: 100 ug membrane protein (suspended in 0.25 M sucrose) in 0.1 ml tris-HCl (0.01M)- NaCl (0.15M), pH 8.0 was mixed with the assay solution and incubated 15 min (37°C). After stopping the reaction with 0.1 ml glacial acetic acid, and spinning for 5 min (1000 x 51 g), the absorbance of p-nitroaniline was determined at 410 nm with a Varian Double Beam Spectrophotometer. Insulin and Epidermal Growth Factor Binding 125 125I Binding of [ IJ-insulin and [ ] epidermal growth factor (EGF) was studied, following essentially the method of O'Keefe et al. (1974). Plasma membranes (50 ug protein) were suspended in 0.2 ml of Kreb's Ringer bicarbonate buffer “Hi 7.4) containing' 0.1% ibovine serum albumin (BSA) and incubated with or without the native ligands (insulin 2.0 ug, EGF 0.5 ug) for 10 min at 24°C before the addition of 0.25 ng of the labeled ligand (insulin Sp. Act. 100 uCi/ug, EGF, Sp. Act. 150-200 uCi/ug). All of the tubes were incubated for an additional period of 20 min. At the end of the incubation, the reaction mixture was diluted with 3.0 ml of chilled Kreb's Ringer bicarbonate buffer containing 0.5% BSA and rapidly filtered through a bfillipore filter (type HAWP, 0.45 u) under vacuum. The filters were washed twice with 5.0 ml aliquots of the same buffer, air dried, and counted for radioactivity. Specific binding was calculated by subtracting the amount of radioactivity bound in the presence of native ligand from that in the absence of it. Concanavalin A Binding Binding of [3H]-Concanavalin A (Con A) was studied by the method originally used by Chandramouli et al. (1977). The following modifications were made: 25 ug of plasma 52 membrane protein were suspended in a 0.2 ml volume of 0.1 M tris-HCl buffer, pH 7.4 containing 0.1% BSA. Duplicate tubes were preincubated with or without alpha-methyl D-mannopyra- noside (alpha-mm, 10 mM) for 10 min at 370C before the addition of 0.25 ug of 3H-Con A (Specific Activity 25-45 Ci/mmol, New England Nuclear, Boston, MA). At the end of an additional 10 min of incubation at the same temperature, 8.0 ml of cold tris-HCl (0.1 M, pH 7.4) containing 0.5% BSA was added to each tube and the contents were quickly filtered over ea Millipore :filter (HAWP, 0.45 u). The filter' was washed with an additional 8.0 ml of the same buffer and the radioactivity was counted as described above. Specific binding was defined as the difference between the amount of radioactivity in the presence and in the absence of alpha-mm. The binding assays were performed both by B.V. Madhukar and myself. Protein concentration was determined by the method of Lowry et al. 1951. RESULTS Time Course of Plasma Membrane Protein Alterations To study the biochemical characteristics of the plasma membrane, rats were treated with 25 ug/kg of TCDD (single dose) ‘through. intraperitoneal injection (i.p.), enui their hepatic plasma membrane isolated after 2, 10, 20, or 40 days as before. When these plasma membrane preparations were analyzed with SDS-polyacrylamide gel electrophoresis, qualitative differences in protein composition became 53 apparent. In the electropherotogram shown in Figure 7 the most significant effect of i_n 3312 administered TCDD was observed in the pmeparations obtained from rats sacrificed at 10 and 20 days after treatment where many of the bands between 14 and 30 K dalton were completely abolished. After 20 days it appeared as if the effect was reversed; densitometric measurement of these bands confirmed this visual conclusion. The band at 48 K, which is a structural protein comprising 8-13% of the 'total membrane protein, served as a good internal standard (Brewster et al. 1982) and remained constant throughout the treatment period. Alterations of Enzymatic Activity and Receptor Binding To study the qualitative nature of the altered plasma membrane, activites of several membrane bound enzymes and receptor proteins were measured (Tables 6 and 7). Na-K, Mg, and Ca ATPase were all significantly reduced in the treated animals. There was little change in gamma-glutamyl trans- peptidase activity. The level of protein kinase activity in the plasma membrane from treated rats was significantly higher than that of the control, indicating that TCDD treatment does not always cause a reduction in enzymatic activity. Both c-AMP stimulated and nonstimulated protein kinases from the TCDD-treated animals showed higher enzyme activity than control. Examination of receptor binding (Table 7) revealed the EGF receptor to be most affected by TCDD. In agreement with 54 Table 6. Difference in enzyme activities between hepatic plasma membrane preparation from control and 1a vivo TCDD- treated rats. Results are expressed as mean + standard deviation of the number of animals in parenthgsis. Each preparation was tested twice. Enzyme activities (nmoles product/mg protein/hr) Enzyme Control TCDD-treateda Na-K ATPasec 1496 1 142 (3) 890 1 178 (3)b* Mg ATPaseC 1820 1 10 (2) 1110 1 149 (2)* Ca ATPaseC 608 1 65 (4) 340 1 140 (8)* Gamma-Glutamyl transpeptidase 887 1 231 (4) 658 1 35 (3) Protein kinasee in the absence of c-AMP 36 1 12 (4) 147 1 67 (7)* in the presence of c-AMP 58 + 25 (4) 217 1 110 (7)* aTCDD at 25 ug/kg single dose intraperitoneal injec- tion. Plasma membrane collected at 10 days post- treatment. Data were analyzed using a two tailed student's "t" test; *P<0.05. n moles P. libgrated/mg plasma membrane protein/hr at pH 7.1, 37 C. n moles p-nitroaniline liberated/mg plasma membrane protein/hr at pH 8.0, 37 C. n moles Pi incorpgrated/mg plasma membrane protein/ hr at pH 8.0, 37 C. 55 Table 7. Effect of 1a vivo TCDD treatment on ligand binding to cell-surface membrane receptors in the rat liver. Results are expressed as mean 1 SD of the number of animals in paren- theses. Specific binding Dose (ug/kg Ligand single i.p.) Control TCDD-treated 3H-Con Aa 25 15.2 1 2.5 (4) 11.7 1 0.4C*(3) lZSI-EGFb 25 32.0 1 8.6 (5) 3.8 1 0.3*(3) 125I-Insulinb 25 8.1 1 1.2 (7) 11.7 1 0.9*(3) Eng of iHECon A bound/25 ug protein/10 min. pg of I-EGF, or insulin-bound/50 ug protein/20 min. Data were analyzed using two tailed student's "t" test; *P<0.05. 56 previous observations (Brewster et al. 1982) Con A binding was also reduced, however, insulin. binding was slightly increased at this dose and time regimen. The time course of TCDD's effect was also studied following' a single i.p. dose of 25 ‘ug/kg (Figure 12). During the 40 day observation period, TCDD-treated rats gained consistently less body weight than did 13mg control rats. Insulin binding was little affected at the beginning, but by 20 days after treatment it was significantly reduced (P<0.05). Con A and EGF binding were continuously suppressed with the maximum effect occurring 20 days after treatment. TCDD's effects normal control values at day 40 but the c-AMP dependent phosphorylation remained elevated throughout the 40 day observation period. To ascertain whether reduced food consumption and lowered body weight could affect these membrane parameters, rats were maintained on a limited diet for 10 days. Na-K ATPase activity, EGF, and insulin binding were no different from ad 111 control levels (Table 8). Mg ATPase activity was reduced somewhat but not to the extent as that caused by TCDD; Ca ATPase was actually increased. It is interesting to note that the behavior seen in these animals reflected that of the TCDD treated; pilo erection, cage huddling, lack of interest, and uncommon aggressiveness were obvious. 61 Table 8. Activity of liver plasma membrane bound enzymes and receptors from rats maintained on limited diets or fed ad libitum for 10 days after dosing with acetone:corn oil. Activity Membrane Parameter Control Starved Na-K ATPasea 1496 1 142 (3)b 1524 1 101 (3) Mg ATPasea 1820 1 10 (2) 1413 1 19 (3)* Ca ATPasea 608 1 65 (4) 829 1 42 (3)* EGF bindingC 32 1 9 (5) 37 1 1 (2) Insulin bindingc 8 + 1 (7) 13 + 12 (2) :Enzyme activity is as defined in Table 6. Results expressed as mean 1 SD of the number of animals in parenthesis. Data analyzed with a two tailed student's "t" test. *P<.05. Receptor binding is as defined in Table 7. 62 Effect of TCDD on Enzymatic Activity in the Guinea Pig and Hamster To ascertain whether the liver is affected by TCDD in other species, 4 enzymes were measured in the guinea pig and 3 in the hamster 10 days after treatment with either corn oil:acetone or TCDD (Table 9). Neither ATPase nor protein kinase activity in either animal was significantly affected except guinea pig Mg ATPase which tripled with dioxin treatment. TCDD treatment to the rat decreased Na-K, Mg, and Ca ATPase in a dose dependent manner (Figure 15). In the guinea pig only Ca ATPase was dose dependent, however only at the higher doses was activity below control levels (Figure 16). The Na-K and Mg activity showed no dose dependence in this species. The hamster was unique in that increasing doses of TCDD increased activity of all 3 enzymes (Figure 17). DISCUSSION TCDD caused many functional changes in the rat hepatic plasma membrane. Na-K, Mg, and Ca ATPase were decreased 41, 39, and 44% respectively, 10 days after a single dose of 25 ug/kg. Con A binding declined 23% and EGF binding 88%. However, insulin binding showed an initial increase of 44% before becoming 60% of control levels at 20 days after treatment. The largest change produced by this chemical was in protein kinase activity which increased 274% and 308% in 63 ozu ch3 coux_mcm mum: Mama .mo.vm zuHS HOHDCOU m>Huomdmmu eoum ucmuwmeo aHmmoHumHumumc .cmcHEumumn yo: I 020 human .0: m.u:mosum cmmamu .mHmmcucmumd cH mHmEHcm mo Hones: ecu no“ 0m + come no ommmmumxm muHsmmm .o mHnme :H mm pmmmmumxw >HH>HHUHuoa nonmem: umm mH mocHso oHumEhucm moHowmm “mama when oH .ocmunEmE .mHo>Huoodmou nave mx\ma oooo no .mm .H nqu acmEummuu mamde oHumdm: uwumEm: no .umu .mHm mmcHsm H0uucoo mo >HH>Huom mexucm .m QHDMB 64 Figure 15. Dose response of rat hepatic plasma membrane Na—K (A), Mg (I), and Ca ATPase (0). 32. 3.3383 65 I] 8 mucosa e a... % Cogtrol 1"} 0" —T——’ "——i so .‘ Dose TC DD (pg/kg) E Figure 16. Dose response of guinea pig hepatic plasma membrane Na-K (o ) , Mg ( 0 ) , and Ca ATPase ( O ) . 67 .1 I 063.: mo cam . A . V m: . A o a 8762 0:38:05 MEmMHm UHHmmmn HoumEmn mo mmcommmu moon .hH musmHm 645.: coup 030 82 080— 0000 00— o— _ H A L 0 ll mm .% H 5 I m ml (1 mu 4 H III IA III. F IIH IIIRI“ MET-TH H mm. 68 the presence and absence of c-AMP respectively. At day 10 only gamma-glutamyl transpeptidase activity was not significantly different from control levels. All of these differences were noted at 2 days, reached their maximum at 20 days post treatment then appeared to begin a return to normal levels as mirrored by protein bands in the electrophoretogram in Figure 7. This may or may not be a true reversal since at 40 days after administration only a fraction of the treated population survived. An approximate LD50 dose was used in these experiments, therefore these animals could be a subgroup of rats more resistant to the effects of dioxin. Rats exposed to higher doses should be used to answer this question in future studies. To answer the question of whether these changes in plasma membrane function are associated with any toxicant which causes general stress, the effects CM? 5 other toxic chemicals on these membrane parameters were examined by D.W. Bombick. Rats were treated i.p. daily, for 10 days with 9:1 corn oil/acetone, 3-methylcholanthrene (20 mg/kg), Aroclor 1242 (50 mg/kg), phenobarbital (120 mg/kg), DDT (0.3 mg/kg) or Firemaster B-6 (i.e., PBB, 50 mg/kg). At the end of the 13 y1yd treatment, hepatic plasma membrane was isolated and the extent of Con A binding was measured. Levels of specific binding as expressed in terms of ng/SO ug plasma membrane protein were: control 17.0 1 2.0, 69 3-methylcholanthrene 21.0 1 4.9, Aroclor 1242 18.4 1 2.1, phenobarbital 10.6 1 1.8, DDT 13.9 1 1.2 and Firemaster B-6 19.5 1 6.6 (mean 1 standard deviation of 3 animals except for DDT and Firemaster experiments where 2 animals were used). Only phenobarbitol significantly reduced Con A binding from control levels (P10.05). Therefore, these results are not due to general stress or microsomal induction 3J1 these animals. Moreover, they are not due to severe losses of body weight since membrane preparations from starved animals show enzymatic activity and receptor binding similar to those from ad 111 controls. The purpose of this investigation was to survey how widely such TCDD-induced changes occur, following an observation where the levels of many protein constituents of the rat liver plasma membrane were altered as a result of “TCDD exposure (Brewster et al. 1982). It is difficult to correlate many of the i_n 111.19 changes observed here with the 1a 1119 symptoms of dioxin toxicity. It is postulated that many other changes are occurring that have not been elucidated in this study which may result in toxicological consequences if they occur at critical biochemical sites. Although the results of this investigation do not indicate the cause of these changes or which of the changes are causally related to TCDD's toxic action, the phenomenon of the alterations in plasma membrane functions as a result of TCDD exposure merits further consideration. For 70 instance, some of the altered enzymes and receptors found in the current investigation are known to carry out very important physiological and biochemica 1 functions . Ca-ATPase and Na-K ATPase are involved in the transport of Ca2+ and Na+ across the plasma membrane, respectively. Insulin receptor certainly is a vital system controlling carbohydrate and lipid metabolism and body weight homeostasis. The observed large sustained membrane phosphorylation induced by TCDD could be critical since the effect of protein kinase activity on cellular biochemistry is well known. In addition to the regulation of many critical pathways, kinase activity is important in the cellular response to growth factors and hormones (Schlessinger et al. 1982; Hayden and Severson 1983). Some of the kinase actions regulate surface receptors which in turn control total cellular homeostasis. The change in EGF binding is thought to be due to down regulation of the receptor as a result of increased receptor phosphorylation within the cell after TCDD treatment (see Madhukar et al. 1984). Therefore some of the symptoms of TCDD toxicity may be associated with changes in the EGF receptor which is critical in the hyperplastic response and in the inhibition of terminal keratinocyte differentiation as shown by Rheinwald and Green (1977) and Knutson and Poland (1980). Other symptoms of TCDD toxicity which are 71 associated with EGF include: fatty invasion of the liver (Heimberg et al. 1965; Gupta et al. 1973), promotion of skin cancer (Rose et al. 1976, Poland et al. 1982), conjunctival cell proliferation (Cohen. and. Elliott 1963, Norback and Allen 1973), inhibition of gastric secretion (Todaro and DeLarco 1978, Norback and Allen 1973), and serum hypertriglyceridemia (Heimberg et al. 1965, Albro et al. 1978). No explanation is offered at this time for the cause of the plasma membrane alterations. TCDD is known to bind with a cytosolic receptor (Poland and Glover 1976; Poland and Kende 1976; Nebert 1979) and transported into the nucleus. Therefore, it may be reasonable to assume that the changes in the plasma membrane enzyme and receptor activities are also triggered by such induced DNA pleiotropic responses (or altered gene expression) rather than the result of TCDD's direct interaction with. the plasma membrane. At least, there is evidence that the reduction of Na—K ATPase activity, as a result of 1a 1113 administration of TCDD, is not due to a direct interaction of this chemical with the enzyme itself (Peterson et al. 1979a) since the amount of TCDD found in the plasma membrane after in vivo administration is not sufficient to cause inhibition of this enzyme 11 vitro. 72 Conclusions In conclusion, many physiological homeostatic mechanisms are dependent upon cellular surface functions; therefore, alterations in the cell surface could conceivably lead to some toxic manifestations of TCDD. Because cell surface membranes are functionally different in different tissues, species, and sex, it is reasonable to expect differential manifestations of toxicity in different organs and animals. This conclusion is at least partially confirmed with the results of the enzyme assays in the guinea pig and hamster, where very few changes were seen nor was a dose response evident. This is in stark contrast to the rat where a good dose response relationship was observed and all the enzymes were reduced. The guinea pig and the hamster liver did not show many signs of toxicity after TCDD administration, therefore these results are not totally unexpected. CHAPTER IV EFFECTS OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN ON THE THYMUS INTRODUCTION Thymic involution is one of the most consistent pathological changes observed. in different species after administration of TCDD (Gupta et al. 1973, Harris et al. 1973, V05 et al. 1973, Kociba et al. 1976, Neal et al. 1979, Van Logten et al. 1980). Zinkl et al. (1973) noticed a loss of lymphocyte numbers in the thymus and theorized this to occur from a decrease in precursor cells or a direct cytotoxic effect on mature cells. They further hypothesized that reduced precursor cells could be the result of depressed stem Cell division or an inhibition and blockage of the maturation steps leading to mature T lymphocytes. However, Kociba. et (al. (1976) concluded. that involution resulted from increased lymphocytic migration to the thymic medulla followed by secretion of cortical thymocytes into the cardiovascular system. Faith and Moore (1977) observed that TCDD caused an impairment of the thymus dependent immune system through differential suppression of T cell subpopulations. No effect was found on the humoral system. This confirmed the results of earlier studies suggesting that animals 73 .74 administered TCDD showed increased sensitivity to bacterial infections (Vos et al. 1973, Thigpen et al. 1975, V05 et al. 1978). However, this depression of cellular immunity is not responsible for lethality since Greig et al. (1973) found that germ free environments for TCDD treated animals did not prevent death; nor did death occur from massive bacterial infection in animals exposed to the normal atmosphere. 19 11119 evidence does not suggest TCDD to act directly on lymphocytes since cultured cell lines showed no effect when exposed to this dioxin (Knutson and Poland 1980). 19 11919 metabolic activation appears unnecessary since TCDD is capable of producing cytotoxicity in several other mammalian cell types (Niwa et al. 1975, Milstone and IeVigne 1984) without the addition of metabolic activators. Furthermore, there is little evidence in the literature to suggest that TCDD needs activation 19 1119 in order to produce toxicity. The mechanism for this thymic involution has not yet been elucidated. Adrenalectomized animals treated with TCDD did not reverse this atrophy, therefore increased levels of serum corticosteroids do not seem to be involved (Neal et al. 1979, Van Logten et al. 1980). Van Logten et al. (1980) also present evidence eliminating the pituitary and thyroid glands. Because of the marked sensitivity of the thymus to TCDD a brief examination of the thymocyte membrane was undertaken 75 in an effort. to understand how' dioxin. may act in this organ. Perhaps the thymus could then be used as a model to discern the biochemical mode of action of this chemical in other organs showing manifestations of toxicity. MATERIALS AND METHODS Thymocyte Isolation and Plasma Membrane Preparation Male Sprague-Dawley Rats were housed and treated with TCDD as before. Thymocyte isolation and plasma membrane fractionation were performed according to Koizumi et al. (1980). Procedures for Scanning and Transmission Electron MicroSCOpy are outlined by Hooper et al. (1979) and were performed by Dr. Stan Flegler and Dr. Karen Klomparens-Baker respectively, from the Center for Electron Optics at Michigan State University. Biochemical Assays Determinations of Na-K, Mg, and Ca ATPase activity, EGF and insulin binding were as before. Glucagon binding was performed as described by Matsumura et al. (1984). Glucose uptake was determined by adding 0.5 ml of 1 mM D-glucose (in 14C-D- reaction buffer) containing approximately 0.1 uCi glucose to 100 ug protein in 4.5 ml of saline- bicarbonate-acetate buffer pH 7.0. After 15 minutes at 37°C the reaction was stopped by filtration with Millipore HAWP (0.45 u) filters. The filters were then washed with 2-10 ml portions of ice cold buffer, dried, and quantitated by 76 liquid. scintillation.«counting. Final salt. concentrations for reaction buffer were: NaCl 115 mM; KCl 5 mM; CH3COO-Na+ 10 mM; NaH PO 1.2 mM; and 2 4 25 mM. 1.2 mM; CaCl 1.9 mM; MgSO 2 4 NaHCO3 RESULTS Electron Microscopy The thymocyte surface was markedly affected by TCDD 10 days after dosing as shown by the scanning electron micrographs in Figure 18. TCDD caused a reduction in surface pits in all samples examined and increased bleb formation or cellular extensions. Bleb formation resulted in an obvious ruffling of the thymocyte as opposed to the relatively smooth surface of cells from control animals. Transmission electron microscopy revealed a loss of cytoplasmic material and an obvious sloughing off of the outer surface of the cells (Figure 19). Preparations from four different control or treated animals were examined. Biochemistry Two days after treatment, there was no significant change in total thymus weight or thymic weight relative to overall body weight in TCDD treated animals compared to controls (Table 10). After 10 days, thymic weight had been reduced 80% by TCDD. Data shown in Table 11 indicated little effect of TCDD on these biochemical parameters (ME the thymocyte membrane. 77 Figure 18. Scanning electron micrographs of rat thymo- cytes isolated 10 days after administration of either ace- tone:corn oil (A) or 25 ug/kg TCDD (B). Note the pits (ar- rowheads) and lack of cellular extensions in the control cells compared to the treated. 79 Figure 19. Transmission electron micrographs of rat thymo- cytes 10 days after administration of either acetone:corn oil (A) or TCDD (B). Note the reduction in cytoplasmic ma- terial, the tendency for increased cellular projections, and the sloughing off of the plasma membrane (arrowheads) in cells from a treated animal compared to control. 81 .Hoo.vm Hm mHouucoo o>HuummmmH eouw HcmummeU eHucmonHcmHm« .Hmmu =u= mucmcsumlcmHHmu 03» mch: nmNeHmcm .mHmmzucmHmm :H mHMEHcm Ho Henson ecu How am + come we Ummmmumxm mHHsmmmm sumo «AHHLmo.o H mo.o AmcHo.o H 4H.o Loveo.o H a~.o Laemo.o H 8H.o uanmz econ m we msewze IAHHVGo.o H oH.o Amiao.o H Hm.o Asimo.o H mm.o miscmo.o H ov.o Am. uanmz unease memo 0H when m when 0H mama N Dove Houucoo .coHumnuchHEUm “mums mama OH cam N am bamHmz magma» co aaoa mm\m: mm 96 muoommm .cH 0Hnma 82 .cHE om\cHODoue m: om\ccson commosHm no .cHHsmcH .momIH mo em .mHmwnucmem :H mHoEHcm mo Hones: may mo 0m + com: .owcHemmwmo #020 .Q G Asvmq.H H ve.m Lasso.m H 64.4 02 Away :Hmpoum mcmunEmE mEmMHm mo UHOHH oz mmmm mHom Isoov mxmoo: mmooooo mm.H mm.MH oz 92 omCHocHn commusHU Amvnm + H.mm Amvm + m.om oz oz omcHUCHn cHHsmcH m.mm ~.mm «Hm mHH omcHocHo mom om oom om mmH Ame\omumu0oHH Mo zoo mmmoea mo AmomsH H omHH Amoma H NamH mas OHe Aos\omomumoHH .o zoo ammoea oz AmoomH + amaH mANCGOH + mHmH mom mom Ams\omumu8oHH .o zoo mmmoaa xImz oooe Houuooo oooa Honuooo mcmubemz wuwooemze .oooe mx\ms mm no HHo :Hooumcoumom SHHz Hameumouu Hmawm meow OH @mumHomH ocmunEmE mammHm mumooemnu Ho mmuwooemnu mo mnmuwemnmm HMOHEOQOOHQ msoHHm> co Dove mo powwmm one .HH mHnma 83 The increased ATPase activity of the plasma membrane compared to whole cells indicate that membrane was indeed isolated. The lack. of replicates and large variability makes other conclusions difficult. DISCUSSION The thymocyte surface is visibly altered 10 days after treatment with TCDD. The loss of pits in cells from treated animals may indicate changes in gap junctional areas or alterations in membrane regions important for nutrient uptake. If gap junctions are affected, these cells have lost the ability to communicate with each other; if nutrient uptake: occurs through. these ‘pores, these cells would. be nutritionally starved. In either case drastic influences throughout the body may be observed since one of the roles of the thymus is postulated to be trephocytic (Metcalf 1966). Metcalf presents convincing evidence that thymic lymphopoiesis occurs to provide cellular building blocks to tissues throughout the body. Metcalf (1966) and Fisher (1968) explain that under stress and infection T cell derived lymphocytes are released into the bloodstream via thymic dissolution, they are then transported to peripheral areas: of the body' where disrupted ion. occurs and their cellular contents reutilized Eur tissues iJI need. Tracing radiolabeled lymphocytes has confirmed this to occur (Metcalf 1966, Russkanen and Kouvalainen 1973, Ernstrom et al. 1973). Therefore, ii? cellular communication is 84 inhibited and thymocytes are energy deficient, the severe wasting syndrome produced by TCDD is implicated. It is interesting to note that neonatal thymectomy results in severe body weight loss similar to that produced by TCDD (Parrott 1962, Hess 1968, Faith and Moore 1977). The bleb formation and pit disappearance induced by TCDD may be due to disturbances in intracellular calcium regulation. Jewell et al. (1982) report the formation of hepatic cellular extensions as a response to changes in extramitochondrial calcium ions and theorize an inability of the hepatocyte cytoskeleton to maintain surface morphology. Increased levels of cellular Ca also obliterate gap junctions and inhibit cellular communication in hepatocytes as shown by Peracchia (1978). Conceivably, these same mechanisms could be occurring in the thymus. Data presented here indicate: a significant loss of thymic tissue after 10 days of treatment but not after 2 days. Biochemical studies are difficult to perform on isolated plasma membrane from this organ after TCDD treatment as little material is available for study. Perhaps lower doses or earlier time points should be used to ascertain biochemical disturbances in membrane parameters related to involution. Glucocorticoid transport and glucose uptake should be examined in the thymus. Glucocorticoid binding is regulated by Ca and causes the induction of a protein which inhibits 85 glucose transport and thereby results in thymic involution (Foley et al. 1980, Homo et al. 1980). Perhaps dioxin acts in a similar way; either by altering intracellular Ca, inhibiting glucose transport, or inducing the glucose transport inhibitory protein. Since tine thymus contains a very high concentration of the TCDD cytosolic receptor (Carlstedt-Duke 1979, Mason and Okey 1982) effects of TCDD should be able to be demonstrated in this tissue. CHAPTER V 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN INHIBITION OF GUINEA PIG ADIPOSE LIPOPROTEIN LIPASE ACTIVITY AS CAUSE FOR SERUM HYPERTRIGLYCERIDEMIA INTRODUCTION TCDD is unique in that it produces differential responses in different animals and different tissues. The guinea pig has been shown to be the most sensitive mammalian species to this dioxin (Gupta et al. 1973), but its response is unusual because the liver shows very little pathology. Data presented in Chapter II .confirm this observation. Gupta et al. (1973) could find neither, proliferation of endoplasmic reticulum, increased fat deposition, nor edema formation as had been observed in rats. No induction of ndcrosomal enzymes, arylhydrocarbon hydroxylase, or ALA synthetase has been reported (Neal et al. 1979). Therefore, these hepatic biochemical pathways appear not to perform a major role in TCDD's toxicity in this species. Gasiewicz and Neal (1979) and Swift et al. (1981) observed one of the most significant toxic manifestations in guinea pigs dosed with TCDD to be serum hyperlipedemia. Serum triglyceride and cholesterol concentrations began to increase as soon. as 3 days after treatment. Hypertri- glyceridemia is a major factor in coronary heart disease and 86 87 atherosclerosis (Sirtori et al. 1975, Hulley et al. 1980, Cabin and Roberts 1982) and may therefore have a (great impact on TCDD treated laboratory animals. In fact, preatherosclerotic lesions appear in aortic vessels of rabbits as soon as 20 days after treatment with TCDD (D. Bombick, unpublished observation). Injections of heparin containing blood were found to abolish severe lipemia in dogs (Hahn 1943). This observation and other studies (Weld 1944, Anderson and Fawcett 1951) implied that lipemia is reversed by the release of some "factor" into the serum upon administration of heparin (Hamosh and Hamosh 1983). Korn (1955) demonstrated this factor to be an enzyme which hydrolyzed serum trigly- cerides. Subsequent studies showed this enzyme to be synthesized in extrahepatic tissue (especially adipose), secreted into the blood, and attached to capillary endothelial cells where its function is the catabolism of triglyceride carrying lipoproteins (see Robinson 1963a, Garfinkel et. al. 1967, Hamosh. and. Hamosh 1983). For a comprehensive review of lipid metabolism see Nilsson-Ehle et al. 1980, Brown et al. 1981, Oscai and Palmer 1983, and Brown and Goldstein 1984. Because adipose LPL has a critical role in controlling serum triglyceride concentration and adipose uptake of free fatty acids the effect of TCDD on this enzyme was examined in the guinea pig. This is the most sensitive animal to 88 TCDD in terms of lethality, body weight loss, and serum hypertriglyceridemia. MATERIALS AND METHODS Young (200-225g) male Albino English Shorthair Guinea Pigs (Strain MDH:SR(A)) were purchased from the Michigan Department of Health and male Sprague Dawley Rats were obtained from .Harlan. Animals, Haslett, Michigan. Animals were dosed once intraperitoneally (ip) with either TCDD (obtained. as a: gift. from. DOW’ Chemical Company, Midland, Michigan -"- purity >99.99%) dissolved le acetone:corn oil (1:9) or vehicle alone. Animals were housed in suspended stainless steel cages and given food (Purina Guinea Pig or Rodent Chow) and water (supplemented with ascorbic acid) ad libitum. Where pair-fed studies were conducted the control animals received only the amount of food consumed by the TCDD treated animals. At the indicated time periods animals were anesthetized with CO2 and blood was collected via cardiac puncture. The blood was allowed to coagulate, then serum was collected after spinning 15' at 8000 g and stored at ~700C until use. Preparation of Acetone/Ether Fat Powders Abdominal and perirenal fat tissue was removed, washed with cold 0.85% NaCl, and extracted with acetone and ether according to Robinson (1963b) except, the tissue was ground in 20 volumes of cold acetone then extracted with 5 volumes 89 of room temperature acetone followed by 2 volumes of ether. The parchment like filtrate was air dried and stored in a dessicator at -20°C until use. LPL Assay The enzyme source was extracted out of the fat powder, and lipOprotein lipase activity was measured with the procedure described by Nilsson-Ehle and Schotz (1976). Serum triglyceride concentrations were determined. with the colorimetric procedure set forth in Sigma Chemical Company Bulletin #405. Glycerol tri [9,10(N)-3H] oleate CL Ci/mol) was pmr— chased from Amersham, Arlington Heights, Illinois. All other reagents were of analytical grade and purchased from Sigma Chemical Corporation, St. Louis, Missouri. Data are expressed as mean 1 SD for at least 5 animals or as indicated. Statistical analysis was performed using either 1 way or factorial ANOVA and means compared with either Dunnett's or Tukey's test for unconfounded comparisons. In all cases P<.01. RESULTS Wasting and Slobbering TCDD caused the typical wasting syndrome and loss of body weight as described previously. As seen in Figure 20 90 Figure 20. A. Typical appearance of a control and (B) a 1 ug/kg TCDD treated guinea pig 10 days after administration. C. Severe Slobbering effect induced by TCDD. 92 these animals also exhibited abnormal posture, pilo-erection, hair loss, vasodilation of tune extremities, and a severe drooling or slobbering effect. Time and Dose Changes of LPL Activity and Serum Triglyceride Concentration Ten days after treatment with 1 ug/kg TCDD, adipose LPL activity was onLy 18% that of control animals (Table 12). Concurrently, serum triglyceride levels were almost tripled. Body weight and adipose weight were 67 and 31% of control weights respectively. After 20 days of treatment very little .adipose 'tissue :remained. in. the TCDD treated animals compared to the pair-fed control (Figure 21). These changes were both dose and time dependent. Doses greater than 0.5 ug/kg caused significant losses in LPL activity resulting in an EDSO of 0.6810.06 ug/kg at 10 days after treatment (Figure 22, Table 13). The ED50 for loss of adipose weight was approximately 0.5 ug/kg, however for loss of body weight it was 3.2710.27 ug/kg. Enzymatic activity was suppressed as soon as 1 day after treatment when body weight gain began to decline and serum triglyceride levels began to rise (Figures 23-25). As shown in Figure 24 TCDD caused a severe loss of body weight beginning one day. after a single i.p. administration. Because adipose LPL activity decreases upon starvation (Nilsson-Ehle et al. 1980, Hamosh and Hamosh 93 .Ho.v o .Hosuooo emeIsHmo sous nooseeeHo HHHmoHumHomom 4. .Ho.v a .Hosuooo mHH mm sose oomsmeeHo HHsmoHomHomom 4 .<>oz< HeHHouoem ueume ecomHHemeoo eeecsomcooco How Heep m.mexse buH3 UeNHHece mes memo .mHeEHce Adv MOM :oHueH>ee Queeceum H :eee ecu me cemmeumxe eue muHsmem I .H:\He©3om new eeuoeuuxe me\eHoe auuem eeuw meHosc Amvm.mHH + m.m>mH me3 when o He >HH>Huoe HmH .HeueH muson m erHmHuoem mHeEHce cenu AcoHusHom mmn mo HE >.mv mHHeHo ce>Hm mez emoosHU D U .H£\Hee3om pew eepoeupxe mE\©Hoe OHeHo mm meHoscm .AmHos.o H o.H 41amos.o H H.H Aomom.H H o.m Ame nooHez emooHoa I I I AHmHuHoH we «Amom + mOH 44.41HHom + ma AoHom + NsH noonz Hooo I I I AHo\oso msHs .AaosH + am 11.41soas + mmm 141mm + em IwomHmHse sssmm Asso.mmm m m.mmoH Asom.HH m m.osm om ommoosHm Hmso + 4imoa.mmH + m.mHs .HHH14.Hm~ + maHam c.01moH.amm + m.mmH~ mHDH>Hoo< ooo Hosnsoo seeasHmo oooe Hosuooo oHH mm uceeueene .Qooe mx\ms H Ho HHo :Hoo "ecoueoe HecuHe :uH3 uceeueenu ueume when OH mmHm eecHzm Houucoo UeMIHHeQ cam 4eeueeuu Dave .Hogpcoo mme mm :H uanez emomHoe ace .uaneB moon .coHueHuceocoo eeHHeoeHmHnu EDHem .euH>Huoe HmH emomHU< .NH eHneB Figure 21. Abdominal dissection of (A) pair-fed control and (B) TCDD treated guinea pig, (20 days after 1 ug/kg sin- gle i.p. dose). Note severe loss of perirenal and abdominal body fat (arrowhead) in the treated animal. C-descending colon, F-fat, K-kidney. 95 96 Figure 22. Dose response relationship of LPL activity (0) and body weight.( A)a(as % control) 10 days after a single i.p. dose of TCDD. tatistically different from con- trol LPL activity or control body weight at P <.01. 97 38:83 mmoo .-os r «Iow 6-0? om OOH TOHINOO% 98 .Ho.v o as Amx\ms o. .¢>OZH Heume Mme» muueccso >2 ceNeHece even .mHeEHce Ace How :oHueH>ec eueeaeum + sees me eemmeumxe euHHHneHHe>e .UecHEHeHeU uoz Hosuooo sose usesmeeHo HHHmoHumHomum4 n Izvoo.o H m.o 4AeomH + as oz 4isos.HvH H m.asm 0.4 .zmmva.o H o.H «AHHCNH H ma «zooms H mmm 4AHHCH.HmN H a.Hmm o.H IAmCH.o H o.H imom H NHH imam H HH Iimvm.mmH H m.masH m.o Leva.o H m.m zeoo H HHH .mooH H mm isom.aeH H m.moom OH.o Amos.H H o.m AmosH H amH oz Amom.mHm H m.smmH mo.o Ascs.o H s.m Leos H SHH ooz Asia.sv H o.ssm~ Hoo.o onvm.H H mH.m AoHcm H msH 141mm H as siaoH.mmm H m.amHm o Amsmsmo iHmHonH we AHo\os. Aso\ume omuomsuxe zmx\msv uanez uanez eeHueomHmHue ms\Huo< Hog .coHueuu IchHEUe nave Heuwe whee oH uane3 emomHee nae .panez econ .ooHueHuceocoo eeHHeOSHmHHH EDHem .>HH>HHoe HmH mo mHnmcoHueHeH emcommeu emoo .MH THEME 99 .AHO.n my mCOeHHemEoo neecsowcoocs How uweu m.>exse mchs .Houucoo eemIHHem men OH Eoum uceHeHMHe hHuceonHcmHm “Homecoo eemIHHem EOHM HaeuemmHe mHuceonHcmHm uozmU “Houuaoo eewIHHem EOHm uceuemee wHuceonHcmHm «mHoupcoo MMfl mm EOHH pceuemeo eHuceonHcmHm .emzoune.eemv HepeH mason m eeusmees >HH>Huoe eeeuce Use UmuepchHEee mHHeHo me3 emoosHm mmn M NO HE >.m .OH wee :0 .mmHm eecHsm Adv Houucoo new uHmo so Joe Hofiooo .fl m... .AOO 86.8.3 oooe :H memo OH use .N.H.O He AHn\HeU30m wepoeuuxe OE\UemeeHeH UHoe OHeHo mm EGO euH>Huoe HmH emomHe< .mm enamHm Am><9 msE. 0.. m W1 _ m L O P lhnxumw SSOIHHOA‘IOIHI 103 1983) the effect of pair-feeding on these parameters was assessed. After 10 days, enzymatic activity in the pair-fed animals was significantly less than ad 119 animals as expected, and no difference existed between TCDD treated and pair-fed guinea pigs (Table 12, Figure 23). Serum triglyceride levels had increased 8 fold and body weight had decreased by 10% (Figure 25) compared to pair-fed animals. It was observed that pair-fed control LPL activity decreased at a much slower rate than did the TCDD treated (Figure 23), since after 1 day the treated animals had already fallen 83% while the pair-fed only 39% relative to 0 day control levels. At the same time serum triglyceride levels began to rise dramatically in the TCDD animals but not in the pair-fed controls. Glucose Effect on LPL Activity Large doses of glucose 19 1119 are known to reverse the starvation induced depression of adipose LPL by stimulating synthesis within the cell (Cryer et al. 1974, 1976). Therefore, if reduced food intake was causing the decline of activity in the TCDD treated animals, the effect should be able to be reversed by the administration of glucose. No such effect was seen. Although the pair-fed animals were greatly stimulated to near ad 119 control levels, no change was noted in the treated animals (Table 12, Figure 23). 104 Serum Apoprotein Effect on LPL It is well known that LPL is stimulated by some serum apolipoproteins (Apo C-II in rats) and inhibited by others (Brown et al. 1981, Hamosh and Hamosh 1983). To assess whether the TCDD induced decline of LPL resulted from alterations in these serum factors, enzymatic activity was measured using various combinations of control, TCDD treated, and pair-fed control guinea pig or rat serum as the activation source (Table 14). LPL is defined as an enzyme activated by APO-CII and inhibited by high ionic concentrations (Hamosh and Hamosh 1983). Data in Teble 14 clearly indicated that adipose LPL was being measured since enzyme from control, TCDD, or pair-fed animals was markedly increased by the addition of rat serum (compare lines 1 and 2 of Table 14) but significantly inhibited by 1.0 M NaCl. Guinea pig serum alone contained IN) stimulatory factor in agreement with Fitzharris et al. (1981). Serum from TCDD treated guinea pigs (10 day 1 ug/kg) stimulated activity from control, and pair fed control but not from treated guinea pigs. Because serum from treated animals has high levels of triglyceride carrying lipoproteins such as VLDL and chylomicrons, this stimulation was not unexpected since the apoproteins of these lipoproteins can activate LPL. Enzymatic activity of fat powder from TCDD treated animals could not be stimulated using any serum combination, (compared to both controls) nor was control activity 105 .eecHEHeuee uoze .Huez z O.H :oHueupceocoo Hech .mHeEHce AGO How coHueH>ee eueeceum H :eeE may we eemmeumxe muHsmemw .Amwe ou. Esuem mHm eecHsm eewIHHem wee OH H meme «semen OHQ eecHsm mx\ms H wee OH u ewe “Esuem Hen Ame ouv eeMIHHem eee OH u mmm «Esuew Hen QQUB ox\ms mm wee OH n me “EDHem Hen Houpcoo u mo “semen OHQ eecHzm Houucoo u moo .emoe QH eHOQHm e Hepme whee H OH mHouucou eeuIHHem Ho .eeueeuu move mx\ms H .Houucoo new QHH wee Amos.s4 m ~.4mm A4Om.mmH w H.0H4 i4os.mmH m s.em4m ms imio.Ha + 4.mmm AeOH.emH + m.~am L4O~.m~H + e.msmm mo oz oz A40m.m~H m e.esmm mo + memo oz eoz A40s.s4 + m.mm~ memo om Amo4.oa m e.em~ i4oa.s4H m a.mmem mam + owe AHOm.me H m.osm AHOH.mH H H.m4H L4OH.me~ H ~.em4~ ms + mes zmvo.H4H H o.omm A4Oo.amH H 4.Hme A4Oo.mmm H m.HHmN mo + see AHOH.mm + H.4Hm isom.Hm + a.mHH A4OH.NHN + m.eNOH owe imOH.mm M e.H~m Amom.0HH M m.4mm 14.0.msH m m.4aem mam + moo Amoa.oe H ~.mme Amos.mmm H m.mom 14.x.4Hm H H.Noem ms + moo Aeca.HN H m.m4 Amoe.4 H N.mH Amom.o~ H 4.4m eHoez + mo + moo A4Om.mm H H.4ee ANHOm.m4N H H.444 zeOH.mem H m.~emm mo + moo AOHOH.HH + H.mm AHHOe.mN + H.me oimHos.4m + O.HmH 1H8>mH Hemeoo oeo HOHHQOO eewIHHem nave Houacoo QHH ee nuoue>Huoe AH£\Hee3om new eeuoeuuxe OE\eHoe oHeHo mm zcv e>HH>HHo< HmH mHm eecHoO .HmH OHQ eecHsm Honueoo eeMIHHem ece .eeueeup Dove .Houucoo mo muHHHneeeo msHumHouee: eeHHeomHmHHp :o mueu ese mmHm eecHsm Homecoo eeMIHHed eee .eeueeuu Dave .Houecoo QHH ee soum mHOHoew Eduem mo muoewmm .«H eHnee 106 inhibited by treated serum. Therefore the changes in LPL activity are not due to alterations in serum factors. Regression analysis of LPL activity on body weight revealed a coefficient of determination of 0.82 (Figure 26). Therefore 82% of the variance in body weight is explained by variance in LPL activity. DISCUSSION TCDD administration to guinea pigs produced the typical wasting effect described for other laboratory animals. It aso caused a severe drooling or slobbering effect. This slobbering effect, or "slobbers" as discussed by Navia and Hunt (1976) is normally' due to abnormal wearing' of the incisor teeth thereby allowing the teeth to overgrow and prevent normal eating and swallowing functions. As a result the animal usually starves to death. If TCDD does indeed act through the epidermal growth factor receptor as discussed by Madhukar et al. (1984) quite possibly alterations are occurring in dental growth to cause abnormalities in swallowing and drinking. TCDD significantly increases the time to incisor eruption in mouse neonates thereby mirroring the same effect caused by exogenous administration of EGF (Madhukar et al. 1984). Quite possibly, this dioxin also has toxic manifestations leading to "slobbers". TCDD also caused a time and dose dependent depression of adipose LPL activity resulting in 107 O / r 0 / if or / g o * D / 2000 - % i? [3 lfifi * 3.. 3'37 2: Z / '— / 2 /Q m i} M l < / & /'> _a i! z / r: / o 1’ 8 8 / o. 1000 I t— /' I / I . . If ll/ /' f = .91 // A r X l//, ‘A ... . x . ° 0 A xx f. d 100'. // 0 o l . l i _____ 60 100 140 % INIIIAl BODY WEIGHT Figure 26. Relationship of body weight and adipose LPL activity. 10 day ad lib control (c>), 10 day control pair-fed to l ug/kg TCDD (A.), 10 day TCDD treated at 0.001 (¢), 0.05(D),0.1(*), 0.5 (7dr),1.o (0), and 4.0 (x) ug/kg TCDD. 108 the severe serum hypertriglceridemia seen in this species. Associated with this loss of enzymatic activity was inhibition of body weight gain and loss of adipose tissue. Pair-feeding had similar effects on body weight gain and adipose tissue loss. Restricted food intake however, was not. entirely' responsible for loss of LPL activity since pair-fed animals showed a slower decline of activity compared to TCDD treated and were able to be reversed after 10 days by a large oral administration of glucose. Serum from ad lib, pair-fed, and TCDD treated rats showed little difference in their abilities to activate enzymatic activity, therefore inhibitory serum factors (i.e. apoproteins) seemed not to be involved. Neal et al. (1979) concluded that there was no generalized impairment' of glucose absorption from the intestine of guinea pigs 7 days after treatment; with. 2 wig/kg TCDD. Seefeld and Peterson (1984) also iconcluded that. TCDD ihad little influence on intestinal absorption. Therefore it seems likely serum levels in the treated animals contained adequate glucose concentration to induce LPL activity. Serum triglycerides did not increase in the pair-fed animals because functional enzyme was still being produced by the adipocyte. The pair-fed control activity as measured by this assay was low because only the active pool within the adipocyte was assayed. LPL is synthesized as an inactive proenzyme then activated via post translational 109 modifications before or during secretion from the cell (Nilsson-Ehle et al. 1976, Ashby et al. 1978, Borensztajn 1979). This inactive pool can be activated by glycosylation (Hamosh and Hamosh 1983) as seen with the 10 day pair-fed control animals (glucose also increases 92 £922 synthesis of enzyme). Apparently no reserve enzyme Exxfl. is present in the treated animals. Cause for Loss of Adipose Tissue At least 5 possibilities exist for the cause of adipose tissue loss: (a) increased energy expenditure (b) decreased intestinal nutrient absorption (c) decreased production of hepatic and intestinal serum triglyceride-carrying lipoproteins, (d) increased hormone sensitive lipase (HSL) activity leading to lipolysis, and (e) decreased LPL activity and failure to deliver free fatty acids from the endothelial cells to the adipocytes. Neal et al. (1979) and Cunningham and Williams (1972) have indicated no affect of TCDD on overall energy production or consumption in experimental animals. TCDD did not alter l4CO2 production from radiolabelled glucose, oleate, or alanine, nor did it change fatty acid compositions, ATP levels, pyridine nucleotides, or their ratios in guinea pigs. Lucier et al. (1973) also found hepatic' mitochondrial respiratory activity' and subsequent ATP synthesis 1x) be the same: as controls. Consequently increased energy expenditure can not account for fat llO losses. Seefeld and Peterson (1984) concluded as did Neal et al. (1979) malabsorption of intestinal nutrients did not occur upon treatment with TCDD, because fecal energy loss (as a percentage of food intake) was no different between control and treated animals. It appears likely then, that nutrient depletion is not the cause for loss of adipose. Since TCDD increases serum concentrations of very low density lipoproteins and triglycerides, fat deposition should increase as more storage is necessary, therefore the third possibility can be eliminated. Increased lipolysis could account for the loss of adipose tissue since serum free fatty acid concentration is controlled by HSL through a feedback system coupled to LPL activity (Patten 1970). However, Swift et al. (1981) found no difference in serum fatty acid concentration between pair-fed control and treated guinea pigs. As a result, only decreased LPL activity and failure to deliver free fatty acids from endothelial cells tx> adipocytes can explain the loss of adipose tissue exhibited by TCDD treated guinea pigs. LPL and Serum Triglycerides There is little doubt that LPL controls serum triglyceride concentration thereby affecting fat storage (Robinson 1963a, Garfinkel et al. 1967, Robinson 1970, Borensztajn 1979, Nilsson-Ehle en: a1. 1980). Injection of antiserum against LPL totally blocks serum triglyceride removal from chickens in vivo (Kompiang et al. 1976). There 111 is also good evidence that reduction of LPL levels is directly correlated to hyperlipidemia in humans, e.g. among untreated diabetes and Type 12 familial hyper- lipOproteinemia patients (Huttunen and Lindquist 1973, Fredrickson et al. 1978, Walker and Martin 1979). Therefore, reduction in adipose LPL activity upon TCDD administration is responsible for serum hypertriglyceridemia and the loss of fat storage in adipose tissue. TCDD-induced "wasting syndrome" has always been observed to accompany loss of adipose tissue, and present data indicate that in these animals very little adipose tissue remained 20 days after 1 ug/kg TCDD. This loss of fat confirms results found by Swift et al. (1981). LPL is known to be synthesized in adipocytes and delivered to the luminal surface of the endothelial cells, where it is attached through a receptor (heparan sulfate). The quantity of LPL at this site is determined by a feedback system with serum insulin levels being the main determinant of the rate of _d_e_ £9572 synthesis of LPL in adipocytes (Nilsson-Ehle et al. 1980, Hamosh and Hamosh 1983). The failure of LPL from the TCDD-treated animals to respond to high levels of serum triglycerides or to exogenously added glucose in yiyg, as contrasted to LPL from pair-fed animals, indicates that its g2 £929 synthetic process may be inhibited. This could be from an impairment of the above feedback system. The reduction of LPL is not likely to be 112 caused by the direct interaction of TCDD with the enzyme itself as judged by the qualitative and quantitative similarities of LPL from treated and pair-fed animals (both at day 10, Table 14) and by analogy with the well studied case of the TCDD-caused reductions of membrane ATPases (Matsumura 1983, Matsumura et a1. 1984). Since LPL at the endothelial site has a very short halflife (Nilsson-Ehle et al. 1980, Hamosh and Hamosh 1983), the inability of the adipocyte to supply fresh LPL in the treated animals makes them incapable of adapting to nutritional changes and needs. Two major questions arising from this study are whether this conclusion is applicable to other animals and whether this phenomenon is causally related to the lethal action of TCDD. It is well known that patterns of serum lipoprotein metabolism are very different among mammalian species (Brown et al. 1981). Therefore, one must be cautious in drawing a direct analogy to other animal species. However, wherever serum hypertriglyceridemia is observed as a result of TCDD administration, one could at least suspect a reduction in adipose LPL activity as one of the causes. For instance, elevated serum triglycerides, phospholipids, and cholesterol have been frequently reported in. humans (particularly' among' industrial workers) with. a history' of exposure to chlorinated dioxin-containing products (Oliver' 1975, ‘Walker' and Martin 1979, Zack and 113 Suskind 1980, Pazderova-Vejlupkova et al. 1981). While such epidemiological data are often difficult to interpret, results of our current study and the similarities of human hypertriglyceridemia to that of the guinea pig (Huttunen and Lindquist 1973, Fredrickson et al. 1978) suggest the possibility of LPL reduction being the cause for these problems. It is premature to conclude that loss of LPL pe_r _sg causes death in the guinea pig as starvation itself is not expected to lead (x) death under these experimental conditions. However, there are additional factors to be considered. First, adipose LPL of treated animals is irreversibly reduced, as judged by the lack of effect of glucose, indicating that the affected animals have lost the ability to adapt to nutritional changes. Second, the serum of the treated animals contained very high titers of triglyceride carrying lipoproteins, while starved animals did not. Third, there was a concomitant increase in cholesteryl ester carrying lipoproteins in the treated guinea pigs probably as a result of reduction in LDL receptor activities on the plasma membrane of the hepatocyte (Bombick et al. 1984). The combined actions of these lesions could conceivably cause various secondary effects such as arteriosclerosis, hemorrhages, xanthoma and lipemia (Greten et al. 1976) which can be detrimental to the affected animals. In fact, in the above glucose test we 114 have observed that 4g of glucose/animal (instead of 2g/animal as shown in Figure 23) killed all TCDD-treated guinea pigs (4 out of 4) within 2 hrs. None of the pair-fed animals died. The cause of death was probably glucose shock. Such an effect illustrates the overall inability of the treated guinea pigs to readjust to nutritional changes. Conclusions The following mechanism of action is proposed: TCDD causes inhibition of adipose LPL, by some as of yet unknown mechanisnn which results in (decreased serum triglyceride breakdown (from both. dietary sources via chylomicra and hepatic synthesized sources via very low density lipoproteins). Consequently, serum levels of triglycerides increase and levels of free fatty acids being stored by fat decrease. Lipolysis proceeds normally' via HSL 'utilizing previously stored fatty acids and resulting in loss of adipose stores. In time meantime, since serum levels of triglycerides and fatty acids are elevated, normal feedback mechanisms may signal a reduction of food intake thereby contributing to body weight loss. After 20-25 days when adipose stores are exhausted protein catabolism begins, which coupled with secondary effects from hypertriglyceride- mia. and hypercholesteronemia could. conceivably' alter critical biochemical mechanisms and lead to death. This energy depletion is similar to that proposed by McConnell et al. (1978b). LPL is important in providing fatty acids to 115 muscle cells for energy production (Hamosh and Hamosh 1983). If this inhibition by TCDD occurs throughout the body one could then hypothesize severe muscular effects, such as protein. catabolisnt and. energy' loss 'taking' place. Future research should focus on the long term effects of TCDD with respect to both adipose and muscle LPL. Serum concentrations of insulin and glucose will be examined in an effort to determine why LPL activity is decreased by TCDD. Secretion of active enzyme will be decreased if serum insulin is depressed, the adipocyte insulin receptor is altered, or glucose transport across the adipocyte plasma membrane is inhibited. CHAPTER VI DIFFERENTIAL SPECIES RESPONSE OF ADIPOSE LIPOPROTEIN LIPASE TO 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN INTRODUCTION Serum triglyceride concentrations have been reported to be unchanged in the rat (Albro et al. 1978) and hamster (Olson et al. 1980), but significantly increased in the rabbit (Lovati et al. 1984) after acute administration of TCDD. If adipose LPL is responsible for controlling serum triglyceride levels, these changes should then reflect alterations in this enzyme's activity. Dioxin is unique in having a wide differential species response (both in lethality and symptomology) therefore it would not be surprising if the response of fat LPL was different in these animals. It has already been demonstrated that TCDD causes a tremendous differential species response of hepatic plasma membrane bound receptors and enzymes, in terms of binding and enzymatic activity. Little data exists on serum parameters in TCDD treated mice. Poland and Glover (1980) have clearly shown that TCDD toxicity (i.e. cleft palate formation and thymic involution) segregates with the genetic locus responsible for the aryl hydrocarbon hydroxylase (AHH) activity. Nebert et al. (1972) had pmeviously demonstrated that exposure to 116 117 polycyclic aromatic hydrocarbons caused induction. of .AHH activity in certain inbred strains of mice but not others. These strains are termed responsive and nonresponsive, respectively. Poland et al. (1974) showed both classes of mice to be inducible by TCDD but variable in their sensitivity of induction. This induction was found to result from TCDD binding to either a high affinity cytosolic receptor in the responsive mice curaa low affinity receptor in the nonresponsive mice strains (Poland and Glover 1976). They postulated the low affinity receptor to occur from a mutation which produced an altered receptor with diminished binding for polycyclic aromatic hydrocarbons. Therefore one of the aims of this study was to ascertain whether serum hyperlipedemia follows this same pattern in these species. MATERIALS AND METHODS Male Sprague-Dawley Rats (100-125 or; 225-250g) were obtained from Harlan Animals, Haslett, MI; mice (18-25g) from the Jackson. Laboratory, Bar Harbor, Maine; English shorthair Guinea Pigs (200-250g), White Albino Rabbits (1500-3500g), and Golden Syrian Hamsters (75-100g) from the Michigan Department of Health. Mice strains termed AHH responsive (high affinity TCDD receptor) were: C57BL/6J, A/J, BALB/c, SEC/1ReJ, and. CBA/J. Non-responsive strains (low affinity receptor) included: AKR/J, RF/J, DBA/ZJ, SWR/J, and 129/J. Animals were housed as described 118 previously, given a single dose i.p. of TCDD or vehicle alone, and sacrificed 2 or 10 days later by anesthesia. The methods of serum and fat collection, preparation of fat. powder, measurement. of IJE, activity' and serum triglyceride concentrations were as before. Serum cholesterol and protein were measured using the methods of Carr and Drekter (1956) and Lowrey et al. (1951), respectively. Data are presented as the mean 1 standard deviation for the number of animals indicated. Statistical analysis was performed with the 2 tailed student's "t" test or ANOVA; P was always <.05. RESULTS Rabbit Adipose LPL activity was reduced and concurrent serum triglyceride concentrations increased, in the guinea pig and rabbit, in a dose dependent fashion 10 days after TCDD treatment (Table 15). These data indicate the rabbit to be even more sensitive than the guinea pig in terms of LPL inhibition since the rabbit at 1 ug/kg (115 x'less than the reported LD50 by Schwetz et al. 1973) showed a 44% decline compared to pair-fed controls. The guinea pig showed no such inhibition until an approximate LD50 dose was administered (see Table 15). One can conclude that, at least at 1 ug/kg, the rabbit is as sensitive to TCDD as the guinea pig, if not more so. Rabbit serum triglyceride levels .mM4mummm .mumn nzo .mHNHIOOH .mumn mcsox .omoo oohmoHccH on pouluHmm u mm .Ho.v o .zouucoo omo-oomo echo hamummmoo zzzmuHumHumum .4 .Ho.v o .Honucoo some ocmuoooHc zzzmooomHhmum 4 .¢>oz< Hmuwm umou muuoccso cuH3 omumecm com mHmEHcm Acv mo coHumH>w© numccmum H come no ommmmumxm macaw U U n ll9 .4ONHH H 0mm «..4A4OH.GGm H m.oms HmHH ImOHm H mm .m.~.mHH H c.4moH AHmHH on zoo o Amim4 H mm .4.m.4mm H 4.444H mm A40mo + GNH AmON.HmH + m.m4oH o hmumsmz ¥«.¢AOVHOH H ovm ««~«Amvo.mH H m.Nm om AmOmH H Hp «imim.moH H 4.~4m Aom 0» oo. o ;«.«A40om H mom ..m.m.~mH H m.4mm H AmO4m H om Am.o.4mz H H.0O4 AH on mm. o Amoom + mm Am.~.~m + ~.mo4 o uHoomm Amomm H om A4OH.Hm~ H 4.G~GH mm Amomm H mm .4.m.mmm H n.4aoH o cacao. pom Aoomm H 4o «A4oo.~m H H.mooH mm AGOmH + an A4oo.m4 + m.4o~H o oimcsoz. ham ««.¢Amva H NMN «AHva.HmN H m.Hmm H .AmO4H H mm {A40m.HH H m.GHm o.H on mm. o A4Omm + 4m mAmOH.mm~ + m.mmH~ o mHo mocosu Auo\ma\coom huumm mmuw zcv .Ho\me. ooo Amx\mso mmHomom we mmOQHp< mmoo .Qooe mo mwoo cmumoHc IcH mcu umuwm mhmo OH Hmumemn can .annmu .umu .mHm mocHsm on» no A09. :oHumnucwocoo moHHmomHmHHu Esumm 0cm >HH>Huom and omoch< .mH NHQMH 120 increased 125% at 1 ug/kg and 211% at 50 ug/kg compared to the respective pair-fed control levels (Table 15). Serum from the higher dosed rabbits was yellow in color and very milky. This is indicatiwe of high lipid concentrations. Serum cholesterol and triglyceride was increased 1.7x and 3.1x respectively but no change was observed in serum protein concentration (Table 16). Hamster LPL activity of the hamster declined 12% at the lowest dose tested but was 46% of pair-fed control levels at the L050 dose. The decline of activity in both the hamster and rabbit is not 'due to depressed food intake as treated animals in both species showed activity significantly below that of pair-fed control levels. Pair-feeding had no effect on hamster LPL compared to ad lib controls. Hamster serum triglycerides were significantly increased above pair-fed control levels. High variability in the hamster triglyceride levels is believed to be due to the jpeculiar’ food. eating' pattern of these animals. The excess skin around their mouth and necks allows them to stuff their cheeks with large quantities of food, thereby, providing a constant nutritional source. Rat Little change in either LPL activity or serum triglyceride levels were noticed in older rats. However 121 .Ho.v o .ozo zoom ucmnmmmHo zHuomoHoHcmHm I .¢>oz« HMHuouomw soccmu Hmumm Hmmu m.>omsa nuHB omnhHmcm mama .mHmEHcm Acv How :oHHMH>m© pumpcmum + some mum muHsmmmm +| $.va m.mH imim.o + o.mo A40m.o + 4.MH A4O~.H H 4.mH compono I I I I Azo\me. AHOHN + om {AmOmH + HOH A4Omm + we .A4omm + mOH HoumummHooo I I I I Azo\meo AmOmH + AH IAGOHOH + O4m A4O4m + om Imi4eom + mom moHumoszmHna ozo oooe mx\mo om ozo oooe mx\ms H .muHQnmu omumouu on pmeuHmm can HHo :Hoonocoumom no Dave mx\ms Om no H mo GOHpmHuchHEUm Hmuwm wwmo OH mcoHumuucoocoo cHououm ocm .HoumummHoco .moHHmomeHHu EDHmm .OH mHnnB 122 there was a significant increase in LPL activity from younger rats (100-125 g) (Table 15). 1110.: Serum analysis of 5 AHH responsive and ES AHH nonresponsive mice strains 2 days after treatment indicated 3 of the responsive (BALB/c, A/J, and CBA/J) strains to have significantly increased levels of triglycerides while all 5 of the nonresponsive strains remained unchanged (Table 17). One responsive (SEC/1ReJ) and one nonresponsive (SWR/J) strain each showed nonsignificant increases in triglycerides. C57BL/6J, the other sensitive strain, revealed lower but nonsignificant levels of serum triglycerides. None of the strains tested indicated any significant change in serum cholesterol concentrations except 129/J. This result is believed to be due to an artifact since the control samples were highly hemolyzed thereby influencing the colorimetric assay. Furthermore it appears that the normal cholesterol range for these strains is between 38 and 49 mg/dl; values far below that obtained for the 129/J control samples. These triglyceride and cholesterol assays were done by myself and Ms. Laura Anderson. DISCUSSION The rabbit seems to be as sensitive to TCDD as the guinea. pig iJI terms (If a. hyperlipedemic response. 123 .uxmu mom .Esuom owNmHOEmc thmHm .Ho.v m on Houucoo scum ucmumHoHo zHHmoHHmHHmHm 4 .ummu gag mucmcshm omHHmu m nHHz omusHmom momo .mHmeHcm AGO How coHumH>m© oumccmum H coma map mm pmmmmumxm muHsmmmm b Amos H mm Hmvm H mm .40m H Hm A4omm H OOH om\Hmcommchoz A~O4 H mm A4OG H 4m .4OHm H mOH HGON4 H HNH eG\Hono Amvm H mm AOOH H vv «vamh H ONO AmOHm H mmm O\Hmcomwmm a U a U chuum AHo\mEO HozmommHooo AHo\me. moHHmoszHHe .HHo :Hooumcoumom Ho nova mx\ms Om HospHo nqu mchoo Houmm mhmp m mchHum OUHE m>Hmsommmucoc can m>HmcommmH :H AHO\OEO mcoHumuucwocoo HoumummHono com mpHumomHmHHu Ezuom .HH oHnma 124 Significant inhibition of LPL activity as well as serum triglyceride elevation occurred in the rabbit at doses far below the LD value. These responses were not seen to the 50 same degree in. the (guinea. pig until the LD50 dose was attained or exceeded. Furthermore treated rabbits began to die sooner than did treated guinea pigs. Schwetz et an” (1973) reported rabbits died beginning the sixth day after a single lethal intraperitoneal dose of TCDD. Studies in this laboratory support this finding, thus the reason for having LPL data from 8 pair-fed control rabbits but only 3 animals at: 50 ug/kg' 10 Idays after treatment. The earliest any treated guinea pig died was 18 days after injection. As reported previously (Brewster and Matsumura 1984) adipose LPL activity does correlate with serum triglyceride levels in the guinea pig. As demonstrated here the same is true for the rabbit and the hamster. No change was observed in either enzymatic activity or triglyceride concentrations in old rats. Why TCDD caused an increased activity in the younger animals is not known. It appears as if the activity of control animals is underestimated since IHH; is not age dependent and since little difference was observed between the 2 treated groups and old control group. LPL activity does follow a diurnal pattern in rats according to when feeding occurs (Hamosh and Hamosh 1983), therefore, it is possible these control rats were sacrificed after active feeding had ceased. This however offers no explanation as 125 to why, after 10 days, treated animals which have reduced their food intake still have enzyme levels approaching that of ad lib controls. In conclusion TCDD does not inhibit rat adipose LPL thereby causing serum hypertriglyceridemia. This confirms the observations of Albro et al. (1978). Olson et al. (1980) observed no change in hamster serum triglyceride levels 10 days after treatment but did see a 47% decrease in levels 20 days after treatment with TCDD. These were compared to ad lib control animals. Present. observations indicated a: significant. increase in serum triglyceride concentration with a severe inhibition of LPL activity 10 days after an LD dose of TCDD compared to 50 pair-fed control animals. In addition, pair-feeding had no effect on hamster LPL. This may be attributed to the fact that lipid metabolism in the hamster is vastly different from the rat, guinea pig and rabbit and that serum triglycerides are regulated by other controlling mechanisms. In fact, hamster metabolism is quite different from other animals which undergo hibernation since serum glucose concentrations increase rather than decrease during dormancy (Orr 1976). This animal must waken every few days and consume food in order to prevent starvation. Therefore, in order to remove endogenously produced triglycerides (via VLDL) this animal must have other mechanisms available during dormant periods in order to prevent adipose LPL decline due to lack of food intake. One such mechanism 126 could be increased serum glucose which in turn increases pancreatic insulin output and results in stimulation of LPL synthesis. Present results seem to support this since no change was seen in pair-fed control levels. The milky blood serum obtained from the 50 ug/kg dosed rabbits was indicative: of the Ihigh lipid concentrations produced by inhibition of adipose LPL (present study) and hepatic LDL binding (D. Bombick, personal communication). Low density lipoprotein (LDL) is the major cholesterol carrying lipoprotein in the blood. It is produced by the stripping off of triglycerides from VLDL particles by LPL and by direct hepatic synthesis and is efficiently removed by binding to its hepatic receptor (Brown et al. 1981). Both processes are inhibited by TCDD, in the guinea pig (Bombick and Matsumura 1984, Brewster and Matsumura 1984) and. the rabbit, thereby' causing hypercholesteronemia and hypertriglyceridemia. The yellow' color imparted. to this serum may be due to increased levels of bilirubin since in contrast to the guinea pig, rabbit liver is markedly affected by TCDD. Fatty infiltration, hepatomegaly, discoloration, and brittleness or loss of elasticity of blood vessels were seen in livers of rabbits exposed to 50 ug/kg TCDD. Significantly increased blood serum triglycerides were noted in 3 of the 5 AHH responsive strains of mice and none of the nonresponsive strains. If toxicity segregates with 127 the AH locus as suggested by Poland and Glover (1980) this is not surprising. However, the question arises as to why C57BL/&J and to a lesser extent SEC/lReJ (both responsive strains) and SWR/J (a nonresponsive strain) do not show the expected toxic response. These data suggest that these mice are genetically different from the other strains. Knutson and Poland (1982) suggest that TCDD and other halogenated hydrocarbons produce 22 distinct pleiotropic responses: The first is associated with the AHH gene and involves microsomal induction and enzymes involved in drug metabolism, ‘while the second. involves an. additional gene complex manifesting other toxic lesions. These authors conclude this battery of genes to be present but unexpressed in some tissues or species (i.e. in the present study perhaps C57BL/6J or SEC/1REJ). It is simplistic to think that all the pleiotypical responses evoked by TCDD are due to interactions at the Ah locus. This would mean that this locus controls lipid metabolism, microsomal induction, cellular proliferation, epithelial hyperplasia, thymic involution, body weight, and all other toxic symptoms. It appears more logical if other loci are involved. Therefore to understand how TCDD manifests its toxicity at the subcellular level, one must understand how it regulates and deregulates gene expression in different tissues. The same genes are present in all tissues of a given species but their expression depends upon the particular cell type and 128 vice-versa. TCDD in some, as of yet unknown way, must alter this gene expression. No increases in serum cholesterol levels were seen in any mouse strain. Perhaps longer exposure to TCDD or higher doses would result in hypercholesterolemia in the AHH sensitive mice. Previous research with guinea pigs (Brewster and Matsumura 1984, Bombick et al. 1984) indicated serum triglyceride levels to increase at lower doses and after shorter exposures to TCDD than serum cholesterol. Conclusions In conclusion, TCDD reduced adipose LPL activity in the guinea pig, rabbit, and hamster, thereby causing the serum hypertriglyceridemia seen after dioxin administration to these species. No depression occurred in the rat nor did serum triglycerides increase. Hamster adipose LPL was unchanged in pair-fed animals (compared to ad 132 controls) but other mechanisms peculiar to this hibernating species probably prevent excess increased triglyceride buildup. Finally hypertriglyceridemia as a toxic response appeared to segregate with the Ah locus in several responsive and nonresponsive mice strains. The hyperlipedemia observed in the rabbit is of special interest as this species has been extensively used in atherosclerotic studies. Since lipid metabolism in the white rabbit is similar to that of man's this animal has 129 proved to be a good model to investigate defects in lipid metabolisnI leading' to (arteriosclerosis. Further' research should be conducted to examine the role of TCDD in serum hyperlipidemia and the atherogenic process. For example, recent evidence indicates that LPL may be important in the formation of active HDL (high density lipoprotein) by stripping off the surface components of VLDL and chylomicra triglycerides during lipolysis to form active HDL (Dieplinger et al. 1985). HDL is a serum cholesterol scavenger and has a protective role in preventing atherosclerosis (Dieplinger et al. 1985, Brown et al. 1981). Since TCDD was seen to inhibit rabbit LPL in the present study it is suggested that this action may play a role in the formation. of the preatherosclerotic lesions (along with the hepatic inhibition of LDL binding). Although Swift et al. (1981) found no changes in serum levels of HDL, between control and TCDD treated animals, it is not known whether these particles are active in removing serum cholesterol i.e. it is not known whether this HDL was activated or not. It is not surprising that TCDD produces different effects in different animals as regards to lipid metabolism. Their biochemical pathways are quite different. For example the size and composition of the lipoprotein moities and the apoprotein composition varies with species (Chapman et al. 1975, 1980). Linoleic acid is 130 the major fatty acid in the guinea pig while oleic acid is primary in the rat. Furthermore, guinea pigs lack apoprotein C-II but still have efficient triglyceride clearance mechanisms and are normally resistant to hypertriglyceridemia. Guinea pigs have low serum levels of hepatic lipase compared to rats and therefore have only one mechanism to remove serum triglycerides as opposed to two in the rat. Rabbits typically have low levels of VLDL and HDL. Finallyy as previously' discussed, hamsters hibernate and therefore have completely different lipid biochemical pathways compared to guinea pigs, rats, and rabbits. Since LPL is known to be controlled by insulin (Hamosh and Hamosh 1983) quite possibly what may be occurring in these species in regard to TCDD is either an inhibition of serum glucose regulation, an inhibition of pancreatic insulin production or secretion, or an inhibition of insulin action at the fat cell. Any of these would decrease LPL activity resulting in increased blood triglycerides in the guinea pig or rabbit. CHAPTER VII EFFECTS OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN UPON THE GUINEA PIG HEART INTRODUCTION Lipoprotein lipase activity (LPL) has been found to be present in a variety of extrahepatic tissues other than adipose such as lung, skeletal muscle, lactating mammary gland, milk, aorta, corpus luteum, brain, and heart. (Hamosh and Hamosh 1983). To achieve a constant energy source, cardiac LPL is regulated differently than adipose LPL. Fielding (1976) has presented evidence‘ for a high affinity heart lipase enzyme and a lower affinity fat lipoprotein lipase enzyme. Although these enzymes differ in structure and in kinetics their function is to hydrolyze circulating triglyceride-rich lipoproteins, thereby providing fatty acids to either the heart for energy or to fat for storage. Both enzymes are activated by apo-C II and are inhibited by high ionic concentrations. It has been postulated (see Hamosh and HamoSh 1983) that these lipase enzymes have a critical physiological function since the high affinity cardiac lipase provides energy 1x: the heart even when serum triglyceride concentrations are low, such as during fasting periods. Likewise, adipose LPL will provide storage materials to fat only when blood triglyceride 131 132 concentrations are high, such as post prandial. As a result, shunting of potential energy occurs between fat and other active tissues with increased metabolic requirements. Therefore, cardiac LPL is less nutritionally dependent than adipose LPL, and in fact, cardiac activity may rise during fasting to insure adequate nutrition to the muscle. Known Cardiotoxicity of Dioxin Since guinea pig adipose LPL activity has been shown to be by TCDD (Brewster and Matsumura 1984) it was of interest to know of what effects this dioxin would have on heart LPL activity. If lipase activity in this muscle is reduced by dioxin, one would expect severe effects on heart function to occur. This coupled. with. the observed. high serum lipid concentrations could be a contributing factor to death of the animal. Although chlorinated dioxins were shown to cause chick edema disease (characterized as edema of the pericardial sac) in the late 1960's (see Firestone 1973), virtually no investigators have focused their attention on the effects of TCDD concerning heart function. Three reports are found in the literature describing pathological or histological lesions in the heart after dioxin treatment, but these effects are seen only after very high doses of TCDD or after chronic exposure. None has dealt with heart function and all have been described only for the rat (see Table 18). 133 mHmH mhm .Hm #@ MQHUOM H .H8 H8 moose mumom m mx\m: H.O Hmuo .hHHmv mzmo OH Imx\ms OH .Q.H .onch wmmcmno m>Humnmcmmmp HMHUHmoo>z HQEOHLH oochmmHo «mmmcuuoem: mmHHHHoHHoHE Ho coHHMHocmmmp “meonm HmHs>Hm> 0cm Hbsounu OHocHHhHm “moon“ m>Hm> mo conouo NHOH .HO HO Hoousso mzmc OH «OH\OE OH Ho H HOHOHmooocm NmHHHHO>Hm> wocmumwmm mEHa QOHumHuchHE©< conmH whomomxm «moon .QDUB SUfl3 #CGEDMGHH H0#Hfl mCOHmmH omHOHmo pwuuommm .OH mamas I34 MATERIALS AND METHODS Cardiac LPL Assay Guinea pigs were obtained and housed as described before. After 10 days, ad l_i_b_ control, TCDD treated, or pair—fed control animals were anesthetized with ether and their hearts removed after which, cardiac LPL activity was estimated using the same procedure previously described for adipose tissue LPL. In a subsequent experiment TCDD treated or pair-fed animals were given 2.7 ml of a 75% glucose solution 2 hours before sacrifice. Atrial Isolation and Heart Function Tests Heart function was evaluated by measuring the inotropic (force of contraction) and the Chronotropic (rate of contraction) responses in isolated atrial muscle. Animals were administered a single i.p. dose of TCDD (1 ug/kg) or acetone:corn oil (1:9) and either pair-fed or allowed to feed ad libitum. After 10 or 20 days the guinea pigs were stunned with a blow to the head, the hearts rapidly removed and immersed in room temerpature Krebs-Henseleit bicarbonate buffer (118.0 mM NaCl, 27.2 mM NaHCO 4.8 mM KCl, 1.2 mM 3’ 1.0 mM KH P0 11.1 mM glucose). After 4' 2 4' 2’ retrograde perfusion with aerated buffer the left atrium was MgSO 1.4 mM CaCl excised and hung vertically in a jacketted tissue bath (containing buffer at 30°C and aerated with 95% 02-5% CO2 at a minimal rate) in order to measure the inotropic response. The Chronotropic response was determined using the right 135 atrium hung in a similar manner in an adjacent bath. All four atria, from pair-fed control and TCDD treated animals, were hung at the same time and exposed to the same experimental conditions. Platinum electrodes were used for electrical stimulation of the left atria at 1.5 Hz of 5 ms duration approximately 100% above threshold (Grass S9 Stimulator, Grass Instrument Company, Quincy, MA). Resting tension of all samples was adjusted to 1 g whereupon the force of contraction of the left atria, in response to the electrical stimulation and the inherent rate of contraction of the right atria (no electrical stimulation) were continuously recorded on a physiograph recorder (Grass Model 7B Polygraph). The atria were attached 1x3 a force displacement transducer (Grass Instrument Company, FT-03C). Every 15 minutes throughout a 60—70 minute equilibration period the buffer in the baths was changed to prevent excess foaming. The resting tension was maintained at 1 gram throughout equilibration after which isoproterenol (in ethanol) was added to the bathing medium at 5 minute intervals so that final concentrations were 3x10-10M, - — -‘7 - 1x10 9M, 3x10 9 8 8M, 1x10 'M, and 3x10 7M. M, 1x10- M, 3x10- All chemicals used were of the highest quality available and were purchased from Sigma Chemical Company, St. Louis, MO. Statistical analyses were performed by random factorial or one way analysis of variance where indicated 136 followed by the appropriate multiple comparison test as noted. RESULTS TCDD had little effect on cardiac weight 2 days after a single i.p. administration (Table 19). Heart weight was significantly depressed from pair-fed and ad Add controls 10 days but not 20 days after treatment. However, no differences were noted when weight was expressed as -a percentage of body weight, thus indicating no significant loss of cardiac tissue after TCDD treatment. L_P;L_ Cardiac LPL activity was not significantly altered by dioxin treatment (Table 20). No changes were noted after 2 days or 10 days. Glucose administration 2 hours prior to sacrifice significantly increased activity in both TCDD treated and pair-fed control animals. Finally, administration of 4 ug/kg dioxin did not cause a statistically significant depression of lipase activity compared to pair-fed animals, 10 days after treatment. Atrial Function Cardiac atrial force of contraction was significantly depressed 20 days after a single administration of TCDD (Table 21). No effect was noted at 10 days after treatment and little effect was observed on basal rates of contraction in the absence of isoproterenol after 10 or 20 days of 137 .ochoO Ho meHH H4 m OOOIOOm HOOHmz zoom .mchon mo mEHu an m OOmIom~ uanw3 xoom .omcHEkump uoz .mpocuoe pom mHmHHoume 'OQJ‘H :H owbHuomov mm moHuHuomm Daemon wwoosHm «0 once Hmuo oHocHw m pw>Hwomu mHMEHcm wxmp OH Heuuoz< Hmuum ammo mHHmccso nnHz omnsomcm mono .pmuoc woucmummuHc ucmoHuHchm ozb fl on on .HOOO.OMOH.O om . om .HOO4.OMOO.~ OHo .m.no.O.nm.O .mOOO.O+~4.O .m.4O.O+m4.O HmOm~.O+MH.H HmOOm.O.4O.H .HOHH.O.HN.H momsox smo ON I l I l omwouon .4OH0.0+Om.O .4.00.0+~H.O oz .4000.0+M0.0 ...4040.0+M0.0 Ooz . zmo OH Hwom0.0H4m.O .OOM0.0H4H.O ANCH0.0H~4.0 ..OOMH.OHH0.0 ....HHOOOO.OHHO.O .NO4O.OHO~.H Hmo OH .HOHO.Ou4m.O .HOHO.OH4M.O .mO~O.OHHH.O AHO-.Oumm.O .HOm~.OuHO.O .HOHO.OHOO.O H4O ~ noquHmm oooa oHH mm cmHIHHmo oooa oHH mm muanwz hvom a wemuu oEHe wusmoaxm BSUHHS Bm009 :H mmcmno o: mmB whose .oon IHHomm mHommn mnooc m .mmoosHm me no HE H.m omuouchHspm mHMEHc< .mO.v m .mHMEHcm amp OH o>HHoommmH ECHO ucmummep mHucmoHMHcmHm4 .¢>Oz< HmHHouomw Houmm mcowHHmmeoo Omccoowcooco How umou m.>mxsa Ho ummu mupoccso nuHB pwuhHmzm open .UmcHEHmpoo #02 U 0 b m INOO.NMHHm.HOH AmOO.OOHu4.4ONH HmO4.OmHHN.OOOH LOO HO zmo ON oz OHmOO.mHHHH.HMH oz LOO 4O zmo OH I I vomoosHO + m: HO 4A4OO.HmH+H.mmO IoH4OH.4H+m.Nmm oz zoo OH AOOm.OOHHO.Omm HOOO.NNHHO.NON ANOm.mNH~.HNH AOOHO zmo OH HOO4.mmHO.mOm HmOH.OO~HO.mmm moz LOO HO zoo N OOHIHHmo oooe oHH mfl meHe mmsmomxm .Ao ..o.m H cmoev mcon OHDH£0> Ho 0008 mx\m: v Ho H QHHB uzosumwuu .Q.H OHOOHm Hmuom mono OH no N HHH>HHom HoH omHOHmo .O~ oHooa 139 .HOO.v m um HOHHGOO UOMIHHQQ Eoum ucmummeU NHHCMOHchmHm « o<>Oz¢ H0n~mnm quU m.u#®CCSD SUHB UmNNHMCM MHMQ .UocHEHmuwo no: I oz .AMHHHM HSOHH. OHDCHE\HOQE:: :oHHomuucoo .AmHHum uonO :oncmu pmmon>mU mo mamuo fUQO'O HOO4HHO4H AOONHHOHH AmOOHmmH HOO~.OHO.O IOHOOH.OHH.O HmOH.OHm.O zmo Om H4OmHHmH A4OmHH44H oz H4ON.OHO.O A4O4.OHH.O ooz zoo OH ooo oooa oHH OH ooo oooe oHH O4 noumm COHHOMHHEOU mmouom GOHHOMHHCOO .AmHMEHcm c ..Q.m H some. mHmEHcm poummuu move map ou OOMIHHQQ Ho EDHHQHH mm com com cho HHo GHoo Ho nave mx\ms H on musm Iomxm Houmm mxmo ON mHHum mHm mocHom mo :oHuomuucoo mo moon can mouom .HN mamas 140 treatment. Little change was noted in the inotropic and Chronotropic responses of isolated atria to isoproterenol from 10 day TCDD treated or pair-fed control animals (Figure 27). After 20 days of treatment obvious differences were noted in the atrial contraction response to the drug but not in the Chronotropic response (Figure 28). Although atria from pair-fed control and TCDD treated animals both developed a maximal tension of 2-2.5g, those from dioxin treated animals needed higher doses of isoproterenol to reach that response. To correct for atrial size differences the % of maximum developed tension as a function of isoproterenol dose was plotted in figure 29. The response of pair-fed control animals was the same as young ad .lEE control animals; TCDD treated animals followed the response of old ad llE control animals. The % of maximum develOped rate, plotted against isoproterenol dose, 1J5 significantly altered by TCDD (Figure 30). Isoproterenol Dose Response Since atrial size influences contractile force, the % of maximum response as ea function of isoproterenol concentration was plotted and analyzed (Figure 31). The ED50 values are presented in Table 22. After 10 days of treatment, 2.7x more isoproterenol was needed to obtain 50% of maximum contractibility lJl‘thiS muscle preparation from TCDD treated animals. After 20 days this difference was 3.1x that of pair-fed control levels. 141 Figure 27. Chronotropic (A) and inotropic (B) responses of isolated guinea pig heart atria to isoproterenol. Atria were isolated from TCDD treated (o) (1 ug/kg, i.p.) or pair-fed control (0) animals 10 days after administration. Each point represents the mean + S.D. from 5 animals. Split-plot ANOVA revealed no significant differences between treatments. 142 :5 35.3038. «moo .II JO. . O-Ho. H OHO. j \\ .oJOO m HII LIIIHHHHM. ML ... .1 .n \H\ W\ I. H i c : c H .r I H I _ . _ 4 H I \\ M O Y? \HHIIIHH ~an — II can (6) uogsual padolanao (quI/suonaenuoa) 3:23 143 Figure 28. Chronotropic (A) and inotropic (B) responses of isolated guinea pig atria, to isoproterenol, 20 days after treatment with either 1 ug/kg (i.p.) TCDD (O) or acetone:corn oil (0) and pair—fed. Each point represents the mean + S.D. of 5 animals and were analyzed with split-plot ANOVA followed by Tukeys Test for Unconfounded Comparisons with P = .01. *Significantly different from pair-fed control. 144 :5 353333. 030 m .\..H allulliHllliL t l\t_. L\\o .: ...-W 1%! 1W l 11 < _ . _ 4 _ 4 s we H\: IIIII HH :\ H 12. (6) uogsual pecan/sag (mm/momentum 3:23 145 Figure 29. Inotropic response (as a percent of the maximum developed force) of isolated guinea pig atria (from young animals 250-300 g) 20 days after treatment with 1.0 ug/kg TCDD (O) or acetone:corn oil and pair-fed (O), or fed ad libitum (Q). The response was also measured in atria isolated from old animals (500-600 g) allowed to feed ad libitum(A) for 20 days after treatment with vehicle only. Each point represents the mean + S.D. from 3-6 animals. The data was transformed and analyzed with one way random factorial ANOVA followed by Scheffes test for multiple comparisons at P = .05. Signifigantly different from old ad libitum control animals. Not significantly ifferent from old ad libitum control animals. d Significantly different from pair-fed control. Not significantly different from pair-fed control. Significantly different from young ad libitum control. 146 '.‘ _fi 9- ‘ . g \F—_-: 0’ _ so ’m F114 o——-| F—On 4 H12 O m Hm -~ ' §\ 0: 2 u '0' i Q) T"? H51:- — 0 § “U U. i i i (y‘ai 1 ‘7‘" _J o O 100 8 33103 padolaAag wnngew % Dose Isoproterenol (M) 147 .Houucoo cowluflmm Eoum ucmeMMHU wfiucmoHMHcmHm* .Ho.u m um mcowflummeou omccsomcooca now umma mmmxse moans .mm wusmfim CH mm .omumfimcm mnmz cam mfimeflcm m mo .o.m H some map mucmmwummu usaom comm .Adv EsuHQHH mm cow no AOV vowinamm cam ADV Dove mx\ms H mo coapmupmflcflecm Hmumm mmmc om mauum mam mmcasm omumfiomfl mo Amumu pmmofim>mc Eseflst mo mmmucwonmm m mmv wmcommmu oamouuocouno .cm wusmflm as: 3:98.293. .30 u-o_ .o. O O— o- . :“V a1..-- a. colnflumwakfi H\ L H 1.--.-. a H j 11 \\1Il|_ O 8— 3:23 pedopna umngvu g 148 Figure 31. Isolated guinea pig atrial inotropic dose response to isoproterenol 10 or 20 days after treatment. (g5) pair-fed control, 10 day; (y) TCDD, 10 day; (0) pair-fed control, 20 day; (0) TCDD, 20 day; (9 )young ad lib control, 20 day; (A)old ad lib control, 20 day. (Error bars omitted for clarity). 149 ' as 0.0“) —-. E ‘4. O '— ‘3" s. at < o . _g 9 ‘5‘ o\ O ‘ 0‘ _. \\ \\ ‘? o O _- \\ &\ \“\ \ \\ \\\\\ \ \ \ \\\\\\?\ «I 1 - - 0 § 3 o asuodsaa wnngow 9;, Dose Isoproterenol (M) 150 TABLE 22. Effective concentration of isoproterenol to produce 50% of maximal contractile force in isolated guinea pig atria 10 or 20 days after i.p. administration of l ug/kg or acetone:corn oil and pair-fed or fed ad libitum. Ad lib control TCDD PFC young old a -9 ‘ -9 10 Day ED50 ND ND 5.2x10 M 1.9x10 M 20 Day E050 2.3xio'9M 6.8x10’9M 1.1x10'8M 3.5x10'9M aNot determined. 151 The 20 day PFC ED was 1.5x that of the young ad lib 50 control but only half of the old ad lib control ED50 and as stated above about 1/3 of the preparations from TCDD treated animals (Table 23). The ED50 from treated animals was almost 5X that of young ad lib control but slightly more than 1 l/2X the old ad 132 control levels (Table 23). Statistical analysis indicates significant differences between TCDD and PFC, PFC and old ad lib, TCDD and young ad lib but not between PFC and young ad lib or TCDD and old ad lib. DISCUSSION TCDD produced a 51 and 24% decrease in heart weight compared to ad 1_ib_ control and pair-fed control animals respectively, 10 days after treatment. No change in weight was seen after 2 days. Since TCDD causes body weight loss or inhibition of body weight gain, heart weight was expressed as a percentage of body weight and was no different between TCDD treated and control guinea pigs. It is concluded, that this apparent loss of heart weight is a secondary response resulting from body weight loss. Why the absolute weight appears to return to normal levels after 20 days of TCDD treatment is not known at this time. Fielding and Havel (1977) proposed that the high affinity heart LPL allows a constant lipid uptake even when serum triglyceride concentrations are low. Hamosh and Hamosh (1983) indicate that heart LPL increases after fasting to provide sufficient nutrient uptake. Data 152 TABLE 23. Twenty day "dioxin" factor for atrial contraction ED50 as a function of treatment. Young Old Ad lib Ad lib TCDD PFC Young a Ad lib -- 2.96 4.78 1.52 Old Ad lib .34 -- 1.62 .51 TCDD .21 .62 -- .32 PFC .66 1.94 3.14 -- a"Dioxin" factor calculated by dividing the ED value from each 20 day treatment group by eagg other. 153 presented here indicate that high doses of TCDD may inhibit this effect. After 10 days pair-fed and TCDD treated animals showed increases in LPL activity as expected, however those animals given 4 ug/kg TCDD exhibited a 59% decrease in activity compared to pair-fed controls. The difference was not statistically significant, possibly because of the large variability in the assay. Clearly more research needs to be completed before this question can be answered in its entirety. If TCDD does indeed inhibit cardiac LPL activity then one would expect heart function to be compromised thereby contributing to death of the animal. Studies are currently in progress to assess cardiac LPL activity after longer exposure periods. Linder et al. (1976) have shown that heart LPL responds very little to insulin. It is therefore possible that the inhibition in adipose tissue shown by Brewster and Matsumura (1984) is not entirely due to alterations in hormonal insulin production or secretion. Experiments with cultured cells and $2 XEEEQ TCDD exposures are being considered to assess whether inhibition of LPL activity is a direct or indirect effect upon adipose tissue. TCDD produced significant alterations 111 cardiac function after a prolonged exposure to this chemical. Contraction force was 1/6 that of pair-fed controls 20 days after TCDD administration, and the response to isoproterenol was much less than that of pair-fed controls. Furthermore 154 it appears as if TCDD causes an age related change since treated animals exhibited a response similar to that of old guinea pigs, while pair-fed and young animals were similar. Further evidence for this invoked aging effect is discussed in Chapter VIII, where no change was noted in adipose lipolytic activity although the lipogenic actions were severely depressed characteristic of older animals. Conclusions Whether the decreased force of contraction is a direct effect of TCDD or not is not known at this time. The compromised ability of this organ to respond to isoproterenol suggests an alteration in the beta-adrenergic system. Future studies concerned with cardiac membrane binding should be considered to assess whether this effect is due to changes in receptor affinity or receptor number or due to changes post. receptor’ binding. Isoproterenol is known to increase adenylate cyclase and c-AMP levels within the cell. Possibly receptor binding is changed only marginally and the changes in contraction are due to alterations in cyclic nucleotide levels. TCDD is known to influence c-AMP levels in adipose tissue (Brewster - unpublished observation). Contraction changes may also be due to alterations in ionic transport, another manifestation of toxicity which was seen in liver plasma membrane functions (Matsumura et en“. 1984). This possibility could be investigated by studying the action potential and 155 observing changes in different areas of the spike. In this way one could infer whether Na, Mg, or Ca ions are involved. Whatever the underlying mechanism, the depression in heart function coupled with the many and varied other toxic lesions produced kn! this chemical (such as hyperlipedemia) could very conceivably result in lethality. CHAPTER VIII STUDIES ON THE CELLULAR MECHANISM OF LPL INHIBITION CAUSED BY lg glyg ADMINISTRATION OF 2,3,7,8 TETRACHLORODIBENZO-P-DIOXIN INTRODUCTION With the elucidation of the cause for the severe serum hypertriglyceridemia, produced by TCDD in the guinea pig, came a much more complex and difficult problem -- the bio- chemical mechanism for the inhibition of the enzyme lipoprotein lipase (LPL). As discussed by Patten (1970) the two main lipid metabolic pathways in the adipocyte are reciprocally regulated. Lipogenesis, controlled mainly by insulin, and lipolysis controlled mainly by catecholamines, can be said tx> be opposing' pathways and thereby' have opposite physiological functions (see Figure 32). During periods of fasting, epinephrine and norepinephrine stimulate the fat enzyme, Hormone Sensitive Lipase (HSL) responsible for the breakdown of stored triglycerides to free fatty acids. These fatty acid are then transported throughout the body and used where needed for energy via fatty acid oxidation. However when actively eating, insulin induces the synthesis of LPL the function. of which is to remove circulating triglycerides from the blood for storage in the adipose tissue. Agents responsible for stimulating HSL activity 156 157 .wmmaflfi w>flufimcwm ocospocngm: .wpflom xbbmm wouwiJ_On_ _|_ @905 m_mmZm_OOn=-_ £32: 3: :3 0h. EDCQW ._. cause sustained cellular phosphorylation in the liver (Matsumura et al. 1984, Madhukar et al. 1984, Bombick et al. 1985). Along with the observed stimulation of cellular phosphorylation there occur alterations in epidermal growth factor (EGF) binding' due to endogenous phosphorylation of the EGF receptor by TCDD (see Appendix A and Madhukar et al. 1984). Changes in EGF binding have been associated with serum hypertriglyceridemia (Heimberg et al. 1965). 159 / Nucleus Insulin lPL b (proenzyme) Anne ) LPLb' hnochve lPl. a (active) / NUCIeUS 1 Insulin \ Proenzyme glycosylation . . ‘ epinephrine . lnochve _ _ _ __ ? ___ _+Achve Nucleus ( synthesis . glucose Insulin metabolites \ Proenzyme activation gl ycosylat ion secretion Active< Figure 33. Three possible mechanisms for the production and cellular regulation of lipoprotein lipase. Panel A was taken from Cryer et al. (1975), panel B from Ashby et al. (1978), and panel C from Spooner et al. (1979). 160 .Awhma mquCHoum eoum puma :Hv GHHDmGHIH .mcflucmmcflmmim “mmamucm mwx mo coaumfismon Hmcosuoc m>Hummoc cam w>HuHmom mpmuflocfl I.\+ .qmq mo :oaumHsmmH Hmooumflumu wnp cam Amm mmomflom mo coflumflsmou mmmcflx new Houucou Hmfisfiamu .vm whamflm 3282:. r: + +m +_ 4 63 US .r: 32 US .3: n:.< 329.9%; 3233 I _ 3335.: ET?— omex $395+ «was? 5391 39.23% n_<<<-U . n:.< -0490 W m @2355 I A am: m2< _ 3293 +m _XCGWO 161 Lipogenesis There are at least three mechanisms proposed for the synthesis and regulation of LPL (Figure 33). Cryer et al. (1975) suggested that insulin caused both an independent and dependent protein synthetic process which activates LPL. Insulin was shown to produce newly synthesized but inactive LPLb which gained full activation (LPLa) upon post translational modification just prior to being secreted from the cell. However investigators also noted another form of the enzyme, termed LPLb', which could not undergo this modification and therefore could not be activated to LPLa. This "storage form of the enzyme" could be acted upon by insulin to form LPLb. Conversely, adrenalin changed more of the proenzyme to the storage form (LPLb -- LPLb'). Ashby et al. (1978) concluded the proenzyme to be activated, probably through glycosylation, either immediately before or during secretion. These workers also noted an inactive LPL within the adipocyte but determined that adrenalin caused deactivation of the active form, not of the proenzyme as proposed by Cryer et al. (1975). In addition, they demonstrated little reversibility of the deactivation process. Finally, Spooner et al. (1979) ascertained that insulin regulates adipose LPL in three ways: (1) through the normal protein synthetic process, (2) through an energy independent process promoting increased secretion of the active form by either decreasing its binding to the plasma 162 membrane and other storage sites or by activating an inactive precursor and, (3) through stimulation of the hexose transport system and the formation of regulatory glycolytic intermediates. In conclusion, insulin causes the production of an inactive LPL precursor (probably through protein dependent. and independent processes) which must undergo activation (probably through glycosylation) before secretion and that catecholamines (specifically epinephrine) inhibit this process. Glycosylation 'is thought to be responsible for activation (Hf the proenzyme because (1) tunicamycin (a specific inhibitor of glycosylation) inhibits enzyme secretion, (2) Iii. is glycosylated, and (3) glycosylation of proteins is known to be required for secretion in many cases (see Hamosh and Hamosh 1983). There are at least three regulatory sites involved with the production of viable LPL: stimulation of gene expression and energy dependent processes, activation of an inactive precursor, and inactivation. of time active enzyme before secretion. Because the serum half life of LPL is only 7-25 min. (Hamosh and Hamosh 1983) hormonal control would be necessary to insure an adequate enzyme pool in the adipose tissue. This pool can be readily regulated under different nutritional conditions at the 2nd and 3rd regulatory steps. As discussed by Ashby et al. (1978), adrenergic stimuli could quickly and efficiently change LPL activity thereby influencing triglyceride uptake. Such responses would be 163 vital under conditions (ME stress where triglyceride uptake would need to be shunted to working muscles. An alteration in protein synthesis would delay this response. Along with inhibition of LPL, the catecholamines would activate HSL. This once again illustrates the reciprocity between these two systems in the adipocyte. Lipolysis Hormone sensitive lipase has been shown to be regulated by c-AMP dependent protein kinases in both Ea vivo (Steinberg 1976) and $2. XEEEE (Hirsch and Rosen 1984) systems. Epinephrine and ACTH are positive effectors of the system and stimulate membrane bound adenylate cyclase. The resulting c-AMP binds to the regulatory unit of c-AMP dependent protein kinase allowing phosphorylation of HSL to occur. Epinephrine also stimulates fatty acid transport out of the cell (Abumrad et al. 1985). As seen in Figure 34 insulin can inhibit the accumulation of active HSL at any of these points. However the converse is also true. If any of the steps in this pathway are stimulated by TCDD and increase HSL, then LPL would be depressed. The purpose of these investigations was to examine the intracellular events responsibLe for the activation of LPL and HSL and ascertain what affect if any TCDD would have upon these parameters. Perhaps then the biochemical mechanism for LPL inhibition by this dioxin could be explained. 164 Adipose pp60src In the past 10 years many investigators have focused their attention upon oncogenes and the effect of oncogenic products on normal cellular metabolism. Proteins encoded by oncogenes were thought to always cause cellular transformation but within the last few years the normal physiological functions of these proteins in the absence of tumors have been investigated. Since TCDD does promote tumor formation in laboratory animals (Kociba 1978) the effect of this dioxin on tumor promoting oncogenic activity’ was investigated. TCDD ‘was SRC activity in liver homogenates from rats (D. Bombick, personal communication). Since pp60SRC is found to increase pp60 a. tyrosine specific .protein. kinase (see Hunter 1984 for review) as is the insulin receptor, it was of interest to SRC know what effect TCDD would have upon pp60 in adipose C in adipose tissue were measured tissue. Levels of pp60SR in two ways; binding to a specific iodine labeled monoclonal antibody and measurement of its autophosphorylating capability. MATERIALS AND METHODS Shorthair male albino guinea pigs were obtained, housed, and administered 1 ug/kg TCDD or vehicle alone, as before. Adipose acetonezether powders and blood serum were prepared and stored as previously noted until needed. 165 Adipose Plasma Membrane Isolation Fat cell plasma membrane was isolated according to the method of Jarett (1974) utilizing a 9% and 15% discontinuous ficoll gradient (in 0.25 M sucrose). Briefly, perirenal and abdominal fat tissue was homogenized in 3.5 volumes of 10 mM TRIS-HCl (pH 7.4), 1 mM EDTA, .25 M sucrose (Thomas "B" glass-teflon homogenizer, 10 strokes, 1800-2600 rpm). The resulting pellet (16000 g, 15') was resuspended in 4 ml buffer (6 strokes, 1000-1250 rpm, Thomas "A" homogenizer) and centrifuged over the ficoll gradient 15-18 hours. The band formed on top of the 9% ficoll was precipitated with 10 mM TRIS-HCl, 1 mM EDTA, pH 7.4 (20 min., 10,000 g) then resuspended with either the same buffer CM: 50 mM phosphate buffer and frozen at -800C until needed. Intestinal Plasma Membrane Isolation Intestinal plasma membrane was isolated by a modification of Miller and Crane's original 1961 procedure: The proximal 2/3 of the small intestine was excised and flushed with cold saline or homogenizing buffer (0.25 M sucrose, 0.01 M triethanolamine-HCl, 0.5 mM EDTA, pH 7.5), then everted and the mucosal layer removed by scraping with a glass slide. All subsequent steps were at 4°C. The mucosa was collected, homogenized in 50 ml buffer (25 strokes, loose fitting glass-teflon homogenizer, 1200 rpm), and the cellular debris precipitated by centrifugation (2600 g - 15 min). Twice, the supernatant was spun at 10,000 g - 20' and 166 the white fluffy layer on top of the pellet was resuspended by homogenization (50 ml, 5 strokes at 1200 rpm). The resulting homogenate was centrifuged at 20,000 g - 10 min., the white fluffy layer resuspended (10 strokes 12000 rpm), and centrifuged again (20,000 g'- 20 min.). The resulting crude membrane pellet consisting of both basolateral and brush border membrane was resuspended in homogenizing buffer and stored at -80°C until assayed for ATPase activity. Both fat and intestinal membrane preparations were monitored by electron microscopy for contamination by other cellular organelles. Epinephrine Binding Epinephrine binding to fat membrane was as follows: to 50 ug protein in 0.25 M sucrose-10 mM Tris (pH 7.4) was added reaction buffer (50 mM TRIS, 1% BSA, pH 7.4) to a final volume of 500 ul. After a 10 min. preincubation at 30°C 3H-Epinephrine was added (final concentration 10-7M) and the tubes incubated an additional 20 min. at 30°C. Cold reaction (3 ml) buffer was added to stop the reaction. The mixture was then quickly filtered over a 0.45 u cellulose-nitrate: membrane .filter (HAWP-Millipore) and washed with 2 - 5 ml aliquots of chilled buffer. The filters were allowed to air dry and quantified via liquid scintillation counting. Non-specific binding was measured by the addition of 10 ul cold epinephrine (final concentration. 10'4 M) ix: alternate tubes prior to 167 preincubation and specific binding was calculated by subtracting the non—specific from the total amount bound. Phosphodiesterase Assay Fat tissue was prepared for the determination of c-AMP and c-GMP phosphodiesterase activity as follows: Tissue in 5 volumes of homogenizing buffer (0.25 M sucrose, 25 mM :6H TRIS-HCl -- pH 7.4, 0.1 mM EDTA, 5 mM MgCl 0, 5 mM KCl, 2 2 1 mM phenylmethylsulfonyl fluoride -- PMSF, and 100 units/ml Aprotinin) -- was homogenized (6 strokes, medium speed) with a tight fitting glass—teflon homogenizer. The homogenate was then centrifuged at 1000 g (5 min. 4°C) to separate the lipid after which the infranatant was subjected to 2500 g (20 min. 4°C). The supernatant was frozen at -80°C until use. The assay for phosphodiesterase activity was modified from that of Wolff et al. 1977: 50 ug of protein were added to 20 mM imidazole (pH 7.4), 100 uM CaCl (final volume 300 2 ul) containing 25 uM 3H-c-GMP or 3H-c-AMP (New England Nuclear). After a 3 min. incubation at 37°C the reaction was stopped by placing the tubes in a boiling water bath for 2-3 min, and 0.5 units 5' nucleotidase (Sigma Chemical) was added to hydrolyze the non-hydrolyzed cyclic nucleotide. After 30 min. (37°C) 1 ml AG l-X8 ion exchange resin (Bio Rad, Cl form, in isoprOpanol:H20:resin -- 2:2:1) was added, the mixture was vortexed, centrifuged (10 min, 3000 rpm, IEC Clinical Centrifuge), and samples of the supernatant taken 168 for liquid scintillation counting. The assay was performed by Mr. Yoshiro Kanemoto. Hormone Sensitive Lipase Assay Hormone sensitive lipase (HSL) was assayed according to the procedures set forth by Khoo and Steinberg (1975) and Khoo et al. (1976) with the following modifications: 100 ug protein in 50 ul 10 mM TRIS-l mM EDTA (pH 7.4) was added to 50 ul of 10 mM Mg Acetate, 1 mM TRIS-ATP, 0.02 mM c-AMP, and 200 ug/ml c-AMP dependent protein kinase and incubated for 5 min at 30°C in order to activate the enzyme. The addition of 0.1 m1 of a 3H triolein substrate composed of 1 volume of the concentrated substrate used for the LPL assay (see . Nilsson-Ehle and Schotz 1976) and 5 volumes 125 mM phosphate buffer containing 2.5 M NaCl and 3% BSA was fOllowed by a second incubation at 300C (30 min) after which the reaction was stopped with a methanol:chloroformzheptane mixture (1.43:1.25:1.0). KZCO3-KZB4O7 (0.05 M, pH 10.5, 1.05 ml) was added and the aqueous and organic layers separated by centrifugation at 3000 rpm (IEC Clinical Centrifuge), 15 min. Aliquots of the aqueous (top) layer were sampled for labeled hydrolyzed oleic acid. Serum Analyses Serum.lglucose concentrations 'were measured using an enzymatic ‘ultraviolet. procedure (Sigma 'Technical Bulletin No. 15 - UV Sigma Chemical Co., St. Louis, MO), and serum 169 insulin, pancreatic insulin, and serum thyroxine concentrations were estimated via radioimmunoassay (Cambridge Medical Diagnostics, Inc., Billerica, MA). Intercellular c-AMP’ concentrations were also assessed. by radioimmunoassay (Biomedical Technologies Inc., Cambridge, MA). The pellet formed during the final step of all radioimmunoassay procedures was dissolved in 0.5 ml NaOH (0.2 N) of which a 0.4 ml aliquot was sampled for beta emissions. Pancreatic insulin was extracted by the method of Potter et al. (1983). The LPL, protein kinase, ATPase, and insulin binding assays were all performed as discussed previously, on either plasma membrane fractions or the dried acetonezether powders as noted. Protein concentrations were determined according to Lowry et al. (1951). SRC Gene Product, pp6O Assay To get some indication of whether or not the SRC gene product pp60, was present in fat tissue, 1 g of tissue (or 25 mg acetonezether fat powder) was homogenized in 2 ml (1 ml) RIPA buffer in a glass-glass Tenbroek homogenizer. RIPA (Radioimmunoassay Precipitation Buffer) consisted of 1% Triton X-100, 1% sodium deoxycholate, 100 Kallikrein units/ml of aprotinin, 0.1% sodium dodecyl sulfate, 0.15 M NaCl, in 0.05 M TRIS-HCl at pH 7.2. Cellular debris was discarded after centrifugation for 5 min at 1500 g and the supernatant was recentrifuged for 30 min at 100,000 g. To 170 500 ug protein, (in 500 ul 0.25 M sucrose, 1 mM EDTA) obtained from the second supernatant fraction was added 3 ul iodinated SRC-Ab mixture. The SRC-Ab ‘was obtained from Oncor, Inc. and iodinated with Na125 I using the enzymobead and lactoperoxidase method suggested by Bio-Rad, Inc. The mixture was allowed to react 30' at 25°C then stopped with 5 ml chilled 50 mM TRIS-HCl, 1% BSA, quickly filtered with 0.45 u cellulose-nitrate membrane filters (HAWP-Millipore), and washed with 3 - 5 ml aliquots of cold TRIS—BSA buffer. After air drying the filters were dissolved in scintillation cocktail and monitored for residual activity. Immunoprecipitation and quantification of pp60 was as follows: 1 gram of fat tissue (or 25 mg of fat acetonezether powder) was homogenized in 2 ml (1 ml) RIPA buffer in a glass-glass Tenbroek homogenizer. The homogenate was centrifuged 2X as before and 500 ug of protein from the second supernatant fraction was added to 10 ul of the SRC-Ab mixture (in distilled H20). After allowing the mixture to sit for 30 min (4°C), 25 mg of protein A-sepharose (Sigma, Inc.), in 200 ul 50 mM TRIS-150 mM NaCl (pH 7.4), was added and the mixture was vortexed. After waiting 3 min the mixture was centrifuged at 1500 g; (2 min), the supernatant discarded, and the bead complex resuspended with 200 ul RIPA. The procedure was repeated 4X with RIPA then once with 400 ul of TRIS-NaCl buffer. Autophosphorylation. of the protein was accomplished by resuspending the protein 171 A-sepharose bead complexicontaining the attached sarc Ab which was bound with the sarc gene product) in 50 ul kinase buffer (20 mM TRIS-HCl, 5 mM MgCl 2! uCi gamma labelled 32P-ATP (Amersham). After incubating 10 pH 7.2) and adding 20 min (30°C) the reaction was stopped with 100 ul 0.125 M TRIS-HCl (pH 6.8), 4% sodium dodecyl sulfate, 20% glycerol, 10% 2-mercaptoethanol and heated for 1 min at 95°C to separate the Ab-Ag from the bead-Protein A complex. The complex was then precipitated by centrifugation (2 min, 1500 g). To 50 ul of the supernatant was added 3 ml 10% TCA plus 0.1 ml BSA solution (100 mg BSA, 136 mg KH PO4/10 ml H20) to 2 effect c0precipitation. The mixture was centrifuged 4 min, 3000 g and the pellet washed twice by resuspension with 0.2 N NaOH (0.5 m1) followed by reprecipitation with 1 ml 10% TCA and centrifugation. The final pellet was resuspended with 1 ml 0.2 N NaOH and aliquots sampled for phosphorylated protein using liquid scintillation counting. All biochemicals were purchased from Sigma Chemical Co. except where noted. All other reagents were of the highest purity possible. RESULTS Glucose Reversal of LPL Previous results indicated orally administered glucose to have no effect in stimulating adipose LPL activity when given 10 days after TCDD exposure. In an effort to further understand TCDD's toxicity, animals were given glucose at 1, 172 2, and 5 days after dioxin administration and LPL activity was monitored. As can be seen in Figure 35, glucose given 1 day after TCDD restored LPL activity to the same extent as when given to pair-fed animals, however after 2 days glucose reversed LPL activity of TCDD treated animals was 89% of pair-fed controls. After 5 days it was 83% of pair-fed controls and after 10 days it was 23% of pair-fed controls as noted before. TCDD seemed to produce a time dependent inability to provide active LPL upon adequate nutritional stimulation. LPL activity was decreased as soon as 1 hour after dosing with TCDD. The average activity of 1221.4 1'. 394.9 nM 3 H- oleic acid released/mg extracted acetone:ether powder/hour in 5 guinea pigs examined. Serum triglyceride concentration was 88 i 35 mg/dl. The inability of glucose to reverse TCDD induced LPL depression, after 10 days, was not due to abnormal intestinal glucose transport or uptake. No change was noted in serum glucose concentrations from either TCDD treated, pair-fed control or ad _l__i;1_3_ control animals 2 days after treatment (Table 24). Although animals starved for 22 days exhibited levels slightly less than the other groups, they were not statistically significant. Nor was any difference seen between pair-fed control and TCDD treated animals after glucose intubation, however there was ea considerable increase in serum glucose compared to the non-administered animals as was expected. The same was true for the 10 day .Aao.n my mcomflummeoo owc:50mcoucs Mow ammo w.mmxoe mcflm: “mmousam usonua3 onus woo oa eouw "mmooqu usonuflz ucmaummuu umwoosfim ucmumooflo saucmuflcnamflm uoz mafia mflucmonuficmam woman uoz o>Hpummmou scum ucmum + Houucoo pwwluflmm eouw ucmuwMMHo wflucmoflm “Honucoo bowiuflmm Ecuw ucouomwflc waucmuflwflcmflm uozc “Houucoo cowiuflmm Eoum acouomwflo waucmuflwacmam "Houucow mam mm eon“ ucmcmocflo sfiocmoflcacmwm .mmfla III In man .101 mmcflsm AOV Houucou QHH om no Adv Houucoo Umwlu omousam powwow» nous wo hufl>fiuom and whomflom co Amzouumv commuwflcHEUm wdfimuo mo pommwm mmusou oEHB .mm Guzman Am> ld‘l coo. 179 TABLE 26. Serum insulin (uU/ml) concentrations for 8 pairs of TCDD treated (1 ug/kg) or pair-fed control guinea pigs, 2 days after exposure. Egg TCDD % Decline 72 6O 17 58 25 56 35 19 46 32 19 40 98 66 33 47 31 34 40 38 5 58 3O 48 x 58 1 21 uU/ml 39 1 19 uU/ml 35% Ii... 180 .mo.v m .ume =u= pouwmm cuflk Houucou ommluflma scum acouommwo anucmoHMHcmHm‘ .Hc\:fiououm mE\oHum ufioao z: .cHE oa\cop30m Dow uozuoumcoumom mE\coumuomHoucH Hm 2Q. .cHeOfi\cHou0uQ ocmuneoe Hammad me\omumcomuoocH an 2&2 .cfie M\cfimboHd ms\nmm>fiocnan axe-0 no 424-0 :8 .msmmfiu «mm m\mz¢|u saw H o @ ~c\msn .CHE om\mcmuneoe ou>00QHom me\ocson Hammofiwfloodm mcflucdocflam : no :HHDmCHIH mm .cfiououd uHumoumcma omuumuuxomma\aso Ho\szm in. m.m H m.mv Am. 6.H H «.mv Ham: .m. av.mm H ma.mom Ami mv.Ho H am.~am sconcmmmncfl 624.0. .m. Ho.mafi . ms.mosa Ame mfl.mm . mo.smHH unmocmamn 424.0 I l HmHsHHoU Am. ma.m~ H mH.mmH Am. om.m~ H am.oH~ sconcmmmccm 824-0 ..m. mm.m~ . Hm.on Am. me.sa . Av.HHH sconcmmoo mz no anus oxmeA nuzuwo nag: acoEumouu nouwm whoa m moan mmcasm no mommau uou can eauom coonn cw mumuoeoumm oauhfiomfin can uficmmomfifl m30wum> co onus no uumuum ohm Ndmdh 181 Insulin and Epinephrine Binding 125 Specific I-insulin binding tended to be increased in isolated adipocyte membrane preparations from TCDD animals as opposed to pair-fed controls (Table 27). Although the variability was large, binding was increased approximately 3.4x over controls. This tendency for an increased binding and the wide variability in values is reasonable considering the decreased serum insulin concentration values and their variability. Since thyroxine is also known to inhibit LPL activity (Hamosh and Hamosh 1983) it was of interest to know what effect TCDD would have upon serum thyroxine concentrations. Serum concentrations of T4 from animals previously dosed with TCDD were not significantly' different from control animals (Table 27). After 10 days exposure, concentrations were slightly increased above the 2 day values, but TCDD had no appreciable effect (1.4 1 0.5 vs 1.7 1 0.6 ug/dl -- control and TCDD treated respectively). Dioxin reduced binding of 3H-epinephrine to the adipocyte membrane by more than 30% after 2 days of exposure (Table 27). No effort was made to measure adenylate cyclase but the total cellular c-AMP concentration was increased 1.6x by TCDD. Levels of c-AMP phosphodiesterase activity were also increased 158% by dioxin exposure. This phosphodiesterase is one of the main catabolic pathways of cellular c-AMP and is under strong positive regulation by 182 insulin (see Figure 34). This promotes switchimg of a fat cell from lipolytic to lipogenic modes. No change was observed in c-GMP phosphodiesterase activity, total cellular protein kinase activity, total cellular c-AMP independent protein kinase, nor in total cellular c-AMP dependent protein kinase activity. Plasma membrane associated c-AMP independent protein kinase was reduced 11% by TCDD. Membrane bound c-AMP dependent protein kinase was significantly depressed 31% relative to control animals. This c-AMP dependent protein kinase is another key regulatory step in adipocyte biochemical pathways. High serum levels of insulin (such as postprandial when high serum levels of triglycerides also occur) shut off HSL activity by inhibiting activation of c-AMP dependent protein kinase and activating c-AMP phosphodiesterase thereby preventing lipolysis while activating lipogenesis. Cellular c-AMP dependent kinase activity was 15-20X higher than that found in the plasma membrane portion as would be expected (Table 27). SRC pp60 Estimation C in fat tissue from There occurred 2.5x more pp60SR animals treated with TCDD than from pair—fed control animals (Table 28). Activity of the enzyme was estimated by autophosphorylation and found to be increased 114% over the pair-fed controls. It is noteworthy that although the variability was large TCDD treatment resulted in values 183 .mo.v m .umme mxcmm cmcmflm cuHB 0mm Eouw ucmcmcuflo HHH60HHMHn6um4 .cflououm m: oom\cmHMHOQHOOCH mmm Emu .cmufiflm\aflmuoum m: oom\wcson omH-Hmma snow I I l nmcaccfln Houaflw «Ame smma + mmmm «inc mam + meom Ase mmm + smma 6H> soflflmso l l mcoflumfimuocmmocmousm oz 4181 was + Hmmm Ami Hem + msmm 6H> mhflncmso 8m oooa omm .acmEumouulumom wmmc N moan omcHDm Amx\m: moav cmummuu we no .Amx\ms av mmfim mocflsm cwpmoHu QQUB .Houucoo cowluamm mo muH>Huom comm mmomflcfi .am mum‘s 184 ranging from 1.5-18X that of control levels in the ' qualitative assay and from 1-2X that of controls in the quantitative assay. The TCDD treated value was always higher than the control animal in any single pair of animals examined. Administration of 105 ug/kg T also significantly 4 increased the amount of pp60 2.5x above control levels. DISCUSSION As concluded previously (Brewster and Matsumura 1984), TCDD reduced LPL activity thereby decreasing serum triglyceride clearance and causing hypertriglyceridemia. Considerable evidence exists for adipose LPL being the critical regulatory step in controlling serum triglyceride concentration (Robinson 1963a, 1970, Scow et a1. 1972, Kompiang et al. 1976, Nilsson-Ehle et al. 1980). The present study indicated that reduction of the enzyme occurred as soon as one hour after dioxin administration, attained maximum depression after two days, and remained at that level throughout the 10 day observation period. Oral doses of glucose are known to reverse starvation induced depression of this enzyme (Cryer et al. 1974, 1975) and are correlated with rising serum concentrations of insulin. TCDD prevented this glucose reversal effect.1U) days after administration but not after 1 and only marginally after 2 days. TCDD therefore seemed to produce a time dependent inability to provide active enzyme upon stimulation. The synthesis and/or activation of LPL seemed normal early but 185 lost this ability over time. This loss was not due to malabsorption of glucose into the blood but rather quite possibly to an inability of the pancreas to synthesize adequate amounts of insulin. Binding studies of the insulin receptor in the adipose plasma membrane at day 2 indicated it; to» be functioning normally' and even. up regulated in response to the lowered serum insulin levels. One must be cautious in interpreting any of the insulin data reported here as the radioimmunoassay procedure used an antibody produced in the guinea pig against porcine insulin and as such would only bind to "foreign" insulin molecules. Guinea pig insulin is very different from that of other mammals (esp. rat, pig, and human) and therefore is of great use in current RIA procedures. About 1/3 of the guinea pig's insulin amino acid composition. differs from other mammalian insulins and it does not bind Zn, dimerize at high concentrations, or form crystals (Smith 1966, Zimmerman and Yip 1974a, Jukes 1979). Unfortunately purified guinea pig insulin and especially anti-guinea pig insulin is not only very difficult to purchase but also very expensive. However, the guinea pig has been recently found to produce two insulins: classical guinea pig insulin found only in the pancreas and blood and a second insulin distinct from normal guinea pig insulin, produced in all tissues (including pancreas and brain) similar to typical mammalian insulin and at concentrations approximating those in non-pancreatic 186 tissues of other maimals (Rosenzweig 1980). It is this second "normal" insulin which was detected in the assays used here and discovered to be depressed by TCDD treatment. Furthermore the production or secretion of this normal insulin may increase upon food restriction since the ad libitum serum insulin values obtained here were always much less than either the pair-fed control or TCDD treated animals. Rosenzweig et al. (1980) concluded that the guinea pig has retained a typical mammalian insulin gene which is expressed in all tissues at low levels in like manner as other mammals. Since the measurement of serum insulin correlated well with LPL activity (Figure 36) it is concluded from these data that at 2 days post treatment with TCDD pancreatic insulin output is decreased. This caused up regulation of the adipocyte membrane insulin receptor and loss of LPL activity. At 2 days oral glucose reversed this loss in activity, probably by da BEES LPL synthesis, and activated an inactive cellular LPL pool which slowly declined over time. After 10 days this reversal could not occur; either glucose could not elicit the pancreatic inSulin response or the adipocyte membrane insulin receptor was inoperable. In either case the inactive pool has been used by this time. TCDD inhibited the lipolytic pathway' in adipocytes: Epinephrine binding and c-AMP dependent protein kinase activity' were: both. depressed. and. c-AMP ‘phosphodiesterase 187 increased by TCDD treatment. Normally this lipolytic inhibition would cause LPL activity to increase, however LPL has been shown to be depressed by dioxin and HSL to be increased or at least unchanged (Brewster and Matsumura 1984, Swift et al. 1981). Preliminary experiments performed in this laboratory also indicate HSL not to be significantly changed. One may suggest this paradox to be explained by the large increases in c-AMP concentration observed here. However, there is evidence indicating that cellular c-AMP levels are compartmentalized and therefore not indicative of lipolysis (Steinberg 1976, Severson 1979). Severson concludes that changes in c-AMP levels can be dissociated from the antilipolytic action of insulin. The reduction of epinephrine binding was not from a downregulation of receptors in response to high levels of serum epinephrine. The treated animals did not display signs of hyperactivity, dermal vasoconstriction, increased cardiac force of contraction and tachycardia, or signs of central nervous system stimulation. The question of the biochemical cause for the decline of LPL activity still remains. It is proposed that this effect is the result of aberrations at 2 different sites: A primary effect on the pancreas to decrease insulin synthesis and/or secretion and a secondary response in adipose tissue. After two days of treatment serum insulin concentrations are low (from decreased food intake if not 188 from pathological causes) which could account for the depressed LPL activity to some extent. Recent data (see Appendix D) suggests that more than decreased insulin levels are involved. Animals treated with TCDD and starved for 2 days had significantly lower levels of LPL activity than control animals starved for 2 days. These data indicate the cell functioned as if it had received increased insulin, since HSL activity was not changed. The absence of any change in HSL activity may be explained by a finely tuned regulatory system - the increase in activity from decreased food intake may be balanced by the antilipolytic actions occurring within the cell. There is precedence, at least within rat adipocytes, that starvation itself uncouples adenylate cyclase activation from lipolysis (Fain and Garcia-Sainz 1983). Although isolated adipocytes from starved animals were more sensitive to isoproterenol activation of adenylate cyclase than those from control animals, lipolysis was significantly reduced. Conclusions It is interesting that the adipocyte behaves like the hepatocyte in that the cell displays increased responsiveness to a substrate when that substrate to the cell is actually reduced (see Madhukar et al. 1984, Appendix I). It is proposed that as with liver cells and EGF, TCDD induces an immernal phosphorylation, which then causes the antilipolytic actions discussed above. This 189 internal trigger could even be EGF itself, as it has been shown 1x) promote intracellular lipogenic activities ‘when administered 1J1 nanomolar' concentrations 11) isolated adipocytes (Ng and wong 1984). Although sustained cellular phosphorylation via protein kinase does not occur in fat tissue there is sustained phosphorylation via pp60SRC similar to that observed in the liver (D. Bombick - personal communication). This pp60SRC is an autophosphorylating kinase of 60,000 daltons which can be increased by EGF and whose cellular substrates are not fully realized. It is interesting to note that T4 (also known to inhibit LPL) also increases pp60 activity. Administration of EGF 1a 111d has been observed to promote serum hypertriglyceridemia (Heimberg 1965). Therefore it is proposed that TCDD inhibits LPL activity by causing endogenous cellular phosphorylation (possibly through altered gene expression) which promotes the reesterification of free fatty acids to stored triglycerides. Therefore TCDD invokes an energy requiring futile cycle in adipocytes: HSL is normal and breaks down stored triglycerides to free fatty acids for export; the increased EGF like receptor activity and cellular phosphorylation. reesterifies these fatty' acids. to stored triglycerides which require ATP consumption; some of the fatty acids are mobilized thus the reason for loss of adipose stores with time; consumption of ATP limits synthesis of LPL therefore no new fatty acids from serum 190 triglycerides enter the cell and hypertriglyceridemia develops. Patton (1970) concluded that LPL is regulated in just such a manner. Furthermore, if the same process is occurring in other cells, nutrient depletion and atrophy would be expected since the cell would reesterify any free fatty acids it obtains into stored triglycerides. This quite conceivably is the mechanism for the fatty liver observed in many species after dioxin administration. These cells are less likely to deplete their ATP stores because of the much greater concentration of ndtochondria compared to adipocytes. The results presented here and in previous work are consistent with this possibility. Further research concerning cellular substrates of the phosphorylated EGF receptor and its relationship to lipogenesis must be examined. In lieu of the similarity of the insulin and EGF receptors and their resulting kinase activities upon substrate interactions this proposal is attractive. An examination. of tyrosine kinase activity, protein kinase C, and Ca (all of which influence EGF activity) must be undertaken after administration of TCDD. It is also interesting that TCDD again, as discussed in Chapter VII, seems to provoke an aging response in these animals. Fain and Garcia-Sainz (1983) cite studies whereby the lipolytic sensitivity of adipocytes from older animals was markedly reduced from that of younger animals. In the present study IK) evidence (ME increased lipolytic activity 191 was noted after TCDD treatment even though LPL was significantly depressed. CHAPTER IX SUMMARY These investigations have sflunni that TCDD does indeed alter the character of the plasma membrane in different tissues and species. Based on the inferences of earlier investigations it was prudent to investigate this cellular organelle further, and it was soon found that extensive time dependent alterations in protein composition of the cellular membrane occur after administration of TCDD. It was then hypothesized that changes occurrtmg at vital physiological and biochemical sites could indeed be responsible for some of this agent's toxic manifestations. In an effort to link these membrane alterations with specific biochemical pathways, a number of receptor and enzymatic interactions were examined for their response to TCDD treatment. The rat liver underwent time and dose dependent inhibitions in ion transport and regulation, growth factor binding, amino acid transport, and an inability to accumulate energy sources and nutrients such as glucose. There also occurred a sustained increase in cellular phosphorylation; a process critical in homeostatic cellular regulation to hormones, growth factors, and other critical biochemical pathways. Little change was seen in 192 193 the livers of guinea pigs and hamsters, two species showing very little hepatotoxicity in response to this chemical. Next an effort was made to correlate some of these biochemical alterations with specific toxic lesions. One such pertubation examined was thymic atrophy. TCDD produced obvious morphological changes on the surface of the thymocyte which may prevent cellular communication and nutrient uptake and be involved in wasting and immunological incompetence. The biochemical mechanism responsible for the surface changes noted could quite conceivably be related to calcium transport and regulation, however this area needs to be examined further. Investigative efforts were then focused upon the cause for the severe serum hypertriglyceridemia observed in the guinea pig after dioxin exposure. The mechanism for this anomaly was discovered to be almost complete inhibition of the enzyme lipoprotein lipase which is responsible for the removal of serum triglycerides. This depression was found to be time and dose dependent and responsible for the loss of fat stores. It also correlated with rising serum triglyceride concentrations and body weight loss; it could not be reversed with glucose after 10 days and it was not a result of changes in serum inhibitors or stimulators. Decreased LPL results in hypertriglyceridemia. This could contribute to secondary effects such as xanthoma, atherosclerosis, cardiac disfunction and eventually death. 194 In addition, the high serum titer of triglycerides could quite conceivably trigger normal feedback mechanisms to reduce food intake, contributing to weight loss and wasting. The question was posed as to whether TCDD diminished LPL and caused serum hypertriglyceridemia in other animal species. Rabbits and. hamsters resembled guinea pigs in their lipid response to TCDD and the similarity of rabbit and human lipid metabolism make rabbits a useful model to study cause and effect of lipid disorders. Rats had not been reported to have increased serum triglycerides and no change in LPL activity was noted. Mink showed a modest decrease in enzyme activity which did not correlate with serum levels of triglycerides probably' because of their complex lipid metabolism. Mice hypertriglyceridemia, for the most part was observed in the sensitive strains and not in the less responsive strains. The size and small amount of adipose tissue available made LPL determination very difficult in this species. Because of the high serum lipids and the reduction of adipose LPL it was of interest to know if heart function was compromised by TCDD. It soon became obvious that the heart is somewhat protected from nutritional regulation of LPL and showed little response to dioxin treatment. However, the membrane bound beta-adrenergic system was reduced in its response to isoproterenol after prolonged dioxin exposure. 195 The depressed rate and force of contraction (basal levels) of heart atria may have resulted from the increased serum lipid load and are indicative of an aging effect. This and the depressed adrenergic response could very well be linked with lethality. Finally, the task was undertaken to elucidate the biochemical mechanism responsible for LPL inhibition. It can be definitely stated that the cause was not due to HSL activation and a reciprocal LPL depression. Enzymatic activity was decreased as soon as one day after treatment, and TCDD was shown to produce a time dependent inability to reverse this depression, independent of serum glucose levels. This finding implicated an inhibition of insulin synthesis or secretion. Although insulin serum levels and the pancreatic response to glucose are depressed the adipocyte functions as if excess insulin is present and prevents high rates of lipolysis. It is proposed that TCDD promotes phosphorylation of the EGF receptor which in turn causes sustained cellular phosphorylation.cnf any number of substrates including' pp6OSRC. It is; this sustained phosphorylation which in turn blocks LPL synthesis and/or activation. This effect is secondary and probably occurs after the depression of pancreatic insulin synthesis and secretion. As far as LPL depression is concerned there are two mechanisms by which the enzyme is not produced. First is 196 the reduction of pancreatic insulin. Data presented in Appendix D indicate that pancreatic EGF-like binding proteins are twofold greater in TCDD treated animals than pair-fed control, indicating a depression of insulin synthesis. However, since animals treated with TCDD and starved have significantly lower LPL activity than starved animals alone (see Appendix D) another mechanism must be occurring other than simply lack of insulin production (or secretion) by the pancreas. The second critical site is at the level of the adipocyte. Either the increased cellular phosphorylation shuts off synthesis directly, or feedback to the synthetic process prevents synthesis, or the cell simply runs CNN: of' energy. Patton (1970) theorized this third possibility to be the reason why enhanced HSL synthesis decreases LPL activity. HSL produces free fatty acids from stored triglycerides which in the presence of glucose and insulin are re-esterified to triglycerides. This process increases ATP consumption and causes a reduction in protein synthesis. This study has elucidated some plasma membrane alterations responsible for a few of TCDD's toxic manifestations. However, the underlying mechanism of TCDD toxicity (how these alterations come about) is still a matter of conjecture. Direct interactions with the plasma membrane are not indicated as 1a 11139 enzymatic inhibition does not occur, far more toxic lesions occur than the body 197 burden of TCDD allows (the number of dioxin molecules in the body is far less than the number of lesions) and TCDD is known to first interact with a cytOplasmic receptor, then is transferred to the nucleus to produce a pleotropic effect. Proposed Unifying Theory for TCDD's Mode of Action There is now some evidence that TCDD invokes an increased tyrosine kinase activity which is coupled with an increase in an activated EGF receptor synthesis (B.V. Madhukar - personal communicaton). This would account for the increased hepatic EGF receptor phosphorylation and depression of ligand binding (increased internal phosphorylation of the EGF receptor causes down regulation of the membrane receptor' by' a feedback mechanisn1 - see Appendix A). Present results indicate that TCDD increased protein kinase and/or tyrosine kinase activity in the liver, adipocyte, and pancreas. Other studies in this laboratory have shown increased tyrosine kinase activity in the thymus, the liver (D. Bombick - personal communication), and in an 1a; 11119. system of XB cells (B.V. Madhukar - personal communication). In. all tissues displaying toxic effects after TCDD administration, tyrosine kinase activity has been found to be increased. It is therefore postulated that activation of tyrosine and other kinases, ome stimulatory and others inhibitory, is responsible for the manifestations of toxicity in most of the tissues listed in Figure 37. Figure 37. of action. 198 Proposed unifying hypothesis for TCDD's mode coqrazcm cuoou 0cm ocdcmao 0fl_o>o ou 05a» azacmnnomm >ocmd0nuusmcfi OHOumu sufiflonuamm t 11 uowuww mumhfl k cu30uq Hemwucfi-lll k I k ‘1 mmocm>fimcommou cumwh uuooz ufioumcouom .L ‘11 COHUOMUQm 0CD k. mfiemczamcfionxc Esuwmllnll mamocucxw 2:09; 1| 0; OJ 1; x:30uu0 ufisxcu .mcflummz 0D :odusnfiuucou mflEoUHHUUHHoHuuuwmxz EDHOm oHEwoaumuxfimHuuuomx: Eauwm mucouomeoo HoodoOHOCDEEHAM \ COfiuMUACSEEOU A (”A Howanficcfi 0E0m HHOuMHDEHum 060m. QQOuUCQm V mommcflx uwcuo .1 new czamohxu ix mseaca\ 05203056 0325: oomHEOEQEOUTimcosouofim .505 500/ ofleo~0uoumoH020uoaxc Esuom unsmom _mudvo.oww>ca .1) co—:_~ou I mflwwfiur—%m «ng‘l/I .nTl. >u~>auom Amq.l ouxuoma0< .l weanedn do: 14 /uw>aq I COHUocma~< poufleocuowm mounds unusaum ”KEG: ucwHHUNuC/I HO COHUN>HHU< / 285;... cesamumc mom \ QOUH 200 Activation of kinases in the liver system would be responsible for general membrane alterations and at least in some cases compromised hepatic function. Since the LDL receptor is a tyrosine kinase when bound with its ligand, endogenous activation of tyrosine kinase would cause down regulation of the LDL receptor and lead to serum hypercholesterolemia, a common response of TCDD exposure. Another example is the insulin receptor which is organized in a similar manner as the EGF receptor in that activation by a tyrosine kinase is expected to result in down regulation. In the adipocyte, tyrosine kinase activation has also been demonstrated and may be responsible for the depressed LPL synthesis or activation described in the current study. This link has not been proven here but as evidenced by these data the reduction of LPL is responsible for the second major lipid defect produced by TCDD -- serum hypertriglyceridemia. As for the thymus and pancreas, general growth hormone binding, nutrient uptake, and cellular communication are affected. Consequently thymic atrophy, wasting, depressed immunological competence, and serum hypoinsulinemia would be expected. An increase in the phosphorylation activity of the EGF receptor in the pancreas was shown in this study (see Appendix D). In view of recent evidence that increased EGF caused a decrease in insulin secretion from the pancreas 201 (Scott et al. 1985), it is reasonable to assume that the increased phosphorylation of the EGF receptor would cause the reduction of insulin synthesis or secretion by this tissue. As for the heart, EGF can effect the beta-adrenergic system through alterations in GTP metabolism and the GTP binding protein. As discussed. by Fain and Garcia-Sainz (1983) alpha and beta receptors can be modulated by guanine nucleotide binding protein which in turn is influenced by the phosphorylated EGF receptor and tyrosine kinase. The effect of EGF on teeth and epithelial tissue is well known. It has been demonstrated in these studies that both EGF and TCDD administration cause early tooth eruption and eyelid opening in neonatal mice (Appendix A). Significance This research has elucidated some of the mechanisms responsible for TCDD's toxicity. An insight into the biochemistry of toxic symptoms has been established. TCDD is a unique chemical in that it provokes many different changes in different tissues and organs, and as a result it can be used as a probe to study the normal cellular physiology of those tissues. With this understanding one can gain an understanding of how other environmental pollutants cause toxicity and rum: to prevent these biochemical alterations from occurring. APPENDICES APPENDIX A THE INFLUENCE OF 2,3,7,8-TETRACHLORODIBENZO- P-DIOXIN ON EPIDERMAL GROWTH FACTOR RECEPTOR BINDING IN THE HEPATIC PLASMA MEMBRANE OF THE RAT, GUINEA PIG, MOUSE, AND HAMSTER It has recently been found that the plasma membrane protein composition and function of hepatocytes from TCDD-treated rats are quite different from those of control rats (Brewster et al. 1982, Matsumura et al. 1984). Therefore, the current investigation was undertaken with the following objectives: (i) find several biochemical parameters on the plasma membrane that are severely affected by TCDD, (ii) study whether any of the changes occur at low enough doses and at very early stages in susceptible species, and only at high doses in tolerant species, and (iii) make an attempt to relate such effects to some of the toxic manifestations 1a_ 3119. This study was done in collaboration with Dr. B.V. Madhukar and Mr. David Bombick of the Pesticide Research Center, MSU. Dr. Madhukar completed tine dose response relationships, scatchard analyses, EGF binding to mouse plasma membrane and autoradiography, and Mr. Bombick the effect of various chemicals on EGF binding in the rat liver. I examined the time course of changes in specific binding of EGF in the rat, the changes of body and thymus weight after TCDD 202 203 treatment, and aided in ascribing the effect of TCDD on eyelid opening, incisor eruption, and hair growth in neonatal mice exposed to TCDD or EGF. MATERIALS AND METHODS Male Sprague-Dawley rats (150-200 g) and Golden Syrian hamsters (80-90 g) were obtained from Spartan Laboratory Animals (Haslett, MI). Male guinea pigs (200-250 g) were obtained from Michigan Department of Health, Lansing, MI and female BALB/c mice were purchased from Harlan Laboratories (Haslett, MI). Inbred mouse strains C57BL/6J, CBA/J, and AKR/J were obtained from The Jackson Laboratory. Food and water were provided ad 11a. All chemicals used 13 1113 were administered to the animals intraperitoneally (i.p.) as solutions in either corn oil/acetone (9:1; TCDD), 0.85% NaCl (phenobarbital), or corn oil (all others). Control animals received an equivalent volume of the vehicle only. TCDD ‘was a gift from Dow and was >99% pure (GLC examined); 3,4,3',4'-tetrachloroazoxybenzene was generously given to us by M.T. Stephen Hsia (University of Wisconsin, Madison, WI) and was >99% pure; sodium phenobarbital was purchased from NBllinckrodt; 3-methylcholanthrene, and epidermal growth factor (EGF) were from Sigma; Aroclor-1242, a polychlorinated biphenyl mixture containing 42% chlorine, was a gift from Monsanto; 3,4,3',4'-tetrachlorobiphenyl (>99% pure) was obtained from Analabs, North Haven, CT; and 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane >99% pure 204 was a gift from Montrose Chemical (Torrance, CA). Firemaster BP-6 was given to us by Matthew J. Zabik (Michigan State University). lzsI-labeled insulin (specific activity, 80-103 uCi/us: 125 1 Ci = 37 GBq), I-labeled EGF (specific activity, 161-174 uCi/ug), and 3H-labeled. Con 21 (specific activity, 25-50 329] ATP Ci/mmol) were purchased from New England Nuclear. [ (Tris salt, specific activity, 3000 Ci/mmol) was obtained from Amersham, alpha-Methyl D-mannopyranoside and insulin (porcine) were purchased from Sigma. Receptor grade EGF was obtained from Collaborative Research (Waltham, MA). All other biochemicals and chemicals used were of the highest purity available. Liver plasma membrane from normal or TCDD-treated animals was prepared as described by Peterson et al. (1979a) and preparations were periodically examined by 125 electron microscopy. Binding of either I-labeled EGF or 125I-labeled insulin to liver plasma membrane was assayed according to the method of O'Keefe et a1. (1974) and 3H-labeled Con A binding was determined essentially as described by Chandramouli et al. (1977) with minor modifications as described by Brewster et al. (1982). Phosphorylation assay was done essentially as described by Rubin et al. (1982). Females of BALB/c mice (15-18 days pregnant) were housed in plastic cages individually, and food and water 205 were provided ad 1133. The time of delivery was closely followed and within 3 hr, the dams were treated i.p. with a single dose of either TCDD in corn oil/acetone (9:1) at 10 ug/kg or with the vehicle alone (0.5 ml per 100 g of body weight). Prior to treatment, two littermates from each litter were exchanged and marked to identify them from the original littermates. Body weights of the neonates were recorded daily and were checked for incisor eruption and eyelid opening twice daily (between 8 and 9 a.m. and between 5 and 6 p.m.). The day of delivery was considered as day 0. Hair diameter and length were measured on day 14 from samples taken from the mid-right dorsal region. They were mounted on glass slides with glycerol and measured under a microsc0pe. EGF in 0.85% NaCl was injected subcutaneously to newborn mice daily at 2 ug/g of body weight. Body ‘weights and. other developmental parameters were measured as described above. All animals were sacrificed at 22 days of age and thymus weights were recorded. RESULTS We have examined the effects of _i_a yard-administered TCDD on three receptors of the rat hepatic plasma membrane at various doses to assess which of these is most sensitive. It is clear that the effect of TCDD on EGF binding was most pronounced, followed by that of Con A and insulin. It is significant that the effect on EGF binding 206 is observable at a dose as low as 0.1 ug/kg. It was also noted that insulin binding was significantly lower at the highest dose (115 ug/kg) and higher at low doses as compared to the corresponding control preparation at 10 days after treatment. In view of the sensitivity of EGF binding to TCDD treatment, we examined the time course of TCDD effect after a single i.p. dose (25 ug/kg). As observed perviously (Figure 12) during the 40-day observation period, TCDD-treated rats gained consistently less body weight than did control rats. During the same period, the level of EGF binding was continuously suppressed. The decline was noticeable on the second day, reached a maximum on day 20, and by day 40 a trend of apparent recovery was observed. At this dose and treatment, this apparent recovery is believed to result from mortality of the susceptible population. The average mortality was 0 at day 20, but reached 20-30% by day 40; therefore, the data at this time point represent the value from surviving animals. The nature of the changes in the EGF receptor was studied by Dr. Madhukar using Scatchard analysis of ligand-receptor' binding., The results indicated. that. EGF binding generally showed a: biphasic relationship, as shown by Ivanovic and Weinstein (1982) and that the number of high as well as low affinity receptors in the TCDD-treated rats 207 was reduced without any apparent changes in the receptor affinity. To study whether such. biochemical changes are also evoked by other toxic chemicals or only by TCDD, Mr. David Bombick assessed the effect of various xenobiotics 1a 1113 on EGF binding 11 11119. Among nine chemicals tested, only TCDD and Aroclor-1242 caused a significant decrease in EGF binding. In both cases, the effects appear to be dose related. It must be noted that rather high doses were used for other chemicals, resulting in the manifestation of toxicities in most cases. Yet, even under these conditions no reduction in EGF binding was observed. To further understand the relationship between these biochemical changes and susceptibility of different species to TCDD, the dose response of EGF binding was examined by Dr. Madhukar. Ten days after single i.p. treatments, plasma membranes were isolated from each animal, and the levels of EGF binding were quantified. The results indicate that the guinea pig system was most sensitive, followed by that of the rat and the hamster. If one adopts the ISO (i.e., the dose of TCDD to suppress EGF binding to 50% of the control level) as a critical dose, the rat and the hamster may be considered to be “14 and ~32 times less sensitive, respectively, than the guinea pig in this regard. At I75, the species difference was much greater. 208 Inbred strains of mice (C57BL/6J, CBA/J, and AKR/J) known to show different degrees of tolerance to TCDD (Poland et al. 1974) were examined. The results (Table 29) clearly indicate that reduction in EGF binding was more severe in the two sensitive strains (C57BL/6J and CBA/J) than in the resistant strain (AKR/J). Although the CBA/J strain is known to possess a high affinity cytosolic TCDD receptor, it is tolerant to TCDD in terms of cleft palate formation (i.e., a teratogenic effect). It is interesting that this strain is sensitive to TCDD, as judged by EGF receptor assay as well as body weight loss and thymic involution. To study whether some of the TCDD-caused toxic effects are similar to those produced by excess EGF (see Discussion), mouse neonates were treated postnatally with TCDD and various developmental parameters were examined. The most recognized 11 111$ effects of EGF are early eye Opening and tooth eruption in mouse neonates (Heimberg et al. 1965; Cohen and Elliot 1963). The action of TCDD in this regard was clear (Table 30) in that both events occur at an earlier age in treated animals than in controls. Other parameters examined also show that the lesions caused by TCDD are remarkably similar to those occurring in EGF-treated animals (Schlessinger et al. 1983). When Dr. Madhukar isolated hepatic plasma membrane from treated and control rats, incubated it with [gamma-32PIATP, subjected it to gel electrophoresis, and autoradiography, it 209 .mooo.v m .Houucoo EOHN ucocowwflo Hammoflumfiumum« ..ucmHoB zoom mo m OOH com me m.ov wcoam ofloflcm> 0:» mo oEDHo> oumflumonmmm cm om>flmowu mfiouucoo .mx\m= mad um Rana. occuoum\flflo :Hoo :H onus mo omoc .m.H onCHm 0 cu“: coumouu mums mufizm .m.m.cno.mm .m.c.suo.mm .mie.mum.cm im.a.flum.mm *Amv~.maum.mm Avim.fiaflm.~ofi n\c14 .Am.H.~no.HH imim.~uk.mv .imcm.mnm.mm .m.m.mnH.MOH .imefl.o HH.~ resc.4fluc.-4 n\00 ca mcflmuum 00005 :H ucmflmz mashcu 0:0 .ucmfimz x00h .ocmnnEoE mammflm ofiummoc Ou m:w0cfln mom coflmnmmnamma CH momcmcu .om mqmoa Ace mom Ace oooe acmEumouB Ace Hocucoo cam .coflumsum HOmHocH .UOHE o\mq99.99% pure - Dow Chemical Co., Midland, MI) was dissolved in acetone:corn oil (1:9) and was administered intraperitoneally to the animals at doses of either 1 or 50 ug/kg. Ten days later animals were etherized, blood 217 collected via cardiac puncture, abdominal and perirenal adipose tissue collected, and the liver perfused. Serum triglyceride concentrations, adipose Iii; activity and hepatic LDL binding were determined as before (Brewster and Matsumura 1984, Bombick et al. 1984). Serum cholesterol concentration. was determined by the method of Carr and Drekter (1956), protein concentration Via the procedure of Lowry et al. (1954), and insulin concentration via radioimmunoassay' (Cambridge: Medical Diagnostics, Inc., Billerica, MA). Twenty days after treatment sections of aortic arches were prepared for transmission and scanning electron microscopy utilizing the methods put forth by Hooper et al. (1979). All reagents used were of the highest quality available and were purchased from Sigma Chemical Corp., St. Louis, MO. RESULTS At these doses TCDD treated rabbits showed little of the wasting syndrome typically produced in other species after exposure to this compound for 10 days. Body weight at the end of the 10 day exposure was 89 1 8% of initial body weight for animals dosed with 50 ug/kg TCDD and 94 1 3% for the pair-fed controls to this dose. At 1 ug/kg treatment body weight was 96 1 4% while pair-fed controls were 96 1 3% of initial body weight. However, after 20 days at 50 ug/kg pilo erection and hair loss were evident and the animals 218 exhibited a significant decrease in cage movement preferring to remain huddled near the back of the cage. Food and water intake were qualitatively depressed. Abdominal and perirenal fat weight was approximately 12 g; in the treated animals and 14 g in the pair-fed controls (Table 31). Gross observation of the liver revealed hypertrophy, molted appearances, and. occasional fatty infiltration in those animals with the higher dosage. An increased brittleness of the hepatic arteries was also apparent thereby making hepatic perfusion. much more difficult in these animals. One liver from the 1 ug/kg group de- monstrated severe fatty infiltration and hypertrophy. Others from this group resembled controls lJl-all aspects. At the higher dose 6 of 9 animals died between days 7-10, therefore the reason for 9 pair-fed animals but only 3 TCDD treated. Serum from all treated animals was very cloudy, indicative of the high lipid concentration. A yellow to greenish pigment was observable in the serum from the higher dosed animals. As seen by Table 32, LPL activity showed a dose dependent decrease being only 19% of pair-fed levels after 10 days of treatment. No significant change was observed in activity between pair-fed and ad 119 control animals. Likewise, hepatic LDL binding was 50% of pair-fed control levels after 10 days and also showed a dose dependent depression. HmHHOHOML soccmm cuH3 conhHmcm mama .umm HmcoHHHom cam HmcHEocc< .HO.v m cuHB Houucoo cmeHHmm 0>Huooammu souw ucmummch HHHmuHumHumumc c .mCOmHHmmeou cwccsowcooc: How awou m.xmxse an cmonHow K>oz¢ .mHmEHcm Ac. How :oHumH>o© cumccmum + :mozm 219 I I I I I .Hc\me. .m. mm + mmH ..m. mH + HHH .m. MH + omH ..m. H + mmH .m. 4m + cHH 06005.0 I I I I I .Hs\ss. .4. m + mH .m. m + «m .m. H + AH .m. m . HH .4. m + cm cHHsmcH I I I I I .HEHHE. .m. H + 6H .m. H + MH .4. H + HH .4. H + mH .m. H + NH chhocm I I I I I .Hc\me. .m. Hm + co ..m. mH + moH .m. cm . as ..H. mm + moH .m. cm + mo HocmnmmHoco I I I I I .HO\ E. .m. «H + be ..c. HOH + ova .H. «A + cm ..4. OH + mom .m. cm + mm mcHumomHche «ED-HOW .m. m.c H «.HH .m. m.v H o.mH .m. O.H H m.HH .m. m.~ H H.~H c2 .0. .Hz H61 I I I I .meHHcH H. .c. m + Hm .m. m + mm .4. m + cm .m.cv + cm oz .Hz atom 0mm onus ome oooa nHH mm mx\mc om mHHmc H mx\m: om Ho H OCHHouchHEcm Hmuwm .ESHHQHH mm now no pmeuHmm 6:0 move mmmc OH mannmu mo muoumemumm HmonoHonhcm mooHHm> .Hm Ham‘s TABLE 32. powder/hr) 220 Adipose LPL activity (nM and hepatic LDL binding 3 (ng H oigic acid/mg acetone:ether I-LDL bound/200,000 cells/hr) in rabbits 10 days after ad lib feeding or pair-fed to those dosed with 50 or 1 ug/kg TCDD:— LPL Activity LDL Binding Ad Lib 463.2 1 92.2 (3) ND 1 ug/kg PFC 400.1 : 124.0 (3) 134 i 22 (4) TCDD 224.5 1 197.3 (3) 12 1 22 (2) 50 ug/kg PFC 278.9 1 191.9 (9) 138 1 12 (2) TCDD 52.8 i 19.0 (3) 65 : 23 (5) 221 Serum triglyceride and cholesterol concentrations were significantly increased by TCDD treatment 2.6 and 1.5 times above control levels respectively (Table 31). Little change was seen in serum protein concentration. Insulin was non-significantly increased over pair-fed control levels at the higher dosage but was no different from ad lib control levels. Serum glucose however was significantly lower in the treated animals. Scanning electron microscopy revealed severe altera- tions in the surface morphology of aortic arches taken from treated animals 20 days after administration (D. Bombick - personal communication). Ruffling and sloughing off of the surfaces was evident. Transmission electron microsc0py showed again a sloughing off of outer layers and loss of endothelial integrity. DISCUSSION TCDD produced dose and time dependent inhibitions in adipose lipoprotein lipase activity and hepatic LDL binding. Adipose LPL has been shown to be the key regulatory enzyme controlling removal of triglycerides from blood (Kompiang et al. 1976, Borensztajn 1979, Korn 1955, Robinson .l963a). It ii; synthesized. by' the adipocyte and secreted into the blood whereby it is bound by a glycos- aminoglycon heparan sulfate receptor on the endothelial cell surface. Its main function is to hydrolyze triglycerides carried by dietary chylomicrons (produced by the intestine 222 and consisting of ~80% triglycerides) and endogenously produced very low density lipoproteins (60% triglyceride, 22% cholesterol and cholesteryl ester, produced from both the intestine and liver) thereby promoting storage of the resulting free fatty acids by adipose tissue or allowing beta oxidation of these energy sources by muscle tissue. Chylomicron remnants consisting primarily of cholesterol and cholesteryl ester are normally removed from circulation by hepatic uptake. Catabolism of VLDL also occurs via a reduction in triglyceride composition via LPL resulting in a lipoprotein consisting of 50% cholesteryl ester and 10% triglyceride termed. low' density lipoprotein (LDL), which functions to provide necessary cholesterol to tissues and organs throughout the body (Hamosh and Hamosh 1983). Therefore the function of chylomicra and VLDL is to transport dietary exogenous and endogenous triglycerides from the intestine and liver to various tissues for either storage or energy ‘whereas that of LDL is to transport cholesterol, needed for membrane integrity and steroid synthesis. Hepatic LDL binding was used as an indicator to measure removal of LDL particles from the serum. As discussed by Hamosh and Hamosh (1983) the "LDL pathway" consists of binding to a cellular surface receptor, followed by endocytosis and finally lysosomal degradation. The LDL receptor has been shown to be a glycoprotein and is 223 competitively inhibited by ‘VLDL (Anderson et al. 1976, Goldstein and Brown 1974). Therefore it appears as if TCDD causes an alteration in surface' membrane characteristics resulting in an inhibition of LDL binding and LPL binding to their respective receptors. LPL half life in the serum is controlled by its binding to the heparan sulfate receptor. The enzyme-receptor complex has a t 1/2 of 10-25 minutes whereas the free enzyme is rapidly removed from the liver and degraded within 1 minute. The surface alterations reported here and caused by TCDD administration are consistent with earlier reports from this laboratory suggesting changes in protein constituency to occur in the hepatic plasma membrane of the rat thereby resulting in pertubations of critical physiological enzymes and alterations in bindimg of important homeostatic ligands to their receptors such as insulin and epidermal growth factor (Brewster et al. 1982, Matsumura et al. 1984, Madhukar et al. 1984). TCDD has also been shown to cause inhibition of LPL activity (Brewster and Matsumura 1984) and LDL binding (Bombick et al. 1984) in the guinea pig, resulting in the hyperlipedemia seen in this species. As demonstrated here, the same results occur in the rabbit. The yellow imparted to the serum does not seem to be proteinaceous since no change in serum protein concentration was observed. It could be due to an excess production of 224 bile salts, bilirubin, (n: other' products resulting from increased porphyrin synthesis since TCDD has been implicated to cause a large increase in delta-aminolevulinic acid synthetase activity in the chick embryo (Poland and Glover 1973a). This rate-limiting enzyme in the hemebiosynthetic pathway, is not significantly changed in rats (Woods 1973) upon exposure to TCDD. However, various mammalian species have a very wide variability in their response to porphyrogenic agents as discussed by Woods and Dixon (1972) and Woods (1973). Since LPL synthesis is controlled to a large extent by insulin and to a lesser extent by thyroxine an effort was made to determine whether the serum concentration of these agents were altered by TCDD. No changes were seen in insulin, thyroxine, or triiodythyronine concentrations. Glucose was significantly below that of pair-fed control concentrations and may partially account for a decrease in active LPL levels since the enzyme is synthesized as an inactive precursor which needs to be glycosylated for activation before secretion (Hamosh and Hamosh 1983). Recent evidence indicates that LPL may be important in forming HDL (high density lipoprotein) during the lipolysis of chylomicra and VLDL triglyceride (Dieplinger et al. 1985). Since HDL has been implicated in scavenging serum cholesterol and thereby protecting against atherosclerosis (Dieplinger et al. 1985, Brown et al. 1981) the inhibition 225 of LPL as well as the inhibition of LDL binding may play a role in the formation of the preatherosclerotic lesions observed in this study. There is ample evidence to relate hyperlipedemia with atherosclerotic and arteriosclerotic processes. Stein and Stein (1979) cite several reports demonstrating the passage of lipOproteins and cholesterol into the intima, media, and adventitia of the endothelial wall. Results of this invasion include denudation, proliferation of smooth muscle cells, and platelet adhesion to the subendothelial surface. The earliest detectable lesion leading to atherosclerotic plaque formation is a deposition of cholesteryl ester in smooth muscle cells and macrophage foam cells of the intima and media as described by Goldstein et al. 1983. Similar results were produced in the present study with TCDD as observed with electron microscopy. Watanabe (1980) observed severe atherosclerosis within 2 months in rabbits deficient in LDL receptors therefore, it is feasible to notice preatherosclerotic lesions within 20 days after dioxin treatment as the present results indicate. After cholesterol deposition, eventually a necrotic cholesteryl ester filled core and a fibrous cap from the atherosclerotic plaque (Goldstein et al. 1983). A potentially serious implication (n? this research is the production of similar types of effects in humans upon exposure txn TCDD cnr other polyhalogenated aromatic 226 hydrocarbons. Oliver (1975) and Pazderova—Vejlupkova (1981) have reported hypercholesteronemia in humans exposed to either TCDD or 2,4,5-T. Industrial workers exhibiting chloracne, resulting from occupational exposure also exhibited hyperlipedemia (Walker and Martin 1979). In conclusion, we have examined the mechanism by which TCDD produces 2 of its many toxic manifestations. We have studied the mechanism, the result, and long term effects of dioxin in the rabbit. Probably through membrane altera- tions, adipose LPL activity and hepatic LDL binding are depressed by this dioxin, resulting in increases in serum triglyceride and cholesterol concentrations. One serious direct effect of this hyperlipedemia is the changes in endothelial cells of the aortic arch reminiscent of pre-atherosclerotic lesions. Longer effects could conceivably include total arterial blockage, which along with high serum lipids could easily compromise heart function and contribute to lethality. APPENDIX C THE EFFECT OF TCDD ON ADIPOSE LPL AND SERUM LIPID PARAMETERS IN THE MINK This study was done in collaboration with Drs. Aulerich and Bursian in the Department of Animal Science at MSU who had shown mink to be very sensitive to TCDD (LD50 ‘7 ug/kg - personal communication). Because of the similarity in LDSO to that of the guinea pig it was of interest to know what effect TCDD had on various lipid parameters in this species. These animals showed a dose response inhibition of LPL activity 28 and 43 days after treatment (Table 33). Changes in serum triglyceride concentrations in response to TCDD treatment were difficult to evaluate because of the high ad lib. control value. If mink are nocturnal animals quite possibly these 4 individuals had just finished feeding before sacrifice, which occurred at. approximately 8 a.m. Also these animals were sacrificed on day 28 not day 43 like the other groups. Overall, it appeared that serum trigly- cerides decreased with increasing dioxin doses. Little changes were noted in serum cholesterol or protein concentrations. Serum glucose was significantly elevated in the 28 day groups. 227 228 .one Hmummo .uz zoom .coHumH>wt pumocmum H coo: .xumHo muwono HHm .m ooom A .mmoo HmHo mech meE HH< .QU'O m p.mm HSH om mm oz m .H. cm.k o.e~ NHH He ma oz mm AN. o.m v.H H O.Hm mm H «mm m H mm m H VNH oz mm Av. m.m O.H H H.mm H H moH m H on cm H NOH mHm ma Ami O.H m.m H o.mm HH H aHH o H am e H mmH m.~mH H o.mv0H me .~. H.O m.H H O.Hm SH H mHH N H HA no H NNH o.~m~ H a.mmmH me Ami Ho.o m.o H a.mm as H mkH a H as m H mm a.mv H O.HmmH ma om. Hoo.o oz omH ms moH oz m AH. oioooio H.H H a.mm mH H 0mm NH H mm mm H 0mm m.~>~ H m.eHoH mm Hes onH 84. o I I I I z oHH om Hk.o . p.mm Hm + mmH HH . mm hm . mVH oz 0 ... on zum>i o Iflmwmal Iflmflmal H©\me Hc\me un\uumuuxo you me IAMNmmfll Mfll mmmon :wmuoum wmooomo HoHobonozu oUHHmomeHua \oHom onHOIm 2: Had mHSmOQXN m .coHumuumflcHeom Houmm mxmn mv pom .mm OHQHH Esuwm msoHHm> pom >HH>Huom 4mg mmooficm xCHe co onus cououmwcflec m >HHmHo no muommum .m an muomcomeoo .mm Nflmtb 229 Because of the various exposure times, low number of replicates and different color variations of animals it is impossible to make any firm conclusions. The change in LPL and serum triglycerides along with the sensitivity to TCDD displayed in these animals does warrant further investigation. In a second experiment animals were orally dosed with acetone:corn oil, or with 4 ug/kg TCDD, and pair-fed to the treated animals or allowed to feed ad libitum. After 4 days abdominal and perirenal adipose tissue was removed and serum collected for the determination of LPL activity and triglyceride concentration. Results displayed in Table 34 indicate serum triglyceride levels to be severely reduced in those animals exposed to TCDD compared to the controls. Unlike the other species investigated, this did not correlate with adipose LPL activity, since this was also lower than pair-fed control activity but marginally increased over the ad lib controls. 230 TABLE 34. Adipose LPL activity and serum triglyceride concentration of ad lib, 4 ug/kg TCDD or pair-fed mink after acute exposure 0 LPL TG (nM/mg extracted fat/hr) (mg/d1) ad lib 1177.78 1 222.45(5) 138 1 64(7) 4 ug/kg TCDD 1467.43 1 327.98(6) 58 1 30(6) Pair-fed 1917.75 1 308.31(5) 138 + 36(6) Appendix D ADDITIONAL DATA CONCERNING THE BIOCHEMICAL MECHANISM FOR LPL INHIBITION In an effort to determine whether decreased serum insulin levels were the sole cause of depression of adipose LPL after dioxin treatment, five guinea pigs were ad- ministered 1 ug/kg TCDD and starved for two days prior to LPL determination. A second group of animals were administered acetone:corn oil, and also starved for two days as control animals. Food restriction depresses enzymatic activity by altering serum hormone levels. Therefore, if a synergistic effect occurs with dioxin, it must be due to an effect at the level of the adipocyte. Results indicated the TCDD treated group to have significantly lower activity (t test, P <.01) than the control (264.6 1 72.8(5) and 411.5 1 222.2(3) nM/mg extracted fat protein/hr, respectively). Therefore, it is concluded that TCDD not only lowered serum insulin levels but also had a direct effect on the adipocyte to impede LPL activity. In a second experiment in order to ascertain the effects of TCDD on pancreatic EGF, a group of animals was sacrificed after a two day exposure to 1 ug/kg TCDD or acetone:corn oil. Pancreatic tissue was removed and 32P assessed for incorporation into EGF-like binding 231 232 proteins. Results indicated TCDD treatment to increase this activity in the pancreas by two fold (PFC = 11.2 1 4.1; TCDD 22.5 1 3.2 nM 32F incorporated/mg protein/3' -- 5 control and 5 TCDD treated animals). LI ST OF REFERENCES LIST OF REFERENCES Abumrad, N.A., P.R. Perry, and R.R. Whiksell (1985). 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