MSU LIBRARIES RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ABSTRACT STUDIES ON THE RELATIONSHIPS OF TCDD TOXICITY AND vITAnIN A BY Robert Harold Powers The effect of 2,3,7,B-tetrachlorodibenzo-p-dioxin (TCDD) and similarly acting compounds on the mobilization and transport of vitamin A, the depletion of hepatic retinoids and the accumulation of retinoids in the kidneys of Sprague-Dawley rats were investigated in several different experiments. In addition, the effect of the thyroid hormone T3 on the above parameters was also investigated. TCDD was determined to be a non-competitive inhibitor of hepatic retinyl palmitate hydrolase, (RPH). Inhibition of RPH ;Q_ gixg, by treatment with either nonadecafluorodecanoic acid or 3,4,3',4'-tetrachloro- biphenyl resulted in lowered serum retinol levels. Inhibition of RPH was determined not to be of toxicological significance in the case of TCDD treatment. The retinoid material which accumulated in the kidney of TODD-treated rats was determined to be retinyl esters. The activity of renal acyl CoA retinylsacyl transferase (ACARAT) was elevated, and correlated with the levels of retinyl esters. A similar accumulation and enzyme induction was observed in rats fed a vitamin A deficient diet. TCDD treatment increased the rate of hepatic retinoid degradation caused by microsoeal retinol oxidase, and retinoyl UDP-glucuronosyl transferase. Oxidation of retinal to retinoic acid and microsomal oxidation of retinoic acid were unaffected by treatment with TCDD. Inclusion of T3 in the diet of rats treated with TCDD enhanced the toxic response, and also increased the depletion of hepatic retinoids and accumulation of retinyl esters in the kidneys of treated rats. TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . . . vi LIST OF ABBREVIATIONS . . . . . . . . . . . . ix INTRODUCTION . . . . . . . . . . . . . . . 1 LITERATURE REVIEH. . . . . . . . . . . . . . 5 TCDD and Polyhalogenated Aroeatic Hydrocarbons..........5 Structure and Toxicological Significance.............5 Effects of TCDD Toxicity............................12 Vitamin A: Structure and Physiological Significance....16 Intestinal Uptake and Transport.....................20 Hepatic Uptake and Storage..........................20 Hobilization of Retinol.............................23 Degradation and Loss of Retinoids...................25 Clinical Signs and Lesions of Deficiency............26 TCDD and Vitamin A Status..............................30 Effects of TCDD on Vitamin A Storage................30 Sieilarities of TCDD Toxicity and Vitamin A Deficiency.......................................34 Bibliography...........................................36 TERS 1. EFFECT OF TCDD, NDFDA AND PCB'S ON HEPATIC RPH, PLASMA RETINOL LEVELS AND TRANSPORT.................49 Abstract.........................................50 Introduction.....................................53 Materials and Hethods............................56 Animals, treatment and tissue preparation............................56 Experiment 1. (NDFDA)......................56 Experiment 2. (TCB and HCB's)..............57 Experiment 3. (TCDD).......................58 Retinoid levels...............................58 Analysis of RPH activity......................59 Partial purification of RPH...................6O LQ_Vitro inhibition of RPH by NDFDA and TCDD..........................60 LQ_Vitro inhibition of RPH by T08 and Two HCB isomers............................61 Analysis of hepatic NDFDA levels..............61 ii Fractional distribution of retinol in plasma..................................62 Effect of microsomal incubation on RPH inhibition by TCB..........................63 Statistical analysis..........................b4 Results..........................................65 Experiment 1. (NDFDA).........................65 Body and organ weights.....................65 Serum and hepatic retinol levels...........65 Hepatic RPH activity.......................73 LQ_Vitro inhibition of RPH by NDFDA and TCDD..........................73 mpatic mm leveISDCCIOOCOOICIDICII......82 Exwrimt 2. (Ten md $8.5)...CCICUCCCCOOOCIBZ Plasma, hepatic and renal retinoid levels..................................82 Hepatic RPH activity.......................82 L_ Vitro inhibition of RPH by TCB..............89 Fractional distribution of retinol among plasma proteins...................89 Effect of incubation of TCB with microsomes on the inhibiton of RPH ig,vitro.........................95 Experiment 3. (TCDD)..........................95 Plasma retinol levels......................95 Fractional distribution of retinol among plasma proteins...................95 Discussion......................................101 Bibliography....................................109 II. EFFECT OF TCDD DR VITAMIN A DEFICIENCY 0N RENAL RETINOID STORAGE AND ACCCUMULATION.................112 Abstract........................................113 Introduction....................................115 Materials and Methods...........................117 Animals; treatment and tissue preparation........................117 Experiment 1 (TCDD).......................117 Experiment 2 (Vitamin A deficient diet)...117 Retinoid levels..............................118 Analysis of ACARAT activity..................118 Renal RPH activity...........................119 Statistical analysis.........................119 Results.........................................120 Renal retinol and retinyl palmitate..........120 Renal RPH activity...........................125 Renal ACARAT activity........................125 Discussion......................................130 Bibliography....................................133 iii III. EFFECT OF TCDD ON HEPATIC RETINOID STORAGE AND DEGRADATION........................................135 Abstract........................................136 Introduction....................................137 Materials and Methods...........................141 Animals: treatment and tissue preparation........................141 Retinoid levels..............................142 Retinal oxidase activity.....................142 Microsomal retinol and retinoic acid. oxidation.................................143 Retinol and retinoyl and p-nitrophenol UDP—glucuronosyl transferase activty......143 Statistical analysis.........................144 Results.........................................145 Body weight, food intake and organ weights.............................l45 Hepatic retinyl palmitate....................145 Microsomal retinol and retinoic acid NADPH-dependent degradation...............153 PNP, retinol and retinoic acid UDP- glucuronosyl transferase activity.........153 Discussion......................................162 Bibliography....................................165 IV. EFFECT OF TCDD AND/OR DIETARY T3 ON HEPATIC RETINOID ACCUMULATION AND RENAL RETINOID UPTAKE.............167 Abstract........................................168 Introduction....................................169 Materials and Methods...........................173 Animals, diets and treatment.................173 Retinol and retinyl palmitate................174 Statistical analysis.........................175 Results.........................................176 Body weights.................................17b Organ/body weight ratios.....................176 Tissue retinol...............................1BO Hepatic retinyl palmitate....................182 Renal retinyl palmitate......................182 Discussion......................................189 Bibliography....................................192 my I I I I I I I I I I I I I I I I I 195 iv LIST OF TABLES Chapter 1. 1. The Effect of a Single i.p. Dose of NDFDA on Hepatic/ and Thymis/Body Height Ratios in Male Sprague-Dawley R.t. (x10 )IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIba 2. Hepatic NDFDA Level Following a Single i.p. dose in Male Sprague-Dawley Rats............................83 3. The Effect of Incubation with Microsomes and NADPH on the Ability of 3,4,3',4'-Tetrachlorobiphenyl to Subsequently Inhibit Endogenous Retinyl Palmitate Hydrolase Activity..................................96 Chapter 2. 1. The Effect of a Single Dose of TCDD or Feeding a Vitamin A Deficient (VA(-)) Diet on Renal Retinol Levels and Retinyl Palmitate Hydrolase Activity Levels in Male Sprague-Dawley Rats, 12 Days Following Dose, or Start of VA(-) Diet.......................121 2. The Effect of Feeding a Control or Vitamin A Deficient Diet for 26 Days to Male Sprague-Dawley Rats on Plasma Retinol, Liver Retinyl Palmitate, Renal Retinol and Retinyl Palmitate, and Acyl CoAsRetinol Acyl Transferase.......................122 Cheeses}.- 1. The Effect of a Single Dose of TCDD or Feeding a Vitamin A-Deficient (VA(-)) Diet on Organ/Body Height Ratios in Male SpraguevDawley Rats, 12 Days Following Either TCDD Treatment or Start of the VA(—) Diet...152 Chapter 1. 1. The Effect of a Single Oral Dose of TCDD and/or Dietary T3 on Organ/Body Height Ratios in Male Sprague-Dawley Rats................................179 2. The Effect of TCDD and/or Dietary T3 on Tissue Retinol Concentration in Male Sprague-Dawley Rats...........170 LIST OF FIGURES Intrgguctigg. 1. Representative Polyhalogenated Aromatic Hydrocarbons...b 2. Structure and Nomenclature of Selected Retinoids......17 3. Intestinal Hydrolysis, Absorptione, and Esterification of Dietary Retinoids...............................19 4. Hepatic Uptake, Storage, and Mobilization of Retinyl EStIrs and RetinOIIIIIIIIIIIIIIIIIIIIIIIIIIIOII0.0.21 gnggter 1, 1. Body weight changes in control and "NM-treat“ r.t§IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIbb 2. Serum retinol levels in control and "mm-tr.at.d r.tSIIIIIIIIIIIIIIIIIOIIIIIIIOOIIIIIIbq 3. Hepatic retinol levels in control and "NM-treat“ r.t‘IIIIIII.IIIIIIIIIIIIII-III...I'D-71 4. Hepatic retinyl palmitate hydrolase activity of control .nd threatId rats-IIIIIIIIIII-IIIIIIIIIII-OIIO74 5. Correlation of serum retinol levels with hepatic RPH activity in control and NDFDA-treated rats.........76 6. Increase in RPH activity in acetone-extracted hepatic homogenates from control and NDFDA-treated rats....78 7. Inhibition of RPH activity by NDFDA and TCDD..........BO B. Effect of a single i.p. dose of corn oil, or corn oil containing 3,4,3‘,4‘-tetrachlorobiphenyl, 2,4,5,2‘,4',5'-hexachlorobiphenyl, or 3,4,5,3‘,4',5'-hexachlorobiphenyl on plasma retinol concentrations in female Sprague-Dawley rats 24 hours post-treatment............................84 9. Effect of a single i.p. dose of 3,4,3',4'-tetrachloro- biphenyl on the hepatic RPH activity of female Sprague-Dawley rats 24 hours post-treatment........86 V1 LIST OF FIGURES Introductigg. 1. Representative Polyhalogenated Aromatic Hydrocarbons...6 2. Structure and Nomenclature of Selected Retinoids......17 3. Intestinal Hydrolysis, Absorptione, and Esterification Of DiItarY RetinoidSIIIIIIIIIIIIIIIIIIIIIIIIIII-IIIlq 4. Hepatic Uptake, Storage, and Mobilization of Retinyl EStIrs .nd Retinal.IIIIIIIIIIIIIII-IOIIIIIIIIIIIOIOZI 9mm;- 1. Body weight changes in control and mmtreatw ratSIIIIIIIIIIIIIIIIIIIII.I.I.III-Iiibb 2. Serum retinol levels in control and "mm-trIItId r.tSIIIIIIIIIIIIIIIIIOIIIIIIIIIII-I..69 3. Hepatic retinol levels in control and NWWtr9.tw r.t‘IIIIIIIIII.III-III-IIIIIIIIIIIIIO71 4. Hepatic retinyl palmitate hydrolase activity of control .nd "WM-trIItId rats-IIIIIIIIIIIIIIIIIIIIII-IIIIO7‘ 5. Correlation of serum retinol levels with hepatic RPH activity in control and NDFDA-treated rats.........76 6. Increase in RPH activity in acetone-extracted hepatic homogenates from control and NDFDA-treated rats....78 7. Inhibition of RPH activity by NDFDA and TCDD..........BO B. Effect of a single i.p. dose of corn oil, or corn oil containing 3,4,3‘,4'-tetrachlorobiphenyl, 2,4,5,2',4',5'-hexachlorobiphenyl, or 3,4,5,3‘,4',5'-hexachlorobiphenyl on plasma retinal concentrations in female Sprague-Dawley rats 24 mr. mt-tre.tmtIIIIIIIIIIIIIIIIIIIIIIIIIIIIB4 9. Effect of a single i.p. dose of 3,4,3',4'-tetrachloro- biphenyl on the hepatic RPH activity of female Sprague-Dawley rats 24 hours post-treatment........86 V1 10. Relation of plasma retinol levels and hepatic RPH activity in female Sprague-Dawley rats 24 hours following a single i.p. dose of 3,4,3',4'- tetrachlorobiphenyl.......................... ...... 89 11. Inhibition of RPH activity by 3,4,3',4'-tetrachloro- bipmleIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII91 12. HPLC anlaysis of combined plasma samples from rats treated with 0 or 15 mg/kg 3,4,3',4'-tetrachloro- bipmleIIIIIIIIIIIIIIIIIIIIIIIIII-IIIIIIIIIIIIIII93 13. The effect of a single i.p. dose of TCDD, or a vitamin A deficient (VA(-)) diet on the plasma retinol levels of male Sprague-Dawley rats 12 days following either the TCDD dose or start of the VA(-) diet....97 14. Relation between total plasma retinol levels and retinoid fluorescence of the retinol-RBP-TTR complex of rats 12 days following treatment with a single i.p. dose of TCDD, or placed on a vitamin A deficient diet for 12 days.........................99 Qhagter g, 1. Effect of a single oral dose of TCDD or feeding a vitamin A-deficient diet (VA(-)) on kidney retinyl palmitate levels in male Sprague-Dawley rats 12 days following treatment or start of VA(-) diet...123 2. Effect of a single oral dose of TCDD or feeding a vitamin A—deficient (VA(-)) diet on kidney ACARAT activity in male Sprague-Dawley rats, 12 days following either treatment or start of VA(-) diet.12b 3. Correlation of kidney retinyl palmitate concentration with kidney ACARAT activity in male Sprague-Dawley rats 12 days following a single oral dose of TCDD or fed a vitamin A deficient diet for 12 days.....128 Chgggec 3, 1. The effect of a single oral dose of TCDD on the body weights of male Sprague-Dawley rats...............145 2. Effect of a single oral dose of TCDD or feeding a diet deficient in vitamin A (VA(-)) on the average body weight of male Sprague-Dawley rats, 12 days following TCDD dose or start of VA(-) diet........148 vii 3. Effect of a single oral dose of'TCDD on the cumulative food intake of male Sprague-Dawley rats for a 12 day period following dose............................150 4. Effect of a single oral dose of TCDD, or feeding a vitamin A-deficient diet (VA(-)) to male Sprague- Dawley rats on hepatic retinyl palmitate levels 12 days following dose or start of VA(-) diet.....154 5. The effect of a single oral dose of TCDD or feeding a vitamin A-deficient diet (VA(-)) to male Sprague- Dawley rats on hepatic microsomal retinol degradation 12 days following either TCDD dose or start of VA(-) diet...............................156 b. The effect of a single oral dose of TCDD or feeding a vitamin A-deficient diet (VA(-)) to male Sprague- Dawley rats on hepatic microsomal p-nitrophenol UDP-glucuronosyl transferase activity 12 days following either TCDD dose or start of VA(-) diet.158 7. The effect of a single oral dose of TCDD or feeding a vitamin A-deficient diet (VA(-)) to male Sprague- Dawley rats on hepatic microsomal retinoyl UDP- glucuronosyl transferase activity 12 days following either TCDD dose or start of VA(-) diet...........160 W1- 1. Effect of a single oral dose of TCDD and/or dietary T3 on body weight of male Sprague-Dawley rats........177 2. Effect of a single oral dose of TCDD and/or dietary T3 treatment in male Sprague-Dawley rats on the concentration (A), or the total amount of retinyl palmitate (B).....................................183 3. Effect of TCDD and/or dietary T3 on the renal retinyl palmitate concentration of male Sprague-Dawley r‘tsIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIle viii LIST OF ABBREVIATIONS ACARAT..........Acyl CoAaRetinol Acyl Transferase AHH.............Aryl Hydrocarbon Hydroxylase BNF.............Beta-Napthoflavone C...............Celsius CRABP...........Cellular Retinoic Acid Binding Protein CRBP............Cellular Retinol Binding Protein CoA.............Coenzyme A DTe e e a e e e m e e e e e eDithiothr.it°l Dil.............Daltm3 DPPD. e m e m m m m m e e ongu'“diphIflYl‘p’phIflYlIflI-diuine EDTA............Ethylene Diamine Tetraacetate “I I I I I I I I I I I I I IE.iSSim exI I I I I I I I I I I I I IEXCitati-m FigI IIIIIIIIIIIFigure gIIIIIIIIIIIIIIIBr“ HCB.............Hexachlorobiphenyl HPLC............High Pressure Liquid Chromatography hr..............Hour i.p. ...........Intraperitoneal Kd..............Coefficient of Dissociation KI..............Coefficient of Inhibition Kg..............Kilogram Km..............Michalis-Menton Coefficient LPC.............Lipoprotein Complex LDSOemmmemememmeI—ethal DOSE, (501) M...............Molar MC..............3-Methylcholanthrene Me-NDFDA........Methyl Nonadecafluorodecanoate min.............Minute mg..............Milligram ml..............Millilitre mM..............Millimolar mm..............Millimeter MOPS............3-(N-morpholino)-propane-sulfonic acid ix NDFDA...........Nonadecafluorodecanoic Acid nmol............Nanomole nm..............Nanometer PAH.............Polyhalogenated Aromatic Hydrocarbon PCB.............Polychlorinated Biphenyl pg..............Picogram PGE.............Phosphate-Glycerol-EDTA PNP.............(Para) Nitrophenol r...............Correlation Coefficient RBP.............Retinol Binding Protein RPH.............Retinyl Palmitate Hydrolase SD..............Standard Deviation S.E. ...........Standard Error (of mean value) T3..............Triiodothyronine T4..............Thyroxine TCAB............3,4,3',4'-Tetrachloroazobenzene TCB.............3,4,3',4'—Tetrachlorbiphenyl TCN.............2,3,6,7-Tetrachloronaphthalene TCDD............2,3,7,B-Tetrachlorodibenzo-p—dioxin TCDF............2,3,7,B—Tetrachlorodibenzofuran TTR.............Transthyretin UDP.............Uridine 5' Diphosphate UDPST...........Uridine Diphosphate Blucuronosyl Transferase ug..............Microgram ul..............Microliter uM..............Micromolar VI I I I I I I I I I I I I I IVOlm VAI I I I I I I I I I I I I IVit..in A Vmax............Maximum Velocity INTRODUCTION TCDD (2,3,7,B-tetrachlorodibenzo-p-dioxin, dioxin) is often described as the most toxic man-made compound yet identified. This chemical has been implicated as the toxic species in a number of serious incidents of environmental contamination. TCDD also represents the archetypical species of an ubiquitous group of environmental contaminants known as the polyhalogenated aromatic hydrocarbons (PAH's). Despite the fact that TCDD and the PAH's have been recognized as the most significant environmental contaminants yet identified in the biosphere, there has been little progress made in the determination of the mechanism by which these chemicals are toxic. The toxic symptoms caused by these compounds have been well described and characterized, and the ability of many of the structurally diverse PAH's to cause essentially the same toxic response in treated animals has also been clearly demonstrated. Much of the research reported to date has focused on the ability of TCDD and the PAH's to bind to a specific cytosolic receptor, resulting in activation of a particular enzyme locus. Structure-activity relationships suggest that the ability for a compound to bind to the receptor 2 correlates with its ability to cause TCDD—type toxicity. However, despite intensive investigation, no clear linkage between increased activity of any of the induced enzymes, and a plausible mechanism for toxicity has been suggested. The research presented in this thesis has grown out of the observation that the characteristic signs of TCDD-type toxicity are very similar to those expressed by animals suffering from the advanced stages of a vitamin A deficiency. Further, it has been demonstrated that TCDD and similarly-acting compounds cause a depletion of vitamin A levels in treated animals, thereby supporting the hypothesis that some aspects of TCDD-type toxicity are caused by a vitamin A deficiency. The mechanism by which this depletion of vitamin A may be caused by TCDD has become the central focus of this research. This topic, while seemingly straightforward, has nevertheless proven difficult to evaluate in terms of vitamin A metabolism, and the effects of TCDD on that metabolism. Indeed, one bit of common ground that exists between research on TCDD and on vitamin A is that, despite years of in-depth study, there has been no clear mechanism of action elucidated for either chemical, with the sole exception of the role of retinol/retinal in the visual cycle. Therefore, research on the mechanism by which TCDD affects vitamin A metabolism becomes, to an extent, an investigation of vitamin A metabolism. In attempting to further define the problem, it became 3 readily apparent that the effects of TCDD and similarly acting compounds on hepatic vitamin A loss could be thought of in terms of only a few possibilities. Specifically, these compounds could be (a) affecting the uptake of dietary vitamin A, (b) increasing the rate of normal mobilization of vitamin A from the liver, (c) increasing the rate of utilization of vitamin A by target tissues, (d) enhancing the rate of normal degradative pathways of retinoids in the liver, or (e) enhancing or initiating a new, as yet unidentified pathway of degradation of retinoids in the liver. Naturally, the possibility also exists that some combination of any or all of these effects could occur. In Chapter 1, experiments describing the effect of TCDD and TCDD-like toxins on the mobilization of vitamin A from the liver are described. The research presented in this chapter demonstrates the effect of TCDD and similarly acting compounds on serum retinol levels and the activity of hepatic retinyl palmitate hydrolase. In addition, the effect of TCDD on the ternary protein complex responsible for the transport of retinol in serum is examined. One biochemical consequence of both TCDD toxicity and a deficiency of vitamin A is an accumulation of retinoid material in the kidney. In Chapter 2, research is described suggesting that the mechanism of this phenomenon in both cases may be the elevation of the activity of an acyl-CoA transferase enzyme. Further, the dose-response and time course for both TCDD- and vitamin A deficiency-induced 4 renal retinoid accumulation is presented. The results from experiments on the effects of TCDD treatment of rats on the hepatic storage, oxidative degradation, and conjugation of retinoids are presented in chapter 3. Specifically, the effects of TCDD on the storage of retinyl esters, the degradation of retinol, (oxidation to retinoic acid), and microsomal oxidation of retinol and retinoic acid are presented in this chapter. Further, effects of on the conjugation of retinoic acid or retinol with glucuronic acid are presented and discussed. TCDD and similar compounds have been shown to affect the thyroid hormone status of treated animals, and an inverse correlation between thyroid hormone levels and vitamin A storage levels has been reported. In Chapter 4, experiments on the effects of dietary T3 on both the TCDD- induced hepatic depletion of retinyl esters and the accumulation of retinoids in the kidney are presented. Each chapter is written in a format similar to that of many scientific journals, and contains its own Abstract, Introduction, Materials and Methods, Discussion, and List of References. Since this subject involves questions of basic vitamin A metabolism, as well as TCDD toxicity, a literature review of each of these topics, containing its own List of References, precedes Chapter 1. A summary of the research is presented as Chapter 5, as well as a discussion of possible directions for future research. LITERATURE REVIEW TCDD and PAH's; Structure gQg_Toxicologicgl_Significance 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is often described as the most toxic man-made compound yet identified. A more significant fact however, is that the toxic response caused by this compound is also characteristic of the toxicity caused by the “Polyhalogenated Aromatic Hydrocarbons“ (PAH's; Poland and Knutson, 1982). This group of compounds, containing TCDD, is comprised of many structurally diverse chemicals, including such species as the polyhalogenated-dibenzo-p-dioxins, —di- benzofurans, -biphenyls, -naphthalenes, and -azo-, -azoxy-, and -hydrazo-benzenes (Figure 1; Soldstein, 1980). Nearly all the PAH's are man—made, having been produced either intentionally for use in a variety of applications, (i.e., coolant fluids, fire retardants, chemical feedstocks) or as contaminants formed during the synthesis of other chemical species, (i.e., the formation of trace amounts of polychlorinated-dibenzofurans and -dioxins during the synthesis of trichlorophenol (Brinkman and De Kok, 1980; Rappe and Busler, 1980)). The PAH's have become increasingly recognized as significant and widespread environmental contaminants, primarily because of two factors. First, specific PAH's have been implicated as the causative agent in several incidents of localized contamination (i.e., Sevaso, Italy; Missouri, U.S.A.; Yusho, Japan) resulting in acute and/or FIGURE 1. REPRESENTATIVE POLYHALOGENATED AROHATIC HYDROCARBONS CI (3 C1 2.3.7.8-TETRACHL0R0018EN20- DD) P-DIOXIN (TC Cl mo 00 CI 3.4.3'.4'-TETRAcHLoao- BIPHENYL (TCB) Cl Cl (”“0 2.3.6.7-TETRACHLOR0- NAPHTHALENE (TCN) Cl C) Cl 2.3.7.8-TETRACHL0R0018EN20- FURAN (TCDF) Cl Cl CI Cl 3.4.5.3'.4'.5'-HEXACHL0R0- BIPHENYL (HCB) w Cl N CI 3.4.3'.4'-TETRACHL0R0Azo- BENZENE (TCAB) 7 chronic symptoms of toxicity among a significant population (Kuratsune, 1980; Reggiani, 1980). Secondly, there have been significant advances made in the analytical technology and methods used for the identification and quantification of this type of compound at trace levels, and as components of complex mixtures (Rappe and Busler, 1980). Because of the increased analytical capability, many PAH's have now been identified both as ubiquitous environmental contaminants, and also as fairly routine contaminants of many human populations (Landrigan, 1980). Despite both the increased attention, and intense research interest, the mechanism by which these compounds elicit their characteristic toxic response has eluded clear identification. Much of the research on the mechanism of PAH action has focused on the well-defined ability of these compounds to bind to a specific intracellular receptor (Dencker, 1985). This receptor shows a high affinity for TCDD (Kd = 0.27 x 10-9M) that is relatively constant in a number of species (Poland, 1984). No endogenous ligand has yet been identified for this receptor, and it has therefore become commonly referred to as the “TCDD Receptor.“ A model for TCDD toxicity proposed by Poland, gt_ gl., (1979) suggests that the consequence of TCDD binding to the receptor is the translocation of the receptor-TCDD complex into the nucleus, binding to the chromatin material, and resulting in the activation of at least one structural gene. 8 Presumably then, the products of this gene activation cause the altered physiology referred to as TCDD-type toxicity. Significantly, this model for toxicity provides a mechanism whereby a group of structurally distinct compounds can cause essentially the same toxic response, and suggests that the basis for differences in toxicity would be the affinity the receptor has for a particular ligand. Poland‘s model also suggests that since TCDD is the most potent member of the PAH's, it should be the best ligand for the receptor. Therefore, the closer a molecule is to the overall size, shape, and electrostatic configuration of TCDD, the better ligand for the receptor, and the more potent toxin it should be. This is supported by structure- activity data suggesting that the toxic PAH's are laterally halogenated, and either exist in a coplanar configuration, or are not energetically restricted from approaching coplanarity, thereby mimicking the laterally chlorinated, coplanar, TCDD. Hence, 3,4,5,3',4',5'-hexachlorobiphenyl (HCB) is a potent TCDD-type toxin, while the more sterically hindered 2,4,5,2',4‘,5'-HCB isomer is not (Nagayama, gt_g;., 1983; Safe gt_gl,, 1981). The model is supported by experimental evidence showing the translocation of the TCDD-receptor complex to the nucleus both ;Q_xixg (Okey gt_ 1., 1979) and in cultured cells (Okey gt g;,, 1980). Further, it has been shown that TCDD-type toxicity results in the activation of at least one genomic locus leading to the synthesis of a group of 9 enzymes, including aryl hydrocarbon hydroxylase (AHH; Poland and Glover, 1973), several UDP-glucuronsyl transferases (Owens, 1977), DT diaphorase (Beatty and Neal, 1976), ornithine decarboxylase (Nebert, §£.éL-’ 1980), and aldehyde dehydrogenase (Dietrich g;_§l,, (1978). The locus responsible for the production of the cytosolic receptor is referred to as the ”Ah locus.” TCDD sensitivity has been shown to be associated with both the Ah locus, and the ability to induce AHH activity (Poland and Knutson, 1982). Further, the ability of TCDD to cause such signs as thymic atrophy, teratogenicity and mortality in treated animals, also appear to be associated with the presence of the Ah locus (Poland and Glover, 1980; Denker, 1985). The ability for the cytosolic receptor to bind several dibenzo-p-dioxin congeners correlates with AHH induction (Poland, gt al., 1976). Also, there is a strong correlation between the toxicity of specific PAH's and their ability to induce AHH activity (Goldstein, 1980). The primary enzymatic activity linked to the locus controlled by the Ah receptor is the cytochrome P-450- dependent AHH. 3-Methylcholanthrene (MC) has been suggested as a prototypical compound for the AHH induction (Nebert, gt al., 1972): 10 @gb 2‘2‘2 3—Methylcholanthrene 2,3,7,8-Tetrachlorodibenzo— p-dioxin TCDD is a significantly more potent inducer of AHH activity than is MC, although experiments based on simultaneous administration of the two compounds suggest that they act by exactly the same mechanism (Poland and Glover, 1974). Hhile Polands's model provides logical explanations for many of the phenomena associated with TCDD—type toxicity, there are several problems with the proposal. It has been .noted for example, that the dose of TCDD required to give maximal induction of the AHH enzyme activity is substantially lower than that required to cause many of the other aspects of toxicity (Golstein and Hardwick, 1984). This suggests that the products of gene locus activation by the Ab receptor—TCDD complex are not the enzymes or proteins leading directly to the toxic response. Also, the relatively slight change in receptor concentration between species does not at all correlate with the large magnitude in difference of the L050. Specifically, while the oral TCDD-L050 for the (Hartley) guinea pig and (Syrian Golden) hamster are ”1.5 ug/kg (Schwetz et al., 1973) and ”1200 11 ug/kg (Olson, _LH_L., 1980), respectively, there was no significant difference found between the levels of TCDD specifically bound in the liver cytosol from the two species. Perhaps the most serious problem is that one of the more sensitive animals to TCDD toxicity is the guinea pig, yet TCDD does not appear to induce AHH activity in this animal (Goldstein and Hardwick, 1984). Finally, many of the features of TCDD toxicity, including the wasting syndrome and lethality can be caused by a perfluorinated alkanoic acid which shows no affinity for the Ah receptor, and does not induce AHH activity (Olson g;_gl,, 1982). These objections are addressed, with the exception of the last, by the suggestion that TCDD induces a pleiotropic response of which AHH-type induction is only one identified consequence, and that the toxic signs are consequences of protein synthesis not necessarily linked with the activity of AHH or coordinately induced enzymes (Goldstein and Hardwick, 1984). Further, it is suggested that the presence of a functional receptor may not necessarily evoke the pleiotropic response. Clearly, changes in structure that affect either the affinity of the receptor for the ligand, or of the receptor-ligand complex for the nuclear binding site would be expected to have a pronounced effect on the expression of the pleiotropic response. 12 Effects QI_TCDD Toxicity TCDD-type toxicity results in a plethora of pathological and biochemical changes in treated animals which may, in the case of sufficient dose, be lethal. There are pronounced differences in the toxic lesions observed between species in the response to TCDD toxicity, i.e., pericardial edema in chickens, chloracne and acneform eruption in primates, and "X-disease” (a skin hardening) in cattle. However, there remains a group of symptoms that appear to be common to all affected animals, which has become generally referred to as the “wasting syndrome.“ The consequence of this condition is a period of pronounced reduced weight gain, or in the case of near-lethal or lethal dose, weight loss, continuing until the death of the animal. Height loss prior to mortality may be as much as 501 of the body weight at the time of dose (Poland and Knutson, 1982). Results of experiments with animals pair-fed to dioxin treated animals suggest that the weight loss caused by the PAH's is more than simply a function of reduced food intake (Harris, gt_gl, 1973; Allen g__g;,, 1977; Gasiewicz, gt al., 1980; Ball and Chhabra, 1981). However, more recent work suggests that the early experiments were incorrectly interpreted, and that the reduced food intake is an adequate explanation for the TCDD-induced weight loss (Seefeld and Peterson, 1984; Seefeld g;_gl,, 1984; Kelling gt al., 1985). In either case, it is clear that the PAH-induced hypophagia places the animal under severe nutritional stress. 13 Peterson gt_gl,, have suggested (1984) that TCDD acts as an anorectic agent (Stunkard, 1982) and thereby causes treated animals to alter their basic body weight ”set point.“ The set point may be thought of as the body weight the animal tends to maintain through a balancing of energy intake and expenditure (Seefeld, gt 91,, 1984). Therefore, in the case of lethally treated animals, the caloric intake appropriate to the new set point is nutritionally insufficient to keep the animal alive. An objection to this proposal is that replacing the caloric deficit resulting from hypophagia does not protect the animals from all the the toxic effects of TCDD. Gasiewicz _t, al. (1980) demonstrated that rats treated with TCDD and given total parenteral nutrition still died as a consequence of treatment (Gasiewicz, gt_g;., 1980). It should be noted however, that the cause of death has been suggested to be a consequence of the effects of overnutrition (Peterson gt al., 1984). In terms of identifiable pathologic changes caused in organs, or organ systems, TCDD toxicity characteristicly results in hepatic enlargement, lymphoid involution and immunosuppression, and morphological alteration of epithelial tissues in all affected species (Poland and Knutson, 1982). The hepatic enlargement caused by TCDD appears to be a function primarily of smooth endoplasmic reticulum proliferation, resulting in both hyperplasia and 14 1., 1978). The hypertrophy (Fowler, gt a1, 1973; Hinton, gt_ hepatomegaly occurs even at TCDD doses well below lethal, and is seen in all affected species (McConnell, 1980). The endoplasmic reticulum proliferation is correlated with cytochrome P-450 induction. This phenomenon has not been clearly linked to a toxic mechanism, although Stohs g;_ al. (1984) have proposed that a consequence of P-450 induction may be elevated rates of lipid peroxidative damage. Hhile 13 this area is being actively researched, and data suggest that, to some degree, TCDD does induce elevated levels of hepatic lipid peroxidation, the physiological significance of the phenomenon remains to be established. Some degree of hepatotoxicity may also be expressed, although not necessarily in all affected species (Thunberg, 1983). In chickens, rabbits, and to a small degree, certain mouse strains, TCDD causes a pronounced hepatic necrosis of lethal severity. Such a lesion is not observed in rats, guinea pigs, cattle, or non-human primates (McConnell, 1984). The PAH-induced lymphoid involution as observed in the thymus was first described by Buu-Hoi, gt_gl. (1972), and has been characterized primarily as a loss or depletion of cortical lymphocytes, with some lymphocytic necrosis depending on species (Vos, gt_g1,, 1973, Gupta g;_g;,, 1973, McConnell, gt_gl., 1978). This sign of toxicity is observed in all affected species. There is also an associated depression of immune function, which is particulary notable in young animals (Vos, gt_gl,, 1980; McConnell, 1980). 15 TCDD-induced lymphoid involution segregates with the Ah locus, and is suggested to be mediated by the Ah receptor (Poland and Glover, 1980). Recent work by Greenlee gt_ al,, (1985) suggests that in addition to the effects described above, TCDD exerts a direct effect on thymic epithelium, and a consequence of this effect may be an altered maturation of thymus-dependent T-lymphocyte precursors. Other lymphoid tissue may also be reduced in size in treated animals, but the most pronounced changes are observed in the thymus (McConnell and Moore, 1979). Many of the effects of TCDD intoxication are reflected by changes in epithelially-derived tissues, and are generally either hyperplastic/metaplastic or hypoplastic, and have been extensively reviewed (Poland and Knutson, 1982; McConnell t al., 1978; McConnell, 1980). The most prominent lesion of TCDD-type toxicity observable in man is chloracne (Taylor, 1974), which may be associated with hyperkeratosis of the dermis, a function of transformation of sebaceous epithelium to squamous tissue (Gunlife and Cotterill, 1975). This condition was first linked to TCDD in 1957 (Kimmig and Schultz, 1957), and similar hyperkeratotic acneform eruptions have been observed in rabbits (Jones and Krizek, 1962), and monkeys (Allen, g; al., 1977; (Poland and Knutson, 1982). 16 Vitamin Q;_Structure gflg_Physiological_Significance ”Vitamin A" is a catch-all term used to designate the particular nutrient activity exhibited by a group of structurally related compounds: the “retinoids.“ Vitamin A was first isolated and described as "fat soluble factor A" by McCollum and Davis (1915) and has been recognized as a discrete and essential nutrient since that time. Various forms of vitamin A have long been incorporated in folk remedies and records exist describing the use of preparations rich in the vitamin for the treatment of specific disease states in cultures as early as the Egyptian (Holf, 1980). The history of the use and recognition of vitamin A, as well as the early biochemical research, has been extensively reviewed by Moore (1957). The free alcohol form of the vitamin, retinol (Figure 2) is able to support all vitamin A dependent processes in the body, and is the basic retinoid to which the activity of other vitamin A active species are compared. Vitamin A activity may be demonstrated, in whole or in part, by a number of different compounds, all of which may be thought of as derivatives or oxidation products of retinol. As researchers discovered or designed other retinoid species with the ability to exhibit aspects of vitamin A activity, confusion over both structure and nomenclature led to the adoption of a standard nomenclature for the retinoids, selected examples of which are shown in Figure 2. Despite early discovery and extensive research, the 17 FIGURE 2., STRUCTURE AND NOMENCLATURE or SELECTED RETINOIDS (ALL-IRANS [SOMERs SHowN) BETA-CAROTENE E W/k [CH 0" . WCHpCdfl'J" CH' RETINOL RETINYL PALHITATE :5 : /L A ,L ,cHo WWW RETINAL RETINOIC ACID 0 L. HOOC 0 OH HO OH RETINOYL-BETA-GLUOURONIDE 18 exact biochemical roles for the various vitamin A species have not yet been elucidated, with the notable exception of the function of retinal/retinal in the visual cycle, as detailed by Hald, (1960). It is clear however, that the vitamin is required to maintain growth, and more specifically, that it functions in the processes of epithelial cell differentiation, and facilitates the maintenance of epithelial structures (Zile and Cullum, 1983). One other major physiological function involving vitamin A is reproduction, where a sufficient quantity of vitamin A is essential for the maintenance of both spermatogenesis and oogenesis, as well as for structural growth of both the placenta and embryo (Thompson, g;_ al,, 1964). Retinoic acid and other C-15 carboxyl-retinoid species are able to maintain the epithelial growth and differentiation functions dependent on vitamin A, while only retinol or retinyl esters appear to be able to support the reproductive function (Zile and Cullum, 1983). Interestingly, there is a well defined toxicity associated with excessive intake of vitamin A. However, because uptake of the vitamin is at least partially modulated by the nutritional status of the individual, this is an extremely rare phenomenon (Underwood, 1984). Further, the toxicity seems to be a function of the membrane- disrupting effects of excessive retinyl esters, rather than the consequence of excessive retinal-dependent metabolic processes (Smith and Goodman, 1976). Figure 3. Intestinal Hydrolysis, Absorption and Esterification of Dietary Retinoids Dietnrx,flgtinnida_ Retinyl Palmitate Hm Retinol Intestinal Lumen \ Intestinal Hucosal Cell B-Carotene Retinol B:annlfinfi.l§ilhiz. Palmitozl ' _C_Oé. CLAW (I Chylon'icron ‘v We l i Chylomicron, containing retinyl esters in lymphatic circulation. 20 Intestinal Ugtake ggg_Transgort tg_Liver 91_Vitamin B. Vitamin A is not synthesized by animals, rather, it is gained directly or indirectly from plants, in the form of carotenes, (primarily beta). In the intestine, beta—carotene is absorbed into the intestinal mucosa and cleaved by the action of a 15-15' dioxygenase, yielding two molecules of retinal (Figure 3; Goodman gt_gt., 1967). Retinal thus formed is reduced by the action of a non-specific aldehyde oxidase to retinol (Fidge and Goodman, 1968). Another major source of retinoid material for carnivorous or omnivorous animals is pre-formed retinyl esters. These compounds are broken down in the intestine into retinol and free fatty acids by hydrolases secreted by the intestinal mucosal cells. Retinol thus formed is absorbed by the intestinal mucosal cells (Goodman and Blaner, 1984). Retinyl esters are not absorbed to any significant extent from normal physiological doses of vitamin A (Mahadevan gt, 1., 1963). The process of hydrolysis/esterification is thought to proceed several times before the retinal is eventually absorbed (Lawrence gt_gt,, 1966) and both hydrolytic and ester synthase activities have been identified in the intestinal lumen (Mahadevan gt_gt., 1961). Hegatic ggtake, Storage and flpbilizgtion gj_Vitamin fl_ Retinol in the intestinal mucosal cells is esterified with fatty acid residues, (primarily palmitate, or stearate with small amounts of oleate and linoleate by the action of 21 zflhum>1hmz5: <8 45:25 22 055$ 625m "<8 #2 2555 + wzcoza Jozzuméam 625m A ) uzcfifl #mmflwum uezakum 5% we? \ a“ m dzEm me2Hkmm 3.288»: HESS; #2:me £885 “Ecfifljz 48% Gm 3% gang: {531% 80d ._oz:.u~_ Lo zo:.981 Me-NDFDA, by total ion current. Fractional thtribution gt_Retinol ;Q,Plasma Plasma proteins were separated on the basis of molecular weight on an HPLC using a Spherogel TSK 63000 SW column (Beckman, Berkeley, CA) and a mobile phase of 1.152 potassium chloride, 0.04% sodium azide. Aliquots of plasma (100 ul) from each rat per treatment group were combined and mixed with an equal volume of mobile phase. Aliquots of 20 ul of the combined diluted plasma were injected into the HPLC and column effluent was monitored for absorbance at 280 nm with a Schoeffel SF770 UV/visible detector and for 63 fluoresence (Rex = 330 nm, Aem = 470 nm) with a Shimadzu RF-530-S detector, connected in series. The peak corresponding to the retinol-RBP-TTR complex was identified by retinoid fluoresence and estimated molecular weight. The chromatographic system was standardized by the use of molecular weight marker proteins analysed on separate chromatographic runs. Effect 91, Microsomal Incubgtion gg,tflg,Ability 91, IQB, tg Inhibit BEH_LQ_Vitro Microsomes were prepared from the livers of rats treated for 3 days at 40 (mg/kg)/day, oral, with the methyl cholanthrene (MC)-type inducer B-naphthoflavone (BNF), as previously described (Millis gt_ gt., 1985). Reaction mixtures consisted of 40 ul of resuspended microsomes (25 mg protein/ml), 1 ml PGE buffer (10 mM phosphate, pH 7.4, 20% glycerol and 0.1mM EDTA), TCB (100 uM), and/or 100 ul of an NADPH-generating system, and were incubated for 1 hr at 370 C in a shaking water bath, conditions previously shown to produce metabolic degradation of TCB (Millis _t gt., 1985). Following this incubation, the mixture was evaluated for endogenous RPH activity by the addition of 100 ul 20% sodium cholate and 20 ul 10 nmol/ul retinyl palmitate and incubation in the dark at 370 C for 20 min in a shaking water bath. The reaction was terminated, retinol extracted, and the amount of retinyl palmitate hydrolysis determined as described above for RPH activity analyses. 64 Statistical Analysis Statistical analyses were performed using Student's t- test with the Bonferroni correction for multiple comparisons (Godfrey, 1985). RESULTS Exgeriment t,(NDFDA) ngy,ggg_0rgan Heights A rapid and pronounced dose-related effect was observed on the body weight of the NDFDA-treated animals, with the high dose group showing a progressive decline in weight (Figure 1). However, while the low dose group initially lost weight, they seemed to recover to a near normal growth rate from day 5 through day 11. An increase in the liver wt./body wt. ratio and lymphoid involution, as measured by a decline in the thymus wt./body wt. ratio, were observed in treated animals, as shown in Table 1. Serum ggg,HeQatic Retinol A significant effect of NDFDA treatment on serum retinol levels was observed (Figure 2). 8y two days post- dose, the average serum retinol concentration in the high- dose treated group was decreased to only 181 that of the control animals. Serum retinol concentration in the low dose treated group was similarly depressed to 392 of the control level. Hhile the low-dose treated animals showed some recovery by day 11 (to 78% of control values), the high dose group was unable to significantly increase serum retinol levels. Hepatic retinol levels did not correlate with serum values, and were only significantly depressed relative to control animal values at day 11, as shown in Figure 3. 65 66 Figure 1. Body-weight changes in control and NDFDA-treated rats. Values are means for groups of n = 3 rats treated with 0 (—Q—), 50 (—A—), or 100 (—|:]—) mg/kg. AVERACE BODY HEIGHT (C) 67 I j 1 I I l I 3N961&f o . A . A . A 0 A zoob .. ,- I I a ll IC7C)- L -6 -3 0 2 5 8 DAYS FOLLowlNC NDFDA TREATMENT 11 .flcuc v Q “OOSHNP HOHHBOO NORM HGOHOMMMU hHHEflOMHMflUfim "I um.oflm.o ab.oHN.n N.ofim.N IIII .ul hoom\.uu 05-559 shHmo hflom deem IIII .uB hoom\.ul unbwa ug- ad N.QHN.N H.QHV.N m.oflm.m IIII .HB hvom\.uu solace enema steam «How IIII .ak hoom\.ul Rehwm "as. d... m.oHN.N m.ofin.N N.QHQ.N m.oflb.m .aB hoom\.a3 uslhch «Hoe neon Memo nHNn .uB hoom\.ul nebmg "Adam—ad an m N o macho gages .A OHNV DH.“ FOHGUOIOQIUHQW can: :« oceans geese» Room ceases can agenda: to «one: no coon .n.A oAucmm a no access one .A oAaaa 69 Figure 2. Serum retinol levels in control and NDFDA-treated rats. Values are means :_S.E. for groups of n = 3 rats treated with 0 (-{)—), 50 (-1§-), or 100 (-th-) mg/kg. Significantly different from control value: a = p (0.025, b = p <0.010, c = p (0.005. 70 AzuzpA_>_ho< Ida o_pz:.m~:\H .m\H v.0 N6 ‘6 0.0 0 $0: 1 _ u a w H .A. 00.0 m m “NUT. 82 competitive inhibitors, with a KI = 450 UN for NDFDA and 750 uM for TCDD. Hegatic NDFDA Levels Hepatic NDFDA concentration was inversely correlated with serum retinol levels (Day 2, r = -0.66; Day 8, r = -0.93; Day 11, r = -0.92) and liver RPH activity (Day 2, r = -0.64; Day 8, r = -0.58; Day 11, r = -0.85). NDFDA levels in the treated animals were found to decline throughout the study, as shown in Table 2. Exgeriment g,(TCB and g_HCB isomers): PlasmaI Hegatic, gQg_Renal Retinoid Levels A single i.p. dose of TCB caused, at the two highest dose rates, at 24 hours, a depression in plasma retinol levels that was dose-dependent and significantly different from control rat levels, as shown in Figure 8. Neither the 2,4,5,2',4‘,5',- nor 3,4,5,3',4‘,5'-HCB congeners caused a similar depression of plasma retinol levels. No significant differences were noted in hepatic or renal retinol, or renal retinyl palmitate levels in any of the groups treated with 3,4,3',4'-tetrachlorobiphenyl when compared to control group values (data not shown). Hgggtic RPH Activity The TCB dose caused a significant and dose-dependent depression of hepatic RPH activity of all treated groups when compared to control group animals, as shown in Figure 9. Further, the hepatic RPH activity was positively 83 .am.H .m n c no museum Hon damn! 2mm Geo: . onflonn OQHONH onHOBN I UN\U: can .dnhal memo omflomm oNHQNH I mu\u: on .domoz no: no: no: no: aowucoo nacho mm m N o acclauowh 4%.. .audm hoHBdoIDSUswam can: cm 0009 .m.« Duncan 0 newBOHuom un~o>oq some: canons: .N Ounce 84 Figure 8. Effect of a single i.p. dose of corn oil ([:::b. or corn oil containing 3,4,3‘,4'-tetrachlorobiphenyl ([flflflb, 2,4,5,2',4‘,5'-hexachlorobiphenyl (m) or 3,4,5,3',4',5'— hexachlorobiphenyl ( gfifii) on plasma retinol concentrations in female Sprague-Dawley rats 24 hours post-treatment. Each bar represents the mean : SD of three rats and asterisks denote significant difference from controls (p < 0.05). Numbers in parentheses at the top of each bar represent the percentage of control value. Plasma Retinal (ug/ml) 85 0./ I- ([00) [.0 5.0 I50 PCB DOSE (mg/kg) 86 Figure 9. Effect of a single i.p. dose of 3,4,3',4‘- tetrachlorobiphenyl on the hepatic RPH activity of female Sprague-Dawley rats 24 hours post-treatment. Each bar represents the mean :_SD of 3 rats and asterisks denote significant difference from control animals (p < 0.05). Numbers indicated at the top of each bar represent percentage of control value. RPH Activity (pmol/min-mg) 87 80 I- ([00) 60 - 40 . (5% TI“ 20 - (I24 (5) 0 I'I‘Ir-t. 0 [.0 [5.0 TC B DOSE (mg/kg) 88 correlated (r = 0.90) with plasma retinol, as shown in Figure 10. This relationship could be considered as a linear (plasma retinol = 0.164 + 3.69 x 10—3 (RPH Activity); SD of fit = 3.56 x 10—2) or a second degree polynomial function (plasma retinol = 0.133 + 8.81 x 10‘3 (RPH Activity) + 9.08 x 10‘5 (RPH Activity); SD of fit = 7.47 x 10‘3). Lg,Vitro Inhibition gt,flEfl_Qy_IQ§_ TCB significantly inhibited the activity of retinyl palmitate hydrolase when included in an RPH assay incubation at 100 uM, as shown in Figure 11. The Km for retinol (8.6 uM), determined by analysis of a double-reciprocal plot of the substrate-velocity data, appeared to be unaffected by TCB. However, the apparent Vmax (17 pmol/min-mg) was significantly lower than that for control incubations (41 pmol/min-mg), indicating that the inhibition was non- competitive with a KI of 91 uM. Fractional Qigtribution gt_Retinol Among Plasma Proteins The analysis of combined plasma samples from the different treatment groups by HPLC yielded a fluorescent peak corresponding to the ternary complex of retinal-RBP- TTR. It appeared that the difference in retinol levels between the control and treated groups could be accounted for entirely by changes in the retinol-RBP-TTR peak (Figure 12). No peaks corresponding to free retinal-RBP were observed. 89 Figure 10. Relation of plasma retinol levels and hepatic RPH activity in female Sprague-Dawley rats 24 hours following a single i.p. dose of 3,4,3',4'-tetrachlorobiphenyl. Each point represents the mean :,SD of 3 rats for each parameter. 90 AoE.c_E\ .083 33:04. InE ow 0m. by OM. ON 9 o . q a _ a . 32.. 2 . £6 I 0N6 I nNG .. Onto (lLu/bn) louuaa owsold 90 BE.c_E\ BEE 33:04. mom 0m 0% OM ON 0\ £30 _ — _ . a as... e 338 A... 33.5 9 .0 ON .0 wwd Onto (lw/bn) louglaa DwSDId 91 Figure 11. Inhibition of RPH activity by 3,4,3',4'- tetrachlorobiphenyl. Values are means 1, SD of 1/RPH activity (1/pmol minTlmg-l) versus 1/retinyl palmitate concentration (1/[retinyl palmitate], uM) for three replicate RPH assays, as described in "Methods." Insert values are the corresponding means 1_SD of RPH activity (pmol/min mg) versus retinyl palmitate concentration (uM retinyl palmitate) for three replicate assays. 92 8 pmol/min-mg B 6 Control +|25 uM TCB I L I I0, 20 , 40 pM retinyl palmllale +|25uM TCB _ Control 1 l L - 0.! 0 0.! 0.2 1/[RETINYL PALMITATE]. UM 1/PMoL MIN-lMG-I; x102 93 Figure 12. HPLC analysis of combined plasma samples from rats treated with O (A) or 15 mg/kg (8) 3,4,3',4'- tetrachlorobiphenyl. Samples were 10 ul aliquots of combined plasma diluted 1:1 with mobile phase, as described in ”Methods.” Column effluent was monitored for absorbance at 280 nm (—“—‘—) and fluorescence at 330 nm excitation, 470 nm emission (""'"-'"). 3c; 25s 8:53.. 94 Fluorescence (330+ 470 nm) cee-dmm 12:... 12.2% (mu 0 92) oouoqmsqv 95 Effect 91_ Incubation 91. TCB with Microsomes 9Q. the Inhibition QI_RPH iQ_vitro Inhibition of endogenous microsomal RPH activity by TCB was significantly reduced by an initial incubation of the microsome-TCB mixture with an NADPH-generating system, as shown in Table 3. Experiment §,_§TCDD] Plasma Retinol Levels A single dose of TCDD in male Sprague-Dawley rats caused a dose-dependent elevation of plasma retinol levels through the 100 nmol/kg dose level 12 days after treatment, as shown in Figure 13. Animals treated with 300 nmol/kg did not have an elevation of the plasma retinol level, and had actually a lower average level than did controls, although the difference was not statistically significant. Qigtribution Qj_Retinol Among Plasma Proteins. The retinoid fluorescence of the ternary complex of combined plasma samples from both control and treated groups were strongly and positively correlated (r = .978; Figure 14). There was no evidence of retinoid fluorescence in the region of the HPLC chromatogram corresponding to the retinol-RBP complex. 96 .mm_aemm moe .Ima_ucmoouccwom . :.mUo:umz: “mm mm .AXONV mum_oco Esmoom _: OOH «0 couuaoom mcu wca30._0m xmmmm ucmscmmnsm a :« nmgnmmme mm: xufi>fiaom Imm .cuon go A2: code mop .ememxm acmumemcmm Imowuo< lam + xv mcouuwucoo ~09ucoo x wEu:«E cofiumnzoc_ _oea xyw>wyo< mmm_oeuxx mumufie_mm .xcwpmm .mxHH>fiu0< mmm_OQU>I medufie.mm .xcwumm msocmmoocm aun«£c_ >_ucmsvmmn3m 0» _>cm£aunouo_5009umel.¢..m.¢.m uo sum—mn< mcu co ImQ_ucmuM+.cmfim "a .muML m *0 mQJOLu L0+ Qm.H mcmmE mLm mmzqm> um om H 5.0“ HM n.0H aw au “.0“ m.ou .oe\cxe\~05a ~.n n 0.0 n a m.¢ m.m "rem dmcmc m.ou n.QH ¢.QH N.QH N.QH ~.QH no.0“ am.“ .tee m.o an.« n¢.q na.~ «.0 m.o 00.0 “mocwumm Awe—mm umwo oom cod on o. o.q AI.¢> .o o .mx\~05c. mmoo nook m.ummn .n.¢> +0 utmum Lo .wmoo mcwzo__ou m>ma Nd .mumm >m~3mnlmaomLom mum: CM m~w>md >uw>wuuc mmmMOLU>I mumuflE~Ml a>cwumm Dcm m~m>m4 _OcHumm amcmm c0 uwwn AAIV¢>V ucmwuw+mo ¢ Cwemuw> m mewvmmu Lo name +0 mmoo muocAm m +0 uumwem 05% .H mmomh 122 .mo.o v a .a30Lo umfiu .+V ¢> sate acmtm++fiu >-mufiummumum u m N ¢.QH m.ou N.oH mu no.0“ o m.o m.~ a.« me mm.o .+. ¢> ¢H nu ¢H mo.QH mu . H m an mmm mm~.~ mmq mwm w .1. «> Ana“. .omu. _I I .me\cwe .ae\cwe .om+:em. .l .nm+zem. u \aoem. \_05n. mumuwe_mm .om+zmm. mucuwexmm .nm+:amo acmacou :mm h¢m¢u¢ ~>cfiumm mocfiumm ~>cwumm Hocwumm ¢ cwemuw> >MCUH¥ Lm>w4 memo~m >Lmamwa .wmmethMLh ~>u¢ unawummu¢ou ~>U¢ Dcm mumuwemmm ~>cwumm ucm aocwumm nmcmm .mumqu—mm ~>cwumm Lm>w4 .uocwumm mammal c0 mumm >wmxmolmdmmLom mam: Du m>mu 0N LOe amen ucmwuwvmn ¢ cwfiauw> LO _OLHCOU m Dcwnmmu *0 Humsvw 03% .N m~00h 123 Figure 1. Effect of a single oral dose of TCDD or feeding a vitamin A-deficient diet (VA(-)) on kidney retinyl palmitate levels in male Sprague-Dawley rats 12 days following treatment or start of VA(-) diet. Values are means :_SD for groups of n --- 3. (-—O—): TCDD treated, ( —O— ): VA(-) diet. 124 J 1(1) 3(1) TCDD dose, nmol/kg body weight T- l. I 30 r 10 1.0 0.1 (L 1}! l I l l as o to o to d- + n n N (WC-Id) ‘emiwlod Munea Keupm 104 5 0 I I!) F 204 125 Renal RPH Activity No effect of the single dose of TCDD on renal RPH activity was observed (Table 1). Neither was there an effect caused by a 26 days of feeding a diet deficient in vitamin A, as shown in Table 2. Renal ACARAT Activity Renal ACARAT Activity in the TCDD treated animals was elevated with increasing dose, as shown in Figure 2. Further, this enzyme activity was strongly and positively correlated with kidney retinyl palmitate, r = .93, as shown in Fig. 3. The renal ACARAT activity was also elevated in in rats fed a vitamin A deficient diet for 26 days, when compared to animals fed a control diet, as shown in Table 2. 126 Figure 2. Effect of a single oral dose of TCDD or feeding a vitamin A-deficient (VA(-)) diet on kidney ACARAT activity in male Sprague-Dawley rats, 12 days following either treatment or start of VA(-) diet. Values are means :_SD for groups of n = 3. (—O-): TCDD treated, (+): VA(-) diet. 127 299,) xvon mx\_oEc .mmoo cove 8m Q: L Om _ 9 h o.— 5 P6 _ o care LL 3 'umuov wavov um!» m-NIw/m 128 Figure 3. Correlation of kidney retinyl palmitate concentration with kidney ACARAT activity in male Sprague- Dawley rats 12 days following a single oral dose of TCDD or fed a vitamin A deficient diet for 12 days. Values are the means of groups of n = 3. 129 2.55.9: .>:>:.o< Em O 104 E o. g; .1- .2 8- 7- +1 0 <( l..— 8 c3 6‘ T D _t_ '0 C .2- H 4__ _l-_ .9. ° I .E t 4.1 Li? L Zfr—I—L r r T F F r ' l T O 0.1 1.0 10 30 300 TCDD dose, nmol/kg DISCUSSION The effect of a single oral dose of TCDD on both the overt signs of toxicity, as well as the storage of retinoids, in treated rats was quite pronounced, but in agreement with previously published data. Particularly interesting was the correlation between weight of the animals 12 days following TCDD treatment and their cumulative food intake for the period, supporting the hypothesis of Peterson gt_gt., (Seefeld, gt_ gt., 1984a, 1984b) that hypophagia is the primary cause of the TCDD- induced weight loss. The results of this experiment however, also provide mechanistic evidence for at least some of the pathways by which vitamin A may be so rapidly lost from TCDD treated animals. The microsomal oxidation of retinoic acid (presumably) to either the 4-hydroxy or 4-keto compound has received significant attention in the literature. This may be because of the identification of these compounds as metabolic products of physiologic doses of either retinol or retinoic acid in a number of species. The analogous microsomal oxidation of retinol has not been equally investigated. However, our research indicates that microsomes from TCDD-treated rats show a significant increase in the rate of microsomal NADPH-dependent retinol oxidation, as compared to control animals. It is important to note that the same reaction proceeds at a significantly slower rate in microsomes from rats in the early stages of a 162 163 diet-induced vitamin A deficiency. Indeed, the microsomes from rats treated with 30 nmol/kg TCDD degraded retinol at a rate over twice that of the vitamin A deficient control rats, despite the fact that both groups had lost approximately the same amount of retinyl palmitate from the liver. It seems reasonable to conclude then, that the normal reaction to a developing vitamin A deficiency is not being demonstrated in the TCDD treated animals. Our findings with respect to the elevated activity of the microsomal PNP-UDPGT were in accord with previous reports in the literature. Indeed several authors have suggested that elevated UDPGT activities may form part of the pleiotropic response to induction enzymes controlled by the Ah receptor. Several authors have also suggested that induction of UDPGT activity towards retinol could account, in some degree, for the loss of hepatic retinoids that characterizes TCDD-type toxicity. While this type of condensation reaction is carried out by certain UDPGT isozymes, it has been demonstrated 1 vivo only following large doses of retinol (Lippel and Olsen, 1968.) We had hypothesized that if indeed UDPGT activity played a role in retinoid depletion, then it would do so by catalyzing the formation of the retinoyl-glucuronide. This compound, the first retinoic acid metabolite to be indentified (Dunagin gt_ gt,, 1965) has been demonstrated as a significant retinoid metabolite in numerous studies. Our results tend to support this hypothesis, as we have seen a significant 164 elevation of the retinoyl-UDPGT activity in the microsomes from TCDD-treated rats. It again is important to note that the response of the rats to a vitamin A deficiency is to decrease the rate at which degradative reactions may proceed. In this case, the activity of the retinoyl-UDPGT was significantly depressed in rats fed a vitamin A deficient diet for 12 days as compared to controls. BIBLIOGRAPHY Bliss, A.F. (1951). The equilibrium between vitamin A alchohol and aldehyde in the presence of alchohol dehydrogenase. Arch. Biochem. Biophys. 31:197-204. Bock, K.W., Burchell, B., Dutton, G.J., Hanninen, O., Mulder, G., Owens, I.S., Siest, G., and Tephly, T.R. (1983). UDP—glucuronosyltransferase activities: Guidelines for consistent interim terminology and assay conditions. Biochem. Pharm. 32:953-955. Brouwer, A., and van den Berg, K.J. (1983). Early decrease in retinoid levels in mice after exposure to low doses of polychlorinated biphenyls. Chemosphere 12:555—558. Cullum, M.E., and Zile, M.H. (1985). Acute PBB toxicosis alters vitamin A homeostasis and enhances degradation of vitamin A. Toxicol. Appl. Pharmacol. 81:177—181. Frolik, C.A. (1984). Metabolism of retinoids. In:“The Retinoids“ (Sporn M.B., Roberts, A.B., and Goodman, D.S. eds.) Academic Press, Inc. Orlando. Vol 2, pp:177— 208. Godfrey, N.K. (1985). Comparing the means of several groups. New Engl. J. Med. 313:145-1456. Hakansson, H., and Ahlborg, U.G. (1985). The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the uptake, distri ution and excretion of a single oral dose of 11,12— H-retinylacetate and on the vitamin A status in the rat. J. Nutr. 115:759-771. Innami, S., Nakamura, A., Miyazaki, M., Nagayama,S., and Nishide, E. (1976). Further studies on the reduction of vitamin A content in the livers of rats given polychlorinated biphenyls. J. Nutr. Sci. Vitaminol. 22:409-418. Lippel, K., and Olson, J.A. (1968). Biosynthesis of B- glucuronides of retinol and of retinoic acid ;g_ vivo and tg.vitro. J. Lip. Res. 9:168-175. Mahadevan, S., Murthy, S.K., and Ganguly, J. (1962). Enzymic oxidation of vitamin A aldehyde to vitamin A acid by rat liver. Biochem. J. 85:326-331. Miller, D.A., and DeLuca, H.F. (1985). Biosynthesis of retinoyl-B-glucuronide, a biologically active metabolite of all-trans-retinoic acid. Arch. Biochem. Biophys. 244:179-186. 165 166 Nath J., and Olson, J.A. (1967). Natural occurrence and biological activity of vitamin A derivatives in rat bile. J. Nutr. 93:461-469. Seefeld, M.D., Corbett, S.W., Keesey, R.E., and Peterson, R.E. (1984). Characterization of the wasting syndrome in rats treated with 2,3,7,8-tetrachlorodibenzo-p- dioxin. Toxicol. Appl. Pharmacol. 73:311-322. Seefeld, M.D., Keesey, R.E., and Peterson, R.E. (1984). Body weight regulation in rats treated with 2,3,7,8- tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmocol. 76:526—536. Thunberg, T., Ahlborg, U., and Johnsson, H. (1979). Vitamin A (Retinol) status in the rat after a single oral dose of 2,3,7.8-tetrachlorodibenzo-p-dioxin. Arch. Toxicol. 42:265-274. Thunberg, T. (1983). Studies on the effect of 2,3,7,8- tetrachlorodibenzo-p-dioxin on vitamin A: A new aspect concerning the mechanism of toxicity. PhD Thesis, Dept. of Toxicology. Carolinska Institute, Stockholm. Underwood, B.A. (1984). Vitamin A in human and animal nutrition. In:"The Retinoids". (Sporn, M.B, Roberts, A.B., and Goodman, D.S., eds). Academic Press, Inc. Orlando. Vol 1, pp:281-392. Zachman, R.D., and Olson, J.A. (1961). A comparison of Retinene reductase and alcohol dehydrogenase of rat liver. J. Biol. Chem. 236:2306—2313. CHAPTER 4 Exacerbation of TCDD-Induced Toxicity and Hepatic 'Retinoid Depletion in Rats by Triiodothyronine. 167 ABSTRACT The effects of triiodothyronine (T3) on both TCDD toxicity, as measured by rate of weight gain and organ hypertrophy, and hepatic retinoid depletion was determined in a single experiment in male Sprague-Dawley rats. Animals were given either a control or T3-containing (500 ug/kg) diet, and both diet groups were further treated with either 0 or 25 ug/kg TCDD and killed 12 days following treatment. Inclusion of T3 in the diet caused a slight, transitory depression in the rate of weight gain, and a pronounced exacerbation of the depression of rate of weight gain caused by the TCDD treatment. Dietary T3 had no effect on the TCDD-caused hepatic hypertrophy, but caused, unlike TCDD, a moderate hypertrophy of kidney. TCDD treatment also caused a depletion of hepatic retinoids, which was moderately, but significantly exacerbated by dietary T3. TCDD also caused a pronounced accumulation of retinoids in the kidney, which was markedly exacerbated by dietary T3. Therefore, an apparent increase in the toxicity of TCDD caused by inclusion of T3 in the diet, also caused an exacerbation of the vitamin A depletion characteristic of the toxic response to TCDD. We conclude that this finding is in accord with the depletion of hepatic retinoids and onset of vitamin A depletion as being a significant aspect of TCDD-type toxicity. 168 INTRODUCTION TCDD, and related compounds, along with certain polycyclic aromatic hydrocarbons, polyhalogenated- dibenzofuran and -biphenyl congeners, all cause a characteristic toxic response in treated animals commonly referred to as the "wasting syndrome.” Manifestations of toxicity include a delayed time to death, pronounced weight loss, hypophagia, cachexia, epithelial lesions, lymphoid involution, and hepatic hypertrophy (McConnell, 1980; Poland and Knutson, 1982; Peterson gt_gt., 1984). Neither the biochemical mechanism resulting in the wasting syndrome or other toxic mechanism(s) of TCDD and similar compounds have yet been identified. The pronounced similarity between the signs of the xenobiotic—induced wasting syndrome and those of a severe vitamin A deficiency was noted by Kimbrough (1974) and reviewed by Thunberg (1983a). The ability of TCDD and similarly-acting compounds to cause both a rapid depletion of hepatic vitamin A stores, as well as other signs of vitamin A deficiency (e.g., depressed serum retinol levels, thymic involution, failure of growth, and the accumulation of kidney retinoids), has been reported in several species (Innami gt gt., 1975, 1976; Brouwer and van den Berg, 1983, 1984; Spear gt gt., 1986). TCDD toxicity causes a short- lived rise in serum retinol levels followed by a decline to below normal levels, presumably as hepatic stores are exhausted (Thunberg t gt., 1979; Thunberg, 1983a). 169 170 Similar to that observed in a vitamin A deficiency (Morita and Nakano, 1982; Moore and Sharman 1950; Wolf, 1980) TCDD causes an accumulation of retinoid material in the kidney (Thunberg, 1983b). It has been reported that diets containing elevated vitamin A contents ameliorate, but do not eliminate, the toxic response to TCDD-type compounds (Innami gt gt., 1975; Thunberg gt gt., 1980). Therefore, it seems reasonable that some aspects of TCDD-type toxicity may be a function of the onset of an actual vitamin A deficiency caused by exposure to TCDD-type toxins. Vitamin A is an essential nutrient, although with the exception of its role in the visual cycle its exact metabolic function(s) remains unelucidated (Wolf, 1984). It is clear, however, that an adequate supply of the vitamin is required to maintain growth, reproductive capacity, and immunological competence (Zile and Cullum, 1983). Vitamin A is not synthesized by animals, but is available in the diet in several readily utilizable forms (Underwood, 1984). The vitamin is stored in the liver as mixed retinyl esters and is hydrolyzed to the free alcohol retinol, prior to delivery to target tissues by a specific transport protein (retinol binding protein, RBP; Goodman, 1984, Goodman and Blaner, 1984). The control mechanisms for mobilization of the vitamin from the liver and of metabolism in target tissues (including the liver) remain largely unknown. Loss of the vitamin from the body seems to occur primarily when 171 the compound is oxidized to retinoic acid or retinoyl derivatives (Wolf, 1980; Frolik, 1984). In addition to the effects on vitamin A levels, TCDD has also been shown to affect the thyroid hormone status of treated animals. Overtly, TCDD treatment seems to cause hyperthyroidism, as the animals demonstrate both weight loss and elevated metabolic rate (Potter 2£ gt., 1986; Rozman, 1984). Further, thyroidectomy seems to confer a significant degree of protection against TCDD toxicity (Rozman, 1984). Curiously though, TCDD treatment also causes a rapid and pronounced depression of serum thyroxine (T4) levels, normally considered symptomatic of a hypothyroid state (Bastomsky, 1977; Gupta gt gt,, 1983). There is evidence for some degree of interaction between levels of thyroid hormones and vitamin A. Underwood (1984) noted the inverse correlation between hepatic vitamin A reserves and thyroid state in humans. Early nutritional studies suggest that during the induction of acute hyperthyroidism there is also an onset of vitamin A deficiency (Sure and Buchanan, 1937). Bhat and Cama (1977) have suggested that the hyperthyroid state causes signs of a vitamin A deficiency by enhancing plasma clearance rates of retinol, while the rate of both apo-RBP synthesis and retinol-RBP secretion remains unchanged. This hypothesis is particularly interesting with respect to TCDD toxicity. As noted above, TCDD—treated animals express symptoms reminiscent not only of vitamin A deficiency, but of 172 hyperthyroidism also. Further, it has been reported that TCDD in rats increases the fractional clearance rate. of retinol in plasma (Bank et al., 1987). Preliminary experiments in our laboratory suggested that inclusion of triiodothyronine (T3) in the diet of TCDD- treated rats exacerbated the toxic response as measured by growth rate. Therefore, we hypothesized that to the extent that vitamin A depletion plays a role in the expression of TCDD toxicity, its depletion from the liver and accumulation in the kidney should be similarly exacerbated by the inclusion of T3 in the diet of treated animals. Experimentally, we wished to compare the magnitude of the retinol and retinyl ester depletion of the liver, as well as the magnitude of the retinoid accumulation in the kidney caused by a single dose of TCDD in normal and T3—fed rats. While expressly designed to demonstrate the exacerbation of TCDD-induced hepatic retinoid depletion caused by thyroid hormones, this experiment would also demonstrate the effects of dietary ”hyperthyroidism" on vitamin A depletion from the liver, and the accumulation in the kidney, thereby enhancing our knowledge about the interaction of natural hormones and vitamin A metabolism. MATERIALS AND METHODS Animals, Diets, ggg_Treatment Male Sprague-Dawley rats were obtained from Charles River Laboratories (Portage, MI) and acclimated for 2 weeks on standard laboratory rat diet (Rodent-Blox (R); Wayne Feeds, Chicago, IL). Diet containing T3 (500 ug/kg) was prepared by grinding the standard diet in a Wiley mill and mixing with 10 ml of a T3 solution at 50 ug/ml in ethanol per kg of diet in a dough blender until all solvent had evaporated, and a smoothly flowing powder was obtained. Control diet was prepared in the same manner, with the exception that ethanol alone was added. One day prior to TCDD treatment, the rats were randomly assigned to 4 separate groups, and housed 3/cage in polystyrene rodent cages with food and water gg_libitum. On day 0, rats in groups not treated with TCDD ("C“ and ”T3” groups) received a 1.0 ml/100 g oral dose of corn oil and those in the TCDD-treated ("CT" and "T3T") groups received a similar dose containing 2.5 ug/ml TCDD. All treatment groups received their appropriate food and water gg libitum. Body weights of individual rats were determined daily. Three untreated rats were killed on day 0 by decapitation following C02 anesthesia and 3 rats from each group were killed on days 3, 7, and 12. Trunk blood was collected in 10 ml EDTA-Vacutainer tubes (Becton-Dickenson and Co., Rutherford, N.J.) and stored on ice. Plasma was prepared by centrifugation (1500 x g for 10 min at 4° C), 173 174 frozen on dry ice, and stored at -700 C until analysis for retinol. Liver was perfused ;g_situ with 0.902 NaCl, excised, and stored on ice in 0.902 NaCl prior to homogenization. Liver homogenate was prepared by homogenization of an aliquot of liver at 3 ml/g in ”HEDG" buffer (0.025 M HEPES (N-2—hydroxyethylpiperazine-N'~2- ethanesulfonic acid), 1.5 mM EDTA, 1 mM DTT (dithiothreitol), 10% glycerol pH 7.4) in a Potter-Elvehjem homogenizer. Aliquots of liver homogenate were frozen on dry ice and stored at -70°C until analysis for retinol and retinyl palmitate. Kidneys were excised and stored as per liver until freezing. Kidney homogenates were prepared from frozen tissues by homogenization of a single kidney at 10.0 ml/g in 1.15% KCl in a Potter-Elvehjem homogenizer. Aliquots of homogenate were used immediately for analysis of retinol and retinyl palmitate. Retinol ggg_Retinyl Palmitate Plasma, hepatic, and renal levels of retinol and retinyl palmitate were determined by modifications to the method of Dennison and Kirk (1977). Aliquots of liver (0.2 ml), kidney (1.0 ml), or testes (2.0 ml) homogenates or plasma (0.2 ml) were mixed thoroughly with 2 ml NaCl- saturated water and 2 m1 ethanol. Retinoids were extracted by the addition of 1.0 ml (liver and kidney samples) or 0.5 ml (plasma samples) of UV-grade hexane followed by thorough mixing. The samples were centrifuged (2500 x g for 2 min) to facilitate phase separation and the supernatants were 175 assayed for retinol and/or retinyl palmitate by HPLC using a fluorescence detector (Shimadzu RS-530-S, ex = 330 nm, em = 470 nm) and a 4 x 250mm column packed with Lichrosorb SI-60 (Alltech, Deerfield, IL.) Isocratic elution of retinol was performed using 252 hexane/75% CHC13. Retinyl palmitate was similarly eluted with 8% CHCl3/9ZZ hexane. Retinol was eluted by this system at a retention time of 5.5 min, retinyl palmitate at 4 min. Detection limit for each species was 500 pg. Retinol and retinyl palmitate (Sigma Chemical Co., St. Louis, MO) standards were prepared in ethanol and verified by E324(1Z) = 1835 and E326(1Z) = 975 A.U., respectively (Windhols, 1976). HPLC standards of 1.0 and 0.1 ng/ul for both retinoids were prepared by dilution of the stock standard with hexane. All standards were stored at -20°C and remained stable for at least six months. Statistical Analysis Data were analysed using Student's t-test with the Bonferroni correction for multiple comparisons (Godfrey, 1985). All references to statistical significance were at the p < 0.05 level. RESULTS flggy_Weights The group of rats fed a normal diet (C group) and sacrificed on day 12 had gained 38.9% of their day —1 weight, as shown in Figure 1. Inclusion of T3 in the diet (T3 group) caused a short, transient depression in the rate of weight gain when compared to the C group. However, the growth rate was equal to that of the C group by day 3 and the T3 group rats averaged a 32.5% gain of their day -1 weights by day 12 (Figure 1). Typical of the response to TCDD treatment, rats fed a normal diet and treated with TCDD (CT group) failed to gain weight normally when compared to control rats, reaching only a 16.1% increase over their day -1 weights by day 12 (Figure 1). The combination of TCDD treatment and dietary T3 (T3T group) had the greatest effect on the ability of the rats to gain weight, with the day 12 T3T rats averaging an increase of only 0.27% of their day -1 weight (Figure 1). Orggn/Body Weight Ratios Treatment of rats with TCDD caused an increase in the hepatic weight/body weight ratio, regardless of whether T3 was included in the diet (Table 1). Curiously, kidney enlargement was observed in both T3-fed groups and was unaffected by TCDD treatment. The testes weight/body weight ratio difference observed, appears to reflect the changes in the body mass of the rat, since testes weight remained essentially unaffected by any of the treatment regimens. 176 177 Figure 1. Effect of TCDD and or dietary T3 on body weight of male Sprague-Dawley rats. Each point represents the mean :_SD of groups of 3 rats: control diet (-C}); control diet + TCDD (—A—); T3 diet (O); T3 diet + TCDD (+). 3: Significantly different from control rats fed standard diet, p < 0.05. 178 55 no. to\oco EoEeooce 009. 9:30:91 930 .3 NF or w c o .v N o NI L P L L F b e h L F e h tr CNN a ' »\\D . _ NOV 1 08 e! a u‘ .. . ‘3‘. ‘ _. .. 1 1, .. 88 + 35 me I .. U.05 n._. I T ovn 82 + 85 .228 Ta $5 .228 To (6) tqbgeM Kpog efioJeAv 179 .W0.0 v Q .ODHGP ADV nun—Ohm HOHHGOU .OHH “COHOHNwU hHHfiOHMM-fiflfim . me mcwcwaucoo gown vouovfloo tabwooou mach .o hat :0 unaccwmom u alddflnfld.dd Aaowo no mx\m= oonv .Aauo .mx\m: mm "a .seoa no acme an no apnea .6609 see: pee-aaouu eczema weenoeaou what NA .uuau m no Manchu loam nm.H made! one moaud> "a weaved venue axemev nave flnflbcd nflfim AMHV HOHHGOO OMB «new vague axeov egos «HAD flfifim on HOHHGOO HOHHGOO A eexv AmeHMV aeo.e no.e anew». econ use. a neon neuuuu_uuee«u neueuu.uuu«q «ea-uuuuauulll d.uad¢ healdnlosuuuom can: em nomaue gauge. eeomxeumuo no we panacea nexus: gave no once Hugo oeueea a no uoouem can .5 omega 180 Tissue Retinol Treatment of rats with either TCDD (CT group) or dietary T3 (T3 group) resulted in a significant elevation of plasma retinol when compared to control rats (C group) at 12 days post-treatment. The combination of TCDD and dietary T3 (T3T group) produced a slightly greater elevation of plasma retinol values than was observed with either treatment alone (Table 2). Plasma retinol for both TCDD-treated groups (CT and T3T) was significantly different from the control group (C) at day 7, but the level for the dietary T3 group (T3), although elevated, was not statistically significant (data not shown). Retinol levels in the kidneys followed a similar pattern, with the exception that the combination treatment (T3T group) resulted in a significant elevation of renal retinol levels, well above those observed in either of the other treatment groups (CT or T3), and over twice that of the untreated control group (C) (Table 2). In addition, some of the effects of TCDD and/or dietary T3 treatment were evident at 7 days post-treatment for plasma retinol levels and as early as 3 days post-treatment for renal retinol levels (data not shown). Retinol concentration in the liver was unaffected by TCDD or dietary T3 treatment, either alone or in combination. 181 .Aoooum 99v uowo me tom menu Aouunoo no oaaab :dofl loam acououuwv haaadomamcmwm . .uowo Homecoo a new menu voudoualoooa mo uoauub Gael noun unouommwo hducdowuwcmwm no .no.ovn .oaaab :uol Annoum UV Houucoo loam acououuwo haacdowuwcmwm no .3 3 3o... .8 min: oomv ma mcwcwuucoo HOMU vehoouon vo>wooou mean .o hat :0 Newccwmom no .o.e .mxxme we "a .zuon no .uowv me no Hanan .oooa sewn acclauouu uozuwo mewBoHHou what NH .uuuu n no museum noun om.H model one nosaub "d mo.aflom.o m.o.uN.QH>.m v.0.vNo.QHmo.~ Ahmev once mo.QHmb.o oN.QHn.N omo.QHAm.o Amav Aouusoo one «0.9.530 ON.OHN.N vvodflomé nasv nova. N.QHQ.Q A.Qfln.~ oo.aflmn.o on Houunoo Houueoo nebwq hocvwm alumna macaw How: GEE 4.353 3H IllludufluuHHIIll u.nau¢ heuudOIonuauom can: cw cowaduacoocoo Hoeeuoe ounces no my nuance: noxeeu neon no aooeum one .N adage 182 Hgggtic Retinyl Palmitate Treatment of rats with TCDD (CT group) caused a significant decrease in the concentration of hepatic retinyl palmitate to a level 63% that of the control group (C) at 12 days post-treatment (Figure 2A). Dietary T3 alone (T3 group) had no significant effect on hepatic retinyl palmitate concentration, and no additional effect over that seen with TCDD alone. However, as shown in Figure 28, when total hepatic retinyl palmitate was considered, a different pattern emerged. Rats in both the control (C) and dietary T3 (T3) groups accumulated hepatic retinyl palmitate throughout the course of the study. The rate of accumulation for the T3 group was somewhat lower than for the control group; however, this difference was not statistically signifcant. In contrast, treatment with TCDD (CT group) caused a decline in total hepatic retinyl palmitate stores to 712 of the level in the control group (Figure 28). Dietary T3 alone (T3 group) had no significant effect on total stores of hepatic retinyl palmitate; however, there was a small additional effect on the decline in total hepatic retinyl palmitate for the combination treatment group (T3T) to 631 of the C group level. Renal Retinyl Palmitate Control rats maintained renal retinyl palmitate levels in the range of 2.0-3.5 ppm throughout the study period (Figure 3). Rats treated with dietary T3 (T3 group), TCDD (CT group), or both (T3T group) showed a tendency to 183 Figure 2. Effect of TCDD and/or dietary T3 treatment in male Sprague-Dawley rats on the concentration (A), or the total amount of retinyl palmitate (B). Each point represents the mean 1,SD of groups of 3 rats: control diet (-(}-); control diet + TCDD (f); T3 diet (-A—); T3 diet + TCDD (+). a: Significantly different from control rats fed standard diet, p < 0.05. b: Significantly different from TCDD- treated rats fed standard diet, p < 0.05. 184 Ea D co tom 3%.... 38 88. 9.32.8 260 nw — _. m h m n P w I n p L In P p i L L b b - 0.0“ F O I 1 O / 6.8. 1O.OON IO.O+N < (de) azouwlod Mums °!7l°d9H 185 Ea 2 e0 55 3%.... $8 88 $32.0... 2.8 m; _.__. . a x. m n F Fl — . — u _ r _ . _ . 3.“ ‘- I H I o [x 10.0— (fiw) azouwlod Munaa onodaH 10101 186 Figure 3. Effect of TCDD and/or dietary T3 on the renal retinyl palmitate concentration of male Sprague-Dawley rats. Each point represents the mean 1.80 of groups of 3 rats: control diet (-{)—); control diet + TCDD (-£}-); T3 diet (-.- ); T3 diet + TCDD (+ ). a: Significantly different from control rats fed standard diet, p < 0.05. b: Significantly different from TCDD-treated rats fed standard diet, p < 0.05. 187 35 E 10 tom 3%.... $8 88 Eaget 26o 2 2 m e m n F T _ e — . _ . _ n _ n _ n )- CO L IT I u. 62 d 7 ‘ I c I soda no.8 a. 80» + in n» I in n To 89 + in .350 I :5 .380 To (de) atouwlod Munea Kaupm 188 accumulate renal retinyl esters above the concentration seen in control rats. Kidneys from T3 group rats contained, at day 12, about 2.6x more retinyl esters than those from C group rats, however, this difference was not significant. At day 12, retinyl esters in the CT group had increased to a level 5.2x that of the C group. The greatest retinoid accumulation was observed in rats given a combination of dietary T3 and TCDD (T3T group), with renal retinyl palmitate levels reaching 10.2x those of control rats by day 12. DISCUSSION A prominent characteristic of the response of many species to TCDD is either weight loss or depressed rate of weight gain (Thunberg et al., 1983a; McConnell, 1980; Gasiewicz et al., 1980; Rozman et al., 1984; Schiller et al., 1985). We observed a depression in growth rate during the first 6 days after TCDD treatment, followed by increased growth rate during the last 6 days of the study, although the recovery was to rates still less than those of control animals. Dietary T3 alone caused only a brief period Vof reduced growth rate, following which the animals gained weight at a rate approximately equal to that of control rats. However, dietary T3 appeared to markedly exacerbate the TCDD-induced depression of growth rate of treated rats. These data support Rozman‘s (1984) suggestion that the initial phase of weight loss caused by TCDD may be at least partially thyroid hormone mediated. Both treatment groups treated with TCDD (CT and T3T groups) had a similar degree of hepatic enlargement. We did not observe any effect of dietary T3 alone on liver weight/body weight ratio, nor did T3 exacerbate the hepatic enlargement seen in the TCDD-treated groups. This result is in accord with the suggestion that hepatic hypertrophy is primarily a consequence of endoplasmic reticulum (ER) proliferation and a direct response to TCDD, perhaps mediated by the TCDD (Ah) receptor and MC-type induction (Poland and Knutson, 1982; Dencker, 1985). Curiously, we 189 190 observed a T3-dependent enlargement of kidney, as measured by kidney weight/body weight ratio, that did not seem to be affected by TCDD. We observed an increase in the concentration of plasma retinol following TCDD treatment similar to that reported by Thunberg (1979). The elevation of plasma retinol levels was exacerbated by treatment with dietary T3, although such treatment alone was able to cause a significant increase in plasma retinol levels. Similarly, we observed a significant increase in the renal retinol levels of TCDD-treated rats, which was also exacerbated by the inclusion of T3 in the diet. Further, dietary T3 treatment itself caused a significant elevation of renal retinol levels and the effects of the two treatments appeared to be additive. Neither TCDD, dietary T3 or the combination of the two treatments caused a significant change in the levels of hepatic retinol. We believe that the levels of retinoids in the kidney provide a sensitive measure of the vitamin A status of the animal. Hence, the exacerbation of TCDD-caused renal retinoid accumulation caused by T3 supports the hypothesis that the depletion of vitamin A is a significant factor in the expression of TCDD-type toxicity. This argument is further supported by the fact that accumulation of retinoids in the kidney is symptomatic of a vitamin A deficiency as induced by dietary depletion. It is significant that TCDD-treated animals has not only overt 191 signs of vitamin A deficiency, but biochemical signs as well. TCDD and related chemicals are able to cause a rapid depletion of hepatic retinoids (Thunberg, 1983; Brouwer and van den Berg, 1986) In this experiment, TCDD caused a depletion of hepatic retinyl palmitate that was apparent both in concentration and total amounts. Dietary T3 had no additional effect on the decrease in hepatic retinyl palmitate concentration produced by TCDD, but did cause a significant exacerbation of the decline in total hepatic retinyl palmitate. These data further support the hypothesis mentioned above, suggesting the possibility that the worsening of TCDD toxicity by T3 may be a consequence of an increase in the rate of loss of vitamin A. 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SUMMARY The ability of TCDD and similarly acting compounds to cause a depletion of hepatic vitamin A reserves in treated animals, as well as certain signs consistent with those of a vitamin A deficiency state has been thoroughly described in the literature. However, relatively little research has been presented on the role this depletion may play in the development of ”TCDD-type“ toxicity and further, on the mechanism by which the depletion of retinoids might be caused by TCDD treatment. It has been suggested in the literature that TCDD and similarly acting compounds affect the mobilization and transport of vitamin A. The research presented in this dissertation shows that at least one consequence of the inhibition of the hepatic enzyme responsible for the hydrolysis of retinyl esters, retinyl palmitate hydrolase (RPH), is the lowering of plasma retinol levels. This point is demonstrated as a consequence of treatment of rats with either nonadecafluorodecanoic acid (NDFDA) or 3,4,3',4‘- tetrachlorobiphenyl (TCB). Both compounds are able to inhibit RPH both i vivo and ;__vitro. It is also shown, that while TCDD is also able to inhibit RPH ;g_vitro, the KI of this interaction is sufficiently high that significant inhibition of RPH activity would not be an expected consequence of TCDD treatment, even at an L050 dose. That TCDD does not cause inhibition of RPH ;g_y;yg_ is further demonstrated by the fact that TCDD treatment causes an 195 196 increase in the levels of plasma retinol of treated rats. Other results suggest that TCDD treatment does not markedly affect the stability of the retinol-RBP-TTR ternary complex as measured by retinoid fluorescence associated with the complex. Therefore, we have concluded that TCDD does not directly affect either the mobilization or transport of vitatmin A. The accumulation of retinoids in the kidneys has been shown to occur during the development of a vitamin A deficiency, although neither the species accumulated, nor the mechanism by which this accumulation occurs has been characterized. We have presented evidence suggesting that the retinoid accumulation in the case of dietary depriovation of vitamin A is a consequence of elevated rates of esterification of retinol by acyl CoA:retinol acyl transferase (ACARAT). Further, we have demonstrated that a dose of TCDD causes a similar accumulation of retinoids in the kidney, and increase in ACARAT activity. We have concluded that the accumulation of retinyl esters in the case of TCDD toxicity is actually a response to the depletion of retinoid reserves, analogous to the early stages of a vitamin A deficiency. The mechanism by which TCDD toxicity results in the depletion of stored retinyl esters in the liver was investigated. Some pathways of the normal degradation of retinoid materials have been identified in the literature. We have shown that the rate of microsomal retinol oxidation is increased as a consequence of TCDD toxicity. It has been 197 suggested that the hydroxylated, or other oxidized retinoids are subject to more rapid oxidative degradation than the non-hydroxylated analog. We determined that the rate of oxidation of retinol to retinoic acid, via retinal, was not affected as a consequence of TCDD treatment. However, the rate of formation of retinoyl-B-glucuonide was markedly increased, and might be expected to enhance the rate of loss of retinoids from treated rats. Lastly, an attempt was made to determine the extent to which depletion of retinoids caused as a consequence ofnTCDD treatment contributed to the development of the toxic response. It was shown that worsening of TCDD toxicity by inclusion of T3 in the diet caused a concomitant worsening of the depletion of vitamin A reserves. In summary, we conclude that as a function of induction of enzymes responsible for the degradation of retinoids, TCDD-treated animals lose vitamin A at an inappropriate rate. As a consequence of this retinoid loss, the animal responds in a mannar similar to that seen shortly following removal of vitamin A from the diet, specifically, elevated plasma retinol levels, and accumulation and storage of retinyl esters in the kidney. This depletion of vitamin A may be the basis for the development of symptoms similar to those of a vitamin A deficiency state.