Development of a microphysiological skin-liver-thyroid Chip3 model and its application to evaluate the effects on thyroid hormones of topically applied cosmetic ingredients under consumer-relevant conditions

All cosmetic ingredients registered in Europe must be evaluated for their safety using non-animal methods. Microphysiological systems (MPS) offer a more complex higher tier model to evaluate chemicals. Having established a skin and liver HUMIMIC Chip2 model demonstrating how dosing scenarios impact the kinetics of chemicals, we investigated whether thyroid follicles could be incorporated to evaluate the potential of topically applied chemicals to cause endocrine disruption. This combination of models in the HUMIMIC Chip3 is new; therefore, we describe here how it was optimized using two chemicals known to inhibit thyroid production, daidzein and genistein. The MPS was comprised of Phenion® Full Thickness skin, liver spheroids and thyroid follicles co-cultured in the TissUse HUMIMIC Chip3. Endocrine disruption effects were determined according to changes in thyroid hormones, thyroxine (T4) and 3,3’,5-triiodothyronine (T3). A main part of the Chip3 model optimization was the replacement of freshly isolated thyroid follicles with thyrocyte-derived follicles. These were used in static incubations to demonstrate the inhibition of T4 and T3 production by genistein and daidzein over 4 days. Daidzein exhibited a lower inhibitory activity than genistein and both inhibitory activities were decreased after a 24 h preincubation with liver spheroids, indicating metabolism was via detoxification pathways. The skin-liver-thyroid Chip3 model was used to determine a consumer-relevant exposure to daidzein present in a body lotion based on thyroid effects. A “safe dose” of 0.235 μg/cm2 i.e., 0.047% applied in 0.5 mg/cm2 of body lotion was the highest concentration of daidzein which does not result in changes in T3 and T4 levels. This concentration correlated well with the value considered safe by regulators. In conclusion, the Chip3 model enabled the incorporation of the relevant exposure route (dermal), metabolism in the skin and liver, and the bioactivity endpoint (assessment of hormonal balance i.e., thyroid effects) into a single model. These conditions are closer to those in vivo than 2D cell/tissue assays lacking metabolic function. Importantly, it also allowed the assessment of repeated doses of chemical and a direct comparison of systemic and tissue concentrations with toxicodynamic effects over time, which is more realistic and relevant for safety assessment.

To ensure systemic doses approximating the LOECs in the Chip3, the metabolism of the test chemicals by liver organoids (i.e. first-pass metabolism in the Chip) was taken into account. This was achieved by adjusting the nominal doses according to the slope of the correlation of the nominal concentration and the concentration remaining after 24 h incubation with liver spheroids.  Genistein and daidzein were tested in a panel of CALUX® transactivation assays to investigate potential MoAs for reproductive toxicity [1][2][3].

Determination of cytotoxicity
Before analysis on the various bioassays, the cytotoxicity of genistein and daidzein was assessed using the U2-OS based CALUX cytotox bioassay. The cytotox CALUX cells constitutively express luciferase. Exposure of the cytotox CALUX cells to chemicals causing cytotoxicity results in a reduction of luminescence. Chemical concentrations causing >20% reduction of luminescence are considered cytotoxic.

hTPO inhibition assay
hTPO was derived from Nthy-ori 3-1 cells. Cell lysate containing hTPO in Glycine-NaOH buffer (pH 9.0) was incubated for 30 min at 37°C in the presence of serial dilutions of the chemicals in DMSO (1% chemical stock in incubation mixture). The incubation mixture was transferred to 96-well microtiter plates after which luminol (34.8 μM) and H2O2 (1.7 mM) were added. Luminescence was measured on a Berthold luminometer.

TTR-binding assay
Serial dilutions of the chemicals were incubated in Tris-buffer (pH 8.0) overnight at 4oC in the presence of TTR (0.058 μM) and a fixed concentration of T4 (0.052 μM) (3.2% chemical stock in incubation mixture). After incubation, TTR-bound and free T4 were separated on a Bio-Gel P-6DG column. The eluate was added to assay medium after which TRβ CALUX cells were exposed for 24 h. For this TRβ CALUX exposure, serum-free assay medium was used.

Results
The cytotoxicity of genistein and daidzein was tested to ensure that the EATS assay outcomes were not impacted by cytotoxic effects. Neither chemical was cytotoxic up to the highest concentration of 10 mM, in the absence or presence of rat liver S9. The rat liver S9 was incubated with cofactors that mediate phase 1 pathways (i.e. an NADPH and an NADPH-regenerating system); therefore, since genistein and daidzein are only conjugated via phase 2 pathways, the cytotoxicity was not expected to be altered by the inclusion of S9.

Supplementary Figures
Supplemental Figure 1. An overview of the culture methods for freshly isolated and thyrocyte-derived thyroid follicles.    Figure 3A. Viability according to glucose concentrations of freshly isolated human thyroid follicles (500 per incubation), Phenion FT models and liver spheroids in different media. Values are mean ± SD, n= 6 samples were incubated for each organoid. Values denoted with dotted lines indicate the concentrations measured in the corresponding medium in the absence of organoids. The media tested were 100% ALI medium (for skin), 100% HepaRG medium (for liver spheroids) and 100% liver-thyroid co-culture medium (for thyroid follicles) and a mix of liver-thyroid co-culture medium and skin ALI medium (in a ratio of 50:50 (Mix 1) or 70:30 (Mix 2)), all thyroid follicle incubations were with or without the addition of 1 mU/mL TSH. Values are mean ± SD, n= 6 samples were incubated for each organoid. The media tested were 100% ALI medium (for skin), 100% HepaRG medium (for liver spheroids) and 100% liver-thyroid co-culture medium (for thyroid follicles) and a mix of liver-thyroid co-culture medium and skin ALI medium (in a ratio of 50:50 (Mix 1) or 70:30 (Mix 2)), all thyroid dfollicle incubations were with or without the addition of 1 mU/mL TSH. Values are mean ± SD, n= 6 samples were incubated for each organoid. The media tested were 100% ALI medium (for skin), 100% HepaRG medium (for liver spheroids) and 100% liver-thyroid co-culture medium (for thyroid follicles) and a mix of liver-thyroid co-culture medium and skin ALI medium (in a ratio of 50:50 (Mix 1) or 70:30 (Mix 2)), all thyroid follicle incubations were with or without the addition of 1 mU/mL TSH. Values are mean ± SD, n= 6 samples were incubated. The media tested were 100% HepaRG medium and a mix of liver-thyroid co-culture medium and skin ALI medium (in a ratio of 50:50 (Mix 1) or 70:30 (Mix 2)).    Supplementary Figure 11. Morphology of skin, liver and thyroid models in the Chip3 model after different application scenarios. Images were selected to show that (1) the Phenion FT skin models (captured from the first Chip3 experiment) retained their structure over time even after application of the lotion formulation; (2) liver spheroids exhibited some outgrowth of cells (indicating the medium needs optimization) and daidzein exhibited marked precipitation, evident as black particles and (3) thyrocytes formed dense aggregates after 4 days of static culture, and the typical spherical morphology of thyroid follicles with lumen was observed after a few days in the chip. Images of each organoid were captured from all circuits -3-5 circuits per treatment.