Dose-Related Mutagenic and Clastogenic Effects of Benzo[ b]fluoranthene in Mouse Somatic Tissues Detected by Duplex Sequencing and the Micronucleus Assay

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that originate from the incomplete combustion of organic materials. We investigated the clastogenicity and mutagenicity of benzo[b]fluoranthene (BbF), one of 16 priority PAHs, in MutaMouse males after a 28 day oral exposure. BbF causes robust dose-dependent increases in micronucleus frequency in peripheral blood, indicative of chromosome damage. Duplex sequencing (DS), an error-corrected sequencing technology, reveals that BbF induces dose-dependent increases in mutation frequencies in bone marrow (BM) and liver. Mutagenicity is increased in intergenic relative to genic regions, suggesting a role for transcription-coupled repair of BbF-induced DNA damage. At higher doses, the maximum mutagenic response to BbF is higher in liver, which has a lower mitotic index but higher metabolic capacity than BM; however, mutagenic potency is comparable between the two tissues. BbF induces primarily C:G > A:T mutations, followed by C:G > T:A and C:G > G:C, indicating that BbF metabolites mainly target guanines and cytosines. The mutation spectrum of BbF correlates with cancer mutational signatures associated with tobacco exposure, supporting its contribution to the carcinogenicity of combustion-derived PAHs in humans. Overall, BbF's mutagenic effects are similar to benzo[a]pyrene, a well-studied mutagenic PAH. Our work showcases the utility of DS for effective mutagenicity assessment of environmental pollutants.

Keywords: COSMIC signatures; benzo[b]fluoranthene; bone marrow; duplex sequencing; environmental pollutant; liver; mutation spectrum; polycyclic aromatic hydrocarbons.

Figure 1

Frequencies of induced micronuclei in peripheral blood of MutaMouse males after vehicle or BbF exposure for 28 days. Mean micronucleus (MN) frequencies (black bar) with indication of standard errors of the mean (SEM). MN frequencies per 1000 cells are shown for both reticulocytes (MN RET) (A) and red blood cells (MN-RBC) (B). Dots represent individual animal data. *p < 0.05 and **p < 0.01 relative to vehicle control.

Figure 2

Mean minimum mutation frequency (MF_min ± SEM) per bp measured by Duplex Sequencing in mouse bone marrow (A) and liver (B) after exposure to increasing doses of BbF (N = 4 per dose group). Asterisks indicate *p < 0.05 and **p < 0.01 relative to vehicle control. Dots represent individual animal MF_min.

Figure 3

Mean mutation frequency per bp measured by Duplex Sequencing in bone marrow (A) and liver (B) for individual mice within each BbF dose group. The mutation frequency of unique mutations is indicated as MF_min (black). The mutation frequency including clonally expanded mutations is shown as MF_max (light blue). Numbers above the bars represent the observed number of mutations.

Figure 4

Mean minimum mutation frequency (MF_min) ± SEM per locus for BbF-exposed mouse bone marrow (A) and liver (B) alongside vehicle controls. Loci are sorted from the highest top dose response to the lowest (left to right). Intergenic and genic loci are show in red and black, respectively. Statistical testing was done for the top dose with respect to the vehicle control using a generalized linear mixed model; *p < 0.05 and **p < 0.01; N = 4 per dose group.

Figure 5

Mutation spectrum for vehicle controls and BbF dose groups observed in mouse bone marrow (A) and liver (B). Mutation subtypes, insertions, deletions and multinucleotide variants (MNV) are presented as mean MF_min ± SEM. Mutation subtypes are based on the six single nucleotide variants using a pyrimidine reference. Statistical testing was done using a generalized linear mixed model; *p < 0.05 and **p < 0.01; N = 4 per dose group.

Figure 6

Mutation signature analyses for top BbF dose (100 mg/kg/day) in bone marrow (A) and liver (B). The original trinucleotide mutation profile is shown on the top left. SigProfilerAssignment used the single base substitution (SBS) signatures of the Catalogue of Somatic Mutations in Cancer (COSMIC) database to reconstruct the signatures. The SBS signatures and their relative contributions are shown on the right. The reconstructed mutation pattern, number of substitutions used for the assignment, and cosine similarity between reconstructed and observed trinucleotide mutation profile are shown on left below the original profile. The total number of mutations in the original signature are indicated on the left.

Figure 7

Log₁₀ benchmark dose (BMD) for a 50% increase in micronucleus (MN) or mutation frequency (MF_min). 90% confidence intervals based on BMD model averaging analyses using PROAST are shown. Confidence intervals are organized from lowest (top) to highest (bottom) potency. Labels indicate micronuclei in reticulocytes (MN-RET) and red blood cells (MN-RBC), and mutation frequency in liver (MF liver) and bone marrow (MF BM).