title: New approaches to assess the transthyretin binding capacity of bioactivated thyroid hormone disruptors.
abstract: Polychlorinated biphenyls (PCBs) and polybrominated diphenyl-ethers (PBDEs) are metabolized into hydroxylated metabolites (OH-PCBs/PBDEs), which can disrupt the thyroid hormone homeostasis. Binding of these metabolites to transport proteins such as transthyretin (TTR) is an important mechanism of their toxicity. Several methods to quantify the competitive thyroxine (T(4)) displacement potency of pure metabolites exist. However, quantification of the potency of in vitro metabolized PCBs and PBDEs has drawbacks related to the coextraction of compounds disturbing the T(4)-TTR competitive binding assay. This study identifies and quantifies the major coextractants namely cholesterol, saturated and nonsaturated fatty acids (SFA and NSFA) at levels above 20 nmol per mg equivalent protein following various extraction methods. Their TTR binding potency was analyzed in a downscaled, nonradioactive fluorescence displacement assay. At concentration factors needed for TTR competitive binding, at least 10μM of these coextracts is present, whereas individual SFA and NSFA disturb the assay from 0.3μM. The effectiveness of the in vitro metabolism and extraction 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 coextractants. A new method to selectively extract metabolites and limit coextraction of disturbing compounds to less than 5 nmol per mg equivalent protein is presented. It is now possible to make a dose-response curve up to 50% inhibition with bioactivated CB 77 and BDE 47. The toxic potencies of bioactivated persistent organic pollutants (POPs) should be taken into account to prevent serious underestimation of their hazard and risk.
authors.affiliation: Centre de Recherche Public - Gabriel Lippmann, Department Environment and Agro-biotechnologies, L-4422 Belvaux, Grand Duchy of Luxembourg.
authors.forename: Mauricio
authors.initials: M
authors.lastname: Montaño
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Cocco
authors.forename: Cedric
authors.initials: C
authors.lastname: Guignard
authors.forename: Göran
authors.initials: G
authors.lastname: Marsh
authors.forename: Lucien
authors.initials: L
authors.lastname: Hoffmann
authors.forename: Ake
authors.initials: A
authors.lastname: Bergman
authors.forename: Arno Christian
authors.initials: AC
authors.lastname: Gutleb
authors.forename: Albertinka Jacoba
authors.initials: AJ
authors.lastname: Murk
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D052244:Endocrine Disruptors / Q000378:metabolism*; D004785:Environmental Pollutants / Q000737:chemistry / Q000378:metabolism*; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism; D055768:Halogenated Diphenyl Ethers; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D011075:Polybrominated Biphenyls / Q000737:chemistry / Q000378:metabolism*; D011078:Polychlorinated Biphenyls / Q000737:chemistry / Q000378:metabolism*; D011228:Prealbumin / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013974:Thyroxine / Q000378:metabolism*
chemical_list: D052244:Endocrine Disruptors; D004785:Environmental Pollutants; D005227:Fatty Acids; D005231:Fatty Acids, Unsaturated; D055768:Halogenated Diphenyl Ethers; D011075:Polybrominated Biphenyls; D011228:Prealbumin; C511295:2,2',4,4'-tetrabromodiphenyl ether; D002784:Cholesterol; D011078:Polychlorinated Biphenyls; D013974:Thyroxine; C028451:3,4,3',4'-tetrachlorobiphenyl