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In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles
Hornung, M., Pat Kosian, J. Haselman, Joe Korte, K. Challis, C. Macherla, E. Nevalainen, AND S. Degitz. In vitro, ex vivo, and in vivo determination of thyroid hormone modulating activity of benzothiazoles. TOXICOLOGICAL SCIENCES. Society of Toxicology, RESTON, VA, 146(2):254-264, (2015).
This manuscript demonstrates the correspondence between activity of chemicals in an in vitro TPO assay and their activity for thyroid axis disruption in amphibians during metamorphosis. It shows the limitations of the thyroid explant culture and the utility of an amphibian 7d assay for verification of in vitro activity. A sensitive analytical method for quantitation of thyroid hormones and related iodinated tyrosines in small tissue samples is presented. It further supports the use of TPO in vitro assay data as a useful tool for prioritizing chemicals for further testing in EPA’s Endocrine Disruptor Screening Program.
As in vitro assays are increasingly used to screen chemicals for their potential to produce endocrine disrupting adverse effects, it is important to understand their predictive capacity. The potential for a set of six benzothiazoles to affect endpoints related to thyroid hormone synthesis inhibition were assessed using in vitro, ex vivo, and in vivo assays. Inhibition of thyroid peroxidase (TPO) derived from pig thyroid glands was determined for benzothiazole (BTZ), 2-mercaptobenzothiazole (MBT), 5-chloro-2-mercaptobenzothiazole (CMBT), 2-aminobenzothiazole (ABT), 2-hydroxybenzothiazole (HBT), and 2-methylthiobenzothiazole (MTBT). Their rank order potency for TPO inhibition was MBT=CMBT>ABT>BTZ, whereas HBT and MTBT exhibited no inhibitory activity. The benzothiazoles were tested further in a Xenopus laevis thyroid gland explant culture assay in which inhibition of thyroxine (T4) release was the measured endpoint, and all six inhibited T4 release. The activity of the benzothiazoles for disrupting thyroid hormone activity was verified in vivo using X. laevis tadpoles in a 7d assay. The two most potent chemicals for TPO inhibition, MBT and CMBT, produced definitive responses in vivo indicative of T4 synthesis inhibition by induction of sodium iodide symporter mRNA and decreases in glandular and circulating thyroid hormones. The capability to measure thyroid hormone levels in the glands and blood by ultrahigh performance LC-MS/MS methods optimized for small tissue samples was critical for effects interpretation. These results indicate that inhibition of TPO activity in vitro was a good indicator of a chemical’s potential for thyroid hormone disruption in vivo and may be useful for prioritizing chemicals for further investigation.
Record Details:Record Type: DOCUMENT (JOURNAL/PEER REVIEWED JOURNAL)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL HEALTH AND ENVIRONMENTAL EFFECTS RESEARCH LABORATORY
MID-CONTINENT ECOLOGY DIVISION