Identification of Classifiers for Increase or Decrease of Thyroid Peroxidase Activity in the FTC-238/hTPO Recombinant Cell Line

Objective To observe the effect of transforming growth factor β1 (TGF-β1) on thyroid peroxidase activity and the expression of mRNA in primary porcine thyrocytes, and approach its possible mechanism. Methods In vitro cultured porcine thyrocytes were divided into 0 μg/L(control group), 2, 5, and 10 μg TGF-β1/L groups.After cultured for 72 h, the cell survival rate was measured with methyl thiazolyl tetrazolium (MTT) assay and the thyroid peroxidase (TPO) activity was measured by guaiacol method. The expression of TPO mRNA was determined cell survival rates of 2, 5, 10 μg/L groups(85.26%, 75.14% and 63.21%) were significantly lower(all P < 0.05 ).significantly decreased as compared with the control group [(2.143 ± 0.102), all P < 0.05)], and the TPO of TGF-β1, the lower the expression of TPO mRNA [(0.875 ± 0.078), (0.466 ± 0.044), (0.273 ± 0.007)] compared with the control group[(1.419 ± 0.148), all P < 0.05)]. Conclusions The inhibiting effect of TGF-β1 on primary cultured porcine thyrocytes may be related to its mechanism of inhibiting TPO activity and reduced mRNA expression. Key words: Transforming growth factor beta 1 ; thyroid gland; Peroxidase; Genes

Background: Congenital hypothyroidism (CH) is caused by mutations in cysteine residues, including Cys655 and Cys825 that form disulfide bonds in thyroid peroxidase (TPO). It is highly likely that these disulfide bonds could play an important role in TPO activity. However, to date, no study has comprehensively analyzed cysteine mutations that form disulfide bonds in TPO. In this study, we induced mutations in cysteine residues involved in disulfide bonds formation and analyzed their effect on subcellular localization, degradation, and enzyme activities to evaluate the importance of disulfide bonds in TPO activity. Methods: Vector plasmid TPO mutants, C655F and C825R, known to occur in CH, were transfected into HEK293 cells. TPO activity and protein expression levels were measured by the Amplex red assay and Western blotting. The same procedure was performed in the presence of MG132 proteasome inhibitor. Subcellular localization was determined using immunocytochemistry and flow cytometry. The locations of all disulfide bonds within TPO were predicted using in silico analysis. All TPO mutations associated with disulfide bonds were induced. TPO activity and protein expression levels were also measured in all TPO mutants associated with disulfide bonds using the Amplex red assay and Western blotting. Results: C655F and C825R showed significantly decreased activity and protein expression compared with the wild type (WT) (p < 0.05). In the presence of the MG132 proteasome inhibitor, the protein expression level of TPO increased to a level comparable with that of the WT without increases in its activity. The degree of subcellular distribution of TPO to the cell surface in the mutants was lower compared with the WT TPO. Twenty-four cysteine residues were involved in the formation of 12 disulfide bonds in TPO. All TPO mutants harboring an amino acid substitution in each cysteine showed significantly reduced TPO activity and protein expression levels. Furthermore, the differences in TPO activity depended on the position of the disulfide bond. Conclusions: All 12 disulfide bonds play an important role in the activity of TPO. Furthermore, the mutations lead to misfolding, degradation, and membrane insertion.

Effect of triclosan, triclocarban, 2,2′,4,4′-tetrabromodiphenyl ether, and bisphenol A on the iodide uptake, thyroid peroxidase activity, and expression of genes involved in thyroid hormone synthesis

The evolution of peroxidase activity was followed in relation to other parameters of thyroid function in rats when PTU was added to a low iodine diet (LID). Ten days of PTU treatment brings about an equivalent increase in thyroid weight and iodide peroxidase activity at whatever time PTU was added to LID (10, 35, 54 and 80 days). It is shown that the effect of PTU treatment on thyroid weight and peroxidase activity is proportionately higher when PTU is added after 10 days of LID alone than after 80 days. The effect of various durations of PTU treatment was studied more closely when the addition began on day 10 of LID. On day 1 after addition of PTU thyroid weight increased significantly and no significant change was found in peroxidase activity. In contrast 5 days of PTU induced a significant elevation in enzymatic activity which is greater than that of thyroid weight. For a longer period of PTU treatment (10 days or more), the per cent increase of thyroid weight and peroxidase activity evolved parallely. Already on day 1 of PTU treatment very rapid change were noted on T4, T3 and TSH levels; there was a decrease in T3 level (26 ± 5.6 %), while an increase in T4 level (26.4 ± 4.7 %) and TSH level (30 ± 5 %) was observed when the iodine content was still high (about 11 μg/thyroid gland). Thereafter T3 and T4 decreased and stayed at the same level after 10 days of PTU, while TSH increased rapidly and reached a plateau between 18 and 35 days. When PTU was added to LID treatment the increase in TSH was mostly due to a decrease of T3. The per cent increase in serum TSH level is always higher than that of thyroid peroxidase activity.

Mild thyroid peroxidase (TPO) deficiency is rare and can be extremely occult. This study aimed to replenish the phenotypic and genetic spectrum of mild TPO deficiency. Four unrelated patients with progressive goiter were described in this study. Genes associated with congenital hypothyroidism were analyzed and in vitro functional experiments were conducted to evaluate the residual TPO enzyme activities of each mutant. The four patients (age: 5-27 years old) were characterized by progressive goiter, discordant alteration in thyroid hormones with free triiodothyronine (FT3) to free thyroxine (FT4) ratio ranging from 0.557 to 1.012, two with slightly elevated TSH level and two with normal TSH level. Six different mutations of TPO gene were identified including three novel mutations (p.Glu337Lys, p.Ala544Val, and p.Glu641Lysfs∗21). Two mutants (p.Asp224del and p.Ala544Val) with residual TPO activity of 41 and 65% may explain the mild TPO-deficient picture in our study. After levothyroxine (L-T4) therapy, three patients showed gradual decline of FT3 to FT4 ratio and two patients showed reduced thyroid size. Patients with mild TPO deficiency can present with progressive goiter, normal TSH level, and largely reserved TPO activities.

Abstract. Several alterations of thyroid function parameters have been reported in patients treated with phenylbutazone and we have studied the effect of this drug on the intrathyroidal iodine metabolism. An inhibition of the iodide transport expressed in terms of T/M ratios was observed in bovine thyroid slices incubated with high phenylbutazone concentrations. 10−3m produced 72% inhibition whereas lower concentrations showed no significant difference as compared with controls. Iodotyrosine synthesis was affected by 10−4m and 10−5m phenylbutazone. Formation of iodothyronine synthesis was markedly affected between 10−4m and 10−7m phenylbutazone concentrations. Thyroid peroxidase activity was measured by tyrosine-iodinase, triiodide and guaiacol assays. Soluble, pseudosolubilized and crude peroxidase preparations from bovine glands, as well as the soluble enzyme from human thyroids, have shown inhibition of tyrosine-iodinase activity when incubated with phenylbutazone in concentrations ranging from 10−3m to 10−8m, with a Ki of 4 × 10−6m for bovine thyroid peroxidase and of 6 × 10−6m for human soluble peroxidase. Formation of triiodide was affected between 10−3m and 10−8m phenylbutazone concentrations. Guaiacol peroxidation was scarcely affected by the action of the drug. We have concluded that phenylbutazone affects the intrathyroidal iodine metabolism through the inhibition of thyroid peroxidase in concentrations which are usually present in the sera of patients treated with this drug.

The biosynthesis of thyroid hormones requires iodide, thyroid peroxidase (TPO), thyroglobulin (Tg) and H2O2. We have studied two sisters with congenital large goiters and hypothyroidism. Perchlorate tests were negative. Serum T3 and T4 were decreased, TSH was increased and Tg was within the lower limit of normal. Biochemical and molecular studies were performed on goiter samples obtained after surgery. Tg content in both tissues was negligible. Paper chromatography of labeled iodocompounds showed a decrease in T4, and the presence of a pronase/pancreatin-resistant iodoprotein. TPO activity was normal in the tissues. Sephacryl S-300 gel filtration demonstrated labeled iodoalbumin-like protein and the absence of a Tg peak. Salting out studies of soluble protein fraction gave an abnormal pattern. Agarose gel electrophoresis showed the presence of an iodoalbumin-like protein and the absence of Tg in the tissues. This last finding was confirmed by immunoelectrophoresis. The Tg and TPO mRNAs levels were also analyzed. Dot-blot hybridization studies with pM5 (TPO cDNA) and phTgM2 (Tg cDNA) probes showed increased and decreased signals, respectively. The increase in TPO mRNA can be explained as a compensatory mechanism vis a vis an increase in serum TSH caused by decreased serum T3 and T4 due to the impairment in Tg mRNA. The Tg mRNA of both patients was further studied with four different probes covering 5' and 3' regions (phTgM1, phTgB1, phTgB2 and phTgB3). Hybridization was observed with all four probes, thus excluding a dramatic deletion defect. Northern transfer showed a clear signal of hybridization with the phTgB1 probe in the 8-9 Kb range. We may conclude that the biochemical and molecular abnormality of these patients is characterized by a decrease of Tg mRNA and of Tg translation.

In this study we describe a novel mutation of the thyroid peroxidase (TPO) gene that resulted in a total iodide organification defect. TPO activity and thyroxine formation in thyroglobulin in the thyroid gland of the patient were below the limits of detection. However, TPO mRNA was detectable at a similar size and concentration as compared with normal thyroid tissues when measured by Northern blot analysis. Sequence analysis of the TPO gene showed the presence of two mutations, a missense mutation in exon 7 and C insertion in exon 14. These mutations were heterozygous and located in different alleles. The latter mutation has already been reported as one of the mutations of the TPO gene resulting in total iodide organification defect. The former mutation was further analysed by mRNA transfection studies in which mutated mRNA was transfected to CHO-K1 cells by electroporation. The results of transfection studies showed that the cells transfected with mutated mRNA expressed similar size TPO molecules to those of cells transfected with wild-type mRNA but that they lacked TPO activity. The two mutations of the TPO gene resulting in the total iodide organification defect in the patient cosegregated from her parents.

The kinetics of tyrosine iodination and of thyroxine synthesis in thyroglobulin, different reactions catalyzed by the same enzyme (thyroid peroxidase), have been compared. Thyroxine synthesis always began after a lag period of 3-5 min. This lag was constant whatever the rate of iodination; this rate of iodination was increased either by increasing the concentration of iodide or enzyme or by decreasing the concentration of thyroglobulin. Increasing the rate of iodination resulted in increasing the number of iodine atoms incorporated during the lag period. Thus the lag observed for thyroxine synthesis was constant and did not depend on the fact that free iodide or non-iodinated tyrosine residues of thyroglobulin were exhausted before thyroxine synthesis occurred. Finally, it appeared that, whatever the explanation of the lag, the enzyme catlyzes thyroid hormone synthesis at a slower rate than iodination. The existence of a lag also allowed us to prepare thyroglobulin samples with different iodine contents but without thyroid hormones. Thus iodination and thyroxine synthesis could be studied independently and the following results were obtained. 1. Iodotyrosine residues which can couple to form thytoxine are made considerably before coupling occurs. 2. H2O2 is required for coupling of these hormonogenic residues; thus the coupling reaction requires enzymic oxidation of the iodotyrosine residues. 3. In addition a strict requirement for iodide was needed for coupling; the requirement was dependent on the concentration of iodide. Thus iodide, a substrate of the iodination reaction, may also have other effects on the activity of thyroid peroxidase.

Di-(2-ethylhexyl) phthalate (DEHP) is used extensively in many personal care and consumer products, resulting in widespread nonoccupational human exposure through multiple routes and media. Limited studies suggest that exposure to DEHP may be associated with altered thyroid function, but detailed mechanisms are unclear. In order to elucidate potential mechanisms by which DEHP disturbs thyroid hormone homeostasis, Sprague-Dawley (SD) rats were dosed with DEHP by gavage at 0, 250, 500, and 750 mg/kg/day for 30 days and sacrificed within 24 h after the last dose. Gene expressions of thyroid hormone receptors, deiodinases, transthyretin, and hepatic enzymes were measured by RT-PCR; protein levels of transthyretin were also analyzed by Western blot. Results showed that DEHP caused histological changes in the thyroid and follicular epithelial cell hypertrophy and hyperplasia were observed. DEHP significantly reduced thyroid hormones (T3, T4) and thyrotropin releasing hormone (TRH) levels, whereas thyroid stimulating hormone (TSH) was not affected. After exposure to DEHP, biosynthesis of thyroid hormones was suppressed, and sodium iodide symporter (NIS) and thyroid peroxidase (TPO) levels were significantly reduced. Additionally, levels of deiodinases and transthyretin were also affected. TSH receptor (TSHr) level was downregulated, while TRH receptor (TRHr) level was upregulated. Metabolism of thyroid hormones was accelerated due to elevated gene expression of hepatic enzymes (UDPGTs and CYP2B1) by DEHP. Taken together, observed findings indicate that DEHP could reduce thyroid hormones through influencing biosynthesis, biotransformation, biotransport, receptor levels, and metabolism of thyroid hormones.

Thyroid hormone (TH) system-disrupting chemicals can cause adverse effects, including neurodevelopmental deficits. While current in vivo test methods reliably detect disruptions from potent antithyroid drugs such as propylthiouracil (PTU) and methimazole (MMI) through thyroid gland histopathology, they may lack sensitivity for less potent chemicals or those acting through alternative mechanisms. A more systemic, mechanism-based approach may therefore be needed to identify TH system-disrupting capacity. Here, we assessed the effects of the environmental chemicals perfluorooctane sulfonate (PFOS) and tetrabromobisphenol A (TBBPA) on the TH system using in vitro and in vivo approaches. PFOS inhibited the TH transmembrane transporter monocarboxylate transporter 8 (MCT8) and the sodium-iodide symporter (NIS). TBBPA inhibited MCT8, the enzyme activity of all three iodothyronine deiodinases (DIO1/2/3), and thyroperoxidase (TPO). In time-mated rats, in utero and early postnatal exposure to PFOS (0.4 or 0.8 mg/kg bw/day) or TBBPA (250 or 500 mg/kg bw/day) reduced serum TH concentrations in dams and offspring, but neither chemical altered thyroid stimulating hormone (TSH) in offspring. We observed no, or only modest, effects on thyroid gland histology. Both chemicals altered TH metabolism by deiodinase and dehalogenase enzymes and reduced iodine content in the thyroid. In the fetal brain, PFOS increased T3, while TBBPA decreased T4 in the postnatal brain. These findings demonstrate distinct TH-disrupting profiles of PFOS and TBBPA, differing from classic antithyroid compounds PTU and MMI. Our results highlight the need for more comprehensive systems-approaches to capture TH disrupting chemicals acting through diverse mechanisms.

Disruption of the thyroid hormone (TH) system by environmental chemicals poses significant risks to human and wildlife health. Reliable in vitro assays are essential for assessing thyroid peroxidase (TPO) inhibition, which disrupts a key step in thyroid hormone (TH) synthesis, while adhering to 3R principles. This study aimed to compare the sensitivity and specificity of two types of in vitro assays, tyrosine iodination (Tyr-I) detected by HPLC-ICP-MS and Amplex UltraRed (AUR) and its modifications, using human HEK-TPOA7 cells and rat thyroid microsomes. It involved the evaluation of TPO inhibition by 21 chemicals (concentration range 0.002-200µM) from diverse use categories, including industrial pollutants, pesticides, and pharmaceuticals. TPO-inhibition potential was indicated for 14 compounds. The Tyr-I assay, which measures the conversion of L-tyrosine to monoiodotyrosine, demonstrated superior sensitivity by capturing both the peroxidation and iodination steps of TPO activity, with effective concentrations for some human exposure-relevant chemicals (benzophenone 2, resorcinol) in the 15-74nM range confirmed with both human and rat TPO sources. In contrast, the AUR assay detects only the peroxidation step, limiting its ability to fully assess TPO inhibition. The inclusion of sodium iodide (NaI) in the AUR assay significantly enhanced its sensitivity (though it was still lower than in Tyr-I assay), while adding L-tyrosine together with NaI did not. The study documents the advantages and limitations, as well as the application and interpretation potential of the different assays´ variants. It provides valuable information and scientifically sound methodology to support the development of efficient testing strategies for the assessment of thyroid hormone system-disrupting chemicals.

The antithyroid microsomal antibodies found in the serum of patients with autoimmune thyroid disease are directed largely, if not entirely, against thyroid peroxidase (TPO). In this study we used a highly purified, well characterized, large tryptic fragment of porcine TPO (hereafter referred to as purified porcine TPO) to examine possible differences among microsomal antibodies in patients with autoimmune thyroid disease. Antibodies against this TPO preparation and also against a synthetic peptide corresponding to residues 780-793 of the deduced sequence of the native enzyme were compared with microsomal antibodies from patients in immunoblot experiments. The antiporcine TPO and antisynthetic peptide antibodies reacted with crude preparations of human TPO. Binding of serum microsomal antibodies to purified porcine TPO was also found. Purified porcine TPO shows two fragments after gel electrophoresis under reducing conditions: a 59K fragment corresponding to the amino end of the molecule, and two approximately 30K fragments corresponding to the carboxyl end. Using an immunoblot procedure with purified porcine TPO as the antigen, we found that at least two epitopes were involved in microsomal antibody production: one associated with the 59K fragment and the other with the approximately 30K fragment(s). The distribution of serum antibodies against these epitopes differed among the patients, indicating that these antibodies comprise a heterogeneous group. Serum from patients with autoimmune thyroid disease significantly inhibited human TPO activity, raising the possibility that microsomal antibodies may contribute to the impaired thyroid function that occurs in some patients with autoimmune thyroid disease.

BackgroundA case of congenital hypothyroidism with goiter (CHG) in a juvenile French bulldog was identified and hypothesized to be caused by dyshormonogenesis of genetic etiology.ObjectivesTo describe case management, unusual phenotypic aspects, and a CHG‐causing mutation in a French bulldog.AnimalsThyroid tissue and blood from a CHG‐affected French bulldog and 4 normal control dogs and buccal brush samples of 125 French bulldogs were studied.MethodsStandard clinical assessment and laboratory tests were applied. Thyroid peroxidase (TPO) iodide oxidation activity was measured in vitro, and TPO protein was assessed on Western blots. Thyroid peroxidase exons and flanking splice sites were amplified from genomic DNA and sequenced. Thyroid peroxidase cDNA was amplified from thyroid RNA and sequenced.ResultsAt 9 months of age, the affected dog had signs of cretinism, but near‐normal skeletal maturation. The enlarged thyroid glands exhibited noninflammatory fibrosis and aberrant follicular organization. Thyroid peroxidase activity and immunocrossreactive protein were undetectable. There was a T>C mutation of the intron 12 splice donor consensus that caused abnormally spliced mRNA, consistent with absent TPO function. The mutant allele was not observed in 125 clinically normal French bulldogs.ConclusionsPresumptive CHG in a French bulldog with unusual clinical presentation is described. Genetic etiology was confirmed by identifying the underlying TPO mutation.

Relaxin Enhances the Oncogenic Potential of Human Thyroid Carcinoma Cells