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Mutagenicity of N-nitroso-fluoxetine and N-nitroso-varenicline in human HepaRG cell models
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DOI：10.1093/mutage/geag012.png)
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Abstract
En 中文
Nitrosamine drug substance-related impurities (NDSRIs) have raised significant concern among the pharmaceutical industry and regulatory agencies, prompting a need to establish methods to determine the mutagenic risks associated with these substances. As human, metabolically competent HepaRG cells previously showed promise in evaluating the mutagenicity of a small-molecule nitrosamine impurity, N-nitroso-dimethylamine (NDMA), this study evaluated the suitability of HepaRG cells for mutagenesis assessment of NDSRIs. HepaRG cells, cultured in both two-dimensional (2D) and three-dimensional (3D) spheroid formats, were exposed to N-nitroso-fluoxetine and N-nitroso-varenicline for either 3 days or 14 days. DNA damage was evaluated using the comet assay, clastogenicity/aneugenicity was measured with the micronucleus (MN) assay, and mutagenesis was assessed using high-fidelity sequencing (HiFi-seq). N-Nitroso-fluoxetine and N-nitroso-varenicline were cytotoxic, produced DNA damage, and increased mutation frequency in both 2D and 3D HepaRG models. Both compounds increased MN formation only at the highest concentration in 3D spheroids after the 14-day exposure. Benchmark concentration (BMC) analysis indicated that, despite overlapping confidence intervals between 2D and 3D HepaRG cultures, in most instances BMC values tended to be lower in 3D than in 2D cultures after both 3-day and 14-day treatments. In addition, the 14-day treatments of both 2D and 3D HepaRG cultures with N-nitroso-fluoxetine resulted in higher levels of mutagenesis than the 3-day treatments, while 3-day and 14-day treatments with N-nitroso-varenicline produced similar mutagenesis responses. These findings demonstrate the feasibility of employing HepaRG cells with HiFi-seq as a mammalian cell assay for assessing NDSRI mutagenesis.
Keywords:
nitrosamine drug substance-related impurity (NDSRI)
HepaRG cells
mutation
error-corrected sequencing