Integrated Genotoxicity Testing of three anti-infective drugs using the TGx-DDI transcriptomic biomarker and high-throughput CometChip® assay in TK6 cells

Abstract

<jats:p>Genotoxicity testing relies on the detection of gene mutations and chromosome damage and has been used in the genetic safety assessment of drugs and chemicals for decades. However, the results of standard genotoxicity tests are often difficult to interpret due to lack of mode of action information. The TGx-DDI transcriptomic biomarker provides mechanistic information on the DNA damage-inducing (DDI) capability of chemicals to aid in the interpretation of positive <jats:italic>in vitro</jats:italic> genotoxicity data. The CometChip<jats:sup>®</jats:sup> assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip<jats:sup>®</jats:sup> assay in TK6 cells to evaluate three model agents: nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). TGx-DDI was analyzed by two independent labs and technologies (nCounter<jats:sup>®</jats:sup> and TempO-Seq<jats:sup>®</jats:sup>). Although these anti-infective drugs are, or have been, used in human and/or veterinary medicine, the standard genotoxicity testing battery showed significant genetic safety findings. Specifically, NIT is a mutagen and causes chromosome damage, and MTZ and NOV cause chromosome damage in conventional <jats:italic>in vitro</jats:italic> tests. Herein, the TGx-DDI biomarker classified NIT and MTZ as non-DDI at all concentrations tested, suggesting that NIT’s mutagenic activity is bacterial specific and that the observed chromosome damage by MTZ might be a consequence of <jats:italic>in vitro</jats:italic> test conditions. In contrast, NOV was classified as DDI at the second highest concentration tested, which is in line with the fact that NOV is a bacterial DNA-gyrase inhibitor that also affects topoisomerase II at high concentrations. The lack of DNA damage for NIT and MTZ was confirmed by the CometChip<jats:sup>®</jats:sup> results, which were negative for all three drugs except at overtly cytotoxic concentrations. This case study demonstrates the utility of combining the TGx-DDI biomarker and CometChip<jats:sup>®</jats:sup> to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.</jats:p>