Detection of thyroid system-disrupting chemicals using in vitro and in vivo screening assays in Xenopus laevis

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 (TR beta) gene. The inhibitory characteristics of these chemicals were similar for both assays performed, although the assay for T-3-dependent activation of TR beta 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.