From Conventional Methodologies to Error-Corrected Sequencing: High-Resolution Profiling of Dose-, Time-, and Tissue-Specific Benzo[b]fluoranthene-Induced Mutagenesis

Abstract

Accurate assessment of in vivo somatic mutagenesis is essential for human health risk assessment. However, current regulatory testing guidelines rely heavily on short-term exposures and single-gene phenotypic reporter assays. This thesis characterizes the in vivo mutagenic dynamics of the priority polycyclic aromatic hydrocarbon (PAH) benzo[b]fluoranthene (BbF) across multiple tissues and extended exposure durations using Duplex Sequencing (DS). This approach simultaneously addresses existing knowledge gaps and validates DS as a promising tool to modernize in vivo mutagenicity assessments based on quantitative and mechanistic data. BbF exposure induced dose-dependent increases in micronucleus frequency in peripheral blood and mutation frequency (MF) in the liver and bone marrow (BM) of MutaMouse males after 28 days. Benchmark dose (BMD) modeling revealed comparable clastogenic and mutagenic potencies. Mutations were preferentially induced in intergenic loci, suggesting a protective activity of transcription-coupled nucleotide excision repair in transcribed regions. Furthermore, mutagenesis was characterized predominantly by C:G>A:T and secondary C:G>T:A mutations. Investigation of the trinucleotide mutation pattern revealed similarities towards human tobacco-associated cancer signatures, aligning with a canonical route of PAH exposure. Evaluating prolonged exposures up to 90 and 180 days demonstrated that mutation accumulation mechanisms are tissue-specific. The liver exhibited a higher maximum mutational burden than BM, reflecting its primary role in xenobiotic metabolic activation. In the highly proliferative BM, mutagenesis was heavily driven by the clonal expansion of mutant cells, whereas predominantly unique de novo mutations were detected in liver. This indicates that the physiological propagation of mutant cells contributes differently to the overall mutation burden depending on tissue characteristics. Despite these distinct tissue dynamics, the underlying mutation spectrum remained consistent over time, indicating a stable mutagenic mechanism. Furthermore, BMD modeling highlighted that prolonged exposures increase mutagenic potency and yield lower points of departure compared to standard 28-day designs. Direct comparisons revealed strong quantitative concordance, highlighted by robust Pearson correlations between DS-derived MF on the endogenous mouse mutagenesis panel or the exogenous lacZ transgene versus the conventional transgenic rodent (TGR) assay. These endpoints yielded overlapping potency estimates, validating sequence-resolved analysis as robust quantitative alternatives to the phenotypic TGR assay. Mutation spectrum analysis revealed comparable mutation subtypes between the MMP and lacZ loci, hinting towards endpoint independent mutagenic mechanisms. Conversely, targeted sequencing of the endogenous Pig-a locus was less informative and did not detect dose-related responses. Mutation spectrum analysis was predominantly driven by insertions and deletions in the Pig-a locus due to sequencing artifacts within homopolymeric sequences. This underscores the importance of distributed loci selection over single-gene sequencing. To address existing barriers to regulatory adoption, power analyses demonstrated that sample sizes of four animals per dose group provide sufficient statistical power, representing a reduction in animal use that supports 3Rs principles. Further collaborative efforts demonstrated the high inter-laboratory reproducibility of DS across eight laboratories and developed an open-source bioinformatic approach to harmonize mutation frequency calculations, statistical analysis and quantitative modeling. Collectively, this research defines the dose-, time-, and tissue-dependent determinants of BbF-induced mutagenesis and shows that these factors directly influence potency estimates. Sequence-resolved analyses confirm stable mutational mechanisms across endpoints and exposure durations. Concordance with OECD assays, together with reproducibility and standardized workflows, establishes Duplex Sequencing as a sensitive and quantitatively robust approach. It reduces animal use, integrates into existing toxicology study designs, and enables high-resolution mechanistic data generation. These features support its readiness for broader adoption in in vivo mutagenicity assessment.