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

Abstract

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 in vitro genotoxicity data. The CometChip^® assay was developed to assess DNA strand breaks in a higher-throughput format. We paired the TGx-DDI biomarker with the CometChip^® 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^® and TempO-Seq^®). 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 in vitro 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 in vitro 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^® 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^® to resolve conflicting genotoxicity data and provides further validation to support the reproducibility of the biomarker.

Keywords: TGx-28.65 genomic biomarker; genetic toxicology; metronidazole; nitrofurantoin; novobiocin; toxicogenomics.

FIGURE 1

MTT assay results for nitrofurantoin (NIT) (A), metronidazole (MTZ) (B), and novobiocin (NOV) (C). MTT measures cellular metabolic activity as an indicator of cell viability and cytotoxicity for solvent controls and treated TK6 cells (n = 3). The dashed line represents the cytotoxicity threshold of 50% cell viability relative to each chemical’s matched vehicle control. Dimethyl sulfoxide, acetic acid and water were used as vehicle controls for NIT, MTZ, and NOV, respectively. Error bars represent the coefficient of variation. Cytotoxic concentrations were eliminated from the gene expression analysis for TGx-DDI classification (i.e., 500 and 1,000 μM for NIT and MTZ, and 1,000 μM for NOV). All concentrations were run for CometChip^® analysis.

FIGURE 2

TGx-DDI classification by nCounter^® and TempO-Seq^® analysis for nitrofurantoin (NIT), metronidazole (MTZ), and novobiocin (NOV). The heatmap on the left depicts the gene expression profiles of the 28 reference chemicals used to generate the biomarker. The test chemicals assessed using nCounter^® and TempO-Seq^® gene expression technologies in human TK6 cells are shown in the subsequent heatmaps (columns). Gene Symbols corresponding to the GenBank accession numbers for the TGx-DDI biomarker genes are on the right y-axis. The colour scale indicates gene expression fold changes relative to control: up-regulated genes are red, down-regulated genes are green, and genes that are not altered are black. TGx-DDI classification probabilities for all treatment conditions are shown using red (DDI) and blue (non-DDI) bars above each heatmap. Caffeine (CA), bleomycin (BL) and ionizing radiation (IR) were used as negative and positive controls, respectively. Cytotoxic concentrations were not analyzed. The grids above the heatmaps indicate the results of the three different TGx-DDI analyses: Probability Analysis (PA, based on Nearest Shrunken Centroid Analysis), Principal Component Analysis (PCA) and Hierarchical Clustering (HC). The overall call is DDI if any one of these analyses yields a DDI call. Yellow boxes indicate a positive DDI classification, blue denotes a negative non-DDI classification and white signifies an unclassified response (i.e., does not yield a DDI or non-DDI call). Sample size was n = 3, except for NIT DMSO solvent control, 2 μM NIT, 3.9 μM NIT, 15.6 μM NIT, 2 μM MTZ, 2 μM NOV, and 125 μM NOV that had an n = 2 and 250 μM MTZ that had an n = 1.

FIGURE 3

DNA damage in human TK6 cells measured using the alkaline CometChip^® assay. Cells were exposed to increasing concentrations of NIT (A), MTZ (B), and NOV (C) from 2 μM to 1,000 μM. Mean % tail DNA is shown following 4 h exposures. The data are expressed as mean % tail DNA ±SD (n = 3, for three experiments run in duplicate on different days). Cytotoxic concentrations are shown in red (>50% cell death compared to matched solvent control).