We developed a thyroid hormone (TH) inducible primary screening assay for the identification and assessment of man-made chemicals that interfere with the TH-signalling pathway within target cells. The assay was developed in a Xenopus laevis cell line that was transduced with a self-inactivating (SIN) lentivirus vector (LV) containing a luciferase gene. The luciferase activation in this cell line was TH-specific: 3,3',5-L-triiodothyronine (T(3)) > 3,3'5-L-triiodothyroacetic acid (Triac) > 3,3',5-D-triiodothyronine (D-T(3)), > L-thyroxine (T(4)) > 3,3',5'-L-triiodothyronine (rT(3)). The application of the ligand-dependent luciferase assay for screening for thyroid system-disrupting chemicals revealed that three phthalates (dicyclohexyl phthalate, n-butylbenzyl phthalate, and di-n-butyl phthalate), two herbicides (ioxynil and pentachlorophenol) and a miticide (dicofol) had 3,3',5-L-triiodothyronine- T(3)- antagonist activity at concentrations ranging from 10(-6) to 10(-5) M. These chemicals also inhibited the expression of the endogenous primary T(3)-response TH nuclear receptor beta (TRbeta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T(3)-dependent activation of TRbeta gene was more sensitive than the luciferase assay. These results indicate that the luciferase assay was a rapid method with a small intra-assay variation for the primary screening of thyroid system-disrupting chemicals. Of the six chemicals, only n-butylbenzyl phthalate and pentachlorophenol exhibited T(3)-antagonist activity in an in vivo metamorphosis-based assay. It should be noted that chemicals elicited thyroid system-disrupting activity in the luciferase assay did not always interfere with the thyroid system in vivo.

We characterized the 3,5,3 --triiodothyronine (T 3 )-uptake system on the plasma membrane of Rana catesbeiana tadpole red blood cells (RBCs) 125 I]T 3 uptake by 2-fold at 3·2 µM. When RBCs were cultured with 10 nM T 3 at 25 C for 2 days in the presence of monodansylcadaverine, ethinylestradiol, ioxynil or dicofol at the defined concentrations, these compounds inhibited significantly the induction of the thyroid hormone receptor gene by T 3 . However, not all chemicals competed with T 3 binding to the receptor at the same concentrations. Our results raise the possibility that the T 3 -uptake system on the plasma membrane of the tadpole RBCs could be a candidate target site for some EDCs and can modulate cellular T 3 response.

We investigated the effects of medical, industrial and agricultural chemicals on 3,3',5-L-[125I]triiodothyronine ([125I]T3) binding to purified recombinant Xenopus laevis (X. laevis) transthyretin (xTTR), a plasma thyroid hormone-binding protein, and to the ligand-binding domain of thyroid hormone receptor-beta (xTR LBD). xTTR derived from X. laevis serum had about 80 times higher affinity for T3 than for L-thyroxine. The xTTR's relative affinities for diethylstilbestrol, pentachlorophenol and ioxynil were 10(-1)- to 10(-2)-fold less than that for T3. However, all chemicals investigated had either a weak or no influence on [125I]T3 binding to xTR LBD. The concentration of diethylstilbestrol, the most potent chemical, required for 50% inhibition of [125I]T3 binding to xTR LBD was 10(4) times greater than that of unlabeled T3. These results indicate the existence of several chemicals that interact with xTTR but not with xTR LBD.

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Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate the transcription of multiple thyroid hormone (TH)-responsive genes. Our study aimed to identify TH-responsive genes in an estrogen-responsive chicken hepatoma cell line, LMH. RNA was prepared from cells treated with or without 10(-8) M 3,3',5-triiodothyronine (T3) for 24 h and was analyzed by differential display. At least six cDNAs were detected whose transcript increased in the presence of T3, and four of them were cloned. The four candidate TH-responsive genes that were identified had high similarity (>83%) to known chicken or mammalian genes, which included the ribosomal protein L7 gene; the cytoplasmic dynein heavy chain gene; the scaffold attachment factor A (SAF-A) gene, also known as heterogeneous nuclear ribonucleoprotein U (hnRNP U); and a gene for an unknown protein. Real-time PCR confirmed that the transcription of the four genes was responsive to T3; their transcript levels increased from four to eleven times with the administration of T3. The amount of TRbeta transcript did not change with the administration of T3. The physiological reasons for the activation of these genes and the utility of this cell line are discussed.