Mutation as a Toxicological Endpoint for Regulatory Decision‐Making

Heflich, Robert H.; Johnson, George E.; Zeller, Andreas; Marchetti, Francesco; Douglas, George R.; Witt, Kristine L.; Gollapudi, B. Bhaskar; White, Paul A.

doi:10.1002/em.22338

Cited by 56 publications

(51 citation statements)

References 59 publications

(45 reference statements)

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“…Genotoxicity is a key component in a comprehensive chemical toxicity evaluation due to its role in carcinogenicity and other adverse human health effects (Guyton et al, 2018;Heflich et al, 2020). Investigators have numerous in vitro and in vivo assays available for characterizing the genotoxicity of compounds of interest.…”

Section: Discussionmentioning

confidence: 99%

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Li¹,

Chen

Guo³

et al. 2020

Toxicological Sciences

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Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

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Section: Discussionmentioning

confidence: 99%

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Li¹,

Chen

Guo³

et al. 2020

Toxicological Sciences

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“…However, current testing standards are slow and expensive; even in rodents, it takes years to reach the endpoint of tumor formation. Over the past 50 y, a variety of approaches have been developed to more quickly assess biomarkers of cancer risk by assaying DNA reactivity or mutagenic potential as surrogate endpoints for regulatory decision-making (7,8). The most rapid and inexpensive of such methods include in vitro bacterial-based mutagenesis assays (e.g., the Ames test).…”

mentioning

confidence: 99%

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Valentine

Young

Fielden

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10−3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10−7. Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.

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“…In this approach gene mutation has to be considered as a toxicological endpoint. For more detail on this consideration see the review by Heflich et al (2019, in-press) [34].…”

Section: Discussionmentioning

confidence: 99%

“…These will be compared with those derived from other toxicological endpoints, and the most relevant and most conservative metric will be used as the final value from which the risk assessment is carried out. This is based on the recognition that mutation is a relevant endpoint and that the human population should be protected from increased risk of mutation [34]. In addition to this, mutation is linked with cancer, and the development of case studies will potentially show that genetic toxicity based HBGV will be comparable to those from the cancer bioassay, obviously ensuring exposure, tissue, study design, adjustment factors are suitable.…”

mentioning

confidence: 99%

Strategies in genotoxicology: Acceptance of innovative scientific methods in a regulatory context and from an industrial perspective

Steiblen

Benthem

Johnson

2020

Mutation Research/Genetic Toxicology and Environmental Mutagene

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The tests used and the general principles behind test strategies are now often over 30 years old. It may be time by now, given that our knowledge of genetic toxicology has improved and that we also technically are better able to investigate DNA damage making use of modern molecular biological techniques, to start thinking on a new test strategy. In the present paper, it is demonstrated that the time is there to consider a new approach for genotoxicity assessment of substances. A fit for all test strategy was discussed making use of the most recent technological methods and techniques. It was also indicated that in silico tools should be more accepted by regulatory institutes/bodies as supporting information to better conclude which tests should be required for each separate substance to demonstrate its genotoxic potency. Next to that there should be a good rationale for performing in vivo studies. Finally, the need for germ cell genotoxicity testing, essential when classification and labeling of substances is mandatory, was discussed. It was suggested to change the GHS for genotoxicity classification and labelling from in vivo tests in germ cells into in vivo tests in somatic cells. Quantitative genotoxicology was also discussed. It appeared that we are currently at a transition, where the science developing to justify carrying out human health risk assessments based on genetic toxicology data sets supported by mechanistic data and exposure data. However, implementation will take time, and acceptance will be supported through the development of numerous case studies. Major remaining questions are: is genetic damage a relevant endpoint in itself, or should the risk assessment be carried out on the apical endpoint of cancer and which genotoxic endpoint should be used to derive the point of departure (PoD) for the human exposure limit?

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Mutation as a Toxicological Endpoint for Regulatory Decision‐Making

Cited by 56 publications

References 59 publications

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing

Strategies in genotoxicology: Acceptance of innovative scientific methods in a regulatory context and from an industrial perspective

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