colorimetric-assessment-deiodinase-1-activity-human-liver-microsomes

Colorimetric Assessment of Deiodinase 1 Activity in Human Liver Microsomes Using the Sandell-Kolthoff Reaction

This method enables the identification of test substances that inhibit DIO1 activity and may thus interfere with thyroid hormone homeostasis. The protocol ...

Experimental Toxicology and Ecology, BASF SE

The selenocysteine-containing enzyme class deiodinases (DIO) consists of three isoforms. DIOs play a role in regulation of thyroid hormone (TH) signaling through the removal of iodide from TH leading to TH that interact with the hypothalamic-pituitary-thyroid (HPT) axis with differing potency. Some gold-containing organic substances are known to inhibit many selenoenzymes, including DIOs. It is, however, unclear whether the Au-containing substances or the Au ions are causing the inhibition. In this study, five organic and inorganic gold substances as well as three gold nanoparticles (AuNPs) were tested for their potential to inhibit DIO1. The enzyme activity was tested using human liver microsomes as an enzyme source and reverse T3 as a substrate; iodide release was measured by the Sandell-Kolthoff method. The three organic gold substances aurothioglucose, auranofin and sodium aurothiomalate inhibited DIO1 with IC between 0.38 and 0.75 μM while their structural analogues lacking the gold ion did not. Likewise, the two tested gold salts, Au(I) and Au(III) chloride, showed a concentration-dependent inhibition of the DIO1 with IC values of 0.95 and 0.57 μM. Further, AuNPs of different sizes (100, 30 and 5 nm diameter) were tested with only the 5 nm AuNPs leading to inhibition with an IC of 8 × 10 AuNP/L. This inhibition was not caused by the Au ions released by the AuNP into the incubation media. The exact mechanism of inhibition of DIO1 by 5 nm AuNPs should be further examined. In conclusion, the microsomal DIO1 assay demonstrated the inhibition of DIO1 by gold ions originating from different gold-containing substances, but not by Au released from AuNPs; rather DIO1 is inhibited by 5 nm, but not larger, AuNPs.

The thyroid hormone converting enzyme human deiodinase 1 is inhibited by gold ions from inorganic salts, organic substances, and by small-size nanoparticles.

In pharmacokinetics plasma protein binding (PPB) is a well-established parameter impacting drug disposition. The unbound fraction (f) is arguably regarded the effective concentration at the target site. Pharmacology and toxicology, increasingly use in vitro models. The translation of in vitro concentrations to in vivo doses can be supported by toxicokinetic modelling, e.g. physiologically based toxicokinetic models (PBTK). PPB of a test substance is an input parameter for PBTK. We compared three methods to quantify f: rapid equilibrium dialysis (RED), ultrafiltration (UF) and ultracentrifugation (UC) using twelve substances covering a wide range of Log P (-0.1 to 6.8) and molecular weights (151 and 531 g/mol): Acetaminophen, Bisphenol A, Caffeine, Colchicine, Fenarimol, Flutamide, Genistein, Ketoconazole, α-Methyltestosterone, Tamoxifen, Trenbolone and Warfarin. After RED and UF separation, three polar substances (Log P < 2) were largely unbound (f > 70%), while more lipophilic substances were largely bound (f < 33%). Compared to RED or UF, UC resulted in a generally higher f of lipophilic substances. f obtained after RED and UF were more consistent with published data. For half of the substances, UC resulted in f higher than the reference data. UF, RED and both UF and UC, resulted in lower f of Flutamide, Ketoconazole and Colchicine, respectively. For f quantifications, the separation method should be selected according to the test substance's properties. Based on our data, RED is suitable for a broader range of substances while UC and UF are suitable for polar substances.

Rapid equilibrium dialysis, ultrafiltration or ultracentrifugation? Evaluation of methods to quantify the unbound fraction of substances in plasma.

Changes in thyroid hormone (TH) levels in rat brain at early developmental stages are correlated with adverse effects on offspring development. To characterize the ability of substances to interfere with the TH concentrations in, e.g., rat brain, it is essential to know the mean TH concentrations in this tissue under control conditions. In this publication, an online solid-phase extraction (SPE) liquid chromatography (LC) tandem mass spectrometry (MS/MS) method was validated and used to measure TH metabolites (T4, T3, rT3, T2 and T1) in the brains of untreated rats. Data on TH concentrations in the whole brain and separate data from the cerebellum and the cortex are shown. The corresponding samples were gathered from young rats at postnatal days (PND) 4 and 21/22 and from adult rats. The results show inter alia the high accuracy and precision of the method, and LOQs of 0.02 ng/mL were determined for T1, T2 and rT3 and of 0.15 ng/mL for T3 and T4. Technical variability is low, as shown by the relative standard deviations of 7.5-20%. For our rat model, we found that T4, T3 and T2 concentrations rise from PND4 to PND21, whereas the rT3 concentration decreases; as well as there is no statistical difference between TH concentrations in the male and female rat brain. This method is suitable to analyze TH metabolites in the brain and build up a database of historical TH concentrations in control rats. Together, this yields a robust diagnostic tool to detect potentially adverse disturbances of TH homeostasis in the most vulnerable anatomic structure.

Article	Year
The thyroid hormone converting enzyme human deiodinase 1 is inhibited by gold ions from inorganic salts, organic substances, and by small-size nanoparticles. Chemico-biological interactions\| PubMed ID: 34662569	2022
Rapid equilibrium dialysis, ultrafiltration or ultracentrifugation? Evaluation of methods to quantify the unbound fraction of substances in plasma. Biochemical and biophysical research communications\| PubMed ID: 36812744	2023
Thyroid Hormone Metabolites Quantified in Pup and Adult Rat Cerebellum, Cortex and Whole-Brain Samples Using an Automated Online SPE-LC-MS/MS Method. Metabolites\| PubMed ID: 38248864	2024

Dorothee Funk-Weyer

Biography

JoVE Journal Publications

Other Publications