Error-corrected next-generation sequencing to advance nonclinical genotoxicity and carcinogenicity testing

Marchetti, Francesco; Cardoso, Renato; Chen, Connie L.; Douglas, George R.; Elloway, Joanne; Escobar, Patricia; Harper, Tod; Heflich, Robert H.; Kidd, Darren; Lynch, Anthony M.; Myers, Meagan B.; Parsons, Barbara L.; Salk, Jesse J.; Settivari, Raja S.; Smith‐Roe, Stephanie L.; Witt, Kristine L.; Yauk, Carole; Young, Robert R.; Zhang, Shaofei; Minocherhomji, Sheroy

doi:10.1038/d41573-023-00014-y

Cited by 22 publications

(12 citation statements)

References 5 publications

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“…How this technology will perform for various types of mutagens still needs to be determined, and a general understanding of the background spontaneous mutation rate and mutation spectra in untreated animals will be necessary to distinguish the DNA mutations associated with chemical exposure via MF or spectra. These findings and those reported by others [15][16][17] support research to explore the adoption of DuplexSeq for the purpose of creating standardized rodent genetic toxicity tests to meet regulatory needs for the detection of point mutations [13], similar to efforts made to develop OECD test guidelines for the TGR and Pig-a gene mutation assays. Compared to currently used assays, direct detection of mutations and identification of mutational spectra in animal models has greater applicability to understanding human exposures to mutagenic substances and may provide more informative data for human cancer risk assessment.…”

Section: Discussionsupporting

confidence: 70%

“…The ability to directly sequence DNA for rare mutations has many applications, including the detection of point mutations for the purpose of genetic toxicity testing [13,14]. To increase the applicability of DuplexSeq for rodent mutagenesis studies, TwinStrand has created generalizable mutagenesis panels and assays for rats and mice that could supersede the use of specialized transgenic rodent models by directly sequencing DNA for mutations.…”

Section: Introductionmentioning

confidence: 99%

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

Smith‐Roe

Hobbs

Hull

et al. 2023

Preprint

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Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 107nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently usedin vivogene mutation assays.HIGHLIGHTSDuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology

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Section: Discussionsupporting

confidence: 70%

Section: Introductionmentioning

confidence: 99%

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

Smith‐Roe

Hobbs

Hull

et al. 2023

Preprint

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“…On the positive side of things, we can expect rapidly growing knowledge on the relative risk when results from a multitude of in vivo mutagenicity studies with NDSRIs (especially TGR) that have already been started will become available. The TGR data may be complemented with results from highestresolution next-generation sequencing (NGS)/duplex sequencing, 93 which has the potential to replace TGR studies and bring a significant acceleration of mutagenicity data generation in the future. For those NDSRIs that do not test negative/ nonmutagenic, it will be essential to develop an accepted methodology that can derive scientifically justified AIs from the in vivo mutagenicity data, as read-across options may be unavailable and carcinogenicity studies are not feasible from a timing, capacity, nor animal welfare perspective.…”

Section: Discussionmentioning

confidence: 99%

The Nitrosamine “Saga”: Lessons Learned from Five Years of Scrutiny

Nudelman

Kocks²,

Mouton

et al. 2023

Org. Process Res. Dev.

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The onset of the N-nitrosamine (NA) saga in 2018 was chiefly related to certain small dialkyl N-nitrosamines originating from the synthesis of the active pharmaceutical ingredient (API). However, the subsequent comprehensive assessments performed on APIs, formulated drug products, and packaging put a different type of NAs in the limelight: a diverse range of complex so-called nitrosamine drug-substance-related impurities (NDSRIs). They may form due to the presence of potentially nitrosatable secondary or tertiary amine moieties in APIs or API impurities and nitrosating agents formed from low levels of nitrite present as impurities. The unique properties of the amine functional group make it irreplaceable in the synthesis of APIs. While nitrite levels may be reduced, the formation of NAs in drug products cannot be completely prevented, and the class default acceptable intake (AI) of 18 ng/day currently poses significant challenges in terms of both viable control and analysis at such low levels. Even so, NA exposure through pharmaceuticals is expected to be orders of magnitude lower than the exposure via food or endogenous formation. While robust carcinogenicity data are available for many of the small, simple NAs, there is a distinct absence of such data for most NDSRIs. Many working groups have therefore been established to share data and rapidly improve knowledge (whether in terms of toxicity data, structure–activity relationships, or analytical techniques), to define best practices to assess the genotoxic potential of NDSRIs, and to advance methods to calculate AIs based on solid scientific rationales. Ultimately, to protect patients from true cancer risk and secure access to important medicines, it is crucial for manufacturers and health authorities to pursue efforts to implement NA control strategies that are equally effective and realistic. As patient safety is paramount, the pharmaceutical industry is committed to ensuring that the medicines it supplies are safe and effective. Where legitimate safety concerns exist, it is undisputed that appropriate actions must be taken, which could include withdrawal of products from the market.

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“…In the past decade, there have been few genetic toxicology studies that have used Hprt mutations in T lymphocytes as a marker of mutagenicity in mice and rats (Bhalli et al, 2013; Walker, Fennell, et al, 2020; Walker, Walker, et al, 2020) and the rodent lymphocyte Hprt mutation assay has been largely supplanted by a combination of Pig‐a , transgenic rodent, and error‐corrected next‐generation duplex sequencing methods (ecNGS) (Bercu et al, 2023; Heflich et al, 2020; Marchetti et al, 2023). However, the general approaches and methods utilized in this report can be duplicated using other reporter genes where mutant T‐cell clones can be selected and expanded, as is the case for Pig‐a mutant T‐cells.…”

Section: Discussionmentioning

confidence: 99%

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N‐ethyl‐N‐nitrosourea

Judice,

Sussman,

Walker

et al. 2023

Environ and Mol Mutagen

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Mutations in T lymphocytes (T‐cells) are informative quantitative markers for environmental mutagen exposures, but risk extrapolations from rodent models to humans also require an understanding of how T‐cell development and proliferation kinetics impact mutagenic outcomes. Rodent studies have shown that patterns in chemical‐induced mutations in the hypoxanthine‐guanine phosphoribosyltransferase (Hprt) gene of T‐cells differ between lymphoid organs. The current work was performed to obtain knowledge of the relationships between maturation events during T‐cell development and changes in chemical‐induced mutant frequencies over time in differing immune compartments of a mouse model. A novel reverse transcriptase‐polymerase chain reaction based method was developed to determine the specific T‐cell receptor beta (Tcrb) gene mRNA expressed in mouse T‐cell isolates, enabling sequence analysis of the PCR product that then identifies the specific hypervariable CDR3 junctional region of the expressed Tcrb gene for individual isolates. Characterization of spontaneous Hprt mutant isolates from the thymus, spleen, and lymph nodes of control mice for their Tcrb gene expression found evidence of in vivo clonal amplifications of Hprt mutants and their trafficking between tissues in the same animal. Concurrent analyses of Hprt mutations and Tcrb gene rearrangements in different lymphoid tissues of control versus N‐ethyl‐N‐nitrosourea‐exposed mice permitted elucidation of the localization and timing of mutational events in T‐cells, establishing that mutagenesis occurs primarily in the pre‐rearrangement replicative period in pre‐thymic/thymic populations. These findings demonstrate that chemical‐induced mutagenic burden is determined by the combination of mutagenesis and T‐cell clonal expansion, processes with roles in immune function and in the pathogenesis of autoimmune disease and cancer.

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Error-corrected next-generation sequencing to advance nonclinical genotoxicity and carcinogenicity testing

Cited by 22 publications

References 5 publications

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

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

The Nitrosamine “Saga”: Lessons Learned from Five Years of Scrutiny

Clonality, trafficking, and molecular alterations among Hprt mutant T lymphocytes isolated from control mice versus mice treated with N‐ethyl‐N‐nitrosourea

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