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.

Defining the role of the antineoplastic drug bleomycin based on toxicogenomic-DNA damage inducing (TGx-DDI) genomic biomarkers data: A meta-analysis using next-generation knowledge discovery method

“…The TGx-DDI gene expression data was obtained from GEO [(GSE196373 (super series) and GSE196369 (sub series)]. 8 As specified in these GEO datasets, the nCounter® experiments were conducted following the instructions outlined in the NanoString nCounter custom TGx- DDI Assay Panel (GEO platform GPL31920). 8 …”

“… 8 As specified in these GEO datasets, the nCounter® experiments were conducted following the instructions outlined in the NanoString nCounter custom TGx- DDI Assay Panel (GEO platform GPL31920). 8 …”

“…This study was conducted using publicly available gene expression data from the Gene Expression Omnibus (GEO). 8 It was conducted at the Faculty of Applied Medical Sciences, King Abdulaziz University (KAU), Jeddah, Saudi Arabia in October 2022. It was exempt from Institutional Review Board (IRB) approval because it did not involve animal models or human subjects.…”

“…DMSO served as a control while bleomycin (BL; 10 µM) was used as a treatment group in the nCounter® experiments performed as previously reported. 8 , 10 The GEO data was compared using GEO2R based on the GEOquery and Linear Models for Microarray Analysis (limma) R packages obtained from the Bioconductor project, which provides R-based tools for high-throughput analysis of genomic data. The DEGs were identified using the limma R package using a log2 fold change (Log2FC) cut-off of 2 and p value cut off of 0.05.…”

Application of Genomics-Based Methodologies in Genetic Toxicology to Advance In Vitro Chemical Assessment

“…(c) in the HepaRG human hepatocarcinoma cell line (Buick et al, 2021); and (d) on different gene expression platforms (RNAseq (Ampliseq, NextSeq, TempO-Seq), qRT-PCR (Chapter 2), Nanostring) (Cho et al, 2019;Li, H. H. et al, 2017;Chen et al, 2022;Buick et al, 2022). Notably, in external validation using 45 compounds, TGx-DDI classified 10 of 11 known irrelevant in vitro positive chemicals as non-DDI (Li, H. H. et al, 2017).…”