Thyroid Hormone Sulfotransferase Research Articles

Maternal 3,3’-Diiodothyronine Sulfate Formation from Guinea Pig Placenta Perfused with 3,3’,5-Triodothyronine

Objective:Serum 3, 3’,5-triiodothyronine (T3) remains low in near-term fetus to prevent the growing fetus from undue exposure to its active catabolic effect in mammals. The present study was undertaken to gain insight in the role of placenta in T3 metabolism, fetal to maternal transfer of T3, and its metabolites by in situ placenta perfusion with outer-ring labeled [125I]-T3 in pregnant guinea pig, a species showing increased sulfated 3, 3’-diiodothyronine (T2S) levels in maternal serum in late pregnancy (term = 65 days), similarly to humans in pregnancy.Materials and Methods:One-pass placenta perfusions performed on pregnant guinea pigs were studied between 58 – 65 days of gestation. In two separate experiments, the umbilical artery of the guinea pig placenta was perfused in situ at 37°C with outer-ring labeled [125I]-T3. Maternal sera and umbilical effluents were obtained for analysis at the end of a 60-minute perfusion, when the steady-state levels of radioactivity were reached in the placenta effluent after 30-minute.Results:Sulfated [125I]-T2S was readily detected in the maternal serum as the major metabolite of T3 following the perfusion of placenta with [125I]-T3, suggesting that placental inner-ring deiodinase and sulfotransferase may play an important role in fetal T3 homeostasis and in the fetal to maternal transfer of sulfated iodothyronine metabolites.Conclusions:The expression of type 3 deiodinase (D3) and thyroid hormone sulfotransferase activity in placenta may play an important role to protect developing organs against undue exposure to active thyroid hormone in late gestation in the fetus. The combined activities of D3 and sulfotransferase promoted a placental transfer of T2S into maternal circulation. The maternal circulation of T2S is fetal T3 in origin and its role as a fetal thyroid function biomarker deserves further evaluations and studies.

Investigating the molecular mechanism of hydroxylated bromdiphenyl ethers to inhibit the thyroid hormone sulfotransferase SULT1A1

Hydroxylated bromodiphenyl ethers (OH-BDEs) have raised great concern due to their potential endocrine disrupting effects on humans. In vitro experiments have indicated OH-BDEs can inhibit the activity of thyroid hormone (TH) sulfotransferases (SULTs); however, the molecular mechanism has not been investigated in depth. In this work, we employed 17 OH-BDEs with five or fewer Br atoms, and performed integrated computational simulations to unravel the possible inhibition mechanism of OH-BDEs on human SULT1A1. The molecular docking results demonstrate that OH-BDEs form hydrogen bonds with residues Lys106 and His108, and the neutral OH-BDEs show comparable binding energies with their anionic counterparts. The further hybrid quantum mechanical/molecular mechanical (QM/MM) calculations unravel a metabolic mechanism of OH-BDEs comprised by proton abstraction and sulfation steps. This mechanism is involved in the SULT1A1 inhibition by some OH-BDEs comprised of three or fewer Br atoms, while other OH-BDEs likely only form ternary complexes to competitively inhibit SULT1A1 activity. Moreover, the effect of the hydroxyl group of OH-BDEs on SULT1A1 inhibition potential follows the order of ortho-OH BDE > meta-OH BDE > para-OH BDE. These results provide an insight into the inhibition mechanism of OH-BDEs to SULT1A1 at the molecular level, which are beneficial in illuminating the molecular initiating events involved in the TH disruption of OH-BDEs.

In silico study for inhibiting thyroid hormone sulfotransferase activity by halogenated phenolic chemicals

Thyroid hormones (THs) are essential to proper growth and development of human bodies. Inhibiting the sulfation metabolism of THs has been demonstrated to be an important way for some environmental pollutants, such as halogenated phenolic compounds, to interfere THs homeostasis, thereby causing health problems. However, the important property characteristics that govern the sulfation inhibition of these chemicals are not well understood, and the experimental data on inhibition potential is limited. In this work, an in silico approach was developed to investigate the structure-activity relationship for their sulfotransferases (SULTs) inhibition. A series of quantum chemical descriptors that quantify the electronic and energy properties of 22 halogenated phenolic compounds have been calculated to establish a predictive model and analyzed their corresponding contributions to SULTs inhibition. Density functional theory (DFT) B3LYP/6−31G** has been employed to optimize molecular geometries to obtain a total of 15 descriptors for every compound. The implementation of linear regression shows three descriptors that represent molecular mass, positive charges on hydrogen atoms, and energy of frontier orbitals strongly correlate with SULTs inhibition potential. This indicates molecular size, hydrogen-bond strength, and nucleophilic-electrophilic reactivity may play important roles in SULTs inhibition. The derived regression model has good statistical performance (r2 = 0.84, rms = 0.35), and different validation strategies indicate it can serve as an efficient predictive tool for other chemicals in application domain but with no experimental data, consequently assisting in their THs sulfation inhibition and health risk assessment.

Disruption of thyroid hormone sulfotransferase activity by brominated flame retardant chemicals in the human choriocarcinoma placenta cell line, BeWo

Brominated flame retardants (BFRs) have been shown to disrupt thyroid hormone (TH) homeostasis through multiple mechanisms, including inhibition of enzymes that regulate intracellular levels of THs, such as sulfotransferases (SULTs). The placenta plays a critical role in helping to maintain TH levels during fetal development and expresses SULTs. This is concerning given that disruption of TH regulation within the placenta could potentially harm the developing fetus. In this study, we investigated the effects of two polybrominated diphenyl ethers (PBDEs), two hydroxylated PBDEs, and 2,4,6-tribromophenol (2,4,6-TBP) on TH SULT activity in a choriocarcinoma placenta cell line (BeWo). BeWo cells were exposed to BFR concentrations up to 1 μM for 1–24 h to investigate changes in basal SULT activity and in mRNA expression of several TH regulating genes. 2,4,6-TBP was the most potent inhibitor of basal 3,3′-T2 SULT activity at all exposure durations, decreasing activity by as much as 86% after 24 h of exposure. BDE-99, 3-OH BDE-47, and 6-OH BDE-47 also decreased 3,3′-T2 SULT activity by 23–42% at concentrations of 0.5 μM and 1.0 μM following 24 h exposures. BDE-47 had no effect on SULT activity, and there was no observed effect of any BFR exposure on expression of SULT1A1, or thyroid nuclear receptors alpha or beta. This research demonstrates that total TH SULT activity in placental cells are sensitive to BFR exposure; however, the mechanisms and consequences have yet to be fully elucidated.

Fasting-induced changes in hepatic thyroid hormone metabolism in male rats are independent of autonomic nervous input to the liver.

During fasting, profound changes in the regulation of the hypothalamus-pituitary-thyroid axis occur in order to save energy and limit catabolism. In this setting, serum T3 and T4 are decreased without an appropriate TSH and TRH response reflecting central down-regulation of the hypothalamus-pituitary-thyroid axis. Hepatic thyroid hormone (TH) metabolism is also affected by fasting, because type 3 deiodinase (D3) is increased, which is mediated by serum leptin concentrations. A recent study showed that fasting-induced changes in liver TH sulfotransferases (Sults) and uridine 5'-diphospho-glucuronosyltransferase (Ugts) depend on a functional melanocortin system in the hypothalamus. However, the pathways connecting the hypothalamus and the liver that induce these changes are currently unknown. In the present study, we investigated in rats whether the fasting-induced changes in hepatic TH metabolism are regulated by the autonomic nervous system. We selectively cut either the sympathetic or the parasympathetic input to the liver. Serum and liver TH concentrations, deiodinase expression, and activity and Sult and Ugt expression were measured in rats that had been fasted for 36 hours or were fed ad libitum. Fasting decreased serum T3 and T4 concentrations, whereas intrahepatic TH concentrations remained unchanged. D3 expression and activity increased, as was the expression of constitutive androstane receptor, Sult1b1, and Ugt1a1, whereas liver D1 was unaffected. Neither sympathetic nor parasympathetic denervation affected the fasting-induced alterations. We conclude that fasting-induced changes in liver TH metabolism are not regulated via the hepatic autonomic input in a major way and more likely reflect a direct effect of humoral factors on the hepatocyte.

Inhibition of Thyroid Hormone Sulfotransferase Activity by Brominated Flame Retardants and Halogenated Phenolics

Many halogenated organic contaminants (HOCs) are considered endocrine disruptors and affect the hypothalamic-pituitary-thyroid axis, often by interfering with circulating levels of thyroid hormones (THs). We investigated one potential mechanism for TH disruption, inhibition of sulfotransferase activity. One of the primary roles of TH sulfation is to support the regulation of biologically active T3 through the formation of inactive THs. We investigated TH sulfotransferase inhibition by 14 hydroxylated polybrominated diphenyl ethers (OH BDEs), BDE 47, triclosan, and fluorinated, chlorinated, brominated, and iodinated analogues of 2,4,6-trihalogenated phenol and bisphenol A (BPA). A new mass spectrometry-based method was also developed to measure the formation rates of 3,3'-T2 sulfate (3,3'-T2S). Using pooled human liver cytosol, we investigated the influence of these HOCs on the sulfation of 3,3'-T2, a major substrate for TH sulfation. For the formation of 3,3'-T2S, the Michaelis constant (Km) was 1070 ± 120 nM and the Vmax was 153 ± 6.6 pmol min(-1) (mg of protein)(-1). All chemicals investigated inhibited sulfotransferase activity with the exception of BDE 47. The 2,4,6-trihalogenated phenols were the most potent inhibitors followed by the OH BDEs and then halogenated BPAs. The IC50 values for the OH BDEs were primarily in the low nanomolar range, which may be environmentally relevant. In silico molecular modeling techniques were also used to simulate the binding of OH BDE to SULT1A1. This study suggests that some HOCs, including antimicrobial chemicals and metabolites of flame retardants, may interfere with TH regulation through inhibition of sulfotransferase activity.

Identification of a novel thyroid hormone‐sulfating cytosolic sulfotransferase, SULT1 ST5, from zebrafish

By employing RT-PCR in conjunction with 3'-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (designated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. Its pH optima were 6.0 and 9.0 with 3,3',5-triiodo-l-thyronine (l-T3) as substrate and 6.0 with beta-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of divalent metal cations on the l-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ exhibited dramatic inhibitory effects, whereas Mn2+ showed a significant stimulation. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity.

Bacterial Expression and Functional Characterization of a Rat Thyroid Hormone Sulfotransferase, ST1B1

At least three forms of phenol sulfotransferase (ST) ST1B1, ST1A1 and ST1C1 are contained in rat livers. To identify the form contributing to the metabolism of 3,3',5-triiodothyronine (T3), functional characterization of these forms was performed by expression in Escherichia coli. ST1B1 and ST1C1 were shown to be active on sulfation towards T3 with high affinity (Km: 44.4 and 25.8 [iM, respectively), whereas ST1A1 had low affinity. In Western blotting using antibodies raised against the individual ST, hepatic contents of each ST were quantitatively determined. ST1B1 showed no clear sex-difference, whereas the level of ST1C1 was higher in adult males than adult females. The content of ST1B1 was 1.4, 6.8 and 10 times higher than that of ST1C1 in adult males, adult females and both sexes of immature rats, respectively. The developmental pattern of ST1B1 was similar to that of ST1A1, but differed from that of ST1C1. These results indicate that ST1B1 and ST1C1 are involved in T3 metabolism in rats and ST1B1 is the constitutive form across sexes and ages.

Molecular cloning, expression, and characterization of a novel mouse liver SULT1B1 sulfotransferase.

A mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3,4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3,3',5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.

Characterization of thyroid hormone sulfotransferases

Sulfation is an intriguing pathway of thyroid hormone metabolism since it facilitates the degradation of the hormone by the type I deiodinase (D1). This study reports the preliminary characterization of iodothyronine sulfotransferase activities of rat and human liver cytosol and recombinant rSULT1C1 and hSULT1A1 isoenzymes. All these enzyme preparations catalyzed the sulfation of—in decreasing order of efficiency—3,3′-diiodothyronine (3,3′-T2)>3,3′,5-triiodothyronine (T3)≈3,3′,5′-triiodothyronine (rT3)>thyroxine (T4). 3,3′-T2 sulfotransferase activity was found to be higher in male than in female rat liver, which has also been shown by others for the expression of rSULT1A1 and rSULT1C1. No sulfation of iodothyronines was observed with rSULT1A1. Different phenol derivatives were found to be potent inhibitors of the sulfation of 3,3′-T2 by native and recombinant sulfotransferases, with pentachlorophenol and 2,4,6-tribromophenol being the most potent. The inhibitions exerted by the different phenols on 3,3′-T2 sulfation by rSULT1C1 correlated better with the effects observed in male than with those in female liver. A strong correlation was also observed between the inhibition profiles of human liver cytosol and hSULT1A1. These results suggest that: (1) rSULT1C1 is an important isoenzyme for the sulfation of thyroid hormone in male rat liver; (2) another isoenzyme with similar properties, perhaps rSULT1B1, is responsible for thyroid hormone sulfation in female rat liver and may also contribute to this process in male rat liver; and (3) hSULT1A1 is an important isoenzyme for thyroid hormone sulfation in human liver.

Uptake and metabolism of 3,5,3'-triiodothyronine and 3,3',5'-triiodothyronine by human liver-derived cells: HepG2 cells as a model for thyroid hormone handling by human liver.

The uptake and metabolism of T3 and rT3 was studied in human liver-derived HepG2 cells. The results showed a saturable, time-dependent, and ouabain-sensitive increase in nuclear bound T3. The effects of ouabain (0.5 mmol/L) and unlabeled T3 (10 nmol/L and 10 mumol/L) were much more pronounced at the nuclear level, suggesting the presence of a nonspecific component in total cellular binding. Nuclear binding of rT3 remained below the detection limit in all experiments. Comparison of rT3 metabolism in HepG2 cells and primary cultures of rat hepatocytes showed an approximately 10-fold lower iodide production in HepG2 cells. Iodide production was decreased in the presence of ouabain and almost absent in the presence of propylthiouracil (100 mumol/L). Our data confirmed the presence of a carrier-mediated uptake system for both T3 and rT3. Metabolism data indicated functional type I deiodinase activity in HepG2 cells, the presence of glucuronidating enzymes, and the absence of thyroid hormone sulfotransferase activity. Based on these data, we propose that HepG2 cells provide an appropriate model for thyroid hormone handling by human liver. In addition, we suggest that in human liver sulfation of thyroid hormone, and therefore deiodination of T3 is of only minor importance.