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 (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.
Sugiyama
Shin-Ichiro
S
Department of Biology, Faculty of Science, Shizuoka University, Japan.
Shimada
Naoyuki
N
Miyoshi
Hiroyuki
H
Yamauchi
Kiyoshi
K
D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D001681:Biological Assay; D002478:Cells, Cultured / Q000187:drug effects* / Q000201:enzymology; D004010:Dicofol / Q000145:classification / Q000633:toxicity; D004703:Endocrine System / Q000187:drug effects* / Q000378:metabolism; D006540:Herbicides / Q000145:classification / Q000633:toxicity; D006727:Hormone Antagonists / Q000145:classification / Q000633:toxicity*; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008675:Metamorphosis, Biological / Q000187:drug effects* / Q000502:physiology; D010795:Phthalic Acids / Q000145:classification / Q000633:toxicity; D011988:Receptors, Thyroid Hormone / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013963:Thyroid Hormones / Q000378:metabolism*; D014982:Xenopus laevis
D006540:Herbicides; D006727:Hormone Antagonists; D010795:Phthalic Acids; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D013963:Thyroid Hormones; C032279:phthalic acid; D008156:Luciferases; D004010:Dicofol