A rapid assay of human thyroid peroxidase activity

Impaired synthesis or action of thyroid hormones (THs) during critically sensitive periods of development can have long term adverse effects on health. Development of rapid assays to identify chemicals that impair THs physiology is an important goal for reducing risks from chemical use. Thyroid peroxidase (TPO) is a key enzyme regulating THs synthesis in thyroid gland and a vulnerable target for chemicals that disrupt THs synthesis. To develop a human-relevant, rapid assay for TPO inhibition, we have engineered two cell lines (CHO and LentiX- 293) to express active human TPO (hTPO) enzyme and applied them in a recently-described assay using a stable fluorescent product (Amplex UltraRed). Assay performance was assessed by comparing activity of 19 reference chemicals with known strong, weak or no TPO inhibitory activity. The assay using hTPO from either cell line consistently identified the relative potency of strong to moderate inhibitors and chemicals known to be inactive. Results were less consistent for chemicals reported to be weak inhibitors of rodent TPO, possibly suggesting some species specificity. Our studies support the use of hTPO from stably transfected cell lines to substitute for animal-derived thyroid microsomes for rapid high throughput screening assays to identify and characterize TPO inhibitors.     

Effects of rose bengal on serum levels of thyroid hormones and thyroid peroxidase activity in male mice

The thyrotoxic effect of Rose bengal (RB) (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein disodium salt; Food Red No. 105) was examined in male (C57BL/6N X C3H/N) F1 mice. They were given drinking-water containing RB at levels of 0 (control), 0.125 and 0.250% for 2 weeks. The effect resulted in decreases in serum levels of 3,5,3'-triiodothyronine (T3) and thyroxine (T4), and slight increases in serum 3,3',5'-triiodothyronine (rT3) levels and thyroid weight, but no difference in the values for the body-weight gain, serum thyroid stimulating hormone (TSH) levels and thyroid peroxidase (TPO) activities. However, the in vitro inhibitory effect of RB on TPO activity was observed by addition of RB to the TPO-catalyzed guaiacol oxidation. These results suggest that RB might have weak goitrogenic properties, inhibiting the peripheral conversion of T4 to T3 and/or inhibiting TPO to lead a decrease of T4 and T3 formation.     

Monoamine oxidase activity and triiodothyronine biosynthesis in human cultured thyroid cells.

1. The proposal that monoamine oxidase (MAO) is a source of peroxide in thyroid hormone biosynthesis has been examined by use of isolated cultured human thyroid cells which retain the ability to secrete triiodothyronine (T3) in response to thyroid stimulating hormone (TSH). 2. The results demonstrated the presence of MAO A and B in human thyroid cells which oxidized 5-hydroxytryptamine and 2-phenylethylamine, respectively, and were selectively inhibited by the MAO inhibitors clorgyline and (-)-deprenyl. 3. Addition of propylthiouracil to the culture system induced a 61% reduction in TSH-stimulated T3 secretion, indicating that the bulk of such secretion apparently derives from de novo iodothyronine synthesis. 4. The MAO A and B substrate, tyramine, was ineffective in stimulating T3 secretion. 5. The selective MAO inhibitors, clorgyline and (-)-deprenyl, alone and in combination, and in the presence and absence of tyramine, failed to inhibit basal as well as TSH-stimulated T3 secretion in cultured human thyrocytes. 6. It is therefore apparent that even though thyroid MAO A and B enzyme reactions result in the generation of H2O2, this H2O2 does not seem to play a significant role in T3 biosynthesis.     

Interaction between organophosphate pesticide exposure and PON1 activity on thyroid function

Organophosphate pesticides are widely used in agricultural purposes. Recently, a few studies have demonstrated the ability of these chemicals to alter the function of the thyroid gland in human. Moreover, the paraoxonase-1 enzyme (PON1) plays an important role in the toxicity of some organophosphate pesticides, with low PON1 activity being associated with higher pesticide sensitivity. This study evaluates the interaction between exposure to organophosphate compounds and PON1 enzyme activity on serum levels of TSH and thyroid hormones in a population of workers occupationally exposed to pesticides. A longitudinal study was conducted on a population of floriculture workers from Mexico, during two periods of high and low-intensity levels of pesticide application. A structured questionnaire was completed by workers containing questions on sociodemographic characteristics and other variables of interest. Urine and blood samples were taken, and biomarkers of exposure (dialkylphosphates), susceptibility (PON1 polymorphisms and activity) and effect (thyroid hormone levels) were determined. Interaction between dialkylphosphates and PON1 polymorphisms or PON1 activity on hormone levels was evaluated by generalized estimating equation (GEE) models. A significant interaction was found between serum diazoxonase activity and total dialkylphosphates (ΣDAP) on TSH levels. Thus, when PON1 activity was increased we observed a decrease in the percentage of variation of TSH level for each increment in one logarithmic unit of the ΣDAP levels. This interaction was also observed with the PON1₁₉₂RR genotype. These results suggest a stronger association between organophosphate pesticides and thyroid function in individuals with lower PON1 activity.     

An improved thyroid hormone reporter assay to determine the thyroid hormone-like activity of amiodarone, bithionol, closantel and rafoxanide

A number of environmental chemicals have been reported to exhibit thyroid hormone-like activity. Since thyroid hormones play a crucial role in development, it is important to identify chemicals in the environment that are capable of endocrine disruption of thyroid hormone homeostasis. In order to detect thyroid hormone-like activity, the growth of pituitary cell lines has been commonly used as a sensitive marker, albeit with limited specificity to thyroid hormones. Reporter gene assays using the thyroid hormone responsive element (TRE) connected to the luciferase reporter gene have also been developed. Thus far however, this type of assay appears to have limited sensitivity compared to cell growth assays. In the present study, we developed a highly sensitive TRE reporter gene assay by using a pituitary cell line, MtT/E-2, and by culturing cells in a serum-free medium. Our assay was developed in order to detect T3 activity at a concentration of 10⁻¹¹ M. This assay identified thyroid hormone-like activity from the antiarrhythmic drug, amiodarone, and from three anti-parasitic drugs, bithionol, closantel and rafoxanide, all commonly used in veterinary medicine. Thyroid hormone-like activity of these compounds was further confirmed by the induction of BCL3 gene expression in MtT/E-2, which is known to be regulated by thyroid hormones. Our improved assay was proved to be a sensitive tool for assessing thyroid hormone-like activity of environmental chemicals.     

Inhibition of thyroid type 1 deiodinase activity by flavonoids.

Some dietary flavonoids inhibit thyroperoxidase and hepatic deiodinase activity, indicating that these compounds could be classified as anti-thyroid agents. In this study, we evaluated the in vitro effect of various flavonoids on thyroid type 1 iodothyronine deiodinase activity (D1). D1 activity was measured in murine thyroid microsome fractions by the release of 125I from 125I-reverse T3. D1 activity was significantly inhibited by all the flavonoids tested; however, the inhibitory potencies on thyroid D1 activity differed greatly among them. A 50% inhibition of D1 activity (IC(50)) was obtained at 11 microM baicalein, 13 microM quercetin, 17 microM catechin, 55 microM morin, 68 microM rutin, 70 microM fisetin, 72 microM kaempferol and 77 microM biochanin A. Our data reinforce the concept that dietary flavonoids might behave as antithyroid agents, and possibly their chronic consumption could alter thyroid function.     

Modulation in vitro of monoamine oxidase activity by thyroid hormones

Experiments in vitro using whole rat brain homogenate revealed that l-thyroxine (T₄) and l-triiodothyronine (T₃) can increase indoleacetic acid (IAA) formation from the oxidative deamination of tryptamine. This effect was dependent upon (a) the homogenate concentration, (b) the hormonal concentration, (c) temperature and (d) pre-incubation of the hormones with the homogenate prior to the addition of trypatime. For T₄, the extent of the increase in IAA production was correlated with the specific activity of the particular homogenate under test but no such correlation existed with T₃. The influence of the thyroid hormones was shown to occur at the monoamine oxidase (MAO) step and appears to be mediated indirectly so as to increase MAO activity. The nature of the interaction is reversible but the precise mechanism(s) involved have not been elucidated. It is postulated that the thyroid hormones and/or their metabolites either suppress an endogenous inhibitor or function to dissociate aggregated MAO. A similar augmentation of IAA production was found in the liver, less in the kidney, and only slight increases were obtained in the heart. Thus, a degree of organ selectivity exists. The present finding describe a new effect of the thyroid hormones which may be of importance in the modulation of monoamine metabolism.     

Diamine oxidase activity and gamma-aminobutyric acid formation in medullary carcinoma of the thyroid

The formation of different metabolites from 14C-putrescine via oxidative deamination has been studied in patients with medullary carcinoma of the thyroid (MCT) and in control subjects. The neoplastic tissue from MCT patients produced in addition to delta 1-pyrroline, large quantities of GABA and some unidentified compound(s), whereas the formation of these metabolites in normal thyroid tissue was almost negligible. Plasma from patients with MCT contained higher diamine oxidase activity than plasma from control subjects, but there was a considerable overlap between the two groups. Determination of the as yet unidentified compound(s) could possibly become a sensitive tool in the diagnosis and follow-up of carcinomas associated with a high diamine oxidase activity.     

Effects of diethofencarb on thyroid function and hepatic UDP-glucuronyltransferase activity in rats.

To examine the mechanism and toxicological significance of thyroidal tumor observed slightly in a long-term rat study with diethofencarb (isopropyl 3,4-diethoxycarbanilate), male Sprague-Dawley rats were fed diethofencarb in diets at concentrations of 0, 5,000 or 20,000 ppm for 3 months. Examinations mainly for thyroid functions including thyroid uptake of 125I, serum thyroid hormone and thyroid stimulating hormone (TSH) level, hepatic UDP-glucuronyltransferase (UDP-GT) activity and histopathological examination in thyroid were performed at week 13. Decreases of body weights and food consumptions were observed at and above 5,000 ppm. Under these conditions, decrease of serum free T4 and increase of serum TSH level were observed only at 20,000 ppm, concurrently with liver weight increase at and above 5,000 ppm and increase of hepatic UDP-GT activity at 20,000 ppm. However, no compound related effects were noted in thyroid weight, thyroid uptake of 125I and gross or histopathological examination in thyroid. These results indicate that the administration of diethofencarb leads to an increase in UDP-GT activity and acceleration of thyroid hormone excretion from the liver. The acceleration causes a decrease in serum free T4 level, triggering the feedback mechanism of the pituitary gland, promotion of TSH release and consequently an increase in serum TSH level. Thus, the slightly higher incidence of thyroid follicular cell tumors observed in the chronic and oncogenicity study with non-genotoxic diethofencarb is considered to be caused by these weak pituitary-thyroid hormonal imbalances. The toxicological significance in humans is extremely low according to the well established facts that the chronic TSH stimulating would not induce thyroid tumors in humans and humans may be less sensitive than rats in regard to the response to goitrogenic stimuli.