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Thyroid Receptor Antagonism of Chemicals Extracted from Personal Silicone Wristbands within a Papillary Thyroid Cancer Pilot Study

md_a

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Aug 31, 2015
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The gold standard for measuring human exposure to organic contaminants is via measurement of biomarkers in human blood and urine. However, these assessments are often both difficult and expensive to collect, with high costs and logistical limitations (e.g. scheduling difficulties and small volumes of biospecimens). As such, there is a critical need to develop and validate external, noninvasive sampling devices that can effectively recapitulate human exposures. Silicone wristbands have been increasing in popularity and have shown promise in measuring personal exposure to complex mixtures of volatile and semivolatile contaminants (SVOCs), including polycyclic aromatic hydrocarbons,13 brominated flame retardants and organophosphate esters (BFRs and OPEs, respectively),2,4,5 pesticides,68 and other chemicals,1,7,9 with good performance in recovery and stability tests across a wide range of chemical classes.9 These personal passive samplers can be used to estimate exposure to contaminants via both dermal and inhalation routes10 and can be utilized to create personalized exposure profiles encompassing multiple microenvironments.11 Moreover, measurements from silicone wristband extracts have been demonstrated to correlate well with biomarkers of exposure in human biospecimens (serum and urine),4,5,12 suggesting a robust association between external wristband chemical concentrations and internal chemical concentrations. These samplers also have superior performance in predicting the internal dose for some compounds relative to external exposure methods, such as hand wipes, household dust,5,12 or air monitoring samplers.3 Wristbands have been deployed in several human studies and have been used to evaluate chemical exposures for diverse populations across multiple continents,8,13 suggesting utility in interrogating exposure to chemical mixtures. They also have been successfully utilized with children6,14 and even pets,15,16 demonstrating their versatility.

Many of the contaminants detected by wristbands are considered to be endocrine disrupting chemicals (EDCSs) that can disrupt normal hormone action and contribute to adverse outcomes in humans, wildlife, and laboratory animals.1719 The proper and unimpeded function of hormones is essential to normal development, maturation, and prevention of chronic diseases. While EDCs have been found to disrupt a number of receptor systems, disruption of nuclear receptors is better described than most receptors and has been linked to adverse outcomes at environmentally relevant levels.1925 Notably, chemicals that disrupt thyroid receptor (TR) signaling have been widely reported across the literature. Receptor agonists are infrequently reported in environmental matrices; these typically include endogenous hormones and pharmaceuticals, which have low bioactivities.26,27 Receptor antagonists, in contrast, come from diverse chemical classes and have been widely reported in diverse environmental matrices,28,29 including natural and drinking water sources,30,31 varied source wastewaters,3235 and indoor household dust.36,37 Chemicals that inhibit thyroid signaling have been linked to disrupted thyroid function; impaired neurodevelopment; behavioral modifications;28,3840 metabolic health, adipogenesis, lipogenesis, and thermogenesis;4144 and the development of thyroid cancer.45,46

Thyroid cancer rates have increased approximately 3.6% per year in the US,47 with current incidence rates of greater than 14 per 100,000;48 this is mirrored internationally, with rates rising similarly in most other countries.49 Thyroid cancer is classified into four histological types, including papillary (~80% of cases), follicular (~15% of cases), anaplastic, and medullary.50 While some researchers and clinicians have posited that these increases are due to improved surveillance and diagnostic changes,51 several factors suggest otherwise: (1) there has been an increase in papillary thyroid cancer (PTC) cases even with large tumor sizes that would presumably not be impacted by diagnostic changes, and (2) there has been a parallel increase in thyroid cancer mortality that would be unexpected if there is improved surveillance and diagnosis.52 This worsening public health trend has promoted increased attention to assess potential factors. We have long appreciated a role for environmental contamination in thyroid cancer influence, reporting sharp spikes in incidence following radioactive iodine exposure in Chernobyl53 and Fukushima54 accidents (though importantly, increases in thyroid cancer incidence and mortality were evident before these disasters47) and higher rates of incidence for populations living in volcanic regions.55 These and findings of elevated thyroid cancer incidence near National Priority Contaminated Sites56 have spurred investigations into the role of EDCs, particularly, TR disrupting chemicals. A recent review summarized evidence for elevated thyroid cancer incidence from occupational exposures in various industries (e.g. construction, papermaking and wood processing, agricultural activities, etc.) as well as evidence for diverse SVOCs (particularly, phthalates, bisphenols, and certain heavy metals) in contributing to thyroid cancer incidence.46 The causal mechanism of action for these effects has been posited to be TR disruption.46 In support of this, controlled exposure studies in vivo with well-characterized TR antagonists [including potassium perchlorate, propylthiouracil (PTU), and carbimazole] have demonstrated a causal role in the development and/or progression of PTC.5762 However, research on causative contaminants in the development of thyroid cancer has primarily focused on single contaminants rather than environmentally relevant mixtures.

Thyroid disrupting chemicals also have been implicated in thyroid dysfunction and disease. Similar to cancer incidence, rates of thyroid disease also have been increasing, with >12% of US adults estimated to experience some form of thyroid disease in their lifetimes. In particular, hypothyroidism (characterized by high thyroid stimulating hormone [TSH] and low thyroxine [T4] concentrations) affects approximately 5% of the US population aged ≥12 years,63 and rates have been increasing globally over the last several decades.64,65 We previously demonstrated that >40% of household dust extracts promoted TRβ antagonism in vitro.37 The potency of TRβ antagonism was positively correlated with the serum-free thyroxine (T4) concentrations of residents,37 suggesting that residents in households with more potent TRβ antagonist activities in dust had higher serum T4 levels. Other recent work evaluating companion felines reported that greater tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) concentrations on silicone tags (worn on the collar) were associated with increased incidence of hyperthyroidism in domestic cats (higher free triiodothyronine [T3] and thyroxine [T4] concentrations).15 In sum, these studies provide evidence for a contributory role of TR antagonists in thyroid hormone dysregulation and dysregulated health in exposed humans and animals.

Herein, we conducted a pilot study to evaluate the potential to measure TRβ antagonism of mixtures isolated from silicone wristbands that were worn for one week by adults from central North Carolina who were participating in a research study focused on PTC. One gram sections of wristband were solvent extracted and analyzed via mass spectrometry to quantify a range of SVOCs (BFRs, OPEs, phthalates, and pesticides). Separate wristband extracts were reconstituted in tissue culture media and tested for their ability to antagonize TRβ using a reporter gene assay in human kidney cells. Bioactivity was assessed across a range of extract concentrations and was supported by dual cell viability measurements to ensure toxicity-independent effects. Dose-related responses were observed, and bioactivities (efficacies and potencies) were estimated and utilized to assess associations with SVOCs and PTC case status (i.e. thyroid cancer patient or control). Notably, this is the first study to assess the bioactivity of mixtures of chemicals isolated from silicone wristbands on nuclear receptor activation and may suggest that there are additional benefits from these passive sampling devices.

DISCUSSION​

These results demonstrate for the first time the viability of measuring nuclear receptor bioactivity from silicone wristband extracts worn by study participants. We report that up to 80% of wristband extracts was able to significantly antagonize TRβ at levels up to 100% inhibition of an EC50 T3 agonist, independent of inhibited cell viability. Notably, we measured these bioactivities at extract concentrations of 0.1–1% (of the extract from 1.0 g of wristband) in contact with the cells, as 5–10% concentrations exhibited significant inhibited cell viability. These low concentrations allow for greater use in a range of different bioassays, limiting the need for extracting large sections of the wristbands.

TR antagonism was positively correlated with concentrations of TCIPP, EH-TBB, BBP, DEHP, DEHT, DiNP, and TOTM on wristbands. We also observed significant associations via logistic regressions with BEH-TEBP, TCIPP, and TDCIPP (p < 0.05) promoting increased odds of TR antagonism. A number of phthalate esters have been rigorously demonstrated to act as TR antagonists in diverse models and/or be associated with thyroid dysfunction in humans. DEHT was demonstrated to disrupt thyroxine and TSH levels in a rodent model,72 and direct TR antagonism has been demonstrated in vitro for DEHP,30,73 DiNP,30,74 and BBP.30 While we have not found literature evaluating impacts of TOTM on modulation of thyroid hormone signaling, there is evidence for effects on estrogen receptor α and β activation in a reporter gene assay75 and it demonstrated higher binding affinity to sex hormone binding globulin than dihydrotestosterone did in molecular docking experiments.76 Our laboratory previously reported that these OPEs/BFRs (TCIPP, TDCIPP, EH-TBB, and BEH-TEBP) were incapable of significantly antagonizing TRβ using a stably transfected human construct in human bone cells,37 though there are conflicting results in the literature. Other research supported an absence of activity for both TR α and β in a Chinese hamster ovary cell reporter gene model,77 with some research in a thyroid hormone-dependent cell proliferation assay reporting agonistic effects.78 Conversely, others have reported antagonistic effects for EH-TBB and BEH-TEBP using a stable reporter assay (rat pituitary cells constitutively expressing both TRα/β),79 and the Tox21 database reports TR antagonist activity for TDCIPP. Outside of direct receptor testing, treatment with TDCIPP has been demonstrated to significantly inhibit thyroid hormone concentrations in developmentally exposed80,81 or adult-exposed82 zebrafish and in developmentally exposed chickens83 and disrupt thyroid hormone synthesis and signaling in rodents.84 TCIPP previously has been reported to modulate thyroid-dependent gene expression in chicken embryonic hepatocytes85 and alter thyroid hormone signaling in developmentally exposed chickens.83 BEH-TEBP has been reported to be associated with thyroid hormone (T3/T4) concentrations in humans,86,87 and its metabolite, mono(2-ethyhexyl) tetrabromophthalate (TBMEHP), reduced thyroid hormone concentrations in the rat model88 and deiodinase activity in a rat liver microsome model described previously REF. Developmental exposures to both BEH-TEBP and EH-TBB have been demonstrated to inhibit thyroid hormone (T3/T4) concentrations and thyroid-dependent gene expression in zebrafish.89 As such, while some mechanistic assays do not demonstrate direct receptor antagonism at the level of the receptor, there is evidence for the majority of these phthalates, OPEs, and BFRs directly or indirectly interfering with thyroid hormone signaling as described above.

We also report that concentrations of TCEP, TDCIPP, 4-tBPDPP, B4tBPPP, T4tBPP, DiNP, and TOTM were significantly associated with PTC, with a ~8.5 times greater likelihood of being a case relative to a control with each log increase in DiNP concentrations on wristbands. Multiple studies have demonstrated thyroid cancer cell proliferation in vitro and in vivo90 and also significant associations between DEHP and thyroid cancer incidence and/or malignancy in human cohorts.9193 DEHT was shown to increase thyroid C-cell hyperplasia in female rats chronically exposed to all doses.94 While there is no evidence of direct carcinogenicity for DiNP, it has been demonstrated to promote autoimmune thyroid disease through increased oxidative stress and activation of the Akt/mTOR pathway.95 Notably, previous work from our laboratory reported significant associations between concentrations of TCEP in household dust and PTC, particularly for larger, more aggressive tumors,66 although a separate study measuring urinary TCEP at the time of diagnosis did not report an association.96 We also report a protective effect for BDE-100, which has been observed previously for serum concentrations in two separate studies,97,98 although only in the middle tertile or quartile of exposure and not in the highest exposure groups. We did not observe a significant protective effect in our previous study66 but did report exacerbated risk with increasing BDE-209 exposure, which we did not observe here.

Many of these and/or similar chemicals have been described as TR antagonists. While not statistically significant, the extent of TR antagonism measured herein was also positively associated with PTC. All four TR metrics (efficacy at 0.1 and 1% wristband extract concentration and potency at 10 and 20% antagonism) demonstrated a consistent positive relationship between the PTC status and TR efficacy and potency, suggesting a potential role for TR antagonism in the PTC associations. Thyroid hormones have long had well-appreciated roles in angiogenesis, proliferation, and thyroid cancer,99 with thyroid hormone receptor mutations in particular linked to thyroid hormone resistance and cancer.99 Researchers have previously demonstrated in mice that inducing a dominant negative mutation in the TRβ gene disrupts the thyroid pituitary axis, increases TSH and thyroid hormone concentrations, and subsequently leads to hyperplasia of the thyroid follicular epithelium.100 More detailed analysis of the progression to metastasis of this follicular carcinoma suggested activation of TSH signaling pathways and repression of peroxisome proliferator-activated receptor gamma (PPARγ) signaling,101,102 suggesting that TRβ might act as a tumor suppressor gene. To evaluate a causal role for elevated TSH, a major stimulator for thyrocyte proliferation, wildtype mice were treated with PTU to inhibit thyroid hormones; these mice exhibited enlarged thyroids but no metastatic thyroid cancer,103 suggesting that TSH-induced growth is a prerequisite but not sufficient for metastasis. Notably, a range of thyroid disruption can potentially contribute to thyroid dysfunction, disease, and subsequent development of cancer, including iodide uptake, TSH signaling, deiodination/sulfation/glucuronidation enzyme activity modulation, disruption of transporters, and more.50 There is also an apparent contributory role for PPARγ, as mice with PPARγ insufficiency demonstrated increased cell proliferation and carcinogenesis and treatment with a PPARγ agonist-delayed thyroid cancer progression,41,104 which may help explain the exacerbated cancer risk in obese individuals/animals.105

While no research previously has assessed bioactivities from wristband extracts, a number of studies have measured TRβ antagonism in household dust extracts. Research by our laboratory using a stably transfected human construct in human bone cells reported significant antagonism for 42% of dust extracts,37 while another study from our group reported that 76% of samples exhibited significant TRβ antagonism when tested using the transient transfection reporter assay used in the current experiments, which has a greater dynamic range.36 Other researchers have assessed TR bioactivities in dust extracts, reporting antagonism from both indoor and outdoor environments106 but at considerably higher concentrations than our previous research. We report a similar frequency of antagonism (~80%) to what we detected previously in household dust using the same transient transfection reporter assay, although this was achieved with much lower concentrations of wristband extracts relative to those required for household dust. As such, these extracts could be utilized to interrogate a diversity of nuclear receptors and interactions (agonism and antagonism) that would not be possible using household dust, for which the sample size also can be limiting.

We have previously reported concentrations for a number of these OPEs and BFRs on wristbands in other human cohorts. Levels of BFRs were previously measured on wristbands from a similar geographic region (central NC) in August 2016, with geometric mean concentrations of BFRs ranging from 2 to 56 ng/g wristband.4 Geometric means herein ranged from 3 to 73 ng/g, with ≤2-fold variances observed relative to previous ones. Levels of the OPEs were previously measured in a separate study of children (August 2015 to April 2016) from a similar geographic region.12 This study did not provide geometric means, but comparing median concentrations to those here revealed equivalent levels of TCEP, five-times higher TCIPP levels, two-times higher TDCIPP levels, and three-times lower levels of TPHP on current adult wristbands relative to previous child wristbands.12 Recent collaborative work from our group examined 22 OPEs on wristbands, with concentrations ranging from 20 to 520 ng/g.107 Most OPEs exhibited equivalent concentrations to present ones (within two or three-fold), although we reported 5–15-times higher detection frequencies here relative to previous ones.107 While previous studies have reported the presence and detection of phthalates and pesticides on wristbands, they have not provided quantitative measures per mass of wristband for the purpose of comparison to the values reported here.

We also reported significant inhibition of cell viability by the wristband extracts with increasing concentrations, with 20–40% exhibiting significant inhibition at wristband concentrations of 5 and 10%. Moreover, we reported associations between our indirect measures of toxicity and concentrations of several contaminants in the wristband extracts: TDCIPP was positively correlated with both cell viability measures; DBP, DEHT, and DiNP were positively correlated in the β-gal assay only; and DEHA was positively and cis/trans-permethrin were negatively correlated in the LDH assay only. We previously assessed the toxicity and inhibited cell viability for each of the chemicals examined herein at concentrations up to 10 μM and did not report any significant cell viability impacts, suggesting potent impacts from the mixtures of contaminants isolated from wristbands. Consistent findings of toxicity for TDCIPP between assays suggest a potential causal role for this contaminant. While the use of TDCIPP has increased in the residential indoor environment following the PBDE phase-out in 2005, it has been in use since the 1960s. It was phased out of use in children’s pajamas in 1977 after it was described as mutagenic108 but has become one of the most widely used flame retardants in polyurethane foam over the last 10 years.109

While this research presents some novel information and insights, it is not without several limitations. For cancer associations, it is important to note that PTC is an indolent cancer, with estimated latency believed to be years to decades. Therefore, while we observed significant associations between specific chemicals and the odds of concurrent PTC in this cross-sectional study, these trends should be interpreted with caution. Because our sample size was relatively small and our study population was drawn from central North Carolina, our results may not be generalizable to the broader U.S. population. Nonetheless, we do not anticipate that the sample size or the heterogeneity of our study samples will impact the validity of the comparisons made herein. Moreover, the limited data available for the participants in this study limited our ability to account for a range of other potential confounding variables. Additional research in a larger population with prospective data collection is needed to confirm these findings. While we assessed 49 SVOCs herein, with 36 detected in >60% of wristband extracts, it is likely that there are hundreds to thousands of chemicals that we are exposed to daily. For example, household dust is estimated to contain thousands of chemicals.110,111 As such, it is likely that there are other active constituents that are yet to be identified and measured, and future research should evaluate other potential contributory contaminants via both target and nontargeted analytical methods. A larger cohort would also support the use of a mixture model approach, which might be more informative than examining associations with individual chemicals present in the mixture. It is also possible that some associations observed here are due to co-occurring contaminants or mixture effects, which should be evaluated in future studies. We also appreciate the potential limited application of a direct TR binding screen, particularly given the small ligand binding domain of TRβ relative to other nuclear receptors.112 Previous work has described limited direct TR binding in the Tox21 library,113 though this analysis had less success in evaluating TR binding related to antagonism. These high throughput screens are often limited in species and tissue diversity, potentially limiting their generalizability. However, it should be noted that diverse mechanisms of TR disruption can contribute to thyroid cancer development and progression, as noted above50 and that despite these limitations, we report significant antagonistic effects in our in vitro model.

In closing, this study is the first to demonstrate the potential to measure nuclear receptor bioactivities in mixtures isolated from silicone wristbands. Wristbands provide a completely noninvasive and comprehensive marker for exposure to environmental contaminants present in the home, work, and outdoor environments. Trends observed in TR antagonism are similar to what we observed in our previous study using house dust. However, wristbands may be a better sampling tool, as they help account for exposures across multiple environments (encompassing home, work, and outside life), allow for a greater diversity of bioassay and analytical measurement testing, and are amenable to citizen science projects (stable at room temperature and/or mail back to the study laboratory).9 These passive samplers have been demonstrated to capture both dermal and inhalation exposures10 and reflect significant correlations with internal biomarkers of exposure for a range of contaminant classes. While we have described the ability to utilize wristband extracts to measure TR antagonism, this extraction method and analysis protocol could be broadly applicable to various receptor-based tests and should be explored further in future research. We also report significant associations between concentrations of specific semivolatile chemicals on wristbands and the odds of concurrent PTC and trends between wristband TR antagonism and PTC. These results provide support for the role of TR antagonists in the development and/or progression of PTC. Given the promising literature associating bioactivities derived from human tissues or biospecimens with various human health conditions,114117 using these external noninvasive samplers may present new opportunities to assess potential relationships between contaminant mixtures and human health..

 

Tom K

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Jun 8, 2018
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Thank you for this most interesting paper. While the goal of this research was diagnostic in nature, one cannot help to ponder an intervention application. Specifically, if a silicone wristband can detect levels of chemicals and endocrine disruptors, is there any evidence or hypothetical conclusion that there is a diffusion component to the use of silicone that encourages the release of toxins? If the answer is yes, one can assume there be a to a detoxification benefit to wearing silicone jewelry, patches of silicone on thin skin areas (ventral/flexor surface of the forearm, medial aspect of the upper arm, lower abdomen,etc.?, f
 

catharsis

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Jan 26, 2017
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A place that needs more coconuts and sun
When I was younger, there were these "Live Strong" wristbands that were very popular and in fashion. Some children I knew collected up to 10 or 20 on each wrist in, what I now know, a social hierarchy presenting manner. In my worst schizo tendencies, I would assume that "the powers that be" intentionally encouraged people to buy these intentionally trendy wristbands in a way to collect money and to give all participants EDC-exposure. Scary stuff. Thanks be to Peat for exogenous thyroid and pregnenolone, sun, and good food.
 

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