Prolonged benzo[b]fluoranthene exposure increases tissue-specific mutational burden, expansion of mutant cells, and...

Chronic exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with increased risk of cancer through a mutagenic mode of action. We investigated how exposure duration influences mutagenicity in bone marrow (BM) and liver of MutaMouse males orally exposed to increasing doses of benzo[b]fluoranthene (BbF), a priority PAH, for 28, 90, or 180 days. We applied Duplex Sequencing (DS) across 20 endogenous loci to assess dose- and time-dependent effects on mutation frequency, clonal expansion of mutant cells, mutation distribution, and spectra. Mutation frequencies increased with BbF dose and exposure duration in both tissues. After 90 and 180 days of exposure to 25 mg/kg/day BbF, mutation frequency was 1.7x and 2.1x higher in BM, and 3.8x and 8.6x higher in liver, than 28-day exposures. Clonally derived mutations increased significantly with exposure duration in BM but not liver. Intergenic loci incurred more mutations than genic regions in both tissues across all exposure durations. Mutation spectra were dominated by C:G > A:T transversions and were enriched in SBS4, the mutation signature associated with tobacco-induced human lung cancer. Benchmark dose (BMD₅₀) modeling showed increasing BbF potency with longer exposures. BMD₅₀ decreased from 8.2 (28d) to 3.7 (90d) and 3.0 (180d) mg/kg/day in BM; in liver, BMD₅₀ shifted from 14.3 (28d) to 4.3 (90d) and 3.9 (180d) mg/kg/day. The observed tissue-specific responses may be attributed to differences in proliferation rate, metabolic activity, and cellular lifespan. Our findings provide insights into the mutagenic impacts of prolonged PAH exposure and highlight tissue-specific differences in mutation susceptibility.