Detection of Thyroid System–Disrupting Chemicals Using in Vitro and in Vivo Screening Assays in Xenopus laevis

Abstract

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.

In vitro thyroid disrupting effects of organic extracts from WWTPs in Beijing

“…Phthalates are synthetic compounds widely used as polymer additives in plastics to improve their flexibility (Sharman et al, 1994). Many phthalates e.g., DBP and DEHP can affect the thyroid system in laboratory animals (Hinton et al, 1986;Howarth et al, 2001) or have antagonistic acitivities to TR in vitro (Sugiyama et al, 2005). Due to the widespread application of phthalates, they have been widespread pollution in China (Cai et al, 2007).…”

Environmental chemicals and thyroid function

“…Expression of TRalfa1 was not affected (137). Dicyclohexyl phthalate, BBP and PCP inhibited the expression of the TRbeta gene (138).…”

Endocrine disruptors and thyroid hormone physiology

“…Sugiyama et al [86] 24 developed a stably-transfected luciferase reporter gene assay to study interference with the TR 1-signaling pathway by chemicals. Three types of phthalates, nbutylbenzyl phthalate (BBP), di-n-butyl phthalate and dicyclohexylphthalates (DCHPs), were reported to exhibit T3-antagonistic activity, at concentrations ranging from 10 -6 M to 10 -5 M. The authors noticed that in the absence of TDCs and the presence of T3, the amount of endogenous TR transcripts increased 13.3 fold in X.…”