Investigating benzene-initiated DNA double-strand breaks and recombination after acute and in utero exposure in mice
Lau, Annette Anling
Benzene , DNA double-strand breaks , In utero , Childhood leukemia , Developmental hematopoiesis
Benzene is an ubiquitous pollutant and industrial solvent that has been identified as a human leukemogen. Early exposure to environmental carcinogens such as benzene has been postulated to play a role in the etiology of childhood leukemia, however the association remains controversial. Genotoxic agents such as benzene can cause an increase in the frequency of DNA double-strand breaks, which may remain unrepaired or result in the initiation of DNA recombinational repair mechanisms. The first objective was to investigate the induction of DNA double-strand breaks following in utero treatment to 200 mg/kg and 400 mg/kg benzene i.p. using the phosphorylated histone γ-H2A.X as a marker. Using immunoblotting, treatment with benzene did not increase the formation of γ-H2A.X in bone marrow cells of adult C57Bl/6N male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells following in utero exposure to 200 mg/kg or 400 mg/kg benzene throughout gestational days 7 to 15. Secondly, the study investigated the induction of micronuclei following in utero exposure to benzene. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells. In utero exposure to 400 mg/kg benzene throughout gestational days 7 to 15 also caused a statistically significant increase in the percentage of micronucleated cells in maternal bone marrow and post-natal bone marrow cells. Fetal liver cells also demonstrated a statistically significant increase in the percentage of micronucleated cells following 200 mg/kg and 400 mg/kg benzene. The third objective was to investigate the initiation of DNA recombination following in utero exposure to benzene using the pKZ1 mutagenesis mouse model as a surrogate marker for non-homologous end joining activity. Adult pKZ1 mouse tissue yielded no recombination events; however, post-natal bone marrow cells did contain detectable recombination frequencies. iii In utero benzene exposure did cause an increasing trend in recombination events, and upon analysis of only the samples containing detectable levels of recombination, in utero exposure to 400 mg/kg of benzene caused a statistically significant increase in recombination frequency within this group. These results demonstrate that benzene does not increase the formation of γ-H2A.X after acute and in utero exposure, however, the induction of micronuclei following acute and in utero benzene exposure confirmed that benzene is a genotoxic agent causing chromosomal breaks. In utero benzene exposure increased the frequency of DNA recombination in bone marrow from post-natal day 9 pups exhibiting detectable levels of recombination. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood leukemias.