In Utero Exposure to Benzo[a]Pyrene Increases Mutation Burden in the Soma and Sperm of Adult Mice

Abstract

Background: Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear.

Objectives: We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) has an impact on the burden and distribution of mutations in adult mice.

Methods: We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development.

Results: Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of first filial generation (F1) males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on the reproductive function of F1 males.

Conclusion: In utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently affecting both the genetic health of the F1 and the population gene pool. Citation: Meier MJ, O'Brien JM, Beal MA, Allan B, Yauk CL, Marchetti F. 2017. In utero exposure to benzo[a]pyrene increases mutation burden in the soma and sperm of adult mice. Environ Health Perspect 125:82-88; http://dx.doi.org/10.1289/EHP211.

Figure 1

Male mice exposed transplacentally to benzo[a]pyrene (BaP) during organogenesis exhibited increased mutant frequencies in multiple somatic tissues. Dams were dosed at 0, 10, 20, or 40 mg/kg/day, and mutant frequencies [in 10-week-old first filial generation (F1) males] were determined using the lacZ transgene mutation assay. (A) In utero exposure to BaP induced a significant dose-dependent increase in mutant frequencies in somatic tissues originating from distinct primary germ layers, including the bone marrow (mesoderm), brain (ectoderm), and liver (endoderm). Error bars represent 95% confidence intervals. *p < 0.05, ***p < 0.0001, generalized linear model with quasi-Poisson distribution. (B) Next-generation sequencing of the lacZ reporter gene from animals exposed in utero shows the well-characterized molecular signature of BaP exposure, indicated in the pie charts as an increase in the proportion of G→T mutations. The number of unique lacZ mutations sequenced is noted beneath each pie chart. INS, insertion; DEL, deletion.

Figure 2

In utero exposure to benzo[a]pyrene (BaP) significantly decreases multiple reproductive parameters in first filial generation (F1) adult male mice. BaP caused a significant dose-dependent decrease in the testis weight (shown as percentage body weight) (A), as well as a drastic decrease in sperm concentration (B) and percentage of motile sperm (C). Liver weight (D) was unaffected. *p < 0.05, analysis of variance (ANOVA). Error bars represent 95% confidence intervals.

Figure 3

Mutant frequencies were significantly increased in caudal sperm (A) and whole testes (B) of first filial generation (F1) males exposed in utero to 20 and 40 mg/kg/day benzo[a]pyrene (BaP). Mutant frequencies in sperm at 40 mg/kg/day were measured by pooling several DNA samples because of extremely low sperm counts. Error bars represent 95% confidence intervals. **p < 0.001, ***p < 0.0001, generalized linear model with quasi-Poisson distribution. (C) Next-generation sequencing of the lacZ reporter gene from caudal sperm. BaP exposure (20 and 40 mg/kg/day combined) increased the proportion of G→T and G→C mutations but had no effect on deletions. The number of unique lacZ mutations sequenced is noted beneath each pie chart.

Figure 4

Clonal expansion of the mutations characterized in each somatic tissue and in caudal sperm. The proportion of mutations that underwent clonal expansion is consistently higher in animals exposed to benzo[a]pyrene (BaP) in utero, illustrating the potential for increased mosaicism. As in somatic tissues, the proportion of clonally expanded mutations is higher in BaP-exposed sperm, highlighting the potential for increased germline mosaicism. Compared with exposure in adults (data from Beal et al. 2015 and O’Brien et al. 2016b), in utero exposure results in a higher degree of clonal expansion. Plaques were collected from the 20 mg/kg/day dose group for cadual sperm because of decreased sperm concentration at 40 mg/kg/day. Each circle within a group corresponds to a scored independent mutation, and the area of each circle represents the number of times that mutation was observed per animal. INS, insertion; DEL, deletion.