Development of a micronucleus test using the EpiAirway™ organotypic human airway model

Abstract

Background: The use of organotypic human tissue models in genotoxicity has increased as an alternative to animal testing. Genotoxicity is generally examined using a battery of in vitro assays such as Ames and micronucleus (MN) tests that cover gene mutations and structural and numerical chromosome aberrations. At the 7th International Workshop on Genotoxicity Testing, working group members agreed that the skin models have reached an advanced stage of maturity, while further efforts in liver and airway models are needed [Pfuhler et al., Mutat. Res. 850-851 (2020) 503135]. Organotypic human airway model is composed of fully differentiated and functional respiratory epithelium. However, because cell proliferation in organotypic airway models is thought to be less active, assessing their MN-inducing potential is an issue, even in the cytokinesis-blocking approach using cytochalasin B (CB) [Wang et al., Environ. Mol. Mutagen. 62 (2021) 306-318]. Here, we developed a MN test using EpiAirway™ in which epidermal growth factor (EGF) was included as a stimulant of cell division.

Results: By incubating EpiAirway™ tissue with medium containing various concentrations of CB, we found that the percentage of binucleated cells (%BNCs) almost plateaued at 3 μg/mL CB for 72 h incubation. Additionally, we confirmed that EGF stimulation with CB incubation produced an additional increase in %BNCs with a peak at 5 ng/mL EGF. Transepithelial electrical resistance measurement and tissue histology revealed that CB incubation caused the reduced barrier integrity and cyst formation in EpiAirway™. Adenylate kinase assay confirmed that the cytotoxicity increased with each day of culture in the CB incubation period with EGF stimulation. These results indicated that chemical treatment should be conducted prior to CB incubation. Under these experimental conditions, it was confirmed that the frequency of micronucleated cells was dose-dependently increased by apical applications of two clastogens, mitomycin C and methyl methanesulfonate, and an aneugen, colchicine, at the subcytotoxic concentrations assessed in %BNCs.

Conclusions: Well-studied genotoxicants demonstrated capability in an organotypic human airway model as a MN test system. For further utilization, investigations of aerosol exposure, repeating exposure protocol, and metabolic activation are required.

Keywords: Aneugen; Clastogens; EGF; EpiAirway™; Micronucleus test; Organotypic human airway model.

Fig. 1

Optimization of incubation with cytochalasin B in the EpiAirway™ model. a Schematic of the incubation conditions with cytochalasin B (CB) for 48 or 72 h. b Percentage of binucleated cells (%BNCs) obtained after incubation with at 0, 1, 3, and 5 μg/mL for 48 h (in light blue) or 72 h (in blue). c Schematic of the incubation conditions with CB for 72 or 120 h. d %BNCs obtained after incubation with CB at 0, 3, and 10 μg/mL for 72 h (in blue) or 120 h (in deep blue). Values are means ± standard deviation of triplicate inserts

Fig. 2

Effect of epidermal growth factor stimulation on the percentage of binucleated cells in EpiAirway™ model. a Schematic of epidermal growth factor (EGF) stimulation conditions for 72 h with cytochalasin B (CB) incubation. b Percentage of binucleated cells (%BNCs) obtained after stimulation with EGF at 0, 2.5, 5, and 10 ng/mL for 72 h with CB incubation. Values are means ± standard deviation of triplicate inserts, except for the 2.5 ng/mL EGF with CB incubation condition, for which values were derived from duplicate inserts

Fig. 3

Changes in transepithelial electrical resistance and tissue morphology induced in various experimental conditions. a Schematic of transepithelial electrical resistance (TEER) measurement and histology of EpiAirway™ tissue under various experimental conditions. Group 1, immediately after a 3-day acclimatization period; Group 2, no test chemical treatment, cytochalasin B (CB) incubation, or epidermal growth factor (EGF) simulation throughout the experimental period; Group 3, solvent treatment; Group 4, no chemical treatment followed by EGF stimulation only; Group 5, no chemical treatment followed by CB incubation only; and Group 6, solvent treatment followed by CB incubation and EGF stimulation. b Tissue integrity assay. Values are means ± standard deviation of triplicate inserts. *Significantly different from the ‘Group 2’ (p < 0.05, Welch’s t-test). c Representative images of tissue sections stained with hematoxylin and eosin from samples collected on Day 0 and Day 4

Fig. 4

Effect of cytochalasin B incubation and epidermal growth factor stimulation on cytotoxicity. a Schematic of incubation with culture medium or with medium containing 3 μg/mL cytochalasin B (CB) and 5 ng/mL epidermal growth factor (EGF) for 72 h in an adenylate kinase (AK) assay. b Results of AK assay after incubation with medium containing CB and EGF. Delta relative luminescence units (ΔRLUs) are means ± standard deviations of triplicate inserts

Fig. 5

Percentage of binucleated cells and frequency of micronucleated binucleated cells by test chemical treatment. a Schematic of test chemical treatment followed by cytochalasin B (CB) incubation and epidermal growth factor (EGF) stimulation. b, c, and d Percentage of binucleated cells (%BNCs) and frequency of micronucleated binucleated cells (%MN) after apical treatment with (b) mitomycin C (MMC), (c) methyl methanesulfonate (MMS), and (d) colchicine (COL) in the EpiAirway™ model. Results are plotted with a blue line indicating %BNCs (left axis) and a red line indicating %MN (right axis). %BNCs are means ± standard deviations of triplicate inserts, while %MN is the combined value of triplicate inserts. *Significantly different from the solvent control group (p < 0.05, Fisher’s exact test)

Fig. 6

Cell images of an acridine orange-stained slide specimen. a Representative example of mononucleated and binucleated cells. b Example of a binucleated cell with a micronucleus surrounded by two mononucleated cells