Characterization of the Mechanistic Linkages Between Iodothyronine Deiodinase Inhibition and Impaired Thyroid-Mediated Growth and Development in Xenopus laevis Using Iopanoic Acid.

Iodothyronine deiodinases (DIO) are key enzymes that influence tissue-specific thyroid hormone levels during thyroid-mediated amphibian metamorphosis. Within the larger context of evaluating chemicals for thyroid system disrupting potential, chemical activity toward DIOs is being evaluated using high-throughput in vitro screening assays as part of U.S. EPA's ToxCast program. However, existing data gaps preclude any inferences between in vitro chemical inhibition of DIOs and in vivo outcomes relevant to ecological risk assessment. This study aimed to generate targeted data in a laboratory model species (Xenopus laevis) using a model DIO inhibitor, iopanoic acid (IOP), to characterize linkages between in vitro potency, in vivo biochemical responses, and adverse organismal outcomes. In vitro potency of IOP toward DIOs was evaluated using previously developed in vitro screening assays, which showed concentration-dependent inhibition of human DIO1 (IC50: 97 µM) and DIO2 (IC50: 231 µM) but did not inhibit human or X. laevis DIO3 under the assay conditions. In vivo exposure of larval X. laevis to 0, 2.6, 5.3, and 10.5 µM IOP caused thyroid-related biochemical profiles in the thyroid gland and plasma consistent with hyperthyroxinemia but resulted in delayed metamorphosis and significantly reduced growth in the highest 2 exposure concentrations. Independent evaluations of dio gene expression ontogeny, together with existing literature, supported interpretation of IOP-mediated effects resulting in a proposed adverse outcome pathway for DIO2 inhibition leading to altered amphibian metamorphosis. This study highlights the types of mechanistic data needed to move toward predicting in vivo outcomes of regulatory concern from in vitro bioactivity data.

Placebo Oral Rabies Vaccine Bait Uptake by Small Indian Mongooses (Herpestes auropunctatus) in Southwestern Puerto Rico.

The small Indian mongoose (Herpestes auropunctatus) is a rabies reservoir in areas of the Caribbean including Puerto Rico, but no rabies vaccination program targeting this host exists. We used two derivatives of iophenoxic acid (IPA) to evaluate placebo oral rabies vaccine bait uptake by mongooses in southwestern Puerto Rico. We hand-distributed baits at an application rate of 200 baits/km2 at three, 400 ha, sites during autumn 2016 and spring 2017. Each site contained 90-100 cage traps in a 100 ha central trapping area. We used ethyl-IPA as a biological marker during the autumn and methyl-IPA during the spring. We live captured mongooses for 10 consecutive days, beginning 1 wk following bait application. We obtained a serum sample from captured mongooses and analyzed the sera for ethyl- and methyl-IPA by liquid chromatography-mass spectrometry. During autumn 2016, 63% (55/87) mongooses sampled were positive for ethyl-IPA. In spring 2017, 69% (85/123) of mongooses were positive for methyl-IPA. Pooling seasons, accounting for recaptures between years, and disregarding marker type, 74% (133/179) unique mongooses were positive for IPA biomarker, indicating bait consumption during either the autumn, spring, or both trials. We conclude that distributing baits at an application rate of 200 baits/km2 is sufficient to reach over 60% of the target mongoose population in dry forest habitats of Puerto Rico.

Iophenoxic acid derivatives as markers of oral baits to wildlife. New tools for their detection in tissues of a game species and safety considerations for human exposure.

The bait-marker iophenoxic acid (IPA) and its derivatives are increasingly used for evaluating and optimizing the cost-effectiveness of baiting campaigns on wildlife, particularly on game species such as the wild boar. We aimed to determine whether concentrations of the three main IPA derivatives ethyl, methyl and propyl-IPA measured on thoracic liquid extracts (TLE) of hunted wild boars may be representative of two exposure doses, 40 and 200 mg, from 20 to 217 days after ingestion. Then we developed a method of detection of the three IPA derivatives by LC/ESI-MS-MS in muscle and liver to evaluate the suitability of these two other tissues for monitoring the marked bait consumption and for measuring available residues in the meat of marked animals. Three semi-captive wild boars received 40 mg of each IPA derivative, three received 200 mg, and three, as controls, did not receive IPA. Blood serum was sampled 20, 197 or 217 days after IPA exposure according to animals and to the derivative. Wild boars were shot by gun after the different times of serum sampling times, and TLE, muscle and liver were sampled. Our results suggest that TLE is not a relevant tissue for quantitatively expressing IPA exposure. Due to interference, no analytical method was validated on TLE containing digestive material. On the other hand, quantifications in the muscle and particularly in the liver could discriminate wild boars that had ingested the two IPA doses from 20 days until 7 months after exposure, especially for the two long term markers ethyl and propyl-IPA. So IPA quantifications in the liver sampled on hunted animals appear to be a reliable tool for monitoring bait consumption in the field at a large scale. Nevertheless, whatever the ingested dose, ethyl- and propyl-IPA concentrations measured in the muscle and the liver of tested animals until 217 days after exposure, remained higher than 0.01 mg/kg, the Maximal Residue Limit (MRL) is recommended for molecules for which no toxicological data are available. Based on the range of IPA residues available in these two tissues, implications for humans consuming marked animals are discussed.

Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay.

Enzymatic 5'- and 5-deiodination are key reactions for local and systemic activation and inactivation of iodothyronines and thyronamines. Expression of the three deiodinase (DIO) isoenzymes is regulated by a number of parameters, including thyroid status, genotype, micronutrient availability, and disease-related signaling. In addition, DIO are potential targets of pharmacological as well as environmentally derived substances, which might affect their enzymatic activity (endocrine disruptors). With the classical DIO activity assay, testing depends on the availability of radioactively labeled substrates (e.g. (125)I-rT(3)) to monitor the release of radioactive iodide. Recently, liquid chromatography-tandem mass spectrometry was described as an alternative method apparently resolving this limitation. However, it has a high demand in technical equipment and analytical routine and is limited in sample number by considerable measuring time. We therefore combined the classical deiodination assay with an easily accessible photometric method taking advantage of the Sandell-Kolthoff reaction for measuring iodide release. In brief, iodine works as a catalyst within this redox reaction between Ce(4+) and As(3+) leading to an acceleration of destaining. Furthermore, the protocol was adapted to minimize handling effort and time consumption. Because this method is not dependent on radioactivity, it expands the substrate spectrum of the classical method. Suitability of this assay was tested with tissue samples from animal experiments (hepatic Dio1 activity in hypo- and hyperthyroid mice) and established DIO inhibitors. As a new but not unexpected finding, the alleged inhibitor iopanoic acid turned out to be a DIO substrate. This finding was confirmed by liquid chromatography-tandem mass spectrometry, and its potential clinical impact requires further studies.

Binding of diuretic antihypertensive bendroflumethiazide to human serum albumin studied by ¹9F nuclear magnetic resonance method.

Simultaneous specific and nonspecific binding of bendroflumethiazide (BFZ) to human serum albumin (HSA) and concentration profile of BFZ in HSA buffer (pH 7.40) solution were investigated by ¹9F nuclear magnetic resonance (NMR) method. The ¹9F NMR spectrum of BFZ (200 µM) in a buffer solution showed a sharp signal of its CF3 group at 17.8 ppm from the reference trifluoroethanol. Addition of 0.60mM HSA to the sample solution caused the CF(3) signal splitting into three broadened peaks at 18.4 (A), 17.9 (B) and 17.4 ppm (C). By its chemical shift and spectral behavior, B was assigned to unbound BFZ. Competition experiments with Site I and II ligands lead to C being assigned to Site II bound BFZ. However, the peak intensity (areas) of A was not reduced by these ligands, suggesting that A arises from nonspecific binding. Using the peak intensities at several total concentrations of BFZ, Scatchard plot was performed. The plot for A provided a straight line parallel to the x-axis confirming nonspecific binding and that for C was consistent with specific binding. The binding constants for nonspecific and specific Site II binding were 1.02 and 1.00 × 104 (M⁻¹) (n=1.1), respectively. The presence of 0.10 M Cl⁻ in the sample solution affected the binding constant of Site II binding, but not that of nonspecific binding. The concentration profile of BFZ calculated using the binding constants revealed that nonspecific binding is more effective than Site II binding for the binding of BFZ to HSA. It was also confirmed that considerable amounts of BFZ liberated from Site II by the Site II ligands or Cl⁻ ions bind again nonspecifically.

Electrospray ionization mass spectrometry as a tool for determination of drug binding sites to human serum albumin by noncovalent interaction.

Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoESI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.

The cDNA for the type I iodothyronine 5'-deiodinase encodes an enzyme manifesting both high Km and low Km activity. Evidence that rat liver and kidney contain a single enzyme which converts thyroxine to 3,5,3'-triiodothyronine.

The enzymatic conversion of thyroxine (T4) to 3,5,3-triiodothyronine (T3) by iodothyronine 5'-deiodinase(s) is an obligate step in the physiologic action of thyroid hormones in most extrathyroidal tissues. In the rat liver and kidney, 5'-deiodinase processes having either high Km (micromolar range) or low Km (nanomolar range) values for thyroid hormone substrates have been described. The number of enzymes mediating these reactions, however, remains uncertain and controversial. To examine this question we have compared the 5'-deiodinase activity expressed in membrane preparations of Xenopus laevis oocytes after the injection of either rat liver poly(A)+ RNA or in vitro prepared RNA transcribed using the G21 full-length type I 5'-deiodinase cDNA. In oocytes injected with rat liver poly(A)+ RNA, high Km (i.e. type I) activity was observed when 20 mM dithiothreitol was used as the thiol cofactor, whereas Km values in the nanomolar range were noted with 0.5 mM dithiothreitol, glutathione, or a reconstituted thioredoxin cofactor system. This complex pattern of 5'-deiodinase activity, which mimics that found in homogenates and subcellular fractions of rat liver and kidney, was reproduced exactly in oocytes by the microinjection of G21-derived RNA transcripts. Furthermore, hybrid arrest of translation in oocytes using a partial type I 5'-deiodinase cDNA completely inhibited the expression of both high and low Km activity after the injection of rat liver poly(A)+ RNA. These findings demonstrate that rat liver and kidney contain only a single 5'-deiodinase which manifests either high or low Km activity depending on the reduced thiol cofactor utilized in the reaction.

The plasma pharmacokinetics of iophenoxic and iopanoic acids in goat.

1. The comparative plasma pharmacokinetics of two organic iodine-containing compounds were evaluated in the goat for their suitability as markers in wildlife studies. 2. After oral administration of a single dose, the plasma elimination half-life for iopanoic acid was considerably more rapid (t1/2 of 1-2 days) than that of iophenoxic acid (t1/2 of 81 days). 3. Similar peak plasma concentrations were obtained after administration of iophenoxic acid (1.5 mg/kg) and iopanoic acid (25 mg/kg); however, the AUC0----infinity for iopanoic acid at doses of 25, 50, and 100 mg/kg were 201 +/- 39, 604 +/- 225, and 1292 +/- 721 (micrograms h/ml +/- SD), respectively, which were less than the value of 36,600 +/- 6387 for the oral administration of iophenoxic acid at 1.5 mg/kg. 4. Iophenoxic acid was chosen as a suitable marker because of its persistence at detectable concentrations in the plasma for 5 months.

Synthesis and evaluation of iodinated analogues of diacylglycerols as potential probes for protein kinase C.

Analogues of diacylglycerol containing a 3-(3-amino-2,4,6-triiodophenyl)-2-ethylpropanoyl or 3-(3-amino-2,4,6-triiodophenyl)propanoyl group in the 2-position (1a and 1b, respectively) were synthesized and shown to compete with [3H]phorbol dibutyrate [( 3H]PDBu) for binding in a crude rat brain preparation. Phorbol diesters have been shown to bind specifically to protein kinase C and the PDBu receptor has been copurified with protein kinase C activity. The four diastereomers of 1a (1c-f) were synthesized from chiral starting material and studied in the same assay. The affinities for the [3H]PDBu binding site of 1a, 1b, and two isomers of 1a with naturally occurring L configuration were comparable to that of 1-oleoyl-2-acetyl-rac-glycerol (OAG), but the D isomers of 1a were essentially inactive. The chirality of the side chain did not influence the binding affinity. Activation of protein kinase C by 1a, 1c, and 1e demonstrated the same stereochemical requirements, but none were as active as OAG. For the 1,3-isomers 2, 2a, and 2b, the competitive binding studies gave different results. The racemic mixture and the D isomer, 2b, were able to compete for binding, but the L isomer, 2a, did not compete. These studies demonstrate that diacylglycerol binding to and activation of protein kinase C is stereospecific for the glycerol backbone, but not the side chain. Furthermore, the D-1,3-isomer must exist in a conformation such that the acyl and hydroxyl oxygens assume a spatial relationship similar to that in the L-1,2-isomers.

Evidence for a large and flexible region of human serum albumin possessing high affinity binding sites for salicylate, warfarin, and other ligands.

The relations between the single high affinity binding sites for azapropazone, phenylbutazone, chlorpropamide, sulfathiazole, and iophenoxate and the binding regions of human serum albumin represented by the marker ligands diazepam, phenol red, salicylate, and warfarin were examined by a series of competition experiments. Binding was determined by equilibrium dialysis at pH 7.0. In order to ensure an accurate analysis of the competition experiment, the number of moles of ligand bound per mole of protein was usually 0.4 or less to minimize ligand binding to weaker sites. Furthermore, binding of both ligands was determined in all experiments (except for iophenozate). None of the test ligands competed with diazepam for a common high affinity binding site, but, surprisingly, they were all able to displace two or three of the other marker ligands according to a competitive scheme. These findings show, first, the existence of a particular serum albumin region for high affinity binding of diazepam. Secondly, they imply that it is not necessary to assume the existence of new drug binding regions beyond those existing for phenol red, salicylate, and warfarin. On the contrary, the relatively many examples of competitive binding indicate that the binding regions represented by the last-mentioned three marker ligands are placed quite close to each other in the albumin molecule in a common region, which is suggested to be located at subdomains 1C and 2A-B. The region must be relatively large, because in some cases independent high affinity binding of pairs of ligands is observed. It is probably also rather flexible, inasmuch as no clear relation could be found between the chemical structure of the test ligands and the two or three marker ligands with which they compete. Correlations between primary association constants and partition coefficients for both marker ligands and test ligands, in the unionized forms, between n-hexane or 1-octanol and aqueous media showed that hydrophobic forces are important for the binding processes. However, the data also showed that other attractive forces must be operative as well.

Esters of iopanoic acid as liver-specific CT contrast agents: biodistribution and CT evaluation.

The synthesis and preliminary biodistribution data for a series of sterol-like esters of iopanoic acid having potential value as liver-specific CT contrast agents are described. Structural modification of the sterol portion of the iopanoate ester afforded a group of compounds that displayed tissue specificity similar to cholesteryl iopanoate, the prototype ester of this series, but were rapidly cleared from the target tissues after hydrolysis. From the biodistribution data, the most promising of these agents, pregnenolone iopanoate (PI), was evaluated by CT in rabbits receiving a radiologic dose equivalent to 30 mg I/kg. The hepatic parenchyma was enhanced within 2 h of infusion to a maximal level of 31 HU above precontrast values. Hepatic CT attenuation returned to normal within 24 h. However, CT performed after PI infusion into Vx2 tumor-bearing rabbits failed to provide superior images compared with those acquired following bolus administration of urographic contrast.

Conversion of thyroxine to triiodothyronine in the anterior pituitary gland and the influence of this process on thyroid status.

There are two iodothyronine 5'-deiodinases in the rat anterior pituitary gland. The type II 5'-deiodinase is responsible for the observable T4 to T3 conversion in pituitary tissue, both in vivo and in vitro. This type II enzyme has kinetic characteristics which distinguish it from type I activity, and it is also insensitive to inhibition by propylthiouracil. Iopanoic acid is both a substrate for, and an inhibitor of, the pituitary type II 5'-deiodinase. Studies in the rat and in man using iopanoic acid as a probe indicate that conversion of T4 to T3 within the anterior pituitary is a necessary step in the expression of thyroid hormone effects on the thyrotrophic and somatotrophic cells after T4 administration. Amiodarone, which has effects on the human pituitary-thyroid axis similar to those of iopanoic acid, has little inhibitory activity on T4 5'-deiodination in rat pituitary homogenate. Phenytoin, which lowers the serum T4 after chronic administration without inducing an increase in thyrotropin, could theoretically act by enhancing intrapituitary T3 production. In actuality, phenytoin shows only inhibitory activity in vitro on type II 5'-deiodination in rat pituitary and brain homogenates and on type I activity in rat liver homogenates. Phenytoin treatment in vivo has no significant effect on T4 5'-deiodination in pituitary or liver homogenates either. Evidence is discussed regarding whether the increase in type II 5'-deiodinating activity in hypothyroidism, and the decrease in hyperthyroidism, are beneficial defense mechanisms; this is likely to be so in some parts of the body, but may ot be in the thyrotrophic cells of the anterior pituitary.

Uptake of iopanoic acid and its glucuronide conjugate by rat hepatocytes in primary culture.

Uptake of iopanoic acid (IOP) and iopanoate glucuronide (IOP-G) was studied in 3-day primary cultures of rat hepatocytes isolated by the collagenase perfusion method. 125I activity of cells after incubation with 125I-IOP (10-100 microM) and 125I-IOP-G (10-100 microM) was used as a measure of uptake. At each concentration, uptake was linear for the first 45 sec. In the absence of albumin, the initial uptake velocity was directly proportional to the concentration or IOP or IOP-G and was nonsaturable up to 100 microM. The calculated uptake rate constants for IOP and for IOP-G were 0.059 and 0.048 nmole/(mg protein X min X microM), respectively. IOP uptake was not inhibited by sodium taurocholate nor by the contrast agents iodipamide, ipodate, and iopronic acid. The data indicate that the enhancement of IOP excretion by bile salts noted in vivo does not occur at the uptake step and that the hepatic uptake of both IOP and IOP-G in the absence of albumin is limited by diffusion.

Uptake of iopanoic acid by isolated rat hepatocytes in primary culture.

Uptake of iopanoic acid (IOP) was studied in 3-day primary cultures of rat hepatocytes isolated by the collagenase perfusion method. 125I activity of cells after incubation with 125I-IOP (1.0-100 microM) was used as a measure of uptake. At each IOP concentration uptake was linear for the first 45 s. The initial uptake velocity was directly proportional to IOP concentration and was nonsaturable up to 100 microM. The calculated uptake rate constant was 0.67 nmol . mg prot-1 . min-1 . microM-1. Uptake was only slightly reduced when the incubation was performed at 4 degrees C and was independent of sodium concentration. Albumin in the medium reduced IOP uptake. Uptake, however, was always greater than that predicted from the unbound IOP concentration alone. The data indicate that the hepatocyte uptake of IOP occurs by both a passive process and a saturable process. The saturable uptake component depends on an albumin-IOP-hepatocyte interaction. The influence of albumin on uptake occurs possibly by an undefined specific cell surface phenomenon of albumin that promotes uptake of IOP or by enhancement of the diffusibility of IOP across the unstirred layer.

Potential tumor- or organ-imaging agents. 23. Sterol esters of iopanoic acid.

A series of sterol esters of iopanoic acid was synthesized and evaluated for their potential to selectively localize in liver and steroid-secreting tissues for possible application in either computed tomography or nuclear medicine imaging. Unlike free iopanoic acid (1), which was rapidly cleared following intravenous administration to rats, cholesteryl iopanoate (2) was found to accumulate in liver, adrenal cortex, and ovary. At 24 h, the ovary was found to contain the highest concentration of 2. The ability of 2 to accumulate in the above tissues was attributed to its resistance to hydrolysis. Pregnenolone iopanoate (3) and dehydroepiandrosterone iopanoate (4), on the other hand, were shown to reach unusually high concentrations in the adrenal cortex within 0.5 h of administration but declined to much lower levels by 24 h. Lipid extraction of tissues showed 3 and 4 to be susceptible to in vivo hydrolysis, which undoubtedly was a major factor in their clearance from adrenal tissue.

Physiologic pharmacokinetic model of iopanoic acid metabolism in rats.

The kinetics of iopanoate metabolism have been examined using a physiologic and pharmacokinetic model in rats. The kinetics of iopanoic acid concentration in blood and in eight other major tissue distribution compartments have been determined and fitted to computer-generated concentrations based on a well-established pharmacokinetic model. The results of these studies in nonfasted, conscious rats revealed that after gastric administration of the contrast material tissue concentrations never exceed 30 microgram/g even in the liver. In addition, a clear-cut enterohepatic circulation of the drug was noted in the experimental setting and had to be incorporated into a computer-generated model to account for differences in the predicted model as compared to the experimental data. Such data point out the importance of knowledge of pharmacokinetics of a drug for development of more appropriate dosage regimens of older compounds, theoretical design and testing of new compounds, or to explain clinically observed drug-related phenomenon.

Competition between serum albumin and soluble fraction of liver for binding of warfarin and other drugs.

The binding of Warfarin by human serum albumin (HSA) and subcellular fractions from rat liver was investigated to evaluate the roles of such interactions in the pharmacokinetic properties of the anticoagulant. In vitro intracellular distribution studies showed that Warfarin was bound primarily by the soluble fraction of rat liver. Equilibrium dialysis studies were carried out to test the hypothesis that the hepatic extraction of Warfarin and drug interactions between Warfarin and other drugs involved competition between albumin and the soluble fraction of liver. A three compartment dialysis cell was designed and constructed for such studies. Three types of competitive binding interactions were identified. Iopanoic acid displaced Warfarin from HSA resulting in increased Warfarin in the protein-free compartment and in the compartment containing the soluble fraction. On the other hand, tolbutamide displaced Warfarin from HSA to the liver soluble fraction with relatively little effect on unbound anticoagulant. Sulfinpyrazone produced a third type of interaction characterized by displacement of Warfarin from HSA with an increase in the concentration of unbound drug. It was concluded that competitive binding between albumin and soluble liver proteins, is important in the hepatic uptake of Warfarin. The three compartment dialysis cells may be useful to simulate the distribution of drugs and drug combinations between non-dialyzable macromolecules.

Optimal perfusion rate determined for in situ intestinal absorption studies in rats.

Iopanoic acid was used as a model compound to study the effect of the intestinal perfusion rate on the mean absorption clearance. Absorption of iopanoic acid followed first-order kinetics, with a first-order absorption rate constant (ka) linearly dependent on the dry intestinal weight. An absorption clearance--time plot revealed three phases. Phase I represented an equilibration phase, Phase II was a uniform phase, and Phase III was a physiological deterioration of the animal under prolonged anesthesia. The variability in the observations during Phase II of the absorptive clearance--time profiles was assessed statistically, and the minimum occurred at 9.9 microliters/sec (0.594 ml/min). The relation between the coefficient of variance (CV) and the perfusion rate is given by CV = (-5.52 X 10(-5)Q3 + (2.78 X 10(-3)Q2 - (3.87 X 10(-2)Q + 0.243, where Q is the perfusion rate through the intestinal lumen. These studies demonstrate that an optimal flow rate exists for minimizing the variability in in situ absorption studies. The dependency of the absorption clearance on the intestinal perfusion rate appears to conform to the convective diffusion model.