Simultaneous quantification of T4, T3, rT3, 3,5‐T2 and 3,3′‐T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls

Abstract

Environmental toxicants that interfere with thyroid hormone (TH) signaling can impact growth and development in animals and humans. Zebrafish represent a model to study chemically induced TH disruption, prompting the need for sensitive detection of THs. Simultaneous quantification of 3,3',5-triiodo-l-thyronine (T3), thyroxine (T4), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2) in zebrafish larvae was achieved by ultra-performance liquid chromatography-tandem mass spectrometry in positive ion mode. Solid-phase extraction with SampliQ cartridges and derivatization with 3 m hydrochloric acid in n-butanol reduced matrix effects. Derivatized compounds were separated on an Acquity UPLC BEH C column with mobile phases consisting of 0.1% acetic acid in deionized water and 0.1% acetic acid in methanol. The limits of detection ranged from 0.5 to 0.6 pg injected on column. The method was validated by evaluating recovery (77.1-117.2%), accuracy (87.3-123.9%) and precision (0.5-12.4%) using diluted homogenized zebrafish embryos spiked with all target compounds. This method was then applied to zebrafish larvae collected after 114 h of exposure to polychlorinated biphenyls (PCBs), including PCB 28, PCB 66 and PCB 95, or the technical mixture Aroclor 1254. Exposure to PCB 28 and PCB 95 increased the T4:T3 ratio and decreased the T3:rT3 ratio, demonstrating that this method can effectively detect PCB-induced alterations in THs.

Effects of thyroid hormone disruption on the ontogenetic expression of thyroid hormone signaling genes in developing zebrafish (Danio rerio)

“…Stock aliquots of T4, T3, and PTU were diluted to yield final concentrations at the time of exposure. Analytical standards used for LC/MS/MS determination of thyroid hormones in zebrafish larvae were described previously (Chen et al, 2018). (1) Circulating levels of T4, the predominant form of TH in the blood, and T3 are transported into target cells by TH transporters.…”

“…The concentrations of four thyroid hormones, T4, T3, rT3, and T2 were measured in zebrafish embryos and larvae raised in control embryo medium (EM; 15 mM NaCl, 0.5 mM KCl, 1.0 mM MgSO 4 , 150 µM KH 2 PO 4 , 50µM Na 2 HPO 4 , 1.0 mM CaCl 2 , 0.7 mM NaHCO 3 ) (Westerfield, 2000) in the absence or presence of 30 nM T4, 30 nM T3, or 0.01% PTU at 24, 72, and 120 h post fertilization (hpf) using the LC-MS/MS method previously described and validated in zebrafish larvae (Chen et al, 2018). Zebrafish embryos were placed in 6-well plates (BD Falcon, Corning, Lowell, MA, USA) containing 2 mL EM at a density of 30 embryos per well.…”

Mass Spectrometry-Based Determination of Thyroid Hormones and Their Metabolites in Endocrine Diagnostics and Biomedical Research – Implications for Human Serum Diagnostics

“…Considering the very low concentrations of TH in biological samples collected in vivo or ex vivo, some authors have attempted to improve the sensitivity of THM detection by pre-analytical derivatization of the analytes of interest, a practice frequently encountered in other analytical disciplines. The derivatization of TH to form acetyl-, butyl-, or other esters to improve detectability and signal quality have been attempted with some success [33][34][35]; however, experience from steroid, toxin and pesticide analytics, indicates that these derivatization steps have to be as quantitative as possible to recover all endogenous analytes of interest. This is rarely controlled or documented in the THM field and is very difficult to achieve if sets of multiple metabolites with different biochemical properties, solubilities, and chemical structures are to be analyzed [3,36].…”

“…The discussion of gas chromatography-tandem mass spectro metry (GC-MS/MS) approaches to THM analysis will also not be attempted here. Suffice it to say, in common with many LC-MS/MS based analyses [27,33,35], most published methods using this approach [37,38] unfortunately lack adequate QA data.…”

“…However, in contrast to human and higher mammal TBG, TBG of rodents has lower T4 affinity and thus is less problematic during pre-analytical workup. THM analyses in sera of humans, cows, rodents, fish, whales, and seals have been reported as well as for amphibians, especially during their development [12,17,25,26,28,35,62,63].…”