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I, *a‘. .E'afi... ‘pr '4?” '4'; 5 1! 3.: -"‘ ‘a’ ,; ' 5,570.»... 4 "v . - '5?!" .3... j, 1:. '14.ng5’ , _ z~€l§£€h- ;;- 5 / w Lr / MIC HlG NESTAT UNIV ERSITYUB 1 IIII IIIIII I III I IIII I I IIIIIIIIIIIIII 3 1293 00558 4903 LIERARY Michigan State University This is to certify that the dissertation entitled EFFECTS OF Ah-INDUCERS ON THE ACTIVITY OF THYROID-REGULATED ENZYMES \ AND CONTROL OF THYROID HORMONE METABOLISM presented by William Louis Roth has been accepted towards fulfillment of the requirements for Ph.D. degree“! Biochemistry WW Major professor Date 5—. ”/8? MS U is an Affirmative Action/Equal Opportunity Institution 0» 12771 MSU LlBRARlES- —‘—- RETURNING MATERIALS: PIace in book drop to remove this checkout from your record. FINES wiII be charged if book is returned after the date stamped below. £03 0 9 2000 EFFECTS OF Ah-INDUCERS ON THE ACTIVITY OF THYROID-REGULATED ENZYMES AND CONTROL OF THYROID HORMONE METABOLISM BY William Louis Roth A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1988 I, ‘//‘;f"4 Y I :7 ’/ - ABSTRACT EFFECTS OF Ah-INDUCERS ON THE ACTIVITY OF THYROID-REGULATED ENZYMES AND CONTROL OF THYROID HORMONE METABOLISM BY William Louis Roth TCDD (2,3,7,8-tetrachlorodibenzo—p—dioxin) is the most toxic of a class of polycyclic aromatic hydrocarbons which cause the induction of enzymes associated with the Ah locus. Some of the effects of Ah inducers on metabolism appear to be explainable as secondary effects of the induction of the Ah locus enzymes mentioned above. Although these enzymes were discovered by virtue of their catalytic activity towards carcinogenic compounds such as benzo[a]— pyrene and 3—methylcholanthrene, they are known to hydroxylate and or conjugate fatty acids, steroids, and thyroxine (T4). Since (T4) conjugation and excretion was known to be increased in the rat on treatment with Ah—inducers, we attempted to determine whether T3 levels dropped with T4 in plasma or liver. In our experiments T3 levels did not drOp with T4. In pharmacokinetic studies, T3 was produced from T4 in a pulse which lagged T4 uptake by 4 — 5 min. The relative size of this pulse in treated vs control rats was similar to the ratio of specific activities for T4 between the respective groups. In light of reports on the passive and active uptake of T3 and T4, these data suggested that deiodination of T4 is regulated by a saturable uptake system which runs in parallel with a nonsaturable, diffusive uptake of T4, driven by its hydrophobicity. While attempting to determine the thyroid status of Ah- inducer treated rats, we discovered that liver malic enzyme was increased by Ah-inducers in a thyroid hormone dependent manner. The induction of malic enzyme by glucocorticoids is thyroid hormone dependent, ie. thyroid hormones must be present for induction to occur. Certain symptoms of Ah- inducer toxicity, such as thymic involution, hyperlipidemia, and fatty liver development can also be produced by gluco- corticoid administration, and might be explained if Ah- inducers had corticomimetic properties in addition to their interactions with the Ah receptor. Experiments designed to determine whether TCDD could act as a ligand for the gluco- corticoid receptor demonstrated no affinity of TCDD for this receptor, but left Open the possibility that the Ah receptor may be able to regulate some of the same genes as does the glucocorticoid receptor. ACKNOWLEDGEMENT I would like to thank my major professor, Dr. Steven D. Aust, for his generous support, and patience. My guidance committee members, Drs. Shelag Ferguson—Miller, Donald Jump (ad hoc), Justin McCormick, Dale Romsos, and William Wells, have given me sound advice and in several cases, spent their time and/or offered material support to me in completing this work. Many other individuals have contributed time, energy, and materials to these efforts, most notably my student assistants, Paula Mossner and Amy Clark, who performed at least half of the bench work described in chapters 2 and 3. Elizabeth Pulsford, Rich Voorman, and, when I was in a pinch, my friend Mary Witt, also contributed their time, and made my time here more pleasant both as friends and coworkers. I owe special thanks to the following individuals, who provided technical instruction and materials for my work Dr. Greg Fink, Pharmacology - Instruction in surgical techniques for both thyroidectomy and catheter implantation in rats. Dr. Robert Nachreiner and AHDL Staff — Allowed me free access and assistance in Operating their multiple well gamma counter, without which analysis of samples generated by my pharmacokinetic experiments would have been impossible. iv Dr. Drs. Dr. Maija Zile, Food Science - Instruction in jugular vein injection and bile duct cannulation. Jack Holland, Jack Watson, and the Mass Spectrometry Facility staff, who gave me access to and assistance with their instruments, computer terminals, and PDP-ll computer, the last of which were indispensable in using the CONSAM kinetic data analysis program. Loren Zech, and the staff of the Laboratory of Mathematical Biology, National Cancer Institute/NIH, for supplying me with a copy of the CONSAM kinetic analysis program and the instructional materials needed to use it. TABLE OF CONTENTS Page LIST OF TABLES ...................................... viii LIST OF FIGURES ..................................... ix DEFINITIONS AND ABBREVIATIONS ........................ Xi CHAPTER I. LITERATURE REVIEW Interaction of Ah—inducers with the Ah—locus. 2 Thyroxine and triiodothyronine metabolism ....... l9 Ah-inducers and thyroid status ................... 29 CHAPTER II. PLASMA THYROID HORMONE CONCENTRATIONS AND ACTIVITIES OF THYROID REGULATED ENZYMES IN LIVER AFTER TREATMENT WITH TCDD. ABSTRACT ............................................. 34 INTRODUCTION ......................................... 35 MATERIALS AND METHODS ................................ 36 RESULTS .............................................. 44 DISCUSSION ........................................... 61 CHAPTER III. INTERACTIONS BETWEEN LIVER THYROID HORMONES, TCDD, AND THE GLUCOCORTICOID RECEPTOR IN THE INDUCTION OF MALIC ENZYME ACTIVITY. ABSTRACT ............................................. 67 INTRODUCTION ......................................... 68 MATERIALS AND METHODS ................................ 69 RESULTS .............................................. 76 DISCUSSION ........................................... 91 vi Page CHAPTER IV. TRANSPORT AND METABOLISM OF THYROID HORMONES. CONTROL OF T3 PRODUCTION FROM T4 IN THE RAT AFTER TREATMENT WITH TCDD. ABSTRACT ........................................... 96 INTRODUCTION ....................................... 97 MATERIALS AND METHODS .............................. 99 RESULTS ............................................ 105 DISCUSSION ......................................... 127 SUMMARY 136 APPENDIX A. ESTIMATION OF T4 ACTIVE UPTAKE AND DEIODINATION RATES FOR T3 PRODUCTION MODELS. 140 REFERENCES 152 LIST OF TABLES Table 1 Rate constants for uptake, recycling, and metabolism of T3 in the 5 compartment model. 2 Rate constants for uptake, recycling, and metabolism of T3 in the 5 compartment model. 3 Metabolites of T3 and T4 in the bile and urine following injection of 125I-T3 or 125I-T4. viii Page 113 114 115 Figure 1 Proposed mechanism for Ah-locus control and Ah-receptor action 2 Specific binding of [3H]-TCDD to liver cytosolic protein after treatment with H88. 3 Structures of aryl hydrocarbons which displace TCDD from the Ah-receptor. 4 Pathways and metabolites of thyroxine metabolism 5 Pathways and metabolites of 3,5,3’-triiodo- thyronine metabolism. 6 Proposed scheme for control of T3 production. 7 Change in body weight and cytochrome P-450 in rats following treatment with TCDD. 8 Ethoxyresorufin-o-deethylase activity of normal and thyroidectomized rats after TCDD treatment. 9 Concentrations of T4 and T3 in plasma after TCDD. Cross-reactivity of T4 with T3 antibody in RIA. 10 Malic enzyme activity in liver cytosol after treatment with TCDD. ll Liver mitochondrial G-glycerolphosphate dehydro genase after treatment with TCDD. 12 Food consumption of thyroidectomized rats fed T3 or T3 + TCDD. 13 Malic enzyme activity in liver cytosol of thyroidectomized rats fed T3 or T3 + TCDD. 14 Total plasma T3 in thyroidectomized rats treated with T3 or T3 + TCDD. 15 Hepatic malic enzyme and a-glycerolphosphate dehydrogenase activity after 72 hr of T3 in diet. 16 Liver and plasma concentrations of T3 and T4 after 72 hr of treatment with T3 in diet. 17 Hepatic T3 and T4 concentrations 72 hr after LIST OF FIGURES treatment with TCDD. ix Page 11 14 25 27 31 45 47 SO 52 55 57 59 62 78 80 82 18 19 20 21 22 23 24 25 26 27 28 29 30 Specific binding of 3H-triamcinolone to the M04 stabilized glucocorticoid receptor. Total specific binding to the glucocorticoid receptor with TCDD or hydrocortisone competitors. Ethoxyresorufin-o-deethylase and malic enzyme activity in C57BL/6N and DEA/2N mice after TCDD. Four and five compartment models proposed for T3 and T4 uptake and metabolism. Best fit of rate constants to T3 tracer data. Best fit of rate constants to T4 tracer data. Chromatographic profile of T4 metabolites in bile and liver of control and TCDD-treated rats. Chromatographic profile of T3 metabolites in the bile of control and TCDD-treated rats. Production of T3 and I- from T4 tracer in the liver of control and TCDD-treated rats. Integrated 10 compartment model for T3 production from T4 in liver cytosol. Predicted levels of T3 production from T4. Production of T3 and I- from T4 tracer in the kidney of control and TCDD-treated rats. Chromatographic profile of urinary metabolites of T4. 84 87 89 106 108 110 116 119 121 123 125 128 130 Cytochrome P-450 Ah locus Ah receptor B[a]P TCDD T4 T3 DEFINITIONS AND ABBREVIATIONS One of a set of terminal monooxygenases found in the endoplasmic reticulum. Binding of carbon monoxide to the heme iron of these enzymes shifts the normal absorption band (the Soret band), which occurs near 425 nm, to a position near 450 nm. Aryl Hydrocarbon Hydroxylase. The name given to an oxidative activity which appears on induction of cytochrome P-450c. A gene which has been shown to control the synthesis of cytochrome P-450c and other enzymes which appear on induction of AHH activity. A Cytosolic protein which specifically binds chemicals that induce AHH activity. Benzo[a]pyrene, a common carcinogenic compound which induces AHH activity. 3-methylcholanthrene, a carcinogenic compound which like B[a]P, induces AHH activity. 2,3,7,8-tetrachlorodibenzo-p-dioxin — the most potent inducer of AHH activity known. Thyroxine, precursor to the active thyroid hormone, 3,5,3’-triiodothyronine (T3). The active thyroid hormone, 3,5,3’-triiodo- thyronine. xi Tetrac Tetraiodothyroacetic acid - a minor metabolite of thyroxine. Triac Triiodothyroacetic acid - a minor metabolite of T3. xii CHAPTER I LITERATURE REVIEW INTERACTIONS OF Ah INDUCERS WITH THE Ah LOCUS Exposure of animals to xenobiotics frequently results in the induction of components of a class of monooxygenases called cytochrome P—4503. Certain isozymes of cytochrome P—450 are normally present at moderate levels, (apparently acting as catalysts in steroid metabolism) while others only appear on exposure to certain classes of chemicals which have shown to be responsible for the induction of those isozymes (54,55). Other enzymes which may be induced by each class of chemicals include glucuronosyl transferases (78), glutathione—S—transferases(5), and d—aminolevulinic acid synthetase. Some enzymes required for synthesis of the porphyrin prosthetic groups of the P—4503 are also induced by these compounds (127). Two different types of cytochrome P—450 inducers have been studied extensively. For compounds typified by pheno— barbital (PB), which are readily metabolized by the monoxy— genases they induce (cytochromes P—450b and P—450e), induction lasts only a short period of time, and is directly pr0portional to the lifetime of the inducer before its catabolism (84,103). A family of aryl hydrocarbons which includes benzo[a]pyrene (B[a]P) 3-methylcholanthrene (3—MC), dibenz[a,h]anthracene, certain symmetric halogenated biphenyls, and halogenated dioxins, of which 2,3,7,8-tetrachlorodibenzo—p-dioxin (TCDD) is the most potent, induce a second set of enzymes. Cytochrome P—450c (sometimes called aryl hydrocarbon hydroxylase or AHH), epoxide hydratase, and 4-nitrophenol glucuronosyl transferase are the best studied of these aryl hydrocarbon inducible enzymes, and have been shown to be coordinately regulated by a receptor which binds to chromosomal enhancing sequences of the Ah-locus (20,53,67,74) as shown in figure 1. The concentration of a particular compound required to induce a certain level of P-450c is relatively constant across species having a demonstrable Ah receptor. For TCDD, some induction can be observed at 0.1 nmol/Kg (32 ng/Kg or 32 parts per trillion). Fifty percent of maximal induction occurs at about 1 nmol/Kg (ED5 and O)' maximal induction occurs between 10 and 33 nmol/Kg (46,103,111). Halogenated cyclic aromatic compounds vary in their ability to bind to the Ah receptor. Most of these compounds are not good ligands for the receptor, but those that are have estimated binding constants in the nanomolar range (KD = 0.1 - 20 nM), and are often refractory to metabolism by the monooxygenase activities they induce (7,84). In the cases of 3-MC and B[a]P, induction of AHH results in rapid oxidation of 3-MC and B[a]P to products which have reduced affinity for the Ah receptor. Synthesis of cytochrome P-4505 ceases as these compounds are metabolized. An unfortunate result of the oxidation by AHH is that these oxidation products can form adducts with DNA, creating potential sites for mutation (17,64). Figure 1 Proposed mechanism for Ah-locus control and Ah—receptor action. Nucleus TCDD-® + ‘7 C flop/05m C etc. AHH ACTIVITY A B For three widespread environmental contaminants, poly- chlorinated biphenyls (PCB), polybrominated biphenyls (PBB) and 2,3,7,8-tetrachlorodibenzo-p—dioxin (TCDD); the result of exposure is an intense and sustained induction of the aryl hydrocarbon hydroxylase system at 30-70 X the activity found in liver and other tissues of unexposed animals (85,110,111). Chronic exposure to low levels of TCDD results in accumulation of this compound in adipose tissues. It has been suggested that mobilization of these residues may result in AHH induction and activation of common carcinogenic chemicals such as benzo[a] pyrene, that might otherwise be excreted or sequestered in lipids without activation (l7,3l,83,87). Large doses of TCDD produce a distinctive set of symptoms including anorexia, hyperlipidemia, hyperkeratosis and thyroid hyperplasia (2,8,104). In some animals, such as the guinea pig, acutely toxic doses of Ah-inducers coincide with the EDSO for induction of cytochrome P-450c. However, many other species, such as the mouse and hamster, show no morbidity at doses of these compounds which are far in excess of those required for induction of Ah-locus enzymes. At a biochemical level, alterations in the metabolism of thyroid hormones (8,104), glucocorticoids (6,37), and testosterone (85) have all been described following TCDD treatment, but clear biochemical cause -> effect relationships have not been established for these changes, although linkages with cytochrome P-450 and glucuronosyl transferase induction have been suggested. These, and other metabolic alterations produced by Ah inducers such as hyperlipidemia, may be secondary or tertiary to induction of Ah-locus enzymes. Some may, alternatively, be the result of hormone—mimetic properties of Ah-inducers. Rozman et al; (112) demonstrated that thyroidectomy was protective to animals treated with 100 ug/kg TCDD. Treatment of thyroidectomized rats with thyroxine restored sensitivity to TCDD. Later work has shown that thyroid- ectomy does not change the pattern of cytochrome P—450 induction, indicating that symptoms of acute toxicity are not the result of AHH induction alone (60,111,113). Rickenbacker and McKinney (108) Suggested that PCBs and TCDD have thyromimetic properties. Our work (111), and that of Osborne et al.(96) has shown that TCDD is not thyromimetic. Our work (Chapter 2) indicated that the induction of certain lipogenic enzymes (eg. malic enzyme) by Ah inducers is dependent on the presence of thyroid hormones, suggesting a biochemical explaination for Rozman’s results in terms of the multihormonal regulation which has been established for lipogenic enzymes (12,49,135). We hypothesized, on the basis of the similarity between malic enzyme induction by TCDD and reports of a requirement for both thyroid hormones and glucocorticoids in the induction of malic enzyme, that TCDD might be corticomimetic. We have shown, as discussed in chapter 3, that this is not the case. However, we have not been able to rule out the possiblity that the Ah receptor acts as an enhancer towards expression of these lipogenic enzymes, in the same fashion as does the glucocorticoid receptor. The discussion here and in the chapters which follow requires an understanding of the similarities and differences between the Ah receptor, glucocorticoid receptor, and thyroid hormone receptors. The last chapter requires a similar familiarity with thyroid hormone metabolism. For these reasons, the remainder of this chapter reviews the properties of these receptors, and the metabolic pathways of thyroxine and triiodothyronine. General_Characteristics of_the Ah Receptor Several studies of the characteristics of the Ah receptor have been made to date. The identification of a discrete, low—capacity, high—affinity binding protein for 3-MC was first reported by Filler and Litwack (45) in 1974. This report was followed several years later by a study which identified a low-capacity, high affinity binding protein for TCDD (53). Studies by other authors in the last six years have shown that the 3-MC and TCDD binding fractions represent either a single protein, or very similar proteins which perform nearly identical functions (l4,58,90,9l). The specific binding fraction in crude cytosol has been consistently reported to sediment at about 9.0 S on 5-20 % sucrose density gradients (58,90), and 5.0 S on glycerol gradients(20). The estimated molecular weight has ranged from 130,000 to 245,000 (20,45,91), depending on the methods and assumptions used. The reported Kd of the Ah receptor for TCDD has ranged from 3.0 nM to < 1.0 nM, depending on both the assay method and animal species used (46,58,91). Claims have also been made for the existence of other distinct specific binding species in the 4-5 S region of sucrose density gradients or the 60—70,000 Mr fractions from gel filtration (64,128). The mechanism by which the Ah receptor induces cytochrome P-450 synthesis has received considerable attention, but has been limited by the crude nature of the cytosolic and nuclear preparations available for its study. Experiments employing gel filtration, DNA-affinity chromatography, and proteolytic enzymes have shown that the Ah receptor has a single type of aryl hydrocarbon binding site, a DNA binding site, and an additional domain that apparently is required for transport across the nuclear membrane (20). The receptor cannot bind to DNA until it has bound an aryl hydrocarbon molecule. Once it has bound to DNA, it promotes transcription of at least one component of the Ah system, the mRNA for cytochrome P-450c (52,129). Movement of the receptor from the cell cytoplasm and its accumulation in the nucleus after aryl hydrocarbon exposure has been demonstrated by several authors (53,64,74,134). 10 From 4 to 12 hours post treatment with an Ah-inducer, the concentration of receptor in the cytoplasm rapidly declines, while nuclear concentrations rise. This drop in the specific binding of 2,3,7,8-[3H]-TCDD in the cytosol following treatment of rats with the compound 3,4,5,3’,4’,5’-hexabromobiphenyl(HBB) is shown in figure 2. Note the reappearance of specific activity in the cytosol after 24 hours. Although the question remains as to whether or not this is newly synthesized protein, it is clear from our experiments that the concentration of receptor does not increase above pretreatment levels in the cytosol of HBB- treated animals. The induction of cytochrome P-4505 and glucuronosyl transferases is detectable in the- liver at about 12 hours, reaching a plateau at 48 hours. This process can be detected in cultured hepatocytes within 30 min. of exposure. Mutants have been identified in cultured hepatoma cells which are deficient in binding of the receptor to DNA. Mutants have also been found which cannot transport their ligand-receptor complexes from the cytosol to the nucleus (74). Many structure-activity studies have been performed to determine the structural requirements for ligand binding to the Ah receptor (7,14,47,90). The relative affinity of the receptor for a particular ligand is determined by a competitive binding assay, which employs [3H1—TCDD as the ligand with highest affinity. The ability of a 100-fold excess of 8 common Ah inducers to displace TCDD from the 11 Figure 2 Specific binding of [3H]-TCDD to liver cytosolic protein after treatment with 10 mg/Kg 3,4,5,3’,4’,5’-hexabromobiphenyl. 12 m <1- 4» N NlHlOHd 0N/£-01 X NdO ONIONlB OlJlOEdS ‘ OOOI'IHEI 72 24 HOURS AFTER TREATMENT 13 receptor is shown in figure 3. The compounds which bind most tightly are those which are most hydrophobic and which are capable of assuming an energetically stable, planar configuration. Although there appears to be some tendency for these compounds to be capable of forming a phenanthrene - like "bay region", as shown below, it is difficult to see how TCDD would assume such a configuration. bay region Steroid hormones have this type of phenanthrene structure, and were investigated as possible natural ligands for the receptor early on (90). None of the compounds tried were potent competitors when [3H]-TCDD was used as the radio- ligand. However, studies of steroid hormone receptors have influenced the direction of Ah receptor studies. Steriod Hormone Receptors Cytosolic receptors have been identified for glucocorticoid, mineralocorticoid, and sex steroids. All of these proteins have specific binding sites for a single "natural" steroid, and a binding site for DNA. As was the case for the Ah recptor, synthetic ligands, such as dexamethasone and 14 Figure 3 Structures of 8 aryl hydrocarbons which displace TCDD from the Ah receptor and the % displacement with 100 X excess * competitor. * from references 7,14,53. X. 0 3X 2,3,7,8-tetrahalo- 100 dibenzo-p-dioxin (TCDD) X :69." 2,3,7,8-tetrahalo- 98 'dibenzofuran e 9% Benzo[a]pyrene 96 3.4.3',4'-tetrahalo- 95 biphenyl 3-methylcholanthrene 95 Chrysene 82 <°©j 0 Isosafrole 67 B-Napthoflavone 55 16 triamcinolone acetonide, are required as binding assay ligands, because the natural ligands bind less tightly and are rapidly metabolised in the crude cytosolic assay systems (21,139). The molecular weights of steroid hormone receptors have been estimated to lie within the range of 67,000 - 89,000 , and have sedimentation coefficients of 8 - 9 S. As with the Ah-receptor, proteolytic fragments have been identified which bind steroid, but do not bind to DNA. Reports of such low molecular weight binding species created some controversy before definitive evidence of receptor proteo- lysis was shown, and methods to mitigate it were developed (1,136,140). Once a ligand molecule is bound, steroid hormone receptors undergo an activating transformation to a complex of lower molecular weight (4.5 - 5 S) and higher affinity for the ligand. This transformation process is temperature dependent, and can be inhibited by oxidation of sulfhydryl groups with molybdate. The activated form can be produced in vitro by incubating the ligand-receptor mixture with dithiothreitol at 250 to 300 C (21). The Ah receptor binds its ligands with equal efficiency at O0 C and 250 C, does not have a demonstrable "activated" form, and cannot be stabilized with molybdate (35). 17 Thyroid Hormone Receptor Investigation of the mechanism of action of thyroid hormones has been pursued using significantly different strategies and with qualitatively different results from those obtained in glucocorticoid investigations. The action of thyroid hormones has been suggested to be regulated in four steps. Our work (chapter 4), when viewed in light of other reports, suggests that step (c) may preceed (b) and be synchronized with step (a): a) Circulating thyroxine is transported across the plasma membrane both via passive diffusion and by a process that involves association with membrane receptors and endocytosis into endosomes via "coated pits" (23,42,56). b) Thyroxine is then specifically bound by a protein in the cytosol. This protein has been poorly characterized, although reports in the literature have shown it to be specific and distinct from serum binding proteins (32,57,121). c) Thyroxine is deiodinated to 3’,3,5etriodothyronine (T3), which binds specifically to another cytosolic protein which is not well-studied. d) T3 is somehow transported to the nucleus, and binds to a nuclear receptor protein, which is stereospecific in its affinity for L-T3. This stereospecificity is reflected in studies of nuclear uptake of T3, in which L—T3 accumulated in the nucleus, but D—T3 remained in the cytosol (116). 18 The thyroid hormone receptor which actually binds to DNA is thought to be located exclusively in the cell nucleus, has a basic subunit size of 3.8 S, and molecular weight of 54,000 (95,98). This receptor appears to associate with both histone and non—histone proteins of chromatin, and protects about 36 base pairs of DNA from cleavage by DNase I (3). While it has been shown that thyroxine and T3 are transported by endocytosis into the cell from circulating plasma carrier proteins, and across the nuclear membrane to putative nuclear receptors, no careful study of the cytoplasmic or membrane vectors has been made. Intensive study has been made of one of the serum T4 binding proteins transthyretin (prealbumin). This protein has been shown by X—ray crystallography to have a single symmetric binding site for thyroxine, and what appears to be a possible DNA binding site (15,16). Transthyretin also has four binding sites for retinol binding protein, which is a vector for transport of retinol, and is involved in the regulation of retinol concentrations in plasma and tissues (121). The presence of binding sites for both retinol binding protein and thyroxine suggests that this protein is part of a control mechanism for retinol metabolism that involves thyroid hormones(13,88,132). In contrast with studies reviewed above for the thyroid hormone receptor system, no studies have been made of the association of the Ah receptor with chromatin proteins in vivo, or to determine whether identifiable sequences of DNA 19 are protected by Ah receptor binding. Jones et a1; (67) have recently cloned the upstream controlling sequences of DNA coding for cytochrome P—450c from a mouse hepatoma line. Restriction endonuclease cleavage and deletion analysis of these isolated sequences indicated the existence of a TCDD—responsive enhancer region between nucleotides ~1580 and -l310. An inhibitory domain was identified between nucleotides -l310 and —695. A promoter region which was not TCDD— responsive was found near the start of transcription, between nucleotides -45 and —8. Thus the gene may actually be regulated by three different proteins: a) the Ah receptor, (b) a repressor binding protein, and (c) a promoter binding protein. THYROXINE AND TRIIODOTHYRONINE METABOLISM Thyroxine Production Thyroxine (T4) is the principal thyroid hormone synthesized by the thyroid gland or thyroid tissue of all species which have been studied. The available evidence suggests that the thyroid is an evolutionary outgrowth of the salivary glands, which still contain a small number of cells which are capable of concentrating iodide, and may produce small amounts of T4 following surgical thyroidectomy (33). Synthesis of T4 proceeds via iodination of the tyrosine residues of a high molecular weight ‘globulin — thyroglobulin. Formation of the ether linkage of the 20 thyronine structure occurs after iodination. The complete biochemical mechanism of thyronine synthesis is not well understood, but both iodination and phenyl ether formation are known to be catalyzed by a peroxidase. A similar reaction is used to make iodothyronines synthetically, using t-butyl peroxide as the catalyst (119). Thyroxine is transported via the plasma, principally associated with two different proteins - transthyretin, which normally carries 85 % of plasma T4, and thyroid binding globulin (TBG) which carries small amonts of plasma T4 (109). Transthyretin has a moderately strong affinity for T4, with a Kd of 108. TBG has a higher affinity for T4, with a Kd of about 1010. During periods of food deprivation, synthesis of T86 is enhanced. Increasing the concentration of TBG reduces the amount of free T4 in plasma, and reduces the rate of T4 uptake and deiodination by tissues (66). Thyroid Hormone Uptake by Tissues Transport of T3 and T4 into tissues is thought to occur via two parallel mechanisms, one active and dependent on ATP, the other passive and driven primarily by the relative hydrophobic properties of T3 and T4 (42,44,59,72,73). Thyroid hormones possess ionic (carboxyl and immonium groups), polar (ether and hydroxyl groups), and hydrophobic (iodophenyl rings) domains. As a result they are insoluble in both water and nonpolar organic solvents, but are 21 moderately soluble in polar organic solvents such as alcohols and water-soluble ethers. The partition coeffi- cients of T4 measured in n-heptane/water, n-octanol/water (97), and lecithin/water (63) systems are 0.0004, 91.0, and 12,000, respectively, reflecting the polarity of the organic phase. T3 is less hydrophobic, and more water soluble than T4. When T3 and T4 move through a hydrophobic matrix, for example, a reverse-phase chromatography column, the T3 retention time is about 1/2 that of T4 (62). Bulk uptake rates for T3 and T4 from perfusion media follow the same pattern, and were initially thought to be entirely due to diffusion (59,97,106). However, experiments with primary hepatocyte cultures and blood cells have shown that a portion of both T3 and T4 uptake is active, ATP dependent, and saturable (42,44,56,72,73). Cheng gt al;(23) have demonstrated what appears to be receptor-mediated endocytosis of tetramethyl rhodamine-T3 in hepatocytes. This evidence, and the inhibition of the saturable, high affinity uptake of T3 by colchicine, ouabain, and cyanide (56,73) suggest that at least a portion of T3 and T4 uptake proceed by receptor-mediated endocytosis from the plasma. The 5'-deiodinase, discussed below, has been shown to have a plasma membrane location. Leonard et a1; (75) suggested that the plasma membrane location of the deiodinase "...may represent a physiological mechanism to allow efficient production of ... T3, from an intrinsically inactive precursor T4, without the obligatory penetration 22 by T4 of the intracellular space." Our results from pharmacokinetic studies of T4 metabolism (chapter 4) indicate that an association between the active uptake system and the deiodinase might serve to deliver a controlled flux of T4 to the deiodinase, while excluding the flux of T4 which passively diffuses into the cell. Such interactions are known for cholesterol metabolism and may be essential for control of the metabolism of hydrophobic compounds in general, since they cannot be excluded from the cell by the lipid membrane as are water soluble compounds such as amino acids. Production of T3 from T4 Although the concentration of T4 in the plasma is approximately 50 X that of T3 (50 nM vs 1 nM), it is ineffective in stimulating thyroid hormone-dependent processes in yiyg when its deiodination is totally inhibited. The active hormone, L-3',3,5-triiodothyronine (T3), is produced in tissues by deiodination of T4. Two thiol-dependent deiodinases have been identified which catalyze this reaction. Both can use glutathione as a substrate, but are thought to utilize a glutaredoxin for transfer of the reducing equivalents in 1139 (51). Type I deiodinase, which is found in the plasma membrane of liver, and kidney, can be inhibited by several thiourea and thiopyrimidine compounds, the most potent of which is propylthiouracil (PTU) (26). Type II deiodinase, which is 23 found in the pituitary and nervous tissue in general, is not inhibited by PTU and has been found to contribute about 30 % of the T3 in the whole body pool (120,133). Several authors attributed physiological activity to T4 (prior to this discovery) which actually resulted from tissue T3 produced by type II deiodinase. The thyroid hormone nuclear receptor, discussed earlier with the Ah and glucocorticoid recptors, does bind T4 in yitrg. However, the combination of its low affinity for T4 and the transport kinetics for T4 into T3 producing tissues ensure that T4 is ineffective in the absence of deiodinase activity. Several studies have shown that tissue concentrations of T4 are much lower than in plasma. VanDoorn, et a1. (130,131), using an isotopic equilibrium technique, found that T4 levels in the liver and kidney were about one half those in plasma. Conversely, T3 concentrations were higher in tissues than in plasma. Liver and kidney T3 concentrations were 5 — 10 X those in plasma. Catabolism of T3 and T4 Both T4 and T3 are metabolized in the liver and/or simply excreted from the liver into the bile. It has been estimated that 30—50 % of T4 is conjugated with glucuronic acid via a UDP-glucuronosyl transferase in the liver (126). This conjugate appears in the bile, but apparently can be hydrolyzed by bacterial B-glucuronidase in the intestine, as little conjugated T4 appears in the feces (125,126). 24 Another 20 % of T4 is deiodinated to form either T3 or rT3, both of which are more rapidly metabolized and eliminated than T4 (40). About 30 % of T4 in the bile appears as free T4, which can be resorbed from the intestine(27). The catabolic products formed from T3 are similar to those formed from T4, but appear more rapidly (9,40). Two different diiodothyronines can be formed from T3 3,5—diiodothyronine and 3’,3—diiodothyronine. Since these compounds are very difficult to separate chromato— graphically, they are often considered together as T2 (125). The total amount of T2 excreted in bile represents \u about 30 8 of all T3 metabolites. T3—glucuronide a constitutes another 40 %, while unconjugated T3 makes up the balance. Only 3-5 H of the total output of T3 is excreted in the urine. However, the kidneys eliminate approximately 90 % of the iodide which results from deiodination. Ether-link cleavage has long been identified as a minor pathway of thyroid hormone catabolism, but the source of the the iodothyroine metabolites has only recently been identified. Burger et al.(l9) have shown that ring cleavage is catalysed by a peroxidase present in "activated" leucocytes. Unactivated leucocytes do not catalyze ring cleavage. This discovery may explain a long recognized decline in thyroid hormone levels which occurs in patients with certain infectious diseases. The metabolic pathways discussed above are summarized in figures 4 and 5. 25 Figure 4 Pathways and metabolites resulting from T4 metabolism. 26 MAJOR METABOLITES OF T4 l n ’ Ho“ocH;cH-coon l I NH, GLUCURON l DAT ION DEIODINATION DEIODINATION ETHER LINK CLEAVAGE DEAM l NAT 1 ON/ DECARBOXYLAT ION L-THYROXINE (T4) UDPGA T4-GLUCURONIDE OH I l HOHOCHf cH-cooH Nhh _ I I L-3,5,3'-TRIIODOTHYRONINE (T3) l I H0 ’0 .CH; (EH-coon I NH, L-3,3',5'-TRIIODOTHYRONINB (2T3) H 7; I NH, 3,5-DIIODOTYROSINE TETRAIODOTHYROACETIC ACID (TETRACI 27 Figure 5 Pathways and metabolites resulting from T3 metabolism. 28 MAJOR METABOLITES OF T3 I I HOHOCH, I'm-coon NI-I, I L-3 , 5 , 3 ' -TRIIODOTHYRONINE (T3) l I GLUCURONIDATION co/O-HOCHECIIH-COOH HO ’C—I UDPGA 0H 0” Ts-cmcunoumz OH I I DEIODINATION HOHOCHI $HfCOOH NF” I 3,3'-DIIODOTHYRONINE (T2) I I NH, DEIODINATION 3 , 5-DIIODOTHYRONINB I- l I DEAM I NAT I ON/ - DECARBOXYLAT ION KY "0. 0 ,CH2 COOH I N83, C02 TRIIODOTHYROACETIC ACID (TRIAC) I I ETHER LINK K CLEAVAGE . HO @ CH-iC'IH-COOH ' NH, 3 , S-DIIODOTYROSINE 29 Ah INDUCERS AND THYROID STATUS Several authors have noted decreased levels of T4 in the serum of animals treated with Ah inducers, accompanied by increased excretion of T4 and hyperplastic goiter (8,48,89). The hamster may be an exception to this rule, as Henry and Gasiewicz (61) reported increased levels of T4 in a comparative study of rats and hamsters, while confirming the decline in T4 seen by other investigators in rats (104). Reports of T3 levels have been contradictory, and were therefore considered to be unreliable. Increased excretion of T4 has been associated with an increased activity of PNP-glucuronosyl transferase (PNPGT). Studies in our laboratory have shown that glucuronosyl transferase activity towards T4 (T4GT) increases in parallel with PNPGT (unpublished). Qualitatively similar findings have been reported by two other laboratories, but there is a large variance in results reported between laboratories (48,108). The change in T4 levels can be convincingly explained by the increased T4GT activity, but the increase, or lack of change in T3 levels reported in Ah inducer treated animals is paradoxical in view of the kinetics reported for T4 deiodinase in vitro. Several studies agree in reporting low catalytic rates and low Km values for deiodination of T4 (26,28,69,133). As shown in appendix A , estimates of T3 concentrations derived using these binding and rate constants and known concentrations 30 of T4 are directly proportional to these T4 levels. Since this relationship does not hold in £339, in animals treated with Ah inducers , the in_vitro measurements probably do not reflect the kinetics of the deiodination system in A clue to the explaination of this phenomenon, which will be explored further in chapter 4, was the independent observation by several laboratories that the hepatic and kidney deiodinase activities were primarily associated with plasma membrane or lysosomal fractions (28,75,79). Deiodinase activity correlated well with other markers for plasma membrane, such as the Na+-K+ ATPase. It is well known that the the reaction kinetics of enzymes associated with membranes are highly sensitive to the environment in which they are assayed, and that parameters measured in vitro are often only poorly representative of what occurs in yiyg. Plasma membrane enzymes are a special case, in that most enzymes which are found in this membrane are involved in signal transmission and/or amplification, or are involved in the transport of substrates from the plasma into the cell. I believe that the discrepancy between the in_vitro behavior of the deiodinase and the in yiyo results can be explained by an association of the deiodinase with the high affinity uptake system which transports T4 from the plasma into the cell. 31 Figure 6 Proposed scheme for control of T3 production from T4 by controlling flux of T4 to the plasma membrane deiodinase. 32 T4 v 5'-DEIODINASE T3 V HIGH AFFINITY T4 RECEPTOR O . 63+ 6556 :- COATED g P IT ENDOCYTOS I s ENDOSOME Q ATP ADP TRANSTHYRET I N O cwosouc T4 BINDING PROTEINS DIFFUSIVE F237 0 PHOSPHOLIPID MEMIBRANE - HEPATOCYTE CYTOPLASM I 0 ‘ .O <——9 @. O CHAPTER II PLASMA THYROID HORMONE CONCENTRATIONS AND ACTIVITIES OF THYROID-REGULATED ENZYMES IN LIVER AFTER TREATMENT WITH TCDD 34 ABSTRACT 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) caused a depletion of serum thyroxine (T4), but paradoxically did not change T3 levels in serum of rats. The activities of the thyroid regulated enzymes a-glycerolphosphate dehydro- genase (GPD) and malic enzyme (ME) were determined in livers of normal and thyroidectomized (THX) rats treated with 0.1 to 100 nmol TCDD/kg body weight. Mitochondrial GPD activity did not change significantly as a function of TCDD dose in either normal or THX rats. ME activity was induced by TCDD in a dose-dependent fashion, but only in non-THX animals. The absence of ME induction in THX rats treated with TCDD indicates that TCDD is not intrinsically thyromimetic. The dependence of ME induction on thyroid hormones is much like the thyroid hormone dependent, multi- hormonal induction of ME by insulin and glucocorticoids. However, TCDD had no additive or synergistic effects on induction of ME activity in THX rats fed T3. A 30% decrease in steady-state plasma T3 levels of T3-fed animals treated with TCDD relative to T3-fed controls suggested that T3 catabolism was more rapid in TCDD treated rats than controls. Thus a thyroid hormone dependent, multihormonal interaction is suggested as the basis for induction of ME by TCDD, but a strictly T3-dependent process has not been ruled out. 35 INTRODUCTION Large doses of TCDD cause a distinctive set of pathological symptoms including anorexia, transient hyperlipidemia, hyperkeratosis, and thyroid hyperplasia (2,8,105). symptoms often associated with abnormal thyroid function. Rozman et al. (112) recently demonstrated that thyroidectomy was partially protective to animals treated with 100 ug/Kg TCDD. In these experiments, 70-80% of normal or euthyroid animals expired 45 days after treatment with TCDD, while none of the thyroidectomized animals died at this dose of TCDD. In the same experiment, treatment of thyroidectomized rats with thyroxine restored sensitivity to TCDD. TCDD also induces aryl hydrocarbon hydroxylase (cytochromes P—450c and P—450d) and other Ah—locus enzymes (110). Many studies have shown a correlation between an Ah—locus response and toxicity of TCDD (53,110). However, recent studies (60,113) have shown that cytochrome P—450 induction is unaffected by thyroid status, eliminating changes in P—450 expression as a possible explanation for the sparing effect of thyroidectomy. It has been hypothesized on the basis of computer modeling studies (107) that the effects resembling thyroid dysfunction are the result of intrinsic thyro— mimetic pr0perties of Ah—inducers. It was therefore of interest to determine whether TCDD and other Ah—inducers have any intrinsic thyromimetic pr0perties. Several symptoms of TCDD intoxication involve derangement of lipid 36 metabolism, including hyperlipidemia, fatty liver and elevated cholesterol levels (104,105). Since the liver is a major organ for lipid metabolism and thyroid hormone metabolism, in addition to being the most studied tissue with respect to biochemical effects of TCDD, it may be the most important tissue in which to determine the thyroid status of following TCDD exposure. The thyroid status of the liver was studied by measuring the activity of two hepatic enzymes which are inducible by thyroid hormones. Malic enzyme (ME), which can be induced by thyroid hormones, is a major source of NADPH used in fatty acid synthesis. Thyroid hormone induction of this enzyme is inhibited by glucagon (gluconeogenic conditions), and is stimulated by insulin (glucose sufficient, lipogenic conditions) and glucocorticoids (49,114,135). Mitochondrial a—glycerol— phosphate dehydrogenase(GPD) is a thyroid hormone inducible enzyme associated with the mitochondrial electron transport system. Induction of this enzyme is influenced by gluco— corticoids, but is not generally affected by the glucagon/- insulin ratio (18). Our results show that TCDD has no thyromimetic prOperties, but that one of these enzymes can be induced by TCDD only in the presence of thyroid hormones. MATERIALS AND METHODS Chemicals TCDD was a gift from Dr. Fumio Matsumura, Pesticide Research Center, Michigan State University. L—3,5,3’—Triiodo 37 thyronine (T3) was obtained from Chemical Dynamics Corporation, South Plainfield, NJ. Ethoxyresorufin was obtained from Pierce Chemical Co., Rockford,IL. 4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPBS,Ultrapure) was obtained from Mannheim-Boehringer Biochemicals, Indianapolis, IN. Patty-acid free bovine serum albumin, malic acid, DL-a—glycerophosphate, and NADP were obtained from Sigma Chemical Co., St. Louis, MO. Methoxyflurane was obtained from Pitman-Moore, Inc., Washington Crossing, NJ. All other chemicals were reagent grade. Animals Male Sprague-Dawley rats weighing 150-175 g obtained from Charles River Laboratories were used in all experiments. Surgical thyroidectomy was performed on age and weight matched rats using methoxyflurane for anesthesia. Thyroid- ectomized rats were allowed 5-7 days recovery before further treatments. The success of thyroidectomy was assessed by measuring total plasma T4 and T3 by radioimmunoassay. All animals were housed in large (20"x16"x8") polycarbonate cages, 3 rats per cage, with hardwood bedding. Pood (Wayne Lab Blox, Chicago, IL) and water were provided ad libitum, except as noted. Treatments All operations involving the use of TCDD were performed in isolation rooms under negative pressure. Solutions of TCDD 38 in corn oil were made up by mixing a concentrated solution of TCDD in benzene (1 ml) with an equal volume of corn oil. This mixture was warmed to 700 C in a water bath for 6-10 hr to remove the benzene. The remaining corn oil solution of TCDD was then diluted serially to obtain concentrations such that each rat received 1 ml corn oil/kg body weight. All rats were given a single dose of either corn oil alone, or TCDD in corn oil by gavage on day 0 of each experiment. In some experiments rats were treated with T3 in the diet by mixing powdered food (Wayne Lab Blox) with either ethanol alone (10 ml/kg diet) or appropriate concentrations of T3 in ethanol (10 ml/kg). Powdered food was weighed and presented to rats in small metal cans(100 gm capacity) which were placed in a larger metal dish to catch spilled food. Each morning, the contents of these two containers was sifted and weighed. The small can was refilled with fresh food and returned to the cage. Buffers and Solutions HEG buffer consisted of 25 mM HEPES, 1.5 mM EDTA, and 10% glycerol, adjusted to pH 7.4 with NaOH. HEDG buffer was made by adding dithiothreitol to MPG buffer to 1 mM immediately before use. HE (25 mM HEPES, 1.5 mM EDTA, pH 7.4) buffers containing glycerol, sucrose or dimethyl sulfoxide (DMSO) were used to prepare and store mitochondria and microsomes. Phosphate buffer, used in the a-glycerolphosphate dehydrogenase assay, contained 100 mM Na HPO 5 mM EDTA, and 10 mM MgCl pH 7.4. Ferricyanide 2 4’ 2' 39 solution was 1.6 mM KCN and 1.6 mM K3Fe(CN)6. Solution A consisted of phosphate buffer plus ferricyanide solution, 5:4, with 162 mg Na2 a-glycerolphosphate added per 10 ml immediately before use. Solution 8 (blank) consisted of phosphate buffer plus ferricyanide solution 5:4, without a-glycerolphosphate. Tissue Preparation Except where noted, rats were sacrificed by C02 asphyxiation and decapitation 72 hr after treatment with TCDD. Trunk blood was collected into EDTA-containing tubes (Vacutainer; Becton-Dickinson, Rutherford, NJ) on ice. Livers were perfused with ice-cold saline in situ using a .blunt, plastic-tipped syringe, and then excised into cold saline on ice. Individual livers were blotted, weighed, and homogenized in a glass Potter-Elvehjem homogenizer with a Teflon pestle using 3 ml of cold HEDG buffer/g tissue for preparation of mitochondria, microsomes, and cytosol. Homogenates were centrifuged at 10,000 x g for 20 min to give a nuclear pellet, a crude mitochondrial layer, and a supernatant layer. The supernatant layer was carefully removed and centrifuged for 70 min at 105,000 x g to give "cytosol" (clear supernatant) and a crude microsomal pellet. Lipid was aspirated, and cytosol collected into polystyrene tubes which were frozen on dry ice immediately after collection. Crude microsomal pellets were resuspended in O HE buffer containing 20 % glycerol, and stored at -80 C 40 for later processing. Crude microsomes were washed by resuspending in HEG buffer followed by centrifugation at 105,000 x g for 90 min. The resulting pellets were then resuspended in HE buffer containing 20% glycerol prior to cytochrome P—450 and ethoxyresorufin—O-deethylase (EROD) determinations. The crude mitochondrial layers from the 10,000 x g centrifugation were gently poured off the nuclear pellet into separate tubes, resuspended in 25 m1 of 0.25 M sucrose in HE buffer, centrifuged at 10,000 x g for 20 min, and the supernatant discarded. The resulting mitochondrial pellets were resuspended in HE buffer containing 20% DMSO for storage at -800. Standard Thyroid Hormone Radioimmunoassays Aliquots of plasma or thyroid hormone standards made up in 10 mg/ml albumin were extracted by placing 400 ul of sample or standard in 1.5 ml microfuge tubes, followed by 1 ml methanol. The tubes were then centrifuged for 10 min at 8,000 rpm in a microcentrifuge, after which time 1 ml of the resulting supernatant was cooled to about -6OOC, and vacuum dried. In later experiments, a high pressure liquid chromatography (HPLC) separation was added between extraction and drying as described in the next- section. The residue was dissolved in 200 ul of 10 mg/ml fatty acid-free bovine serum albumin. Total T4 and T3 concentrations were determined by radioimmunoassay (RIA). Samples (100 ul) were incubated with T4 or T3 tracer solutions in antibody -coated tubes (Becton-Dickinson) for 41 90 min, washed with water, and counted on an LKB 1271 gamma counter. Plasma concentrations were computed from the logit regression coefficients obtained from RIA of extracted T3 and T4 standards in albumin. Extraction efficiencies were determined by extracting spiked samples, and comparing extracted to unextracted standards. The efficiencies were 65 - 70% for T4, and 95-100% for T3. Stock solutions of T3 and T4 were prepared by sonicating about 10 umol of the sodium salt of each hormone in 10 m1 of HPLC solvent, followed by dilution to a final volume of 100 ml with HPLC solvent. Standards of T3 and T4 in albumin were prepared from these stocks by serial dilution of stocks with a solution containing 10 mg/ml albumin and 1% NaCl and were frozen between uses. Periodically the purity of T3 and T4 stock solutions was checked by HPLC using the procedure described below. HPLC Separation of Thyroid Hormones Thyroid hormones were separated by HPLC using a method similar to one of the methods of Hearn gt a1. (62). The stationary phase used was an Econosphere C-18 reverse- phase column (Alltech/Applied Science, Deerfield, IL). The mobile phase consisted of methanol:water:glacial acetic acid, 575:452:1, with 5 ml of 5 N NaOH added for each 3 liters of solvent. A flow rate of 1.5 m1/min at 3300 psi was routinely used. Under these conditions T3 eluted at about 8 minutes, while T4 was retained for about 13 minutes. 42 Plasma T3 and T4 were separated as follows: methanol extracts of plasma (1 ml) were adjusted to the same water and acetate content as the HPLC solvent by adding an acetate buffer to the extract. One milliliter of adjusted extract was then loaded onto the HPLC column, and fractions collected for 20 min. Fractions containing T3, and T4, respectively, were pooled in separate scintillation vials, cooled to about -600 C, and vacuum dried. The residues were then taken up in 200 ul of albumin solution and analysed as in the standard RIA procedure. During method development stages and for checking the purity of standards, ' a Bioanalytical Systems (W. Layfayette, IL) LC-4B amperometric detector set‘ at 0.875 volts was used to detect thyroid hormones. The presence of electrochemically active contam- inants in plasma extracts, and a maximum sensitivity of about 10 nM (10 pmol/ml) made this device unsuitable for direct measurement of plasma T3 and T4. Malic Enzyme Determinations ME activity was determined essentially according to Hsu and Lardy (65). In brief, 100 ul aliquots of cytosol were mixed with a 1 m1 aliquot of either 20 mM Malate-Tris and 1 mM MnCl2 (blank) or the same solution with 2 mM NADP (sample) added immediately before use. The difference in absorbance between the two mixtures was recorded on a Perkin—Elmer model 124 double beam spectrophotometer. The rate of formation of NADPH was determined using an 43 extinction coefficient for NADPH of 6.23 X 103 O.D. units/molar-cm and specific activity expressed as nmol NADPH/min-mg protein, following determination of cytosolic protein by the method of Lowry gt gt. (77). a-Glycerolphosphate Dehydrogenase Determination Mitochondrial GPD activity was determined essentially by the method of Bottger gt gt. (18) which employs ferricyanide as an electron acceptor for the enzyme. Disappearance of the 420 nm absorbance of ferricyanide is proportional to the oxidation of glycerolphosphate. Frozen mitochondrial preparations were rapidly thawed and washed by adding 25 ml of 250 mM sucrose-HE buffer per 2 ml of suspension. These suspensions were centrifuged at 10,000 X g for 20 min. The resulting supernatants were poured off and the mitochondrial pellets resuspended in 2 ml sucrose- HE buffer. Activity was assayed by mixing 1 ml of solution A with 50-100 ul of the mitochondrial suspension, and recording the change in absorbance at 420 nm with 1 ml of phosphate buffer and an equal amount of mitochondrial suspension in the reference cell. Absorbance was generally recorded for 5 min, after which time a blank was run using solution 8 rather than solution A in the above procedure. Protein content was determined by the method of Lowry gt gt. (77). An extinction coefficient of 914 O.D. units/molar-cm for ferricyanide was used in activity calculations. 44 Cytochrome P—450 and Ethoxyresorufin-O—Deethylasg Cytochrome P—450 levels were determined by the method of Omura and Sato (93). EROD activity was determined by the method of Pohl and Fouts (102). 9.6.1.21, firesen tafien All points represent the mean f standard deviation of measurements from three individual animals, unless otherwise noted. Where apprOpriate, 9 % confidence limits and statistical significance were determined using Student’s distribution. Values which were significantly different from controls at the 95% confidence level are designated with asterisks (*) or are noted in the figure legends. RESULTS Figure 7 shows body weight gain and the induction of cytochrome P—450 in rat liver microsomes as a function of TCDD dose. It is important to note that almost full induction of cytochrome P-450 was achieved at a TCDD dose of 10 nmol/kg, while weight gain did not begin to decline until doses of 33 nmol TCDD/kg and above were reached. Figure 8 shows the induction of EROD activity in normal and thyroidectomized rats treated with TCDD. As reported by others (60,113), the pattern of induction of this cytochrome P-450 activity was not significantly affected by thyroid status. 45 Figure 7 a) Change in body weight of rats following treatment with TCDD. Each point represents the average daily weight gain of three rats as determined by linear regression analysis of of body weights between days 5 and 12 after dosing. (b) Total liver microsomal cytochrome P-450 content 72 hr after TCDD treatment. All points corresponding to doses greater than 1 nmol/kg were significantly different from controls with P<0.05 CYTOCHROHE P-4SO NNOL I no PROTEIN HEIGHT GAIN - G/DAY 46 IS - A 10- i fi§_\§ 5- \\\\I‘ \i. ,_T—{%" , , I _1 0 0.1 1.0 10.0 100 TCDD DOSE (NHOLIKG 8H) 2.0- 0 1.5‘ /}’—'{—f—} / I.O« o—f/' 0.5« ~v-I; . . _r r , r , 0 0.1 1.0 10.0 100 TCDD DOSE INHOL/KG 8H) 47 Figure 8 Ethoxyresorufin-o-deethylase activity of hepatic microsomes of normal (H) and thyroidectomized (O—O) rats 72 hr after TCDD treatment. All values of EROD activity greater than 2.5 nmol/min were significantly different from controls with P<0.05 48 10.0- z_ue0ma oz-z_z\sozz »H_>~Ho< mm<4r1~mmo-o-z_msmomum>xozpm ///, u I I O '4. 0". IO. / 1| I 0 O i. f . / 1 TIIOl TI X, // I H ’fl- _./_ '0 11L I s 0 5 7 5 n/U I00 TCDD DOSE (NNOL/Kc BW) 49 Thyroid hormone levels were initially measured by a standard RIA technique, but the accuracy of this method for total T3 was found to be unreliable because of antibody cross-reactivity from T4 (figure 9a). For this reason, extracts of plasma were subjected to HPLC prior to RIA to separate T3 from T4. This procedure did not change T4 measurements significantly (figure 9b), but changed T3 measurements dramatically (figure 9c). Total plasma T4 decreased following TCDD treatment in a dose—dependent fashion, with noticable changes occuring even at doses where no grossly observable symptoms of TCDD exposure were seen, ie. 1 nmol TCDD/kg body wt. (figure 9b). Almost half of the T3 measured by the standard RIA actually reflects cross—reactivity from T4, and the decrease noted using the standard assay was almost entirely due to loss of T4. Despite drastically decreased plasma T4 levels, treatment of rats with TCDD was found to be correlated with increased activity of cytosolic ME activity in liver (figure 10). In general, ME activity followed the same trend as microsomal cytochrome P-450 and EROD activities, until the onset of anorexia at TCDD doses of 33 nmol/kg and above. However, induction of ME activity did not occur in THX rats at any of the TCDD doses tested. starvation, hypoglycemia, and glucagon treatment have all been correlated with supression of ME synthesis(49,ll4,l35), and may be responsible for the decline in ME activity observed at high TCDD doses. The activity of mitochondrial GPD was also measured in these 50 Figure 9 a) Cross-reactivity of T4 with the T3 antibody used for RIA. Concentrations are of T4 (H) and T3 (H) standards added to the T3 RIA tubes as described in methods. Lines drawn through the data points were determined from the expression Logit(B/Bo) = ln(B/(Bo-B)) = m*ln[ligand] + b where B = counts per min in sample, Bo: CPM in blank m slope of line, b = intercept. b) Total extractable plasma T4 in rats 72 hr after treatment with TCDD. Standard RIA (H), HPLC-RIA (0—0) (see Methods). All points corresponding to doses greater than 1 nmol/kg were significantly different from controls with P<0.05 c) Total extractable plasma T3 72 hr after treatment with TCDD, as measured by RIA. Standard RIA (H), HPLC-RIA (0—0) (see Methods). 551 1.0 2.0 3.0 0.0 '100 .62.: :9: L06 CONCENTRATION (NH) ID . .m. . T .....TIOII. G . o . 0 79.. . I . m a o .- m . 7n. .IInYIi mm I. . . m . . . .. m TOI. ll.- 0. . . . . 0 101 as am nu mw( .w . 3 2. ... =1. 8:5:598 !.§>=~§.-_~=Z_ .Mw rIImw\oII. I. I . fl II. \c. #0 ‘1 ll. 0. a \ . .. ~ u I . I m ~ \ ~ 0 I I OI. ran \ .. m ‘ V II .. m s I II .00 - II I _ D D D b mw mm “w mu mu m» mm mm :1. 5:5.ng U332»:— 52 Figure 10 Malic enzyme activity in liver cytosol 72 hr after treatment with TCDD. For thyroidectomized rats (CF-4D), each point represents the average : standard deviation (S.D.) for 3 animals. For normal rats, each data point represents either the average _+_ S.D. for 3 rats (H) or the pooled average ( u ) _+_ Sp for two separate experiments (H). Data were pooled using the formulas: (n -1)S + (n -l)S u = (ul + u2)/2 Sp = --l-——-l --------- 2 “1+“2" where ui = mean of sample i, ni = number of values in sample i, S = sample standard deviation. 53 * 0 T I . TO; ..muu. \ m u ..x.‘ . “O //// m nu *- L TIOIH. r m / u u . *I _ ".0. .. " /// n. _ Jul jmw / u . 1: n I 6.. j nm / . r. I i To... fl q a d 0 n m u 100 l Az_UHO¢i az-z_z\:aoNzu o_u38 . zoZmszoo 80... DAY 30 20 10 / m m w :58 - 225528 c8 DAY DAY 59 Figure 13 Malic enzyme activity in liver cytosol of thyroidectomized rats treated with dietary T3 alone (CF43) or dietary T3 + 10 nmol-TCDD/kg-body wt (H), as described in figure 6a. All animals were sacrificed on day 3 (72 hours). 6O 100 - 5 0 5 7 S 2 Az_uhoma az-z_z\zdoNzu o_4< o.N m.— o._ m.o F P H" ‘ [El] VNSV1d 64 greatly reduced in animals treated with TCDD. We have extended the studies of Rozman e_ a1. by showing that at least in the liver (a primary control point of fat metabolism and Ah-inducer action) changes in ME activity (which supplies NADPH for fatty acid synthesis) were dependent on T3 levels, and did not increase as the result of any intrinsic thyromimetic action of TCDD. However, this data does suggest that induction and suppression of malic enzyme may be explained by a thyroid—dependent, multi- hormonal mechanism. In the induction of thyroid-controlled proteins (malic enzyme, phosphoenolpyruvate carboxykinase, aZ-macroglobulin, growth hormone) thyroid hormones often play a permissive role, ie. thyroid hormones enable a basal level of protein synthesis, while other hormones such as insulin, glucagon, and glucocorticoids are responsible for short—term regulation, but are inactive in the absence of T3 (86,92,118,123,135). While our studies show that factors other than the disturbance of thyroid status are involved in TCDD toxicity, it is also clear that separation of thyroid status from other factors is essential to under— standing the total picture. Several investigators have shown decreased serum thyroxine levels in animals treated with Ah—inducers, including benzo[a]pyrene (48), methyl— cholanthrene (89), hexachlorobiphenyl, and TCDD (8,104,105). This decrease of T4 in serum has been shown to be associated with the induction of a hepatic glucuronyl transferase which can conjugate thyroxine (8,108). Plasma 65 T3 levels have previously been reported to be decreased (113), unchanged (104), or elevated (103) after treatment with TCDD. Our results show that plasma T3 levels do not change significantly, and show that some of the conflicting reports in the literature can be explained by cross- reactivity Afrom T4. The failure of T3 to change when T4 levels are declining is a paradoxical situation if normal deiodination mechanisms are Operative (25,28), i.e. T3 concentrations should be decreasing with those of T4. In experiments where we attempted to control T3 levels in THX rats, steady-state T3 concentrations in plasma suggested that T3 catabolism was increased in TCDD—treated animals. This finding further compounds the paradox of finding normal T3 levels in the presence of declining T4 It is important to note that endogenous T3 is produced from T4, primarily in the tissues where it acts (40,131); thus the concentrations of T3 in these tissues (as Opposed to plasma concentrations) is of primary importance in under standing the induction of thyroid—controlled proteins such as ME. Recent studies in normal rats by Van Doorn et al. (131) have shown that T4 concentrations in liver are less than half that in plasma, while T3 levels were 5 - 10 X those found in plasma. Preliminary studies in our laboratory (data not shown) are largely in agreement with these results. While measurement of plasma thyroid hormones has helped in understanding the influence of thyroid hormones on TCDD 66 toxicity, measurement of thyroid indices in tissue gives a different picture, suggesting that thyroid hormone concen- trations and thyroid hormone metabolism must be measured in individual tissues to develop a complete understanding of the relationship between symptoms of TCDD exposure, effects of TCDD on thyroid status, and direct effects of TCDD which may combine with these changes in thyroid status to produce the observed symptoms. CHAPTER III INTERACTIONS BETWEEN LIVER THYROID HORMONES, TCDD AND THE GLUCOCORTICOID RECEPTOR IN THE INDUCTION OF MALIC ENZYME ACTIVITY. 67 ABSTRACT Hepatic malic enzyme can be induced in rats in a dose- dependent fashion by exposure to TCDD. Although this process requires thyroid hormones, measurements of hepatic 3,5,3’-triiodothyronine (T3) levels indicated that malic enzyme induction does not result from local changes in T3 concentration. Similarities between this process and the induction of malic enzyme by glucococorticoids suggested that TCDD might be act by a corticomimetic mechanism. Competitive binding assays of TCDD with the synthetic glucocorticoid [6,7-3H1-triamcinolone indicated that TCDD was not an effective ligand for the glucocorticoid receptor at toxicologically significant doses. Although it is possible that TCDD can enhance the synthesis of malic enzyme in the rat via a mechanism involving the Ah- receptor, no malic enzyme induction was observed when Ah- receptor sufficient (C57BL/6N) or deficient (DEA/2N) mice' were treated with TCDD. We conclude that induction of malic enzyme by TCDD in the rat does not directly involve the glucocorticoid receptor, nor does it result from changes in hepatic T3 levels. 68 INTRODUCTION TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most potent of a class of polycyclic aromatic hydrocarbons which cause the induction of cytochromes P-450c, P—450d (53) at least one glucuronyl transferase (78), glutathione—S— transferase (5), and other enzymes which have not been as well characterized. The induction of cytochrome P-450c has been shown to result from the binding of TCDD to an intracellular receptor, called the Ah—receptor, for which a specific enhancer region upstream of the cytochrome p—450c gene has been identified (67). Although the induction of cytochrome P-4SOs by TCDD and many other Ah-inducers is well understood, many symptoms of Ah-inducer exposure do not correlate well with cytochrome P—450 induction, or cannot be easily explained as secondary effects of the induction of Ah—locus enzymes. In particular, dosages of TCDD which cause acute toxicity do not correlate well with occupancy of the Ah receptor, as measured by cytochrome P— 450 induction. The guinea pig begins to show morbidity at doses of TCDD of less than 1 nmol/kg body weight, which is approximately the ED50 for cytochrome P-450 induction (103). The Sprague Dawley rat shows no gross morbidity until doses of approximately 20 nmol/kg - a dose of TCDD at which cytochrome P—450 is maximally induced. Little mortality occurs in the Sprague Dawley rat until a dose of 100 nmol/kg is reached. Other strains of rats and mice likewise show no signs of acute toxicity at doses of TCDD 69 which are in excess of those required for cytochrome P—450 induction. Certain symptoms of Ah-inducer toxicity, such as thymic involution, hyperlipidemia, and fatty liver development, can also be produced by glucocorticoid administration, and might be explained if TCDD had corticomimetic properties. We recently discovered that hepatic malic enzyme, which is normally regulated by thyroid hormones (114), insulin, glucagon, glucocorticoids (49,50), and dietary carbohydrate (80), was induced on exposure to either TCDD or 3,4,5,3',4’,5’-hexachlorobiphenyl (HCB) (111). We have also shown that this induction is thyroid hormone dependent, ie. that it does not occur in thyroidectomized rats. In the experiments reported here, we have attempted to determine whether local tissue levels of T3 change enough to account for this induction, and if not, whether it can be explained by a corticomimetic property of TCDD. MATERIALS AND METHODS Reagents [6,7-3H]-Triamcinolone acetonide (3H-TA), (43.6 Ci/mmol) was purchased from New England Nuclear, Boston, Mass. 2,3,7,8-Tetrachlorodibenzo-p-dioxin(TCDD) was a gift from Dr. Fumio Matsumura, Pesticide Research Center, Michigan State University. 3,4,5,3',4’,5’-Hexachlorobiphenyl(HCB) was prepared by Pathfinder Laboratories Inc., St. Louis, MO. L-3,5,3’-Triiodothyronine (T3) was obtained from Chemical Dynamics Co., South Plainfield, N.J. 7O 4-(Z—Hydroxyethyl)—l—piperazine—ethanesulfonic acid (HEPES) was purchased from Boehringer Mannheim Biochemicals, Indianapolis,IN. Bovine serum albumin, dextran, DL—a— glycerolphosphate, NADP, L(-)—malic acid, hydrocortisone, dexamethasone, triamcinolone and L—thyroxine were all obtained from Sigma Chemical Co., St. Louis, MO. All other chemicals were reagent grade. Animals Male, 275—300 g Sprague-Dawley rats, male, 25—30 g C57BL/6N and 25—30 g DBA/ZN mice were obtained from Charles River Labs. Adrenalectomized male Sprague—Dawley rats were obtained from the same supplier. Rats were housed in large (20"x16"x8") polycarbonate cages with hardwood bedding, and were provided with food (Wayne Rodent Blox, Wayne Feeds, Chicago,IL) and water ad libitum, except as noted . Adrenalectomized rats were provided with normal saline in place of water to prevent sodium depletion. Treatments All Operations involving the use Of TCDD were performed in isolation as described in chapter 2. TCDD and HCB were given to animals by gavage in corn Oil ( 1 ml/kg ). In some experiments rats were treated with T3 in the diet by mixing powdered food with either ethanol alone (10 ml/kg diet) or appropriate concentrations of T3 in ethanol (10 ml/kg diet), followed by mixing for 10-15 min to ensure homogeneity and evaporation of ethanol. 71 Buffers and Solutions HEG buffer consisted Of 25 mM HEPES, 1.5 mM EDTA, and 10% glycerol, adjusted to pH 7.4 with NaOH. HEDG buffer was made 1 mM in dithiothreitol(DTT) by adding DTT to HEG buffer immediately before use. HE (25mM HEPES, 1.5mM EDTA, pH 7.4) buffers containing glycerol, sucrose, or dimethyl sulfoxide (DMSO) were used tO prepare and store mitochondria and microsomes, as described under Tissue Preparation. Phosphate buffer, used in the a-glycerol- phosphate dehydrogenase assay, contained 100mM Na HPO 5 2 4' mM EDTA, and 10 mM MgC12, pH 7.4 . Ferricyanide solution was 1.6 mM KCN and 1.6 mM K3Fe(CN)6. Solution A consisted Of phosphate buffer plus ferricyanide solution, 5:4, with 162 mg Na2 OPglycerOlphosphate added per 10 ml immediately before use. Solution B (blank) consisted of phosphate buffer plus ferricyanide solution 5:4, without a-glycerolphosphate. Tissue Preparation All animals were sacrificed by C02 asphixiation and decapitation 72 hr after treatment with TCDD. Trunk blood was collected into EDTA—containing tubes (Vacutainer; Becton-Dickinson, Rutherford, NJ) on ice. Livers were perfused with ice-cold saline in situ using a blunt, plastic-tipped syringe, and then were excized into cold saline on ice. Individual livers were blotted, weighed and homogenized in a glass Potter-Elvehjem homogenizer with a 72 Teflon pestle using 3 ml Of cold HEDG buffer/g tissue for preparation of mitochondria, microsomes and cytosol. For preparation Of "stabilized" glucocorticoid receptor, the same procedure was used except that 20 mM molybdate was added, and 1 mM DTT deleted from the buffer. Homogenates were centrifuged at 10,000 x g for 20 min to give a nuclear pellet, a crude mitochondrial layer, and a supernatant layer. The supernatant layer was carefully removed and centrifuged for 70 min at 105,000 x g to give "cytosol" (clear supernatant) and a crude microsomal pellet. Lipid was aspirated, and cytosol collected into polystyrene tubes which were frozen on dry ice immediately after collection. Crude microsomal pellets were resuspended in HE buffer containing 20 % glycerol, and stored at -800 C for later processing. Crude microsomes were washed by resuspend ing in HEG buffer followed by centrifugation at 105,000 x g for 90 min., prior to ethoxyresorufin-O-deethylase (EROD) determinations. The crude mitochondrial layers from the 10,000 x g centrifugation were gently poured Off the nuclear pellet into separate tubes, resuspended in 25 ml Of 0.25 M sucrose in HE buffer, centrifuged at 10,000 x g for 20 min, and the supernatant discarded. The resulting mitochondrial pellets were resuspended in HE buffer containing 20 % DMSO for storage at -800 c. 73 Thyroid Hormone Assays Aliquots Of plasma or thyroid hormone standards made up in 10 mg/ml albumin were extracted by placing 400 ul of sample or standard in 1.5 ml microfuge tubes, followed by 1 ml methanol. The tubes were then centrifuged for 5 min at 8,000 rpm in a microcentrifuge. Following centrifugation, 1 ml Of supernatant was mixed with 280 ul buffer (0.03 M Na+ acetate + 0.03 M acetic acid) to adjust the water and acetate content Of the extract to match the HPLC solvent described in the next section. Tissue thyroid hormones were extracted by homogenizing 1 to 5 grams Of tissue with 5 volumes of ice-cold methanol in a Teflon-pestle homogenizer immediately after excision. Five milliliter aliquots of the methanol extracts were placed in 20 ml glass scintillation vials, cooled to -600 C, and vacuum dried. The residue was resuspended in 1.5 ml of HPLC solvent, centrifuged, and chromatographed as described in the next section. Following HPLC separation, the T3 and T4 containing residues were dissolved in 200 ul of a 10 mg/ml fatty acid- free bovine serum albumin solution. Total T4 and T3 were then determined by radioimmunoassay:p 1.0 TCDD DOSE. NMOL/KG 84 Figure 18 4 and activated glucocorticoid receptor with the following 3H-triamcinolone binding to the MOO stabilized additions to the incubation mixtures: no competitor (IF—4|), 325 nM cortisol (CF—43), or (E}—{j) 100 nM cold triamcinolone. 85 mmmzaz zc_po 7 days difference between lst and last samples). The resulting values were used to compute either CPM/m1 (plasma, bile, and urine), or CPM/g (liver & kidney). These values were then multiplied by the plasma volume, bile or urine flow/hr, or tissue weights to give whole organ values, after which they were divided by the dose determined from diluted tracer samples for each experiment to obtain results as % of dose. Crude transport and metabolism rates were determined for each compartment 105 using this data, and were then used as starting points for determination of refined rates and variance information by a nonlinear least-squares Optimization and kinetic modeling program (CONSAM 29, references 10 and 11). A four compartment model comprized of plasma, liver, kidney, and the residual carcass was used initially for analysis of T3 and T4 tracer data. These models were later modified by the addition of separate hepatocyte membrane and cytOplasmic compartments to allow data fitting. Computation of expected T3 production levels assuming dependence of the 5’-deiodinase on cytOplasmic or plasma membrane T4 concentrations was accomplished by integrating the five compartment models for T3 and T4 metabolism into a interconnected ten compartment model (Appendix A). RESULTS The overall model structure and rate constants determined for transport between and metabolism in its compartments are shown in figure 21, table 1, and table 2. We initially attempted to fit our data to a four compartment model, illustated in figure 21, but found that the liver data could not be fit for either T3 or T4 unless the liver was divided into a retentive membrane compartment and a cytOplasmic compartment, as shown in figure 21. Our best fit results for each of these schemes are shown in figures 22 and 23, with the predicted masses in the individual and 106 Figure 21 Four and five compartment models for uptake and metabolism of T3 and T4. First order rate constants were fit to plasma, liver, and kidney data. The modified five compartment model of thyroid hormone metabolism contains separate membrane and cytoplasmic compartments in the liver. 107 u;—. u:_u: M O H --—------> - -------«> . 3 K x x was LIIIII T| nlllll‘ nus "fix ... l C:— cm_§ 154 pmol/hr) caused by TCDD treatment results in only a 2 fold increase in the total biliary excretion of T4 (124 pmol/hr -> 246 pmol/hr). The transport of T46 from the liver into the bile is apparently very rapid, as the glucuronide was only detectable in the bile (figure 24). Transport and total metabolism of tracer T3 was similar in control and treated rats (table 3). 113 TABLE 1 RATE CONSTANTS GIVING OPTIMAL FIT FOR FIVE COMPARTMENT T3 MODEL PROCESS T3 RATES (X 103per minute) Control TCDD—Treated Uptake k21 280 277 (liver) (252 - 302) (260 - 296) k31 98.0 101 k41 542 515 (500 - 703) (510 - 751) (carcass) Recycling (CONSAM estimates only) k13 40.4 46.9 (kidney) qu 30.5 31.3 (carcass) k52 368 434 k15 770 641 Metabolism K05 22.5 i 6.5 22.9 i 3.0 (liver -> bile) As approximately equal quantities of T2, T3, & glucuronide K03 3.47 i 0.88 2.52 i 0.40 (kidney -> urine) K04 0.75 0.92 carcass residual) 114 TABLE 2 RATE CONSTANTS GIVING OPTIMAL FIT FOR FIVE COMPARTMENT T4 MODEL 3 PROCESS T4 RATES (X 10 _per minute) Control TCDD-Treated Uptake k21 21.1 32.2 (liver) (11.6 - 34.1) (15.6 - 44.7) k31 7.28 7.58 * (kidney) (2.20 - 4.10) (1.40 - 6.50) k41 94.1 122.0 (carcass) Recycling (CONSAM estimates only) kl3 97.9 85.3 (kidney) kl4 29.1 43.03 (carcass) k52 247.0 206.0 k15 35.8 78.7 Metabolism K05‘ 7.99 i 1.70 19.23 i 0.27 (liver -> bile) glucuronide 2.23 : 0.45(30 %) 10.70 i 3.3(60 %) K03 1.11 i 3.9 1.78 i .61 (kidney -> urine) 2.19 3.87 K04 (carcass/residual) * CONSAM estimates provided better fit, original estimates were based on very low counts ( about 2 X background ). 115 TABLE 3 Metabolites of T3 and T4 in Bile and Urine after 6 Hours Control : S.D. % Total TCDD : S.D. % Total Control :.S.D. % Total TCDD : S.D. % Total T3 Disposition (% of dose/hr) Urine Bile 1- T3 1— Glucuronide T2 T3 2.667 0.0256 0.329 0.547 0.431 0.445 1.636 0.0017 0.050 0.067 0.032 0.016 (89 %) (5.4 %) (10.9 %) (38 %) (30 %) (32 %) 5.619 0.0205 0.284 0.761 0.346 0.421 0.251 0.0038 0.004 0.086 0.004 0.021 (99 %) (0.3 %) (15.6 %) (49 %) (22 %) (27 %) T4 Disposition (% of dose/hr) Urine Bile I- T4 1_ Glucuronide T4 1.634 0.0719 0.107 0.377 1.125 0.459 0.0339 0.0097 0.0098 0.011 (96 96) (4.2 SIs) (5.9 96) (20.9 96) (62.4 %) 1.257 0.0909 0.127 1.092 1.125 0.708 0.0266 0.013 0.148 0.063 (93 %) (6.7 %) (5.3 %) (45.6 %) (41.3 %) 116 Figure 24 Upper figure : Chromatographic profiles of 100 ul bile samples from normal (———), and TCDD-treated (u--) rats 6 hours after T4 tracer injection. Lower figure: Profile of 250 mg equivalent liver extracts obtained from the same animals as bile samples in the upper figure. For this figure fractions were collected every 15 sec. for 25 min. Mobile phase was 500:498:1, methanol:water:acetate, with 1 ml of methanol injected onto the column at fraction 64 to wash out T4. For routine separations, fractions were collected every 30 sec for 20 min, with methanol injection at fraction 32. IZSI-CPM/FRACTION 117 5000- , :; BILE 4000‘ u I :: T4-GLUCURONIDE 3000‘ I: l I: T4 2000- :. I I 1000- -1 0 T‘MEOH O S 10 IS 20 25 ZOOOW MINUTES LIVER 1000- 0 I fMEOH 0 5 10 15 20 25 MINUTES 118 A small increase in glucuronide metabolites was observed in the bile, but was less interesting than an apparent shift from a metabolite with a slightly longer retention time than T3 (triac ?) to T3 as a major product of beta- glucuronidase digestion (figure 25). Although Hearn gt 31; (62) have achieved separation of the thyroacetic acids from their thyronine counterparts using an acidic, unbuffered mobile phase, the difference in retention time between T3 and triac in our buffered system (pH 6) is only about 1 min. (18 vs 19 min. @ 1.5 ml/min.). Our measurements of hepatic T3 produced by deiodination of T4 may reflect reports that type I deiodinase is a plasma membrane enzyme, rather than an enzyme contained in the endoplasmic reticulum or cytoplasmic compartment (24,75,79), for reasons described below. Liver T3 is produced in a rapidly rising peak that lags, but falls off as quickly as does plasma T4 (figure 26). A pulse of iodide (and thus rT3, since the tracer is 3,5- labeled) appears at the same time, in agreement with reports that T3 and rT3 production rates are about equal. If the deiodinase acted on T4 in the cytoplasmic compartment, T3 levels should follow the slowly rising and falling T4 levels in the liver. The results of simulations of this process using the integrated 10 compartment model (figure 27) are shown in figure 28. Attempts to model the pulse of T3 production by the addition of an endocytosis process to the ten (making it 11) compartment model were 119 Figure 25 (a) Chromatographic profile of bile samples of control rats 6 hrs after injection of T3 tracer, before (H), and after «343) incubation with beta-glucuronidase. (b) Chromatographic profile of bile samples from TCDD- treated rats 6 hrs after injection of T3 tracer, before (H), and after (0-0) incubation with beta-glucuronidase. 120 352 E 9 cm ms . o h? e \ . fibd. j . .a a .. . O.u<_x._. _ .. ,. ... H l. s.-. — . _ a . 2: .od. 2 __ .. M. . o2 __ N._. . __ _ _ . . _ u . . _ . . a "0%.. ... w u u DON mm r. 012. I CON 1 m o _. . IJ _ . W 1 l m o l. 0 < . 8m . 8m m < . oov . 8v 121 Figure 26 Hepatic uptake of T4, and production of T3 after injection of T4 tracer. Each symbol represents the average of samples from three rats. Controls are indicated by open symbols, TCDD-treated rats by filled symbols. Note that both I_ and T3 have maxima at 15 minutes. The peak for uptake of plasma T3 tracer occurs at 5—6 min (figure 22). Data for iodide in controls has been omitted for clarity. 122 20¢ HBAIT NI 3300 JO 2 ' 1921 60 80 100 I 20 MINUTES 40 20 123 Figure 27 Integrated model for production of T3 from T4 . Compartments 1 -> 5 are associated with T4 uptake and metabolism, 6 -> 10 are assigned to T3 metabolism. Dashed lines labeled with the rate constant kdi represent deiodination pathways from T4 —> T3. Other dashed lines represent routes of hormone inactivation. 124 TEN COMPARTIENT I‘DDEL 0F T4 AND T3 METABOLISM 125 Figure 28 Predicted levels of T4 (-———-) and T3 (----) in the liver assuming that cytosolic T4 is the deiodinase substate. Rate constants used to simulate T3 production were Vmax = 438 fmol/min-mg protein and Km = 3.6 uM (ref. 68) A first order mass rate constant was computed for the whole liver using these values (appendix A). The final rate constants for deiodination were A) kdi = 0.0027/min B) 10 X (A), kdi = 0.027 Note the change in the T4 curve caused by the increased deiodination rate in (8) relative to (A). ‘25! - 2 OF DOSE 1251 — 2 OF DOSE 20 IS 10 IS 10 126 ..- llll l’---1---1---1---- 20 40 60 80 100 120 MINUTES MINUTES 127 unsuccessful, presumably because of insufficient information about intracellular transport of T3. Although the T3/T4 ratio was much higher in the kidney than in the liver, a pulse of T3 production like that observed in the liver was not observed (figure 29). Very little T3 or T4 was excreted by the kidney . The only significant metabolite of these compounds observed in the urine was I— (figure 30), in agreement with earlier studies of thyroid hormone metabolism (41). DISCUSSION Many biochemical, physiological, and histological changes are associated with exposure to Ah-inducers (34). Most of these changes have been observed only at doses of these compounds which produce gross pathology accompanied by anorexia. Thus, from the literature, it is difficult to distinguish between primary effects of Ah locus enzyme induction, and effects which are secondary to the anorexia, fatty liver, and other pathology which occurs at high doses. One effect which was thought to be well explained by induction of these enzymes was the drop in plasma T4 following Ah inducer treatment. Several investigators have reported increased fecal and/or bile excretion of thyroxine, accompanied by a 50—75 % drop in serum T4, in rats treated with various Ah-inducers (8,48,89). It was assumed that the change in T4 levels was a secondary result of the induction of glucuronosyl transferase . 128 Figure 29 Uptake of T4, production of T3 and 1‘ in kidneys of control (H), and TCDD—treated (O—O) rats following tracer injection. The arrow indicates the time ‘at which a pulse of T3 production occurred in the liver. 129 cm— u m/ oc— mu~=z_z ABNGIN NI 3300 30 z ’ ISZI 130 Figure 30 Chromatographic profile of urine from a control rat 6 hr after injection of T4 tracer. No significant difference was observed between urine from control and TCDD—treated. rats. The urinary profiles following T3 injection were qualitatively similar to those for T4. Mobile phase for urine was methanol:water:acetate, 575:452:l '4000- 3000- 2000« 1251- CPM/FRACTION 1000- 131 MINUTES 132 However, these studies were all performed at inducer doses which result in anorexia, fatty liver, and other gross pathology. As reported in studies from our laboratory (111) and others (78,103), induction of cytochrome p—450c and the glucuronosyl transferases is maximal at a TCDD dose of 10 nmol/Kg. Food consumption and body weight gain are normal at this dose, and no acute morbidity is observable. Nonetheless, we have shown (111) that plasma T4 drOps by 40-50 % at this dose. Liver concentrations dr0p by 10—20 %, and fall precipitously at higher doses (chapter 2). In the present study we have shown that glucuronidation is already as high at 10 nmol/Kg as reported by others at larger doses, suggesting that the continuing dr0p in T4 at higher doses is brought on by anorexia, rather than by a further increase in glucuronidation. We pr0pose that the high rate of thyroxine diSposal creates a moderate iodine deficiency, which is aggravated by the reduced iodine intake and negative protein balance (including tyrosine) associated with anorexia. A lower thyroxine production rate might also account for the failure of earlier studies to find the large amount of free T4 excreted in the bile with T4 glucuronide. The appearance of a pulse of T3 with a magnitude prOportional to the specific activity of T4 in the plasma, rather than being a constant fraction of the tracer dose, indicates that a constant amount of T4 was converted to T3 rather than an amount which was proportional to the total 133 flux of tracer into the liver — which was the same for control and TCDD—treated rats. We suggest that liver deiodination is controlled by a component of the plasma to liver T4 flux which is constant, rather than by the total T4 flux or liver T4 concentration. Such a scheme would explain the ability of our animals to maintain T3 levels as T4 concentrations are dropping. The net T4 flux from plasma to liver is actually larger in TCDD treated rats than controls, as evidenced by the bile disposal rate. It has been reported that both T3 and T4 are transported by two parallel uptake systems: 1) a high affinity, low capacity system that can be inhibited by colchicine, ouabain, and cyanide, which is saturated well below physiological concentrations of T4, and (2) a low affinity, high capacity system which is not saturated at 10 X the normal physiological levels of T3 or T4 (42,56,73). The high affinity system is thought to represent receptor—mediated endocytosis of thyroid hormones (23). The lag time for appearance of the pulse of T3 which we observed in liver is similar to the time required for endocytosis of epidermal growth factor (22) and lysosomal marker proteins (76). The low affinity, high capacity system probably represents passive diffusion of thyroid hormones through the plasma membranes driven by their lipophilic character (59,98). Leonard gt gt; (75) suggested that the plasma membrane location of the deiodinase "...may represent a physiological mechanism to allow efficient 134 production of... T3, from an intrinsically inactive precursor T4, without the obligatory penetration by T4 of the intracellular space." Our results indicate that an association of the high affinity uptake system and the plasma membrane 5’—deiodinase might serve to deliver a controlled flux of T4 to the deiodinase, while excluding the much larger flux of T4 which passively diffuses into the cell. Control of T3 production from T4 is an area of controversy. DiStefano gt gt; have recently found that as much as 75 % of the total mass of T3 in the rat is contained within the intestinal lumen, only 1/6 of which is conjugated (41). T4 does not accumulate in the intestine, but an interesting transformation does occur. Of the T4 entering the intestine from the bile, 30—50 % is conjugated in a normal rat. During its passage through the intestine, most of this glucuronide is hydrolysed by bacterial beta— glucronidase, and can be partially resorbed as T4 (27), rather than simply excreted, as our model and many others have assumed. T3 is even more readily absorbed, and the large T3/T4 ratio in the intestine relative to the plasma (1:2 vs. 1:50 in plasma) suggests that deiodination of T4 to T3 could be occuring in the lumen, although deiodination in the intestine has yet to be shown. Even if the large amount of T3 in the intestine is simply the result of the high rate of disposal of T3, the existance and recycling of this large, previously ignored pool of T3 dramatically 135 differs from accepted models for thyroid hormone regulation. Our results suggest an association between two processes whose pr0perties, independently, could not explain the behavior of the integrated control system in vivo. While our results are not conclusive, the association we suggest between uptake and deiodination better explains the relationship between T4 concentrations and T3 production observed in animals treated with Ah— inducers, than does the model assuming independence of uptake and deiodination, and may provide a rationale for the the plasma membrane location of the 5’-deiodinase. SUMMARY 137 SUMMARY TCDD (2,3,7,8—tetrachlorodiben20*p-dioxin) is the most toxic of a class of polycyclic aromatic hydrocarbons which cause the induction of enzymes associated with the Ah locus. This set of enzymes includes cytochrome P-450c, P-450d, at least one glucuronosyl transferase, glutathione— S—transferase, and other enzymes which have not been as well characterized. Some of the effects of Ah inducers on metabolism appear to be explainable as secondary effects of the induction of the Ah locus enzymes mentioned above. Although these enzymes were discovered by virtue of their catalytic activity towards carcinogenic compounds such as benzo[a]— pyrene and 3—methylcholanthrene, they are known to hydroxylate and or conjugate fatty acids, steroids, and thyroxine (T4). Since (T4) conjugation and excretion was known to be increased on treatment with Ah—inducers, we attempted to determine whether either plasma or liver T3 levels dr0pped with T4. Our initial experiments demonstrated that T3 levels did not change with T4 levels. Since studies of T3 production and metabolism i2 Zi££9 (liver and kidney homogenates or microsome suspensions) indicated that deiodination of T4 to produce T3 was linearly dependent on T4 concentration, we decided to perform experiments 12 yivo to determine whether transport 138 mechanisms destroyed by tissue homogenization could explain the discrepancy between ig_yit£g and ig.yiyg results. In pharmacokinetic studies, T3 was produced from T4 in the liver in a pulse which lagged T4 uptake by 4 - 5 min. The relative size of this pulse in treated vs control rats was similar to the ratio of specific activities for T4 between the respective groups. In light of reports on the passive and active uptake of T3 and T4, this data suggested that deiodination of T4 is regulated by a saturable uptake system which runs in parallel with a nonsaturable, diffusive uptake of T4, driven by its hydrophobicity. Our results suggest an association between two processes whose properties, independently, could not explain the behavior of the integrated control system lg yiyg. While our results are not conclusive, the association we suggest between uptake and deiodination better explains the relationship between T4 concentrations and T3 production observed in animals treated with Ah—inducers, than does the model assuming independence of uptake and deiodination, and may provide a rationale for the the plasma membrane location of the 5’-deiodinase. While attempting to determine the thyroid status of Ah- inducer treated rats, we discovered that liver malic enzyme (normally regulated by insulin, glucagon, glucocorticoids, and thyroid hormones) was increased by Ah-inducers in a thyroid hormone dependent manner. The induction of malic enzyme by glucocorticoids is thyroid hormone dependent, ie. 139 thyroid hormones must be present for induction to occur. Certain symptoms of Ah-inducer toxicity, such as thymic involution, hyperlipidemia, and fatty liver development can also be produced by glucocorticoid administration, and might be explained if Ah-inducers had corticomimetic properties in addition to their interactions with the Ah receptor. For these reasons, we performed experiments to determine whether TCDD could act as a ligand for the gluco— corticoid receptor. These experiments demonstrated no affinity of TCDD for the glucocorticoid receptor, but left open the possibility that the Ah receptor may be able to regulate some of the same genes as does the glucocorticoid receptor. APPENDIX A ESTIMATION OF T4 ACTIVE UPTAKE AND DEIODINATION RATES FOR T3 PRODUCTION MODELS. 141 ESTIMATION OF T4 ACTIVE UPTAKE AND DEIODINATION RATES FOR T3 PRODUCTION MODELS. Studies of T3 production from T4 have generally have been performed in homogenous mixtures of 5'-deiodinase, T4 , and glutathione or a synthetic thiol supplied in vitro. Michealis-Menten rate constants have been determined using whole liver homogenates , aqueous suspensions of liver or kidney microsomes, or cytosol-microsome mixtures. The maximal velocities (Vmax) determined in these preparations are generally very low : 0.2-2.0 pmol/min. The half- saturation concentrations (Km) have been very high(l-S uM), relative to the physiological concentrations of T4. Because of the high Km values, the observed deiodination rates are essentially first—order with respect to T4 concentration. Since specific steps in the metabolism of thyroid hormones occur in specific tissues and/or discrete subcompartments of these tissues, the precise relationship between steady- state T4 and T3 concentrations i3 yiyg cannot be predicted from £2 vitro deiodination rates alone. Crabtree and Newsholme (29) have developed nomenclature and a theoretical treatment for the control of metabolism which describe the fluxes of substrates and metabolites in multi- compartment/multienzyme systems, and have shown that these fluxes, rather than concentrations of metabolites, are the controlling variables in complex metabolic systems. Using 142 their notation, the metabolism of T4 can be described as follows T4 ,2 T4 -——> T4-GLUCURONIDE T4 -—~*T3 -->T2 + T3-GLUCURONIDE In the above scheme, each arrow represents the flux of metabolites from one compartment to another or its conversion into a new metabolite. The symbol r+—> indicates a process which is flux limited, ie. in terms of Michealis— Menten kinetics, the enzyme (or in this case, transporter) is saturated. Thus beyond a certain concentration of T4 (about 5 nM using the data from Doctor, reference 42) the flux from T4 in the plasma to the membrane and to T3 is constant. Since both deiodination and glucuronidation/- catabolism are dependent on the fluxes of T4 and T3 provided by the parallel passive and active transport systems, analysis of ig_yiyg production data requires the determination of these transport rates before an integrated model of the process can be developed. We determined total liver uptake rates (diffusive + active) from our tracer data, but did not measure saturable uptake rates. We therefore used literature data to estimate the ratio of active to passive T4 uptake, and compared these values with the observed T3 production as described below. 143 Determination of the Uptake of T4 by the High Affinity System According to Doctor gt gl.(42) the uptake of T4 by cultured parenchymal cells can be described as the sum of two processes represented with Michealis-Menten kinetic constants as Vmax1[T4] Vmax T4] where the subscripts denote the high and low affinity systems, respectively. For the low affinity system, Km = 500 nM, and Vmax = 1.9 nmol/.1488 gm-min. Since the concentration of T4 in the plasma of a normal rat is about 50 nM, the transport rate would be Vmax 500 x 10' + 50 x 10‘ which indicates that the velocity is well below saturation with substrate, and will behave as a first—order process. However, this number cannot be used directly as a rate constant to predict behavior of this system in yiyg. Given a liver weight of 12.5 gm/300 gm rat, Vmax = 159 nmol/liver-min. Substituting this value to determine the uptake velocity gives v = 0.0909*Vmax or v = 14.45 nmol/liver-min. The total plasma pool (8 ml) contains only 416 pmol of T4 ! 144 High Affinity System Similar calculations can be performed using rate constants for the high affinity system Km = 900 pM Vmax = 3.2 pmol/0.1488 gm v 50 x 10’9 ---- = "'---"":'9"-"--"-":§' = 0.982 Vmax 0.9 X 10 + 50 X 10 Note that the velocity has almost reached the maximal velocity (v/Vmax = 1.00 when the enzyme is saturated). Again using a liver weight of 12.5 gm/300 gm rat, the forward velocity is v = 264 pmol/liver-min. As before, this number is too high, but the ratio of the low and high affinity systems can be used with $2 ytyg total rates to estimate fractional rates. In rats treated with 10 nmol TCDD/Kg-BW, the concentration of T4 in the plasma is about 32 nM. When this value is substituted into the velocity equation for high affinity uptake, very little change in rate is observed Vmax 0.9 x 10' + 32 x 10' To get a 30 % drop in T4 uptake by this system, the plasma T4 concentration must drop to about 2_gM. The passive uptake of T4 by the low affinity system meanwhile, is dropping linearly with T4 concentrations. These values were determined by Doctor gt al. in an incubation medium containing 1 % albumin. In later work (72), it was shown 145 that albumin concentration was critical to the transport of I; by its high affinity uptake system. Increasing the albumin concentration raised the Vmax 5-fold. Increased albumin raised the rate for low affinity (passive) uptake only about 2-fold. Increases in temperature had a similar effect - the high affinity system uptake increased 3-fold between 25 and 370C, while passive uptake was increased 1.24-fold. Assuming that the T4 high affinity system responds to these parameters in the same way as the T3 system, the ratio between the two under physiological conditions can be estimated Low Affinity System @ 370 + 20 mg albumin/ml 14.45 nmol 35.8 nmol ---------- x 2 x 1.24 = ------——- High Affinity System @ 370 + 20 mg albumin/ml 264 pmol 3.962 nmol which implies that high affinity uptake of T4 is about 11 % of the total uptake i2 vivo. Since the concentrations of T4 in both normal and treated animals are at steady-state during the experiments (as opposed to the concentrations of 125I—T4), the uptake velocities are constant with respect to the T4 pools. In the high affinity transport process, uptake of T4 is a constant percentage of the total mass of tracer in the plasma pool. Since the amount of tracer is declining during the experiment, uptake of tracer is the product of % high 146 affinity uptake and the % of Dose remaining in the plasma pool. The specific activity of tracer in TCDD treated animals is higher than in normal rats (since total T4 is lower), hence the amount of tracer taken up when a constant mggg of T4 is transported will be greater for TCDD treated animals than controls if the system is unaffected by TCDD. This effect does not occur with the passive system since it is operating well below saturation, and all uptake is described by the equation dx. __1 = k'iXi dt 3 _ 125 . Xi — mass of I-T4 in plasma compartment Xj = mass of 125I-T4 in tissue compartment Specific activity calculations indicate that the high affinity uptake of tracer (but not total T4) would be 1.62 X controls, using known plasma T4 concentrations Normal 52 nM X 8 ml plasma 416 pmol/pool TCDD 32 nM X 8 ml plasma 256 pmol/pool 328 fmol tracer @ 4400 Ci/mmol injected implies 0.07878 % of total T4 Specific Activity (SA) normal Specific Activity (SA) TCDD 0.12796 % of total T4 or SA TCDD = 1.624 X SA normal Although the total 125 I-T4 uptake was the same in treated and control rats, as expected from the above analysis, with passive uptake predominating, the pulse of tracer T3 which appeared in the livers of TCDD-treated rats (figure 26) was 147 1.5 - 2.0 X larger than that for controls, as would be expected if the same gggg of T4 was deiodinated, but with the higher specific activity in treated animals outlined above. The ratio of active/passive uptake estimated above was used with the total uptake rate determined for the five compartment sub-model for tracer T4, in an attempt to simulate the pulse of T3 production using a modification of the 10 compartment model. The high affinity uptake was modeled as an endocytosis process with the active component comprizing 11 % of the total uptake in controls, and 1.62 X 11 % = 17.82 % of the total T4 uptake in treated rats. A pulse of T4 uptake could be obtained with the same delay as was observed in the data, but T3 did not fall off rapidly after the pulse maximum as did the observed T3 levels. At this point it was clear that there was insufficient information available about the mechanisms of T3 transport in the cytoplasm and nucleus to intelligently modify the ten compartment model so that the T3 production pulse could be fitted. Derivation of a First-Order Deiodination Rate From lg vitro Data for the $2 vivo Model. First-order deiodination rates for whole liver (as opposed to a Michealis-Menten rate in mass/mg-protein units) were derived in the same fashion as the transport rates derived above. The most complete set of $2 vitro Michealis-Menten 148 rate constants found in the literature were those of Kaplan (reference 69). These rate constants were obtained in studies of deiodination of T4 to T3 in liver homogenates and microsomes from rats kept in hypothyroid, euthyroid, or hyperthyroid states, respectively. Homogenization of these livers resulted in a 1:3 dilution of the original liver activity. Computation of the activity in the whole liver was determined by using this dilution factor along with the protein concentrations determined for whole homogenates (45 mg/ml), and microsomal suspensions (5.7 mg/ml when diluted to the starting homogenate volume). Correcting these values for dilution gives 45 mg protein/ml X 4 = 180 mg protein/gm liver (total homogenate) 5.7 mg/ml X 4 = 22.8 mg protein/gm liver (as microsomes) For euthyroid rats, the Vmax and Km determined in the microsomal mixture were Vmax = 438 fmol T3/mg—min Km = 3.6 uM Using this Vmax value, the estimated microsomal protein concentration in the liver, and the liver mass for a 300 gm rat (12.5 gm/liver), a maximal velocity for the whole liver was computed Vmax (438 fmol T3/mg-min)(22.8 mg/gm)(12.5 gm/liver) 124 pmol T3/min—liver The expected velocity at the physiologic T4 concentration (18 nM in liver — chapter 3) is then computed from the 149 Michealis-Menten expression d[T3] Vmax[T4] (124 pmol/min)[18 nM] V = ————— = ————————-—— = ————————————————————— dt Km + [T4] 3600 nM + [18 nM] = 617 fmol/min-liver Since [T4] is at steady state in a living animal, this velocity does not change. Changes in tracer activity are not indicative of changes in reaction velocity as the tracer concentration changes. For this reason a first-order tggg rate constant can be computed from the above whole liver reaction velocity, since the mass of T4 in the liver is known Liver T4 mass 18 nM X .0125 liter (12.5 gm liver) 225 pmol (control) The mass balance equation for the deiodination reaction is v = ng - k 'X dt deiodination 1 where X1 = mass of T4 in liver. X2 = mass of T3 in liver Therefore the mass rate constant, k . . . is deiodination 617 fmol/min-liver kdeiodination: V/Xl = TTTTTTTTTTTTTTTTT = 2.74 X 10—3/min—liver This value was used in the ten compartment model of thyroid hormone metabolism to simulate T3 production, configured so that cytosolic T4 was the 5'-deiodinase substrate 150 (figure 28). Rate constants for uptake, recycling, and metabolism were determined by fitting tracer data to the 5- compartment sub-models for T3 and T4, as described in chapter 4. 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