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Sulfated metabolites of polychlorinated biphenyls are high-affinity ligands for the thyroid hormone transport protein transthyretin.

Grimm FA, Lehmler HJ, He X, Robertson LW, Duffel MW - Environ. Health Perspect. (2013)

Bottom Line: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4.Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding.All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Graduate Program in Human Toxicology, Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, College of Public Health, The University of Iowa, Iowa City, Iowa 52246, USA.

ABSTRACT

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored.

Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4.

Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB-dependent formation of adenosine-3',5'-diphosphate, an end product of the sulfation reaction.

Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8-16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1.

Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption.

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Chemical structures of PCB sulfates, OH-PCBs, and T4. PCB sulfates used in this study were synthesized as ammonium salts.
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f1: Chemical structures of PCB sulfates, OH-PCBs, and T4. PCB sulfates used in this study were synthesized as ammonium salts.

Mentions: ANS displacement assay. ANS displacement studies have frequently been used to determine dissociation constants for potential ligands of TTR (Cao et al. 2010; Cheng et al. 1977; Smith et al. 1994). We determined Kd values for PCB metabolites and T4 utilizing a modified version of a previously published procedure (Cheng et al. 1977). A solution containing 0.5 µM TTR and 5 µM ANS (total volume 1,000 µL) in phosphate buffer [50 mM sodium phosphate, 100 mM NaCl (sodium chloride); pH 7.4] was titrated with small aliquots of PCB metabolites or T4 (structures shown in Figure 1) using a glass syringe (Hamilton, Reno, NV). The displacement of ANS was monitored by measuring the decrease in fluorescence intensity at 470 nm upon excitation of the molecule at 410 nm in a Spectramax M5 fluorimeter (Molecular Devices, Sunnyvale, CA) [for details, see Supplemental Material, Figure S1 (http://dx.doi.org/10.1289/ehp.1206198)]. Three fluorescence measurements were averaged per determination, and at least three separate determinations were made at each ligand concentration. The protocol was optimized for ligand concentrations of ≤ 2,000 nM, and assays were conducted at 25°C (± 0.2°C) in quartz cuvettes with a 1-cm path length. The fluorescence was corrected for dilution (≤ 4.6% of the total volume) and was found to be unaffected by the solvent of the ligands [0.5 mM NaOH (sodium hydroxide)] and the duration of the assay (see Supplemental Material, Figure S2). The total change in pH was ≤ 0.01 pH units. The concentration of ANS in phosphate buffer was determined spectrophotometrically at 350 nm using a molar extinction coefficient of 6.3 × 103/cm/M (Kolb and Weber 1975). Binding data were evaluated by fitting the means of each determination to both a one-site plus nonspecific binding equation [y = (Bmax1 × x)/(Kd1 + x) + Ns] and a two-site binding equation [y = (Bmax1 × x)/(Kd1 + x) + (Bmax2 × x)/(Kd2 + x)]. In these equations, Kd1 is the dissociation constant for the TTR-ligand complex and Kd2 is the dissociation constant for the TTR complex with two bound ligands, Bmax1 and Bmax2 are the relative changes in fluorescence required to saturate the respective binding sites and Ns is a constant representing low-affinity interactions. In this case, the Ns term includes both the low-affinity second T4 binding site in TTR and any other low-affinity interaction with the protein. The variables x and y represent ligand concentrations and changes in fluorescence (Δ fluorescence), respectively. Best fits for Kd1 were obtained by fitting data in the 0–100 nM range to the one-site plus nonspecific binding equation. In order to determine Kd2 values, all available data points were fit to the two-site binding equation.


Sulfated metabolites of polychlorinated biphenyls are high-affinity ligands for the thyroid hormone transport protein transthyretin.

Grimm FA, Lehmler HJ, He X, Robertson LW, Duffel MW - Environ. Health Perspect. (2013)

Chemical structures of PCB sulfates, OH-PCBs, and T4. PCB sulfates used in this study were synthesized as ammonium salts.
© Copyright Policy - public-domain
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672920&req=5

f1: Chemical structures of PCB sulfates, OH-PCBs, and T4. PCB sulfates used in this study were synthesized as ammonium salts.
Mentions: ANS displacement assay. ANS displacement studies have frequently been used to determine dissociation constants for potential ligands of TTR (Cao et al. 2010; Cheng et al. 1977; Smith et al. 1994). We determined Kd values for PCB metabolites and T4 utilizing a modified version of a previously published procedure (Cheng et al. 1977). A solution containing 0.5 µM TTR and 5 µM ANS (total volume 1,000 µL) in phosphate buffer [50 mM sodium phosphate, 100 mM NaCl (sodium chloride); pH 7.4] was titrated with small aliquots of PCB metabolites or T4 (structures shown in Figure 1) using a glass syringe (Hamilton, Reno, NV). The displacement of ANS was monitored by measuring the decrease in fluorescence intensity at 470 nm upon excitation of the molecule at 410 nm in a Spectramax M5 fluorimeter (Molecular Devices, Sunnyvale, CA) [for details, see Supplemental Material, Figure S1 (http://dx.doi.org/10.1289/ehp.1206198)]. Three fluorescence measurements were averaged per determination, and at least three separate determinations were made at each ligand concentration. The protocol was optimized for ligand concentrations of ≤ 2,000 nM, and assays were conducted at 25°C (± 0.2°C) in quartz cuvettes with a 1-cm path length. The fluorescence was corrected for dilution (≤ 4.6% of the total volume) and was found to be unaffected by the solvent of the ligands [0.5 mM NaOH (sodium hydroxide)] and the duration of the assay (see Supplemental Material, Figure S2). The total change in pH was ≤ 0.01 pH units. The concentration of ANS in phosphate buffer was determined spectrophotometrically at 350 nm using a molar extinction coefficient of 6.3 × 103/cm/M (Kolb and Weber 1975). Binding data were evaluated by fitting the means of each determination to both a one-site plus nonspecific binding equation [y = (Bmax1 × x)/(Kd1 + x) + Ns] and a two-site binding equation [y = (Bmax1 × x)/(Kd1 + x) + (Bmax2 × x)/(Kd2 + x)]. In these equations, Kd1 is the dissociation constant for the TTR-ligand complex and Kd2 is the dissociation constant for the TTR complex with two bound ligands, Bmax1 and Bmax2 are the relative changes in fluorescence required to saturate the respective binding sites and Ns is a constant representing low-affinity interactions. In this case, the Ns term includes both the low-affinity second T4 binding site in TTR and any other low-affinity interaction with the protein. The variables x and y represent ligand concentrations and changes in fluorescence (Δ fluorescence), respectively. Best fits for Kd1 were obtained by fitting data in the 0–100 nM range to the one-site plus nonspecific binding equation. In order to determine Kd2 values, all available data points were fit to the two-site binding equation.

Bottom Line: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4.Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding.All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1.

View Article: PubMed Central - PubMed

Affiliation: Interdisciplinary Graduate Program in Human Toxicology, Department of Pharmaceutical Sciences and Experimental Therapeutics, College of Pharmacy, College of Public Health, The University of Iowa, Iowa City, Iowa 52246, USA.

ABSTRACT

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored.

Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4.

Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB-dependent formation of adenosine-3',5'-diphosphate, an end product of the sulfation reaction.

Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8-16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1.

Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption.

Show MeSH
Related in: MedlinePlus