A rodent thyroid-liver chip to capture thyroid toxicity on organ functional level

Abstract

Endocrine disruption by environmental chemicals continues to be a concern for human safety. The rat, a widely used model organism in toxicology, is very sensitive to chemical-induced thyroid perturbation, e.g. histopathological alterations in thyroid tissue. Species differences in the susceptibility to thyroid perturbation lead to uncertainty in human safety risk assessments. Hazard identification and characterization of chemically induced thyroid perturbation would therefore benefit from in vitro models addressing different mechanisms of action in a single functional assay, ideally across species. We here introduce a rat thyroid-liver chip that enables simultaneous identification of direct and indirect (liver-mediated) thyroid perturbation on organ-level functions in vitro. A second manuscript describes our work toward a human thyroid-liver chip (Kühnlenz et al., 2022). The presented microfluidic model consisting of primary rat thyroid follicles and liver 3D spheroids maintain a tissue-specific phenotype for up to 21 days. More precisely, the thyroid model exhibits a follicular architecture expressing basolateral and apical markers and secretes T4. Likewise, liver spheroids retain hepatocellular characteristics, e.g. a stable release of albumin and urea, the presence of bile canalicular networks, and the formation of T4-glucuronide. Experiments with reference chemicals demonstrated proficiency to detect direct and indirect mechanisms of thyroid perturbation through decreased thyroid hormone secretion and increased gT4 formation, respectively. Prospectively this rat thyroid-liver chip model, together with its human counterpart, may support a species-specific quantitative in vitro to in vivo extrapolation to improve a data-driven and evidence-based human safety risk assessment with significant contributions to the 3R principles. *

Boscalid shows increased thyroxin‐glucuronidation in rat but not in human hepatocytes in vitro

“…Up to now, appropriate test systems to investigate, for example, species differences on serum hormone binding and transportation are not in place for routine assessment. Further, approaches include liver organoids (Plummer et al, 2019; Plummer et al, 2021), thyroid organoids (Deisenroth et al, 2020; Moroni et al, 2020), and combinations thereof in multi‐organ on a chip system comprising the liver thyroid axis (Karwelat et al, 2022; Kühnlenz et al, 2022). Furthermore, physiologically based pharmacokinetic (PBPK) modeling of chemical exposure mediated alterations in thyroid hormone levels (Clewell et al, 2022) (poster presented at SOT 2022, personal communication) is another more holistic approach currently under development.…”

Complex In Vitro Model Characterization for Context of Use in Toxicologic Pathology: Use Cases by Collaborative Teams of Biologists, Bioengineers, and Pathologists

“…16 Both rodent and human 3D thyroid and liver models have been created to understand species translation of thyroid toxicities. 28,34 The thyroid can be directly affected by chemicals inhibiting the production of thyroid hormones. Alternatively, xenobiotic induction of phase 2 enzymes in the liver can result in increased clearance of thyroid hormones via the formation of inactive glucuronidated and sulfated forms, which can be significantly different in rats and humans.…”

“…This was tested with 4-day methimazole treatment which decreased the T3 and T4 secretion in the human and rat models, respectively. 28,34 Reproduction of thyroid hormone metabolism in vitro requires characteristic morphological features in the liver model. Liver spheroids were derived either from fresh primary hepatocytes from Ham Wistar rats or the HepaRG cell line for the human model.…”

“…The rodent-derived liver spheroids responded with increased glucuronidated metabolite formation when treated with pregnenolone-alpha-carbonitrile (10 uM), whereas the human model was not responsive to this ratspecific nuclear receptor activator. 28,34 Once the models were characterized functionally and for toxicity, the co-culture of the thyroid and liver organ models was evaluated over 21 days in the HUMIMIC Chip2 to demonstrate the maintenance of morphology and function. In summary, morphology and function must be confirmed to recapitulate key in vivo endpoints to interrogate the model for the questions being asked.…”

“…16 Both rodent and human 3D thyroid and liver models have been created to understand species translation of thyroid toxicities. 28,34…”