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A Multi-Analyte Method for the Quantification of Xenobiotic-Induced Thyroxine Metabolism
Citation:
Richardson, V., J. Strasser, M. Mocanu, AND D. MacMillan. A Multi-Analyte Method for the Quantification of Xenobiotic-Induced Thyroxine Metabolism. International Society for the Study of Xenobiotics (ISSX), NA, NC, September 13 - 17, 2021. https://doi.org/10.23645/epacomptox.20292402
Impact/Purpose:
Poster presented to the International Society for the Study of Xenobiotics (ISSX) meeting September 2021. Examining changes in the xenobiotic metabolizing enzymes (XMEs) involved in throid hormone metabolism is important in understanding thyroid hormone clearance; therefore a simple multi-analyte LC/MS/MS method was developed to quantify and qualify thyroid hormone metabolites in cell culture media.
Description:
Thyroid hormones (THs) are essential to normal fetal development and particularly to the development of the nervous system. Because of the importance of THs to the developing fetus, xenobiotics that disrupt TH homeostasis are of great concern. Exposure to polybrominated diphenyl ethers (PBDEs), a major class of brominated flame retardants, has been shown to decrease TH serum concentrations in rodents by inducing the hepatic metabolism of thyroxine (T4). TH metabolism is carried out by deiodinases, UDP-glucuronosyltransferases, and sulfotransferases in the liver; therefore, assessing the expression (activity or mRNA) changes of these enzymes in the presence of xenobiotics is key to predicting TH clearance. However, enzyme activity assays can be inactivated or inhibited by the test compound and mRNA levels does not always correlate with protein levels; therefore, metabolite quantitation for induction studies is considered a desired approach. Here, we developed a simple and efficient analytical method to identify and quantify TH metabolites in cell culture media. Using human sandwich cultured hepatocytes, we examined the effects of 2,2′,4,4′-tetrabromodiphenyl ether (BDE 47), a major PBDE congener, on T4 metabolism. Human hepatocytes were treated with 0, 0.3, 3, or 30µM BDE 47 (0.1% DMSO final concentration) for 72 hours. After treatment with BDE 47, hepatocytes were incubated (24 hours) with 0.1µM T4 (median physiological concentration in humans). After 24 hours, media were collected and prepared for extraction through protein precipitation. Extracts were analyzed using electrospray ionization with positive ion scheduled multiple reaction monitoring (sMRM) on a Sciex 6500+ Qtrap LC/MS/MS. The metabolites analyzed include: T3, reverse T3, and T2 (deiodination), T4G and T3G (glucuronidation), and T4S and T3S (sulfation). In controls, T3 was the predominate metabolite found in media, followed by rT3, T4G, and T2. T3G, T4S, and T3S were not detected. Compared to controls, 30µM BDE 47 increased T4G and T3 formation by 12.4- and 2.5-fold, respectively. The results indicate not only the pathways for BDE 47-induced hepatic metabolism of T4, but also that deiodination is a favored pathway in human hepatocytes and xenobiotic-induced glucuronidation can play a key role in T4 clearance. These results highlight the utility of hepatocytes and this method as an in vitro screening tool to yield a mechanistic understanding of TH clearance and to evaluate potential TH disruptors. (This abstract of a proposed presentation, does not reflect USEPA policy.)
URLs/Downloads:
DOI: A Multi-Analyte Method for the Quantification of Xenobiotic-Induced Thyroxine Metabolism
RICHARDSON_ISSX 2021_FINAL.PDF (PDF, NA pp, 635.169 KB, about PDF)