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Thyroid in a jar: towards an integrated in vitro testing strategy for thyroid-active compounds
Jomaa, B. - \ 2015
University. Promotor(en): Ivonne Rietjens, co-promotor(en): Jac Aarts; Ad Peijnenburg. - Wageningen : Wageningen University - ISBN 9789090290089 - 187
schildklierziekten - schildklierhormonen - hormoonverstoorders - in vitro - assays - celgroei - thyroid diseases - thyroid hormones - endocrine disruptors - cell growth

Jomaa, B. (2015). Thyroid in a Jar: Towards an Integrated In Vitro Testing Strategy for Thyroid-Active Compounds. PhD thesis, Wageningen University, the Netherlands


The aim of this thesis was to find in vitro and toxicogenomics-based alternatives to in vivo thyroid hormone disruption tests. In vitro alternatives can help reduce the amount of animal testing required under the European Union regulation for the registration, evaluation, authorization and restriction of chemicals (REACH). Moreover, with the use of human cell lines and human-identical synthetic proteins, interspecies differences can be reduced and in some cases eliminated. This thesis has shed light on the relevance of current in vitro assays for thyroid and pituitary cell proliferation, has led to the development of the TSH screen, a luminol-based thyroid peroxidase inhibition assay and the zebrafish-based general development score (GDS) for the detection of developmental toxicants, including those that act through the thyroid hormone system. Moreover, the microarray assay for real-time coregulator-nuclear receptor interaction (MARCoNI) assay was used to reveal the modulating effects of thyroid-active compounds on TRα and TRβ interactions with a peptide array representing 66 different coregulators. These developments along with an in-depth analysis of the thyroid hormone system and the presentation of the state of the art in thyroid disruption testing have highlighted the progress made and at the same time have underlined the challenges that lay ahead.

Towards a realistic risk characterization of complex mixtures using in vitro bioassays
Montano Garces, M. - \ 2013
University. Promotor(en): Tinka Murk, co-promotor(en): A.C. Gutleb. - [S.l.] : s.n. - ISBN 9789461736598
risicoschatting - persistente organische verontreinigende stoffen - verontreinigde sedimenten - mengsels - biotesten - in vitro - toxiciteit - schildklierhormonen - risk assessment - persistent organic pollutants - contaminated sediments - mixtures - bioassays - toxicity - thyroid hormones

This thesis aims to better understand and further improve the relevance and reliabilityof in vitro bioassaysfor a biobased risk characterisation of complex mixtures, with special focus on persistent organic pollutants (POPs) in sediments.

In Chapter 1 the importance of complex mixture characterization in modern society is introduced. The methods available, their current advantages and their disadvantages for complex mixture testing are described. With the shift from policy oriented chemical testing towards the inclusion of in vitro bioanalysis, important challenges have to be overcome to ensure a relevant and reliable quantification of the toxic potency of complex mixtures. These challenges are explained in the introduction, including the status of development and validation of those aspects for reliable testing. One of the main advantages that in vitro bioanalysis has to offer is the possibility to quantify the toxic potency of compounds for which chemical analytical methods have not or hardly been developed, for example because standards do not yet exist. Hydroxylated metabolites of POPs are an example of a toxicologically relevant group of compounds that can exert endocrine disrupting effects, but they cannot yet be routinely analysed. A selection of yet unsolved issues are further studied and discussed in this thesis, as outlined in the “approach and structure of the thesis”.

In Chapter 2 a meta-analysis is performed to study the occurrence and relevance of hydroxylated (OH) compounds in humans and wildlife. Reported body burdens of halogenated phenolic contaminants (HPCs), including OH-POP in different tissues from humans and wildlife species, are reviewed in relation to the concentration of their putative parent compounds to be able to reveal relevant exposure routes and sub-populations at risk. Highest OH-POP levels were found in blood plasma, and highly perfused and fetal tissues. Plasma concentrations of analysed known HPCs ranged from 0.1-100 nM in humans and up to 240, 454, 800 and 7650 nM for birds, fish, cetaceans and other mammals, respectively. Reported metabolite blood plasma levels also are compared with relevant toxicological threshold concentrations from toxicological studies, and appeared to fully fall within the in vitro (0.05–10000 nM) and in vivo (3-940 nM) effect concentrations reported for OH-POPs. Given the sensitivity of early developmental stages, and information lacking about the general population, it is advisable to determine HPC background blood levels in children and fetal tissue .

Given the toxicological relevance of the OH-POPs, Chapter 3 aims at providing solutions to the long standing problem of the in vitro production and analysis of OH-POP metabolite thyroid hormone disrupting (THD) potency via binding to plasma thyroid hormone binding proteins (THBPs). In sediments and for example seafood, the POPs occur as parent compounds that would only become THD after metabolisation (hydroxylation). Several methods have shown the competitive thyroxine (T4) T4 displacement potency of pure metabolites. However, in vitro metabolization of, among others, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers(PBDEs) followed by in vitro quantification of their potency has encountered drawbacks related to the co-extraction of compounds disturbing the T4-TTR competitive binding assay. The present study identifies and quantifies the major co-extractants, cholesterol and saturated and non-saturated fatty acids (SFA and NSFA), at levels above 20 μM (20 nmol per mg protein in the incubation mixture) following various extraction methods. A new method is presented to in vitro metabolise parent compounds into OH-metabolites followed by selective extraction of metabolites while four-fold reducing co-extraction of the disturbing compounds. In addition a microplate-format non-radioactive fluorescence displacement assay was developed to quantify the TTR binding potency of the metabolites formed. The effectiveness of the in vitro metabolism and extraction of the OH-metabolites of the model compounds CB 77 and BDE 47 was chemically quantified with a newly developed chromatographic method analyzing silylated derivatives of the OH-metabolites and co-extractants. Due to the mentioned improvements, it is now possible to make a dose-response curve up to 50% inhibition with OH-metabolites extracted from bioactivated CB 77 and BDE 47. Without taking the toxic potencies of bio-activated POPs into account with bioanalysis, the hazard and risk posed by POPs will be seriously underestimated.

The chapters 4 and 5 are committed to tackle the issues of supramaximal (SPMX) responses and sample extract concentration which are crucial to reliably quantify of the toxic potencies of complex mixtures with in vitro bioassays.

A SPMX effect is the phenomenon that compounds induce a maximum response in an assay that is significantly higher than that of the positive control. As the positive control is used to quantify the toxic potency of a sample, this could result in over-estimation of its toxic potency. As this has been most elaborately reported for in vitro estrogenicity assays, a meta-analysis was performed of such assays, compounds and conditions in which the effect is observed (Chapter 4a).For the 21 natural and industrial chemicals that could be identified as SPMX inducers, the culture and exposure conditions varied greatly among and between the assays. Relevant information on assay characteristics, however, sometimes lacked. Diethylstilbestrol (DES), genistein (GEN) and bisphenol A (BPA) were selected to build a database. The meta-analysis revealed that the occurrence of SPMX effects, could be related to a number of specific assay characteristics: 1) the type and concentration of the serum used to supplement the exposure medium; 2) the endpoint used to quantify the estrogenic potency (endogenous or transfected reporter gene), 3) the number of EREs (estrogen responsive elements) used before the reporter gene, and 4) the nature of the promoter’s. There were no indications that solvent concentration in culture, exposure period or cell model influenced the occurrence of SPMX. It is important to understand the mechanism behind this phenomenon because in vitro assays for estrogenicity are used extensively to characterize and quantify the estrogenic potency of compounds, mixtures and environmental extracts.

Several SPMX inducers also have been reported to block cellular efflux pumps in vivo and in vitro (Anselmo et al. 2012; Georgantzopoulou et al. 2013). Therefore it was hypothesized that efflux pump blockers present in environmental matrices could increase the internal concentration of bioassay agonists and thus cause the SPMX. In Chapter 4b this hypothesis was tested by adapting a 96-well plate cellular efflux pump inhibition assay (CEPIA) to the H4IIE rat hepatoma cell line used for the DR.Luc reporter gene assay for dioxin-like compounds. The influence of various environmentally relevant efflux pump inhibitors on the 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) response was tested. Under the DR.Luc assay conditions there was no evidence that P-gp efflux pump inhibitors modified or potentiated the activity of TCDD. Neither genistein nor quercetin, two potent SPMX inducers on ER-mediated assays, induced any signal on the DR.Luc assay, nor influenced the luciferase induction by TCDD. Future work should be focused on testing the consequences of efflux pump inhibition with an AhR-agonist which is a P-gp substrate, as this could result in intracellular accumulation of this AhR-agonist.

It is standard practice to use a high single stock concentration of extracts to further dilute test concentrations from and perform the analysis. However, a high contaminant load in an extract may oversaturate the solubility of the extracted compounds in carrier solvents and overload the clean-up columns which may reduce the efficiency of polyaromatic hydrocarbons (PAHs) elimination from the extract. These problems may cause respectively under- or over-estimation of the quantified dioxin-like toxic potency. Therefore Chapter 5 focuses on the effects of initial stock concentrations, including sonication assisted dissolution and exposure time, on the quantified dioxin-like potency of cleaned nonpolar sediment extracts. Indeed, more than 20 g sediment equivalents (SEQ)/mL DMSO) as initial stock concentrations resulted in underestimation of bio-TEQ levels in the sediments as observed for cleaned nonpolar sediment extracts from various locations in Luxembourg. An overload of extract on clean-up columns caused an over-estimation of the dioxin-like potency at 24 hours of exposure, probably due to limited removal of PAHs that can induce false positive responses in the in vitro assays. Sonication assisted dissolution of the stock before serial dilution strongly reduced the standard variation of the outcomes. Taking into account the aspects revealed in this study, in addition to already described important issues for quality control, the in vitro bioassays based bio-TEQs can be applied in a comprehensive monitoring program to determine whether sediments comply with health and safety standards for humans and the environment. For the generally applied sediment quality criteria, advices are given about maximum initial stock concentrations to achieve reliable bioassay outcomes.

The methods and concepts developed for metabolic activation of compounds in non-polar sediment extracts and in in vitro analysis of the TTR-competitive binding are applied in Chapter 6 to extracts from highly or less contaminated sediments collected in Luxembourg. Nonpolar fractions of sediment extracts were incubated with S9 rat microsomes, and the metabolites were extracted with a newly developed method that excludes most of the lipids to avoid interference in the non-radioactive 96-well plate transthyretin (TTR) competitive binding assay. Metabolic activation increased the TTR binding potency of nonpolar fractions of POP-polluted sediments up to 100 times, resulting in potencies up to 240 nmol T4 equivalents/g sediment equivalent (nmol T4-Eq/g SEQ). Without bioactivation, medium polar and polar fractions also contained potent TTR-binding compounds with potencies from 1.6 to 17 nmol T4-Eq/g SEQ. This demonstrates that a more realistic in vitro sediment THD risk characterization should also include testing ofboth polar and medium polar sediment extracts for THD, as well as bioactivated nonpolar sediment fractions. Without bioactivation THD potency is not observed in nonpolar sediment extracts, although in in vivo experiments PCBs and PBDEs, and not with dioxins or PAHs, have shown to be thyroid hormone disrupting (THD), demonstrating this bio-activation is toxicologically relevant and therefore required for sediment hazard characterisation.

Chapter 7 discusses the implications of our results to improve the relevance and reliability of in vitro bioassay applied for risk characterisation of complex mixtures from sediments and other matrices. The evidence obtained to support the relevance of POP bio-activation is considered both from the exposure perspective as well as the toxicity perspective. Various features of the newly developed methods and knowledge acquired within this PhD project are discussed in relation to in vitro bioassay risk characterization of sediments towards a realistic in vitro bioassay-based risk characterization of complex mixtures. Some important aspects for the inclusion of metabolizing systems within in vitro bioassay are discussed. In addition, alternatives to deal with the SPMX effect and the definition of suitable sample amounts to improve in vitro bioassay reliability are offered. The suitability of the developed approach application is considered for the risk characterization of sediments. Furthermore, an analysis is made to decide whether this thesis have made in vitro bioassays more reliable and relevant for risk characterization of complex mixtures. Finally, it provides some concluding remarks and aspects for further applications and research.

Microsphere-based binding assays for organic pollutants
Meimaridou, A. - \ 2013
University. Promotor(en): Michel Nielen, co-promotor(en): Willem Haasnoot. - S.l. : s.n. - ISBN 9789461735768 - 139
persistente organische verontreinigende stoffen - toxische stoffen - vis - schildklierhormonen - doorstroomcytometrie - screenen - assays - persistent organic pollutants - toxic substances - fish - thyroid hormones - flow cytometry - screening

In this thesis the “proof of principle” of a preliminary “prototype kit” for the screening of dioxin-like PCBs, PBDEs and PAHs in fish combined with two different simplified generic cleanup procedures, according to the fish fat content, is presented. Toxicants such as the PCBs, PBDEs and PAHs and POPs mixtures (such as the technical mixtures of PCBs (Aroclors)) can be detected at relative low levels (with IC50 values 55±15, 2±0.4 and 4±0.5 ppb for the PCB77, PBDE47 and BaP, respectively) in fish samples in a multiplex, simple and inexpensive manner compared to existing techniques. The 3-plex immunoassay can be performed in two different detection platforms: the traditional flow cytometer- and the new imaging-based bead analyzers. The latter system offers a cheaper analysis and it is an easier transportable platform than the flow cytometer.

Development and validation of in vitro bioassays for thyroid hormone receptor mediated endocrine disruption
Freitas, J. de - \ 2012
University. Promotor(en): Tinka Murk; Ivonne Rietjens, co-promotor(en): J.D. Furlow. - [S.l.] : s.n. - ISBN 9789461734372 - 192
hormoonverstoorders - biotesten - in vitro - schildklierhormonen - endocrine disruptors - bioassays - thyroid hormones

Thyroid hormones regulate crucial processes in vertebrates such as reproduction, development and energy metabolism. Endocrine disruption via the thyroid hormone system is gaining more attention both from scientists and regulators, because of the increasing incidence of hormone-related cancers and developmental defects, and the requirement that newly marketed compounds are tested for thyroid hormone disruption. To reduce the number of experimental animals used and to increase the insight into the mechanisms of toxic interference with the thyroid hormone receptor function, we developed and validated functional in vitro bioassays for thyroid hormone receptor-mediated toxicity. These assays enable quick identification and quantification of specific thyroid hormone receptor disrupting potency of compounds and contribute to the further establishment of a battery of in vitro tests for hazard identification of thyroid active compounds.

Thyroid hormones and adult-type Leydig cell development
Rijntjes, E. - \ 2008
University. Promotor(en): Wouter Hendriks; D.G. de Rooij, co-promotor(en): Katja Teerds. - [S.l.] : S.n. - ISBN 9789085049593 - 184
ratten - schildklierhormonen - leydigcellen - testes - biologische ontwikkeling - volwassenen - hypothyreoïdie - hyperthyreoïdie - endocrinologie - dierproeven - rats - thyroid hormones - leydig cells - biological development - adults - hypothyroidism - hyperthyroidism - endocrinology - animal experiments
Alterations in thyroid hormone levels are well known to influence key functions in growth and development. Although in many countries the diet is fortified with iodide, essential for thyroid hormone synthesis, still not all humans have access to fortified diets, leaving a substantial part of the population at risk of developing hypothyroidism. To study the effects of thyroid hormones on testicular development, and Leydig cell development in particular, we prenatally induced hyper- and hypothyroidism in rats and studied Leydig cell development from the neonate up to adulthood. Earlier studies, using 6-propyl-2-thiouracil or high doses of tri-iodothyronine, were discontinued before puberty due to side effects. We used a different approach to investigate the role of thyroid hormone on these processes; hypothyroidism was induced by feeding an iodide poor diet in combination with 0.75 w/v% sodium perchlorate added to the drinking water to inhibit the uptake of iodide by the thyroid. Hyperthyroidism was induced by supplementing the diet with low doses of thyroxine.
Differentiation of stem Leydig cells into the progenitor-type Leydig cells was delayed in both the hypo- and hyperthyroid rats. Peak levels in proliferative activity of the Leydig cells in the hypothyroid rats were reduced, and in both the hyperthyroid as well as the hypothyroid rats the Leydig cell proliferation was prolonged as compared to their euthyroid controls. In the adult hypothyroid rats the size of the Leydig cell population was decreased, while testis weight and seminiferous tubule diameter were not influenced. These results were confirmed by injection of adult hypothyroid rats with ethane dimethyl sulphonate, an alkylating agent which specifically destroys the Leydig cell population, followed by regeneration of the original Leydig cell population to its original size.
If the hypothyroid status was alleviated before 14 days postpartum the Leydig cell proliferation and development followed the pattern of the euthyroid control rats. If the hypothyroid status was alleviated at 28 days postpartum, at the time progenitor-type Leydig cells differentiate into immature-type Leydig cells, the peak levels of proliferative activity were increased as compared to the continuously hypothyroid rats. The period of proliferation, however, was prolonged, as seen in the continuously hypothyroid rats. Mating this first generation hypothyroid males with hypothyroid females resulted in the production of viable offspring, though pregnancy rate and litter size were reduced.
In conclusion, the results of the present studies suggest that euthyroid levels of thyroid hormones are essential for normal neonatal Leydig cell differentiation and proliferation. Taking into account the incidence of hypothyroidism, and the number of young people at risk of developing hypothyroidism due to insufficient iodide uptake, the clinical relevance of hypothyroidism in male gonadal development needs additional attention.

Novel in vitro, ex vivo and in vivo assays elucidating the effects of endocrine disrupting compounds (EDCs) on thyroid hormone action
Schriks, M. - \ 2006
University. Promotor(en): Ivonne Rietjens, co-promotor(en): Tinka Murk; J.D. Furlow. - Wageningen : S.n. - ISBN 9085044847 - 159
in vitro kweek - schildklierhormonen - xenopus laevis - hormoonverstoorders - in vivo experimenten - in vitro culture - thyroid hormones - endocrine disruptors - in vivo experimentation
Detecting the effects of environmentally relevant concentrations of thyroid hormone disrupting compounds on amphibian development
Gutleb, A.C. - \ 2006
University. Promotor(en): Ivonne Rietjens, co-promotor(en): Tinka Murk. - Wageningen : s.n. - ISBN 9085043549 - 168
schildklierhormonen - amphibia - embryonale ontwikkeling - foetale ontwikkeling - xenopus laevis - hormoonverstoorders - thyroid hormones - embryonic development - fetal development - endocrine disruptors
Nog steeds schildklier
Heide, D. van der - \ 2005
Wageningen : Wageningen Universiteit - 18
schildklier - schildklierhormonen - schildklierziekten - jodium - fysiologie - thyroid gland - thyroid hormones - thyroid diseases - iodine - physiology
Tumour promotion by complex mixtures of polyhalogenated aromatic hydrocarbons (PHAHs) and the applicability of the toxic equivalency factor (TEF) concept
Plas, S.A. van der - \ 2000
Agricultural University. Promotor(en): J.H. Koeman; A. Brouwer. - S.l. : S.n. - ISBN 9789058083463 - 150
polychloorbifenylen - toxiciteit - carcinogenese - onderhuidse injectie - schildklierhormonen - retinol - ratten - polychlorinated biphenyls - toxicity - carcinogenesis - subcutaneous injection - thyroid hormones - rats
<p>The aim of the project described in this thesis consisted of two main objectives, first, to examine the tumour promotion potential of complex, environmentally relevant mixtures of polychlorinated biphenyls (PCBs), polychlorinated dibenzo- <em>p</em> -dioxins (PCDDs) and polychlorinated dibenzo- <em>p</em> -furans (PCDFs) and secondly, to evaluate the applicability of the Toxic Equivalency Factor (TEF) concept for the tumour promotion potential of complex mixtures of PCBs, PCDDs and PCDFs. In addition, the effect of sub-chronic exposure to these complex mixtures was determined on endocrine parameters, i.e. the vitamin A and thyroid hormone status, which play an essential role in normal tissue growth and fetal development and are possibly involved in the process of carcinogenesis.</p><p>Carcinogenicity is one of the toxic endpoints in risk assessment of PCBs, PCDDs and PCDFs (WHO, 1992). PCBs, PCDDs and PCDFs are considered as tumour promoters rather than as initiators of carcinogenicity (Safe, 1989; Silberhorn <em>et al.</em> , 1990; Whysner and Williams, 1996). So far, most studies on tumour promotion by PCBs have investigated the potency of single, mostly planar dioxin-like congeners, based on the presumption that the Ah-receptor pathway is also involved in mediating the tumour promoting effects of PHAHs (Safe, 1989; Silberhorn <em>et al.</em> , 1990). There is much less information available on the tumour promoting effects of complex mixtures of PHAHs after sub-chronic exposure.</p><p>The approach in this thesis was to focus on mixtures of polyhalogenated hydrocarbons (PHAHs), both dioxin-like and non-dioxin-like, relevant for the human intake. To reveal underlying mechanisms of possible interactions between PHAH congeners and to determine the toxic potential of the PHAH mixtures, the ethoxyresorufin- <em>O</em> -deethylase (EROD) and the AhR-dependent luciferase reporter gene (DR-CALUX) bio-assays were performed. Both assays are indicators for an Ah receptor mediated, dioxin-like toxicity. The tumour promotion potential of complex PHAH mixtures <em>in vivo</em> , was studied in female Sprague Dawley rats, using the development of altered hepatic foci (AHF) as a parameter in a two-stage initiation/promotion bio-assay introduced by Pitot <em>et al.</em> (1978).</p><h3>Chapter 2</h3><p>In chapter 2, the results of <em>in vitro</em> experiments are described. Interactions between individual mono- or di- <em>ortho</em> PCB congeners and 2,3,7,8-TCDD were studied in the EROD and the DR-CALUX bio-assay, using mouse and rat hepatoma cell lines. In addition, the dioxin-like potential of the PHAH mixtures, designed for the animal experiments, and possible interactions between congeners within the mixtures, was determined in the CALUX assay. Preliminary data are presented on the inhibition of the gap junctional intercellular communication (GJIC), which is seen as an <em>in vitro</em> parameter for tumour promotion, by the PHAH mixtures.</p><p>When individually dosed, the mono- <em>ortho</em> PCBs induced both the EROD and CALUX activity but to a lower maximum and at higher concentrations as compared to TCDD. Co-administration of mono- <em>ortho</em> PCBs and TCDD, decreased the TCDD induced EROD and CALUX activity dose-dependently, with increasing concentrations of the partially antagonistic mono- <em>ortho</em> PCBs. The residual level of the EROD and CALUX induction in case of co-administration, was equal to the maximum inducible activity level of the individual mono- <em>ortho</em> PCB congener. None of the tested di- <em>ortho</em> PCBs was capable of inducing the EROD or CALUX activity. However, all di- <em>ortho</em> PCBs antagonised the TCDD-induced EROD and CALUX activity in a dose-dependent manner and with different potencies. A couple of combined exposures were tested for the inhibition of the GJIC. The results indicated that similar non-additive interactions, as observed in the EROD and CALUX assay, were seen here.</p><p>The PHAH mixtures designed for the first animal experiment ( <em>Chapter 3,4</em> ), induced the CALUX activity up to the maximum activity level as induced by TCDD. These PHAH mixtures were also potent inhibitors of the GJIC. No interactions between individual congeners in the PHAH mixture could be observed, neither in the CALUX assay or on the inhibiton of the GJIC. Interactive effects were shown in the CALUX assay between the PCB fractions designed for the second animal experiment ( <em>Chapter 5</em> ). The 0- <em>ortho</em> and the 1- <em>ortho</em> substituted PCB fraction induced the CALUX activity up to 40% and 9% of the maximum level induced by TCDD respectively, while the 2-4 <em>ortho</em> fraction did not show any induction of the CALUX activity. Co-administration of the fractions inhibited the CALUX activity down to 3% of the maximum level induced by TCDD. The GJIC was only slightly inhibited by the 2-4 <em>ortho</em> and the reconstituted 0-4 <em>ortho</em> fractions.</p><h3>Chapter 3 & 4</h3><p>In the first animal experiment ( <em>Chapter 3,4</em> ), the development of AHF by a complex synthetic mixture of dioxin-like compounds was studied. The composition of this mixture was based on the presence of and relative ratio's between the six most relevant PHAHs in Baltic herring and covered over 90% of the TEQs present. To study possible interactive effects, PCB 153 (2,2',4,4',5,5'-HxCB) was added to the mixture as a representative of the non-dioxin-like, di- <em>ortho</em> substituted PCBs.</p><p>In chapter 3, the toxicokinetic properties of the PHAH congeners are presented. Gas-chromatography and mass-spectrometry (GC-MS) analysis of PHAH concentrations in the liver showed considerable differences in hepatic retention (as percentage of the given dose) between congeners, thereby changing the relative ratios of congeners between external and target dose in favor of the planar compounds. Further, it was shown that addition of PCB 153 to the PHAH mixture increased the hepatic retention of all dioxin-like PHAH congeners in the mixture. This observation is explained by the capacity of PCB 153 to induce Ah receptor levels in the liver, and consequently increase the hepatic level of CYP1A2, which is known to possess a high binding affinity for planar PHAHs.</p><p>In chapter 4, the AHF data are shown. The promotion of AHF was significantly increased after exposure to the PHAH mixtures, but to a lower extent than expected on the basis of the TEQs calculated from the TEF values as proposed by the WHO (Ahlborg <em>et al.,</em> 1994). A difference between the WHO TEF values (Ahlborg <em>et al.,</em> 1994) used for the calculation of the TEQ of the PHAH mixture, and the relative potency (REP) values of the individual congeners that are actually based on AHF data, may partly explain the observed differences in AHF induction between the rats exposed to the equipotent doses of TCDD and the PHAH mixtures. In addition, differences in toxicokinetic properties of the congeners and interactive effects on deposition of the congeners ( <em>Chapter 3</em> ) may have influenced the predicted toxic potency of the PHAH mixture as well. No interactive effects of PCB 153 on the AHF development or EROD induction could be observed.</p><p>It is concluded that the TEF approach predicted the tumour promotion potency of the investigated PHAH mixtures quite well, within a factor of two. An interactive effect between PCB 153 and the planar PHAHs occurred at the kinetic level.</p><h3>Chapter 5</h3><p>Chapter 5 describes the second animal experiment, in which the contribution of non-dioxin-like as well as dioxin-like PCB congeners to the total induction of AHF by a complex PCB mixture was studied. For this purpose the commercial PCB mixture Aroclor 1260 was fractionated into a 0-1 <em>ortho</em> and a 2-4 <em>ortho</em> PCB fraction, which were tested separately and as a reconstituted 0-4 <em>ortho</em> PCB mixture.</p><p>GC-MS analysis of the Aroclor 1260 fractions confirmed that there were no planar, dioxin-like compounds present in the 2-4 <em>ortho</em> PCB fraction. In addition, the 2-4 <em>ortho</em> PCB fraction did not show luciferase induction in the <em>in vitro</em> DR-CALUX bio-assay, indicating that this fraction had no dioxin-like potential ( <em>Chapter 2</em> ). A remarkable finding in the rat study was that the 2-4 <em>ortho</em> PCB fraction explained approximately 80% of the total observed effect on the development of AHF by the 0-4 <em>ortho</em> PCBs present in Aroclor 1260. In contrast to what is generally accepted, the dioxin-like PCB congeners did not significantly contribute to the effect on AHF development. No interactive effect on AHF development or the toxicokinetics was observed for the 0-1 and the 2-4 <em>ortho</em> PCB fraction. PCB 153, incorporated as additional treatment, showed a similar potential to induce AHF development as the 2-4 <em>ortho</em> PCB fraction in Aroclor 1260.</p><p>It was concluded that the TEF concept largely underestimates the tumour promotion effect of complex PCB mixtures, since the tumour promotion potential of the non-dioxin-like PCBs is not taken into account.</p><h3>Chapter 6</h3><p>In chapter 6, the results are shown on the vitamin A and the thyroid hormone status of the rats of the first and second tumour promotion experiment ( <em>Chapter 4,5</em> ).</p><p>From the first experiment it appeared that hepatic retinyl palmitate is a rather sensitive parameter for exposure to dioxin-like PHAHs, as the retinyl palmitate levels were severely decreased after treatment with the PHAH mixtures and to a similar extent as compared to TCDD treatment. However, an opposite effect was observed on the plasma retinol concentration after treatment with the PHAH mixture and TCDD, respectively. In addition, the PHAH mixture caused a relatively strong decrease of the thyroid hormone levels in plasma and decreased the ratio of total thyroxin and free thyroxine as compared to TCDD. The most likely reason for these observations is the formation of hydroxy-metabolites of PCB 118, present in the PHAH mixture, which are known to disrupt the transport-protein complex (RBP-TTR) of retinol and thyroxine and thereby drastically reducing plasma levels of both vitamin A and thyroxine. This situation does not occur in the case of TCDD exposure, which effects the vitamin A and thyroid hormone status mainly via interference with liver metabolism.</p><p>In the second experiment, the retinoid and thyroid hormone levels were not affected significantly. This indicates that in case of exposure to PCBs at environmental levels, no or at best only marginal effects can be expected on the retinoid and thyroid hormone status.</p><p>It was concluded on the basis of these observations that the effects on plasma retinol and thyroxine by complex mixtures of PHAHs are not well predicted by the TEF concept, due to involvement of several different mechanisms and mechanistic interactions depending on the composition of the PHAH mixture.</p><h3>Concluding remarks</h3><h4>Applicability of the TEF concept</h4><p>The most striking finding of this thesis work is that the non-dioxin-like PCB fraction in the commercial mixture Aroclor 1260 explained over 80% of the observed effect on AHF development ( <em>Chapter 5</em> ). On the basis of these results it was concluded that the TEF approach was inadequate in its prediction for the tumour promotion potential of a complex PCB mixture as used in the second animal experiment. However, the tumour promotion potential of the complex dioxin-like PHAH mixture used in the first animal experiment ( <em>Chapter 4</em> ) was quite well predicted by the TEF approach, e.g. within a factor of two. The observed kinetic interaction between the congeners ( <em>Chapter 3</em> ), had apparently no significant consequence for the tumour promotion potential of the PHAH mixture.</p><p>Further it was apparent that the TEF concept failed to predict the effect of the dioxin-like PHAH mixture on plasma retinol and underestimated the effect on the thyroid hormone concentrations ( <em>Chapter 6</em> ). The lack of predictability by the TEF approach for these endocrine effects is possibly due to additional toxicity of hydroxylated PCBs, formed of PCB congeners present in the dioxin-like PHAH mixture.</p><h4>Interactive effects</h4><p>The <em>in vitro</em> experiments ( <em>Chapter 2</em> ) indicated the possibility of interactive effects between dioxin-like PHAHs and mono- and di- <em>ortho</em> PCBs, at the level of Ah receptor binding. However, competition between compounds is only likely to occur under conditions of Ah receptor saturation and a large concentration difference between the dioxin-like PHAH and the mono- and/or di- <em>ortho</em> PCBs. These conditions can be easily reached <em>in vitro</em> but will be seldomly observed <em>in vivo</em> . In the animal experiments the major interaction was observed at the kinetic level, namely, PCB 153 enhanced the hepatic deposition of the dioxin-like PHAHs ( <em>Chapter 3</em> ), most likely by induction of CYP1A2 which is known to have a high binding affinity for dioxin-like PHAHs. No interactive effects, at any level, were observed between the dioxin-like and the non-dioxin-like PCB fraction of Aroclor 1260. This may be explained by the low hepatic retention of the congeners, possibly due to the low TEQ level of the dioxin-like fraction in Aroclor 1260, i.e. no or <strong>a marginal induction of hepatic binding proteins</strong> . At such a low level of hepatic retention interactive effects will not be seen. In terms of TEQs the dioxin-like fraction was certainly more close to environmental levels of exposure as occurs in wildlife and human, leading to the conclusion that kinetic interactions do not play a role at environmental exposure levels.</p><h4>Implications for risk assessment</h4><p>A remaining question is if, on the basis of these results, it can be concluded whether the current approach for risk estimation of complex mixtures of PCDDs, PCDFs and PCBs is appropriate or not. On the basis of a chronic carcinogenicity study performed by Kociba <em>et al.</em> (1978) for TCDD a no-observed-adverse-effect level (NOAEL) of 1 ng/kg/day was derived. A NOAEL for the synthetic dioxin-like PHAH mixture might be close to the NOAEL of TCDD ( <em>Chapter 3,4</em> ), if it is assumed that the dose-response curves for the carcinogenic potential of the PHAH mixtures and TCDD have a similar shape. This indicates that the TEF approach sufficiently predicts the potential risk of exposure to dioxin-like PHAHs.</p><p>For the non-dioxin-like PCBs it is more complicated, since the TEF concept is not applicable for the risk estimation of non-dioxin-like PCBs nor is there another tool available for this purpose. In addition, the total PCB intake is not well known since no congener specific analysis of the occurrence of PCBs in foodstuff is available as well as any information about differences in congener patterns between food items. An extensive survey was done in the Netherlands by Liem and Theelen (1997), who reported an intake of 20 ng/kg/day (sum of 29 PCBs) in 1994, based on a Dutch diet. Given the assumption that a level of 20 ng/kg/day in foodstuff covers approximately 20-30% of the total dietary intake, a daily intake of PCBs of 60-100 ng/kg/day can be estimated of which &gt;90% consists of 2-4 <em>ortho</em> PCBs. In the tumour promotion experiment, a NOAEL for the 2-4 <em>ortho</em> PCBs in Aroclor 1260 was not achieved; the lowest experimental dose of 1 mg/kg bw/week (~140 µg/kg bw/day, <em>Chapter 5</em> ) still enhanced the development of AHF two-fold. Using a factor of 5 for extrapolation from the lowest-observed-adverse-effect-level (LOAEL) to NOAEL, a NOAEL of approximately 30 µg/kg/day can be deduced. For the calculation of a Tolerable Daily Intake (TDI) for dioxin-like compounds, the WHO uses a safety margin of 100. When the same margin is used for the non-dioxin-like compounds a TDI of 300 ng/kg/day can be calculated, which is a factor 3-5 above the presumable daily intake. Although there are many uncertainties in this calculation, there is probably no reason for immediate concern as large safety margins were applied. However, the results of this thesis work demonstrate the necessity for risk assessment to look at both the dioxin-like and non-dioxin-like PCBs.</p><h3>Overall conclusions</h3><p>Overall, the most important conclusion which can be drawn from this thesis work is that the majority of the effect on tumour promotion by PCBs is caused by a non-dioxin-like mechanism of action. Therefore the TEF approach, although useful to predict effects of dioxins and similar compounds, does not predict the tumour promotion potential of complex mixtures of PCBs as being present in the environment. This may have important implications for the risk assessment of complex mixtures of PHAHs as occur in e.g. foodstuff.</p>
Interactions of polyhalogenated aromatic hydrocarbons with thyroid hormone metabolism
Schuur, A.G. - \ 1998
Agricultural University. Promotor(en): Peter van Bladeren; T.J. Visser; A. Brouwer. - S.l. : Schuur - ISBN 9789054859406 - 173
gechloreerde koolwaterstoffen - schildklierhormonen - chlorinated hydrocarbons - thyroid hormones
<p>This thesis deals with the possible interactions of polyhalogenated aromatic hydrocarbons and/or their metabolites with thyroid hormone metabolism. This chapter summarizes firstly the effects of thyroid hormone on the induction of biotransformation enzymes by PHAHs. Secondly, the results on the inhibition of thyroid hormone sulfation by hydroxylated metabolites of PHAH are summarized. Some conclusions and remarks on the overall implications of the results are given at the end of this chapter.</p><p><strong>The effects of thyroid hormone on the induction of biotransformation enzymes by polyhalogenated aromatic hydrocarbons</strong><br/>The first part of this thesis focussed on the question whether or not the PHAH-induced decrease of plasma T4 is an adaptive endocrine response of the animal to cope with the onset of toxic effects by PHAHs. For this purpose, the possible regulatory effect of thyroid hormones on biotransformation enzymes was investigated, using rats differing in thyroid state which were exposed to TCDD or PCBs as model inducers of biotransformation enzymes.</p><p>In <em>Chapter 2</em> , the thyroid state of euthyroid (Eu), thyroidectomized (Tx) and Tx rats in which T3 or T4 levels are restored using osmotic minipumps were compared. The decreased circulatory levels of plasma T4 and T3, the increased pituitary feedback response (plasma TSH levels), as well as changed functional responses (decreased hepatic D1 and malic enzyme activities, and increased brain D2 activities) in Tx rats were largely restored to Eu levels in Tx+T4 rats and, except for plasma TT4 and brain D2 activity, in Tx+T3 rats. These results indicated that the thyroid hormone-replaced Tx rats were valid models to study peripheral effects of TCDD. Three days after exposure to 10 mg TCDD/kg body weight, plasma TT4 and FT4 levels were significantly reduced in Eu rats and in Tx+T4 rats, and plasma T3 was significantly reduced in Tx+T3 but not in Eu or Tx+T4 rats. Hepatic T4 UGT activity was induced by TCDD while T3 UGT activity was only slightly increased in the different exposed groups. These results strongly suggest that the thyroid hormone-decreasing effects of TCDD are predominantly extrathyroidal and mediated by the marked induction of hepatic T4 UGT activity.</p><p>The effects of thyroid state modulation on the induction of detoxification enzymes by TCDD in experimental animals are described in <em>Chapter 3</em> . In all rats, TCDD largely induced CYP1A1/1A2 activity (EROD), CYP1A1 protein content, and CYP1A1 mRNA levels. TCDD exposure also resulted in higher total hepatic cytochrome P450 content, hepatic p-nitrophenol UGT activity, and GST 1-1 protein levels, but had no effect on hepatic NADPH cytochrome P450 reductase activity, overall GST activity and GST 2-2, 3-3, and 4-4 protein levels and iodothyronine sulfotransferase activity. Thyroid state did not affect the total cytochrome P450, and GST activity and protein levels, but slightly decreased CYP1A1/2 activity, NADPH cytochrome P450 reductase activity, PNP UGT activity and iodothyronine sulfotransferase activity were demonstrated in Tx rats, as compared to Eu rats.</p><p>In the second animal experiment, the interaction between thyroid state and PCBs in the regulation of CYP1A1 and CYP2B expression is described ( <em>Chapter 4</em> ). Male Tx Sprague-Dawley rats, Eu rats, and rats made hyperthyroid by infusing T3 were treated with a single ip dose of the CYP2B inducer PCB 153 and the CYP1A inducer PCB 126. The thyroid states of the rats were confirmed by measurement of plasma T4, T3 and TSH and of functional parameters such as hepatic D1 activity, malic enzyme activity and a-glycerolphosphate dehydrogenase activity. Total hepatic cytochrome P450 content was increased by PCB treatment in all groups, but was not affected by thyroid state. NADPH cytochrome P450 reductase activity was decreased in Tx rats and increased in hyperthyroid rats, while PCB treatment had no effect. PCB 126 specifically induced T4 UGT activity, measured in the absence of detergent, and CYP1A activity, protein and mRNA levels, whereas PCB 153 induced T4 UGT activity, measured in the presence of the detergent Brij 56, and CYP2B activity, protein and mRNA levels. Thyroid state, neither hypo nor hyper, significantly affected T4 UGT activity or CYP1A and CYP2B activities, protein or mRNA levels.</p><p>The almost complete lack of response of basal and PCB- or TCDD-induced activities of biotransformation enzymes to changes in thyroid state observed in our studies is in contrast to effects published by others (Kato and Takahashi <em>et al.</em> , 1968; Rumbaugh <em>et al.</em> , 1978; Leakey <em>et al.</em> , 1982; Müller <em>et al.</em> , 1983a/b; Skett, 1987; Yamazoe <em>et al.</em> , 1989; Arlotto and Parkinson, 1989; Murayama et al., 1991; Chowdhury <em>et al.</em> , 1983; Moscioni and Gartner, 1983; Pennington <em>et al.</em> , 1988; Goudonnet <em>et al.</em> , 1990; Williams <em>et al.</em> , 1986; Pimental <em>et al.</em> , 1993). This may be due to differences in strain and sex of the animals, the severity and duration of the hypo- and hyperthyroid states induced as well as the duration and dose of TCDD/PCB treatment. Overall, it can be concluded that hepatic NADPH cytochrome P450 reductase activity is dependent on thyroid state, whereas total cytochrome P450 as well as CYP1A1 and CYP2B together with UGT, GST and sulfotransferase activities show little or no thyroid hormone dependence. These slight effects are unlikely to represent an endocrine adaptation to a chemical stressor (TCDD). Therefore, the PHAH-induced decreased T4 levels , as well as other aspects of PHAH-induced alterations in thyroid hormone metabolism, are most likely a direct reflection of the developing toxicological response of the animals toward PHAH exposure.</p><p><strong>Inhibition of thyroid hormone sulfation by hydroxylated metabolites of polyhalogenated aromatic hydrocarbons</strong><br/>The second part of this thesis focussed on the question whether or not hydroxylated metabolites of PHAHs (PHAH-OHs) are able to inhibit thyroid hormone sulfation <em>in vitro</em> as well as <em>in vivo</em> .</p><p><em>Chapter 5</em> presents the investigations concerning the possible inhibitory effects of PHAH-OHs on iodothyronine sulfotransferase (SULT) activity. Rat liver cytosol was used as a source of sulfotransferase in an <em>in vitro</em> assay with <sup>125</SUP>I-labelled T2 as a model substrate. Hydroxylated metabolites of PCBs, PCDDs and PCDFs were found to be potent inhibitors of T2 SULT activity <em>in vitro</em> with IC50 values in the low micromolar range (0.2-3.8 mM). The most potent inhibitor of T2 SULT activity within our studies was the PCB metabolite 3-hydroxy-2,3',4,4',5-pentachlorobiphenyl with an IC50 value of 0.2 mM. A hydroxyl group in the para or meta position appeared to be an important structural requirement for T2 SULT inhibition by PCB metabolites. Ortho hydroxy PCBs were much less potent and none of the parent PHAHs were capable of inhibiting T2 SULT activity. In addition, the formation of T2 SULT-inhibiting metabolites from individual brominated diphenyl ethers and nitrofen as well as from some commercial PHAH mixtures (e.g. Bromkal, Clophen A50 and Aroclor 1254) by CYP450 catalyzed hydroxylation was also demonstrated.</p><p>Consequently, the inhibition of thyroid hormone sulfation by PHAH-OHs was studied in more detail, investigating isozyme specificity and inhibition kinetics ( <em>Chapter 6</em> ). The difference in inhibition pattern demonstrated for SULT activity present in rat liver and brain cytosol, is probably caused by a difference in isozyme pattern. It was shown that PCB-OHs inhibited T2 sulfation by interacting with the rat isozyme SULT1C1 and an additional isozyme responsible for T2 sulfation in female liver cytosol, probably rat SULT1B1, but not SULT1A1. On the other hand, human phenol SULT1A1 was inhibited by PCB-OHs, but not the human isozyme SULT1A3. In conclusion, we suggested that at least human SULT1A1, and rat SULT1C1 and perhaps rat SUL1B1 are involved in the inhibition of T2 sulfation by PCB-OHs. However, more information is needed about the various isozymes involved in iodothyronine sulfation in humans as well as in rats, before definite conclusions can be drawn.</p><p>Furthermore, it is shown that T2 is a good model substrate for the active hormone T3 when investigating the inhibition of thyroid hormone sulfation by hydroxylated metabolites of PHAHs. The inhibition kinetics strongly suggested that the nature of the T2 sulfation inhibition by PCB-OHs is competitive. To obtain more decisive information, tests with purified isozymes should be performed. It was also demonstrated that PCDD-OHs and PCB-OHs themselves are substrates -albeit poor- for SULT enzymes, which further supports the competitive inhibition of thyroid hormone sulfation by PHAH-OHs.</p><p>To bridge the gap between <em>in vitro</em> experiments using cytosol and the <em>in vivo</em> situation, we investigated the inhibition of thyroid hormone sulfation in hepatoma cell lines ( <em>Chapter 7</em> ). Two PCB-OHs, 4-hydroxy-2',3,3',4',5-pentachlorobiphenyl and 4-hydroxy-3,3',4',5-tetrachlorobiphenyl, together with the known sulfation inhibitor pentachlorophenol (PCP) were tested in the rat hepatoma cell line FaO and the human hepatoma cell line HepG2. PCP inhibited T2 sulfation <em>in vitro</em> in FaO and HepG2 cells, although it was 1000 times less potent in whole cells than in rat liver cytosol. Micromolar concentrations of the two tested PCB-OHs hardly affected T2 conjugation in FaO cells, but reduced T2 sulfate formation in HepG2 cells. Inhibition of T2 sulfation was more pronounced using medium without FCS than in medium with 5% FCS, due to a lower uptake of inhibitor by the cells in the presence of serum, as demonstrated using radiolabeled PCP.</p><p>These <em>in vitro</em> results indicate that hydroxylated PHAHs are potent inhibitors of thyroid hormone sulfation. Since thyroid hormone sulfation may play an important role in regulating "free" hormone levels in the fetus, and hydroxylated PCB metabolites are known to accumulate in fetal tissues after maternal exposure to PCBs, these observations <em>in vitro</em> might have implications for fetal thyroid hormone homeostasis and development.</p><p>The <em>in vivo</em> experiment in which was tested if PHAH-OHs are able to inhibit T2 sulfation, was described in <em>Chapter 8</em> . Pregnant rats were exposed to 25 mg Aroclor 1254/kg body weight or to the well-known phenol sulfation inhibitor PCP (25 mg/kg body weight) from day 10 till day 18 of gestation. Fetuses and dams were sacrificed on gestation day 20 (GD20). PCP and PCB metabolite levels in fetal serum and tissues were high. Aroclor 1254, but not PCP exposure resulted in an induction of hepatic EROD and T4 UGT activity in dams.</p><p>PHAHs are known for their disrupting effects on thyroid hormone metabolism, as shown in Figure 9.1. In this animal experiment, Aroclor 1254 exposure caused an increase in T4 UGT activity, resulting in decreased TT4 levels. Treatment with PCP also resulted in decreased serum TT4 levels, but increased FT4 levels, in dams and fetuses. The ratio FT4/TT4 was increased indicating a reduced plasma TTR binding capacity in fetuses and dams following both treatments. D1 activity in liver decreased in dams and fetuses after treatment with Aroclor 1254 and PCP. This decrease is probably caused indirectly by the lowered T4 levels. D2 activity in brain decreased by exposure to PCP in dams but no effect was found in fetuses, and increased by exposure to Aroclor 1254 in fetuses, with no effect in dams. The increasing D2 activity is a response of the brain to low T4 levels, to maintain the T3 homeostasis.</p><p>The positive control PCP was shown to increase the T2 SULT activity measured in maternal liver and brain cytosol. Studies using varying T2 concentrations and different protein concentrations suggested competitive inhibition of PCP carried over in the <em>in vitro</em> assay as well as true induction of T2 SULT activity. This effect of PCP on thyroid hormone sulfation <em>in vivo</em> apparently did not result in lower levels of the product T4S, since fetal and maternal serum levels of T4S were not changed after treatment with PCP. This negative answer may be explained by an increased availability of substrate (FT4; maternal) together with a reduced D1 activity by PCP treatment, resulting in a reduced enzymatic breakdown of T4S.</p><p>Exposure to Aroclor 1254, which resulted in the formation of hydroxylated metabolites, did not significantly change the T2 SULT activity in maternal or fetal brain or liver cytosol, nor the serum levels of T4S.</p><p>Remarkably, the T3S and T4S levels were very low in fetal rat serum in this study, especially when compared with the reported high iodothyronine sulfate levels in fetal human and sheep serum. This can not be explained by low SULT activity levels or high D1 activity levels in rat fetuses on day 20.</p><p><strong>Overall implications of the observed PHAH effects on thyroid hormone metabolism</strong><br/>PHAHs induce a wide spectrum of toxic effects in rats. Some effects have been suggested to be linked to a hypothyroid situation, such as the "wasting syndrome", decreased feed intake, and increased cholesterol concentrations. Indeed, reduced serum T4 concentrations have been observed following exposure to PHAHs (Bastomsky <em>et al.</em> , 1977; Gorski and Rozman, 1987; Hermansky <em>et al.</em> , 1988; Brouwer, 1989; Beetstra <em>et al.</em> , 1991), and it is tempting to speculate about a relationship between the hypothyroxinemia and the observed toxic responses. However, induction of a hypothyroid situation or a hypothyroxinemia by PHAHs could also be regarded as an adaptive endocrine response to diminish the PHAH-induced toxicity. One argument in support of this interpretation is the observed protective effect of thyroidectomy on TCDD-induced lethality and immune toxicity (Rozman <em>et al.</em> , 1985).</p><p>In this study, it is proposed that the T4 decrease could well have a regulatory role in the induction of hepatic biotransformation enzymes, as was reported before (see <em>Chapter 1</em> ). The present investigations suggest that the lowering effects of PHAHs on T4 levels are only a toxic effect of PHAHs and not an adaptive response to regulate the induction of biotransformation enzymes. The differences with other reports on modulating effects of thyroid hormone state on biotransformation enzymes may be explained by differences in the time and dose of inducers as well as by a difference in hypo- or hyperthyroid state. Nevertheless, the T4 decreases in the hypothyroid animals in our study are similar to the PHAH-induced T4 decreases. Therefore, the model was good enough to investigate our hypothesis.</p><p>The second part of this thesis demonstrated that the sulfotransferase enzyme is another thyroid hormone-binding protein, besides D1 and TTR, which can be competitively inhibited by hydroxylated metabolites of PHAHs. In a relatively narrow range of low micromolar concentrations, PHAH-OHs were able to competitively inhibit T2 SULT acttivity <em>in vitro</em> , in a SULT isozyme and tissue specific manner.</p><p>Studies using a perinatal exposure setup were performed to test inhibition of T2 sulfation <em>in vivo</em> . It was demonstrated that the well-known sulfation inhibitor PCP was able to indeed competively inhibit T2 SULT activity, but also was able to upregulate the sulfotransferase protein amounts. Aroclor 1254 exposure resulted in a slight inhibition of T2 SULT activity, probably caused by hydroxylated metabolites formed. This inhibition, together with lower substrate (FT4) levels found after Aroclor treatment did not result in decreased serum T4S levels, which is probably caused by a concomitantly decreased inactivation route, i.e. a decreased D1 activity, together with a higher availibility of substrate (FT4) after PCP exposure.</p><p>Remarkably, the serum T4S levels in fetal rat are low compared to the levels in sheep and human fetal serum samples (Wu <em>et al.</em> , 1992a/b; 1993a/b; Santini <em>et al.</em> , 1993). This could not be explained by already higher D1 activities or a relatively low sulfation activity in the control fetus around GD20. For this reason, we concluded that the fetal rat probably is not a very good model for humans in terms of investigating the impact of toxic compounds on fetal thyroid hormone sulfation. However, it should be mentioned that, although PHAHs and their metabolites interfere at many sites with thyroid hormone transport and metabolism, the fetus apparently is able to cope with those changes and can keep its homeostasis in T3.</p><p>Another interesting point deduced from this study, is that PCP, which could be a model for PCB-OHs, itself showed effects on thyroid hormone levels and metabolism, indicating the importance of phenolic organohalogens compounds for disrupting effects on the thyroid hormone system. This also indicates that the disrupting effects of PCBs on the thyroid hormone system are for a large part caused by the hydroxylated metabolites formed. The own toxicity of PCB-OHs and related phenolic organohalogens inducing a separate set of effects together with the recently observed high fetal accumulation of hydroxy-PHAHs, give reason to further investigate the potential toxicity of these compounds on thyroid hormone metabolism and transport (see also Figure 9.1). It is worth mentioning that besides the "old" organohalogen pollutants that have been phased out since the 1980's, there is a wide range of new products on the market, such as brominated diphenylethers (PBDEs), chlorinated benzenes, bisphenol A and so on. PBDEs, which are nowadays used as flame retardants, have been demonstrated at increasing levels in our environment (De Boer <em>et al.</em> , 1989; Sellstrom <em>et al.</em> , 1996), and are probably able to cause similar effects as PHAHs. Serum T4 decreases have already been reported in rats after exposure to PBDEs (Darnerud <em>et al.</em> , 1996) or PCDEs (Rosiak <em>et al.</em> , 1997). Also, hydroxylated metabolites of PBDEs have been found to competitively inhibit the T4 binding to TTR <em>in vitro</em> (Meerts <em>et al.</em> , 1998).</p><p>The human diet contains a diverse spectrum of naturally occuring and xeno-compounds that affect thyroid hormone metabolism. These include the organohalogens and related contaminants, and in addition, a large number of food components. Flavones and flavonoids have been reported to interfere with thyroid hormone binding proteins such as D1 (Auf'mkolk <em>et al.</em> , 1986; Cody <em>et al.</em> , 1989) and TTR (Lueprasitsakul <em>et al.</em> , 1990; Köhrle <em>et al.</em> , 1986). Flavonoids such as quercetin were similarly found to be able to inhibit phenol sulfotransferase activity <em>in vitro</em> (Walle <em>et al.</em> , 1995; Eaton <em>et al.</em> , 1996), and also other food additives were potent inhibitors of phenol sulfation (Bamforth <em>et al.</em> , 1993). The potential adverse human health impact of these compounds depends on a number of factors, including dietary intake, metabolism and pharmacokinetics, compound potency, serum concentrations, relative binding to serum proteins, and interactions or cross-talk with other endocrine pathways. In a risk evaluation, it should be taken into account that humans are exposed to a mixture of compounds with effects on thyroid hormone metabolism. If the mechanism of interference is similar for all these classes of compounds, the effects might very well be additive, or interactive. Additionally, the very persistent PHAHs are probably of more importance from a risk assessment point of view than the natural food components having a higher degradation rate.</p><p>The effects of PHAHs on the thyroid hormone system in this study have been obtained in rats, are the results relevant for the human situation. Occupational or accidental exposure to high levels of PCBs or PBBs results in changes in serum T4 levels as was found by Bahn <em>et al.</em> (1980), Kreiss <em>et al.</em> (1982), Murai <em>et al.</em> (1987), and Emmet <em>et al.</em> (1988). Moreover, in pregnant women exposed to background levels of PHAHs mainly through diet, a significant negative correlation was observed between human milk levels of PHAHs and plasma T4 and T3 levels (Koopman-Esseboom <em>et al.</em> , 1994). In addition, increases in plasma TSH and both increases and decreases in plasma T4 levels were found in newborn babies following exposure to increasing PHAH levels through in utero and lactational transfer (Pluim <em>et al.</em> , 1993; Koopman-Esseboom <em>et al.</em> , 1994). Besides, prenatal exposure to PCBs is related to disorders in neurological development of children, found in some in epidemiologic studies (Rogan <em>et al.</em> , 1986; Jacobson <em>et al.</em> , 1990). It still is however not clear if these effects of PHAHs on thyroid hormone levels and metabolism may have possible effects on (brain) development.</p>
Thyroid hormones and iodide in the near-term pregnant rat
Versloot, P. - \ 1998
Agricultural University. Promotor(en): D. van der Heide. - S.l. : Versloot - ISBN 9789054858065 - 133
metabolisme - schildklierhormonen - jodide - hormonale controle - voortplanting - ontwikkeling - schildklier - bevruchting - zwangerschap - geboorte - ratten - metabolism - thyroid hormones - iodide - hormonal control - reproduction - development - thyroid gland - fertilization - pregnancy - birth - rats
<p>Thyroid hormones, thyroxine (T4) and 3,5,3'-triiodothyronine (T3), are produced by the thyroid gland. To synthesize thyroid hormones the thyroid needs iodide. The uptake of iodide as well as the production and secretion of T4 and T3 by the thyroid gland is regulated by thyrotropin (TSH), which is produced by the pituitary. However, most of the biologically active form, T3, is produced from T4 via monodeiodination in peripheral tissues.</p><p>This reaction is catalyzed by the deiodinases, type I (ID-I) in liver and kidney, and type II (ID-II) in the central nervous system and brown adipose tissue (BAT). T4 and T3 concentrations differ in the various tissues, like the contribution of T3 produced locally from T4. A large portion of the T3 produced in the liver enters the circulation, whereas T3 produced in the brain and cerebellum is mainly used locally.</p><p>The production, distribution and transport of thyroid hormones are influenced by several (patho)physiological conditions. In this study we concentrated on the effects of pregnancy on maternal thyroid hormone metabolism. It is well known that thyroid hormones are very important for normal fetal development, especially of the central nervous system. During development thyroid hormones produced by the mother, mainly T4, contribute to the fetal thyroid hormone pools before and also after onset of fetal thyroid function. Insufficient production of maternal thyroid hormones during pregnancy can result in permanent brain damage in the offspring.</p><p>At the end of gestation the concentrations of T4 and T3 in maternal plasma and tissues have decreased. In order to gain more insight into the effects of pregnancy on the production, distribution, and transport of thyroid hormones in the mother we performed kinetic experiments with T4 and T3 using nonpregnant and near-term pregnant rats (chapter 2). A bolus injection of [125I]T4 and [131I]T3 was given, and blood samples were taken at regular times during the next twenty-four hours.</p><p>Physiological para-meters of the production, interpool transport, distribution and metabolism of T4 and T3 were estimated by means of a three-compartment model. According to this model three compartments can be distinguished: 1. the plasma; 2. the fast pool; and 3. the slow pool. Liver and kidney are considered to be the main components of the fast pool, whereas skin, muscles and brain belong to the slow pool.</p><p>In the near-term pregnant rat the production and distribution of T4 remained unchanged. The transport of T4 from plasma to the fast pool was more than tripled, whereas transport to the slow pool remained constant. We suggest that in the near-term pregnant rat available T4 was distributed between the maternal and fetal compartments by means of very fast transport. This hypothesis is based on the fact that it seems unlikely that the transport of T4 to maternal liver and kidney, which are considered to be the main components of the fast pool, will have increased that much in the near-term pregnant rat. This was confirmed by the results of steady-state, double isotopic experiments using nonpregnant and near-term pregnant rats (chapter 3).</p><p>In this study, the rats received a continuous simultaneous infusion of [125I]T4 and [131I]T3 in order to achieve equilibrium in all tissues. With this method it was possible to calculate the T4 and T3 concentrations, the relative contributions of plasma-derived vs. locally produced T3, the thyroidal T4 and T3 secretion rates, and the plasma-to-tissue ratios for T4 and T3. Indeed, the transport of T4 to liver and kidney, as well as almost all other maternal organs, was diminished. Since the production of T4 remained unchanged this implies that T4 is transported to another compartment, i.e. the feto-placental compartment. This compartment was not measured in these studies.</p><p>The plasma appearance rate for T3 remained constant in the near-term pregnant rat. This was accomplished by an increase in the secretion of T3 by the thyroid and a decrease in locally produced T3. Less T3 was transported from plasma to liver, kidney, BAT and pituitary. ID-I activity in liver, and ID-II activity in the brain both increased during pregnancy. However, this did not result in an increase in the local conversion of T4 to T3 in these tissues. In the liver the contribution of T3 produced locally remained constant, while in the brain even a decrease was found.</p><p>The insufficient availability of T4 in maternal tissues, as demonstrated by the lower T4 concentrations, might explain the discrepancy between deiodinase activities and the local production of T3. The transport of T4 to the feto-placental compartment resulted indirectly in a deficiency of T3 in the maternal organs. We can conclude that pregnancy affects maternal thyroid hormone metabolism. The mother has to share the available thyroid hormones, especially T4, with the fetuses.</p><p>Iodide is an essential element for the synthesis of thyroid hormones. In rats the fetal thyroid is capable of producing thyroid hormones on day 18 of gestation. Iodide is transported across the placenta from the maternal to the fetal circulation. In chapter 4 we assessed iodide uptake by the maternal thyroid, while the iodide uptake by the fetal thyroid was estimated. We measured the in vivo uptake of 125I by the thyroid continuously. By using the specific activity of iodide in the urine we were able to calculate the absolute iodide uptake in the thyroid.</p><p>Pregnancy resulted in a decrease in the absolute thyroidal iodide uptake. On day 20 of pregnancy the fetal thyroid is already capable of concentrating iodide. However, the difference in absolute iodide uptake by the maternal thyroid, compared to nonpregnant controls, cannot fully be explained by the transport of iodide to the fetal compartment and/or the mammary glands. The decrease in iodide uptake by the maternal thyroid has no impact on the thyroidal production of thyroid hormones.</p><p>Iodine deficiency can lead to disturbed physical and mental development. In large populations in the world iodine intake is marginally deficient. For this reason a marginal iodine deficiency, instead of the more common severe iodine deficiency, was induced in our rats. We used this model to study the effects of marginal iodine deficiency on iodide metabolism (thyroidal iodide uptake; chapter 4) and thyroid hormone metabolism (kinetic experiments; chapter 5) in near-term pregnant rats.</p><p>The absolute iodide uptake by the maternal thyroid was not affected by marginal iodine deficiency. The decreased plasma inorganic iodide was compensated by an increase in thyroidal clearance. A similar compensation was not found for the fetus; the uptake of iodide by the fetal thyroid decreased by 50 % during marginal iodine deficiency. During this marginal iodine deficiency plasma T4 and T3 remained constant in nonpregnant as well as near-term pregnant rats. The production rate and the plasma clearance rate for T4 were both decreased.</p><p>No effects of marginal iodine deficiency on pool sizes and transport rates were found for nonpregnant rats. In the near-term pregnant rat marginal iodine deficiency resulted in a marked decrease in the transport of T4 from plasma to the fast pool. For T3 an increase in the production rate and plasma clearance rate was found for nonpregnant, marginally iodine- deficient rats, while these parameters were slightly decreased in near-term pregnant rats. Marginal iodine deficiency induced a 50 % decrease in the interpool transport rates of T3 between plasma and the fast pool in near-term pregnant rats. The hepatic activity of ID-I was increased as a result of marginal iodine deficiency in nonpregnant as well as near-term pregnant rats.</p><p>On the basis of the results of thyroid hormone studies in normal pregnant rats (chapter 2 and 3) we suggest that during marginal iodine deficiency less maternal T4 is available for the fetal compartment. Together with the lower uptake of iodide by the fetal thyroid this can lead to diminished levels of thyroid hormone of maternal and fetal origin in the fetal organs. In this case, marginal iodine deficiency will have a negative effect on fetal development, especially of the brain.</p><p>Another situation which irreversibly affects fetal brain development is maternal hypothyroidism. Two different levels of hypothyroidism were induced in female rats, by giving thyroidectomized rats two different doses of T4 and T3. The effects of hypothyroidism on maternal thyroid hormone metabolism in near-term pregnant rats (kinetic experiment, chapter 6) were studied. Plasma T4 and T3 levels were very low severely hypothyroid animals, whereas only plasma T3 was decreased in the mildly hypothyroid group. Even during this mild hypothyroidism profound alterations in the transport rates of T4 were found compared to intact, pregnant rats. The transport of T4 from plasma to the fast pool was decreased. Therefore, it appears that even during mild hypothyroidism the transport of T4 to the feto-placental compartment is affected.</p><p>In conclusion: Pregnancy seriously affects the maternal thyroid hormone status. Despite an unchanged thyroidal production of T4, all maternal T4 tissue levels are decreased. Less T4 is available for the mother because of the transport of T4 to the feto-placental compartment. Indirectly this results in a T3-deficiency in most maternal organs. During marginal iodine deficiency and maternal hypothyroidism the transport of maternal T4 to the feto-placental compartment is diminished, whereas during marginal iodine deficiency the availability of iodine for fetal thyroid hormone syn-thesis is also decreased. Eventually this can result in impaired development of the fetal central nervous system.</p>
Thyroid hormone binding proteins as novel targets for hydroxylated polyhalogenated aromatic hydrocarbons (PHAHs) : possible implications for toxicity
Lans, M.C. - \ 1995
Agricultural University. Promotor(en): J.H. Koeman; A. Brouwer. - S.l. : Lans - ISBN 9789054854302 - 152
polychloorbifenylen - dioxinen - pentachloorfenol - organische halogeenverbindingen - gehalogeneerde koolwaterstoffen - schildklierhormonen - polychlorinated biphenyls - dioxins - pentachlorophenol - organic halogen compounds - halogenated hydrocarbons - thyroid hormones
<p>Some toxic effects caused by polyhalogenated aromatic hydrocarbons (PHAHs) develop through alterations in the reproductive and thyroid hormone regulatory systems, thereby affecting (brain) development, reproduction and behaviour of several species (Stone, 1995, Birnbaum, 1994, for review: Brouwer <em>et al.</em> , 1995, Peterson <em>et al.</em> , 1993). In this thesis we have focused on the effects of different classes of PHAHs, eg. polychlorinated biphenyls (PCBs), dibenzofurans (PCDFs) and dibenzo- <em>p</em> -dioxins (PCDDs) and their hydroxylated metabolites on thyroid hormone homeostasis. These changes seem to be partly caused by Ah-receptor mediated changes in thyroid hormone glucuronidation, and effects on the thyroid gland affecting hormone production and secretion. However, hydroxylated metabolites of PCBs, PCDFs and PCDDs may have an additional effect on thyroid hormone transport. Previous studies (Brouwer, 1987) have shown that exposure to 3,3',4,4'- tetrachlorobiphenyl (TCB) can disturb the plasma transport of thyroxine (T <sub>4</sub> ) and retinol in rats through specific competition of a hydroxylated metabolite, 4-OH-3,3',4',5-tetraCB, with T <sub>4</sub> for the thyroid hormone binding site of transthyretin (TTR), the major thyroid hormone transport protein in rodents. This observation raised the question if structurally related hydroxylated PHAH- metabolites could interact with TTR in the same way, as well as with other thyroxine binding proteins, like thyroxine binding globulin (TBG) and type-1-deiodinase (ID-1), subsequently disturbing thyroid hormone transport and metabolism. Special attention was also paid on the structure-activity relationships of hydroxylated PHAH metabolites for binding to TTR by using <em>in vitro</em> and in vivo studies and X-ray crystallographic structure analysis,</p> <p><em>In vitro</em> studies on interactions of hydroxylated PHAH metabolites with thyroxine binding proteins</p> <p>In <em>in</em> vitro studies the interactions of several hydroxylated PHAH metabolites with 3 different T <sub>4</sub> binding proteins, eg. TTR, thyroxine binding globulin (TBG) and type-1-deiodinase (ID-1), were investigated (Chapter 2, 3 and 4). The inhibition of T <sub>4</sub> binding to TTR by hydroxylated PHAH metabolites was studied using <em>in vitro</em> T <sub>4</sub> -TTR bindingstudies. These studies revealed the structural requirements for competition of T <sub>4</sub> binding to TTR by hydroxylated PHAH metabolites: para- or meta-hydroxylation on one or both phenylrings, with one or more adjacent chlorine substitutions (Chapter 2). PHAH metabolites with these structural characteristics showed a remarkable resemblance to T <sub>4</sub> the natural ligand for TTR, consequently displacing T <sub>4</sub> from the T <sub>4</sub> binding site of TTR. Both non-planar, ortho-chlorinated hydroxylated PCB metabolites and rigid, planar hydroxylated PCDF or PCDD metabolites could inhibit T <sub>4</sub> -TTR binding. However, the ortho-hydroxylated PHAH metabolites and parent PHAH compounds, like TCDD, 2,3,3',4,4'-pentaCB and 3,3',4,4'-tetraCB could not inhibit T <sub>4</sub> -TTR binding <em>in vitro.</em></p> <p>In subsequent <em>in vitro</em> studies, a wide range of hydroxylated PHAH metabolites did not inhibit T <sub>4</sub> binding to TBG, the major plasma thyroid hormone transport protein in man (Chapter 3). This indicates that ligand interactions with TTR or TBG are clearly different. Additional studies with iodothyronine derivatives, showed that tri-iodophenol and to a lesser extent di-iodotyrosine could inhibit T <sub>4</sub> -TTR binding but not T <sub>4</sub> -TBG binding in vitro. Finally, the enzymatic activity of hepatic ID-1, which plays a role in the (in)activation of thyroid hormones, could be competitively inhibited mainly by di-para-hydroxylated, meta-halogenated PHAH metabolites while mono-hydroxylated PHAH metabolites were 10 to 100 times less potent (Chapter 4). The differences between the structural requirements of hydroxylated PHAH metabolites for interactions with TTR, TBG and ID-1, are in line with previous studies in which related hydroxylated PHAH compounds or iodothyronine derivatives were used. In conclusion, specific hydroxylated PHAH metabolites can disturb T4-TTR interactions, or inhibit ID-1 activity in vitro, indicating that hydroxylated PHAH metabolites may play an additional role in the observed disturbances in thyroid hormone transport and metabolism after PHAH exposure <em>in vivo</em> .</p> <p>In vivo studies on effects of Aroclor 1254 and TCDD on thyroid hormone transport and metabolism</p> <p>Two <em>in vivo</em> experiments were carried out to study role that both disturbances in plasma T <sub>4</sub> transport and hepatic T <sub>4</sub> metabolism caused by hydroxylated PHAH metabolites play in the observed decreases in plasma T <sub>4</sub> levels. Rats were exposed to Aroclor 1254, a commercial mixture containing persistent and metabolisable PCB congeners (Chapter 5) or the persistent 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) (Chapter 6).</p> <p>In adult Wistar rats, that were exposed to a high dose of Aroclor 1254, plasma T <sub>4</sub> levels were decreased on day 3 and day 8 (Chapter 5). In addition, high levels of a single hydroxylated PCB metabolite, eg. 4-OH-2,3,3',4',5-pentaCB, were detected in plasma of the rats on day 8, while lower concentrations of this metabolite were present in the blood on day 3. However, the T <sub>4</sub> binding capacity in plasma was decreased only in the high dosed group on day 8 but not on day 3, indicating a threshold level for the hydroxylated PCB metabolite to disturb T <sub>4</sub> -TTR binding. On both day 3 and 8, hepatic cytochrome P450 1A1 levels and activity were induced, which is essential for the formation of hydroxylated metabolites. Hepatic T <sub>4</sub> glucuronidation was induced simultaneously. The decreased plasma T <sub>4</sub> levels found in all exposure groups could therefore be attributed to both disturbed plasma T <sub>4</sub> transport and/or induced T <sub>4</sub> glucuronidation. No significant changes in plasma T <sub>3</sub><em></em> levels were found following Aroclor 1254 treatment. In addition, hepatic ID-1 activity was not decreased, suggesting that the <em>in vitro</em> inhibition by hydroxylated PCB metabolites does not occur <em>in vivo.</em> However in an earlier study Adams et al. (1990), <em>in vivo</em> exposure to the easily metabolisable 3,3',4,4'-tetraCB or persistent TCDD could inhibit ID-1 activity. The levels of PCB metabolites with the required structure for inhibition of hepatic ID-1 activity were possibly too low (Chapter 4) liver after Aroclor 1254 exposure <em>in vivo.</em></p> <p>Another remarkable finding was the selective retention of a single specific hydroxylated PCB metabolite, 4-OH-2,3,3',4',5-pentaCB, in plasma of rats exposed to a complex mixture of PCB congeners (Fig. 1). This was the result of the presence of PCB-congeners that strongly induce cytochrome P4501AI activity and PCB-congeners that could easily form hydroxylated PCB metabolites in the Aroclor 1254 mixture, and the strict selectivity of the TTR present in plasma retaining only hydroxylated PCB metabolites that meet the structural requirements as described in Chapter 2. Surprisingly the 4-OH- 2,3,3',4',5-pentaCB metabolite which was formed and selectively retained in plasma, has a hydroxy group on the highest chlorinated ring, in contrast to the expected formation of mainly metabolites with a <em>para</em> - or <em>meta</em> -hydroxy group on the least chlorinated ring. However, the inhibition potencies of T <sub>4</sub> -TTR binding for the 4-OH-2,3,3',4',5-pentaCB metabolite (Chapter 5) and the structurally related 4'-OH-2,3,3',4,5'-pentaCB metabolite (Chapter 2) were almost similar. There is no clear indication yet on the mechanism of selective retention of the 4-OH-2,3,3',4',5-pentaCB in rat plasma, although pharmacokinetics and tissue levels of the presumed parent compounds 2,3,3',4,4'- pentaCB (CB 105) or 2,3',4,4',5-pentaCB (CB 118) may play a role.</p> <p>No detectable levels of hydroxylated metabolites were found by GC-MS analysis of plasma extracts of rats at both day 3 and 8 following exposure to the persistent TCDD, although cytochrome P4501A1 levels and activity were markedly induced (Chapter 6). In addition no unequivocal decrease in T <sub>4</sub> -TTR binding in plasma of TCDD-exposed rats was observed. However, hepatic thyroid hormone metabolism was clearly altered: T <sub>4</sub> glucuronidation and brain type-2-deiodinase (ID-2) activity were increased and hepatic ID-1 activity was decreased, which may explain the observed plasma T4 reductions in the TCDD exposed rats. These changes in thyroid hormone homeostasis suggest a hypothyroxinemic state of the TCDD-exposed rats, although no decreased plasma T4 levels were found. The decrease in ID-1 activity after TCDD exposure was not likely to be caused by hydroxylated metabolites, as was described in Chapter 4, but may be caused by direct effects of TCDD on ID-1 activity or by the assumed hypothyroid state of the TCDD exposed rats.</p> <p>While Aroclor 1254 exposure disturbed T4 plasma transport and increase T <sub>4</sub> glucuronidation (Chapter 5), TCDD exposure only enhanced the hepatic elimination of T4 (Chapter 6), leading to decreased plasma T <sub>4</sub> levels. Although the in vivo studies described in this thesis indicate two different mechanisms for decreases in plasma T <sub>4</sub> levels after PHAH exposure, we can not exclude a third possible mechanism since several studies described changes on thyroid gland histology and thyroid hormone secretion after exposure to Aroclor 1254, TCDD and related compounds.</p> <p>Structural basis for interactions of hydroxy-PCB metabolites with TTR</p> <p>The selective retention of a specific hydroxylated PCB metabolite <em>in vivo</em> (Chapter 5), inspired us to look into the interactions of hydroxylated PCB metabolites with TTR in more detail, and to try to find a structural basis for the structural requirements for TTR binding as described in Chapter 2. X-ray crystallographic analysis of a complex of TTR with a hydroxylated PCB metabolite, eg. 4,4'- (OH) <sub>2</sub> -3,3',5,5'-tetraCB, refined to a 2.7 A resolution, revealed a hydrogen bond formation between a para-hydroxy group of the metabolite with the paired Serine 117 amino acid residues, present in the centre of the TTR binding channel (Chapter 7). The location of this hydroxylated PCB metabolite, deep in the binding channel, and the hydrogen bond formation could explain the stronger TTR binding affinity of this metabolite than the natural ligand T <sub>4</sub> . The chlorine atoms present on the meta-positions of the 4,4'-(OH) <sub>2</sub> -3,3',5,5'-tetraCB, metabolite, fitted easily in the T <sub>4</sub> -iodine binding pockets present in the TTR binding channel. Additional computer-assisted graphics modelling studies on the interactions of several hydroxylated PCB metabolites with TTR, showed that hydroxy groups present on meta-positions could also form hydrogen bonds with the paired Serine 117 residues in the centre of the binding channel (Chapter 7). Furthermore, the modelling studies showed no significant differences between the interactions of hydroxylated PCB metabolites and the structurally related pentachlorophenol (PCP) with TTR, although <em>in vivo</em> studies by others indicated the disruption of the complex of retinol binding protein (RBP) and TTR after binding of a hydroxylated PCB metabolite but not by PCP in rodents. In conclusion, the detailed structural studies described in Chapter 7 confirmed the necessity of para- or meta- hydroxylation and adjacent chlorine substituents as structural elements of hydroxylated metabolites of PCBs and related compounds for interactions with TTR (Chapter 2), leading to selective retention of a specific hydroxylated PCB metabolite in plasma of rats exposed to a complex PCB mixture <em>in vivo</em> (Chapter 5).<br/> </p> <p><strong><em>Concluding remarks</em></strong></p> <p>The outcome of the present study clearly reveals the structural requirements that are essential for interactions of PHAH metabolites and other related chemicals for interactions with TTR. Hydroxylated PHAH metabolites can structurally resemble the thyroid hormone T <sub>4</sub> Overall the structural requirements for TTR interaction were hydroxysubstitution on the <em>para</em> - or <em>meta</em> positions of one or both of the phenyl rings, with adjacent chlorine substitutions, herewith confirming some of the suggestions for TTR interactions of related hydroxylated PHAH metabolites by Rickenbacher <em>et al</em> . (1986). Especially the observation by X-ray crystallographic structure analysis that a hydrogen bond could be formed in the TTR binding channel upon binding of a hydroxylated PCB metabolite, provides strong evidence that <em>para</em> - or <em>meta</em> -hydroxylation of the PHAH compound forms an essential prerequisite for binding to the T4 binding site of TTR. No interactions with TTR were found for the tested parent PHAH compounds, contradictory to earlier suggestions of McKinney <em>et al.</em> (1985) which were based mainly on computer modelling and few <em>in vitro</em> binding studies (Rickenbacher <em>et al</em> , 1986). It should be noted that graphics modelling may show that parent PHAH compounds may fit the TTR binding site, but gives little information on binding affinity to TTR. In an affirmative <em>in vitro</em> T <sub>4</sub> -TTR binding assay, several parent PCB congeners (3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 3,3',4,4',5,5'-hexaCB, 2,3,3',4,4'-pentaCB, 2,2',5,5'- tetraCB), TCDD and Aroclor 1254, a commercial PCB mixture, were tested at high concentrations and exhibited no inhibition of T <sub>4</sub> -TTR binding (unpublished data).</p> <p><img alt="Fig. 1." src=""/></p> <p>PHAH metabolites that are predicted to have high binding affinities for TTR are indeed detected in plasma of rodents experimentally exposed to PCBs or PCDFs (Morse <em>et al</em> ., 1995b, Koga <em>et al</em> ., 1990, Kuroki <em>et al.</em> , 1993). For instance, exposure to a complex mixture of PCBs, Aroclor 1254, led to the selective retention in blood of a single PCB metabolite, 4-OH-2,3,3',4',5- pentaCB, which met the structural requirements for TTR binding (Bergman <em>et al</em> ., 1994).</p> <p>On the basis of the proposed structural requirements we can give an explanation for the interactions of related compounds with TTR as found by others, for instance pentachlorophenol (Van den Berg, 1990, Van Raay <em>et al</em> ., 1994, Den Besten <em>et al</em> ., 1991), natural compounds like flavones and halogenated aurones (Cody, 1989; Ciszak <em>et al</em> ., 1992) and certain drugs like milrinone (Wojtczak <em>et al.</em> , 1993). Moreover, these structural insights make it possible to predict whether other related classes of environmental contaminants can interact with TTR. In addition, due to combined exposure in the environment, one may expect additivity of binding to TTR of hydroxylated PHAH metabolites with other structurally related environmental or natural compounds.</p> <p>An important question is whether these experimental data can be extrapolated to other species. Two factors are essential for the TTR mediated selective retention of hydroxylated PHAH metabolites namely the presence of TTR in blood and the formation of relevant PHAH metabolites. It is most likely that hydroxylated. metabolites can be formed in many species other than rodent. Recently hydroxylated PCB metabolites were identified in human and seal plasma, which were environmentally exposed to background PCB levels (Bergman <em>et al.</em> , 1994). The major metabolites were again the 4-OH-2,3,3',4',5-pentaCB metabolite and to a lesser extent 4-OH-2',3,3',4',5-pentaCB in seal and human plasma and 4-OH-2,2',3,4',5,5',6-heptaCB in human plasma. Thus the hydroxylated PCB metabolites detected in vivo completely matched the structural requirements for TTR binding. The PHAH metabolite patterns in plasma of both experimentally and environmentally exposed animals and humans are species specific and depend not only on the structural requirements for binding to TTR, but also on the exposure situation and the capacity of biotransformation of PHAHs of the species.</p> <p>The species-specific metabolism of PHAHs decreases in the order: terrestrial mammals&gt;aquatic mammals&gt;birds&gt;fish (Safe, 1989). Several mammalian and avian species, like rats, seals, porpoises and eiderducks could form hydroxylated metabolites in <em>in vitro</em> microsomal incubations with the model substrate 3,3',4,4'-tetraCB (TCB). However fish, like trout and flounder, could not metabolise TCB, although cytochrome P4501A-like activity, responsible for biotransformation of planar PHAHs, can be induced (Murk <em>et al.,</em> 1994, Morse <em>et al.,</em> 1995c, Ishida <em>et al.,</em> 1991).</p> <p>The selective retention of specific hydroxylated PHAH metabolites in plasma through binding to TTR is expected in species that both can metabolise PHAHs and posses TTR as a plasma thyroid hormone binding protein. TTR is a evolutionary conservative protein present in plasma of not only rodents but also other placental mammals, birds and to a lesser extent in reptiles. No TTR was detected in the lower species like fish and amphibians. In higher mammals like man, however, not only TTR but also thyroxine binding globulin (TBG), is present in blood as a primary thyroid hormone transport protein. In conclusion, hydroxylated PHAH metabolites can be formed and selectively retained by TTR in blood of a wide variety of species.</p> <p>The toxicological consequences of the TTR mediated selective retention of hydroxylated PHAH metabolites in plasma are not yet fully understood. The TTR protein plays a primary role in the transport of thyroid hormones in the blood of many species. Although TTR binds less T <sub>4</sub> than TBG in human serum, TTR may be responsible for much of the immediate delivery of T <sub>4</sub> and T <sub>3</sub> to cells due to the lower binding T <sub>4</sub> affinity. Furthermore TTR is important for the transport of retinol in blood by forming a complex with retinol-binding protein (Robbins, 1991).</p> <p>Disturbances in thyroid hormone plasma levels are found in several species experimentally or environmentally exposed to PHAHs, like rodents, seal (Brouwer <em>et al.,</em> 1989) and man (Koopman Esseboom <em>et al.,</em> 1994), species in which hydroxylated PCB metabolites were also present in plasma. Disruption of thyroid hormone homeostasis after exposure to PHAHs can however be caused by at least two mechanisms, eg. the disturbed plasma T <sub>4</sub> transport through competitive binding of hydroxylated PHAH metabolites to TTR, but also the Ah-receptor mediated induction of T <sub>4 </sub> glucuronidation by parent compounds.</p> <p>The possible disruption of the TTR-RBP complex upon binding of a hydroxylated PHAH metabolite can also markedly decrease plasma retinol levels in rodents (Brouwer, 1987). It was suggested that seals exposed to PHAHs in the environment, have an impaired function of the immune system (de Swart, 1995), possibly resulting from disturbed retinoid levels (Brouwer, 1991, Brouwer <em>et al.</em> 1989). Hydroxylated PHAH metabolites may attribute to these effects on thyroid hormone and retinoid homeostasis through interactions with TTR in plasma. It is not known whether similar effects occur in man.</p> <p>TTR is the major thyroid hormone binding protein in cerebro-spinal fluid (CSF), suggesting a role in distribution of thyroid hormones in the central nervous system. This TTR is produced in the choroid plexus and is present in high concentrations in CSF of rats and humans, even at a very early stage in development. Moreover, in all species where TTR is present in blood, TTR has also been detected in brain. Because TTR is an important carrier for T <sub>4</sub> to target tissues, for instance brain, one may expect that it may also act as a facilitated transport system for hydroxylated PHAH metabolites. This is in accordance with observations of a strong accumulation of hydroxylated PCB metabolites of maternal origin in the plasma and brain of late gestational fetuses from pregnant rats or mice exposed to PCBs (Morse <em>et al.,</em> 1995b,d, Darnerud <em>et al.,</em> 1995). In rat fetuses perinatally exposed to Aroclor 1254 the selective accumulation of the 4-OH-2,3,3',4',5-pentaCB metabolite in maternal plasma and fetal plasma and brain led to decreases in brain T <sub>4</sub> levels, while brain T <sub>3</sub> levels were only lightly changed. In addition plasma and hepatic retinoid concentrations were decreased in fetal and neonatal offspring (Morse <em>et al.,</em> 1995a). The hydroxylated metabolites accumulated to high levels in fetal rat brain and may themselves attributeto observed neurochemical changes (Morse, 1995).</p> <p>Hydroxylated PHAH metabolites have been shown to possess biological activity <em>in vitro</em> (Brouwer, 1994). Hydroxylated PCB metabolites can interfere with mitochondrial structure and function <em>in vivo</em> and <em>in vitro</em> (Lans <em>et al.,</em> 1990, Narashimhan <em>et al.,</em> 1991). Moreover, they can bind to the Ah-receptor and weakly induce EROD activity. In addition, an <em>in</em> vitro marker of tumor promoting potential, the gap-junctional intercellular communication, could be weakly inhibited. Hydroxylated PCB metabolites can also exert (anti)-estrogenic activities <em>in vivo</em> (Bergeron <em>et al.,</em> 1994) and <em>in</em> vitro (Kramer <em>et al.,</em> 1994). No clear structure activity relationships for (anti)- estrogenicity could be found for the tested hydroxylated PCB metabolites. However, the hydroxylated PCB metabolites selectively retained in fetal plasma and brain (Morse <em>et al.,</em> 1995d) do have a weak (anti)-estrogenic activity. The intrinsic capacity to disrupt endocrine systems, eg. thyroid and estrogen status, and the relatively large accumulating levels of hydroxylated PCB metabolites in late gestational rat fetuses, suggests there is a potential risk for adverse developmental effects by these hydroxylated PHAHs. This possible hydroxy PCBmediated route of developmental toxicity should be investigated in a sound <em>in vivo</em> experimental setup.</p> <p>Subtle changes in plasma thyroid hormone levels and parameters for neurological development were described in children exposed to background levels of PHAHs in utero and through lactation (Koopman-Esseboom <em>et al.,</em> 1994, Sauer <em>et al.,</em> 1994, Pluim <em>et al.,</em> 1993). Hydroxylated PHAH metabolites did not interact with TBG, the major T <sub>4</sub> binding protein in human plasma (Lans <em>et al.,</em> 1994). However, the hydroxylated PCB metabolites which are recently detected in human plasma (Bergman <em>et al.,</em> 1994) are mainly bound to TTR, as was found after selective purification of TTR from human plasma (unpublished results). Therefore TTR-mediated accumulation of hydroxylated PCB metabolites or related compounds in fetal plasma and brain and subsequent decreases in T <sub>4</sub> levels, as found in late gestational rat fetuses, may be of concern for fetal growth and (brain) development in a wide variety of species, including man.</p>
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