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. 2021 Jun;62(5):306-318.
doi: 10.1002/em.22444. Epub 2021 Jun 14.

Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing

Yiying Wang #  1 , Roberta A Mittelstaedt  1 , Rebecca Wynne  1 , Ying Chen  1 , Xuefei Cao  1 , Levan Muskhelishvili  2 , Kelly Davis  2 , Timothy W Robison  3 , Wei Sun  3 , Elizabeth K Schmidt  4 , Thomas H Smith  4 , Zachary K Norgaard  4 , Charles C Valentine  4 , Jeffry Yaplee  4 , Lindsey N Williams  4 , Jesse J Salk  4 , Robert H Heflich #  1
Affiliations

Genetic toxicity testing using human in vitro organotypic airway cultures: Assessing DNA damage with the CometChip and mutagenesis by Duplex Sequencing

Yiying Wang et al. Environ Mol Mutagen. 2021 Jun.

Abstract

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.

Keywords: DNA damage; error-corrected next generation sequencing (ecNGS); ethyl methanesulfonate (EMS); human in vitro air-liquid-interface (ALI) airway epithelial tissue model; mutagenesis.

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Conflict of interest statement

E.K.S., T.H.S., Z.K.N., C.C.V., J.F., L.N.W., and J.J.S. are employees and equity holders at TwinStrand Biosciences, Inc. E.K.S., C.C.V., L.N.W., and J.J.S. are authors on one or more Duplex Sequencing‐related patents.

Figures

FIGURE 1
FIGURE 1
Schematic diagrams of the human ALI airway culture, and experimental design. (a) Shows an image of an H&E stained transverse section from a human ALI tissue model that was used in this study. A microporous membrane that provides the permeable support for cell growth, divides the culture plate well into apical (upper) and basolateral (lower) compartments. The human ALI airway culture consists of ciliated cells, goblet cells, and basal cells. The apical side of the cultures is exposed to the air and Maintenance Medium is added to the basolateral compartment. The cultures were treated with various concentrations of EMS for periods of (b) 3‐days or (c) 28‐days. LDH release, cilia beating frequency and mucin secretion were monitored at Day 1, 7, 14, 21 and 28 of the 28‐day treatment period in (c). The CometChip and cell viability assays were conducted following the 3‐day treatment in (b) and the 28‐day treatment in (c). Cultures were collected for Duplex Sequencing or for morphological investigation at Day 28 in (c). Shorter red arrows: dosed medium added at 0, 24 and 45 h in (b) and Monday through Friday for 28 days in (c); longer arrows: assays performed 3 h after the last treatment (b and c). LDH, lactate dehydrogenase; CBF, cilia beating frequency
FIGURE 2
FIGURE 2
Cytotoxicity in EMS‐treated ALI airway cultures. Cytotoxicity was evaluated using the LDH assay in cultures used for Duplex Sequencing. Data presented as means ± SD (n = 3). *,+,# p < .05 was considered statistically significant when compared to concurrent vehicle controls; different symbols refer to comparisons made to controls at different time points. A table with primary data has been included as Supporting Information
FIGURE 3
FIGURE 3
Functional changes in EMS‐treated ALI airway cultures. (a) Cilia beating frequency (CBF; n = 4) and (b and c) mucin secretion (n = 4) were measured in cultures used for Duplex Sequencing assays. Data presented as means ± SD. *,+,# p < .05 was considered statistically significant when compared to the concurrent vehicle controls; different symbols refer to comparisons made to controls at different time points. A table with primary data has been included as Supporting Information
FIGURE 4
FIGURE 4
Histological and immunohistochemical (IHC) observations in EMS‐treated ALI airway cultures. (a) Shows representative images of H&E‐stained ALI airway cultures after a 28‐day treatment with EMS. Magnification is 40×. (b) Shows representative images of ALI airway cultures treated with EMS for 28 days and stained for histological and IHC observations. Magnification is 40×. Cell proliferation was evaluated by IHC with anti‐Ki67 antibody; basal cells were stained with anti‐p63 antibody and goblet cells were stained using periodic acid Schiff (PAS). (c) Shows percentages of PAS‐positive goblet cells, anti‐p63‐positive basal cells and anti‐Ki67‐positive proliferating cells. Data (n = 3) presented as means ± SD. *,# p < .05 was considered statistically significant compared to the concurrent vehicle controls; different symbols refer to comparisons made to different controls. A table with primary data has been included as Supporting Information
FIGURE 5
FIGURE 5
DNA damage in EMS‐treated ALI airway cultures. DNA damage (DNA strand breaks and alkali‐labile sites measured as %DNA in Tail) was detected using the CometChip assay and the relative cell viability (% of control) was measured using the MTS assay after (a) 3‐day and (b) 28‐day treatments. Values for %DNA in Tail for individual cultures (n = 4) (black hollow circles) are plotted on the left Y‐axis, with the mean ± SD shown; corresponding values for relative cell viability (% of control) (red squares) are plotted on the right Y‐axis, along with their mean ± SD. To promote their visualization, the results for different treatment concentrations are spaced evenly along the X‐axis, without regard to relative concentration. *,# p < .05 was considered statistically significant compared to the respective vehicle controls; different symbols refer to comparisons made using different assays. A table with primary data has been included as Supporting Information
FIGURE 6
FIGURE 6
Mutant frequency in EMS‐treated ALI airway cultures. Mutation was measured using Duplex Sequencing after a 28‐day treatment with EMS. The individual data (n = 4, for control, 50 μg/mL and 100 μg/mL; n = 3, for 6.25 μg/mL and 25 μg/mL) are expressed as dot plots, along with the mean ± SD. To promote their visualization, the results for different treatment concentrations are spaced evenly along the X‐axis, without regard to relative concentration. *p < .05 was considered statistically significant compared to the concurrent vehicle control (one‐tailed Dunnett's test). Primary data for the plot shown in this figure are reported in Table 1
FIGURE 7
FIGURE 7
Mutation spectra analysis. (a) Proportions of simple base substitutions are plotted for each sample. *p < .05 was considered statistically significant differences in the proportion of C → T transitions, when compared to the concurrent vehicle control. (b) Trinucleotide spectra of treatment groups show distinct patterns of mutagenesis specific to EMS treatment
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