Error-corrected Duplex Sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter-laboratory consistency

Abstract

Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 µM) and DNA was sequenced 48, 72, and 96 h post-exposure. A DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (R2=0.95). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

Adopting Duplex Sequencing™ Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats

“…A high level of concordance was also observed between DuplexSeq and mutagenesis of the lacZ transgene in the bone marrow of MutaMouse animals exposed to B[a]P [16] or when exposed to procarbazine [17]. An analogous human version of the DuplexSeq mutagenesis assay panel was also used successfully to identify ENU-induced mutations in human lymphoblastoid TK6 cells, an OECDrecommended cell line for genetic toxicity testing [18] and ethyl methanesulfonate (EMS)-induced mutations an in vitro air-liquid-interface airway tissue model used to evaluate the toxicity of inhaled substances [19].…”

“…A change in ENU-dependent mutation spectrum over time was also observed, from mutagenic events arising from alkylation of guanine residues to those arising from alkylation of thymine residues. The C:G > T:A transition, observed at 3 and 24 h, is not considered to be a canonical mutation type for ENU; however, C:G > T:A transitions are the defining characteristic of ENU exposure in human lymphoblastoid TK6 cells, which lack O 6 -alkylguanine-DNA alkyltransferase (AGT) activity [35] , and recently, the DuplexSeq Human-50 Mutagenesis Assay also identified significant increases in the proportion of C:G > T:A transitions in TK6 cells exposed to ENU [18]. AGT is a direct-reversal enzyme that removes O 6alkylation damage to DNA via covalent transfer to a cysteine residue, causing inactivation of the enzyme [36].…”

Interpretation of In Vitro Concentration-Response Data for Risk Assessment and Regulatory Decision-making: Report from 2022 IWGT Quantitative Analysis Expert Working Group Meeting

“…ecNGS is less biased as it does not rely on the use of mutant selection that favors loss-of-function mutations (i.e., frameshifts and premature stop codons). Proofof-principle studies have already demonstrated the utility of investigating mutagen exposures using DS in vitro (Cho et al, 2023;Wang et al, 2021). Parallel investigations using the TGR assay and DS have demonstrated that DS yields comparable results and detects the same types of mutations (LeBlanc et al, 2022;Valentine III et al, 2020).…”