The results of the present study demonstrate that short-term inhalational exposure of mice to the environmental contaminant and potent carcinogen, BD, is characterized by substantial differences in hepatic genetic and epigenetic response among mouse strains. Specifically, although formation of extensive amounts of THB-Gua adducts was observed in the livers of mice from all seven BD-exposed inbred strains, DNA damage was significantly less in CAST/EiJ strain (). Our previous study demonstrated that the presence of THB-Gua adducts in the livers of BD-exposed C57BL/6J mice is associated with disruption in the hepatic epigenetic status (Koturbash et al., 2011
). The present study shows that BD exposure, similarly to the observed differences in genotoxicity response, resulted in substantial variability in epigenetic alterations among strains. Even though many reports have demonstrated strain-specific variability in toxicity in response to chemical exposure (Bradford et al., 2011
; Harrill et al., 2009a
), a full understanding of the underlying mechanisms of such differences remains unresolved.
A detailed analysis of the pattern of genetic and epigenetic alterations caused by BD exposure allowed us to group mouse strains into three categories: (1) mice with high levels of THB-Gua adducts () and a substantial decrease in global and/or LINE1 DNA methylation (WSB/EiJ, PWK/PhJ, 129S1/SvImJ, and C57BL/6J strains; ); C57BL/6J mice were also characterized by a substantial decrease in histone H3K9, H3K27, and H4K20 trimethylation (); (2) mice with high levels of THB-Gua adducts and no significant alterations in global or LINE1 DNA methylation (NOD/LtJ and A/J strains); and (3) mice with low levels of hepatic THB-Gua adducts and increased histone lysine methylation (CAST/EiJ strain). By selecting representative strains from each group, we showed that the strain-specific differences may be due to dissimilarities in chromatin structure in response to a genotoxic insult.
It is well-established that both DNA methylation and histone H3K9, H3K27, and H4K20 trimethylation play a crucial role in the maintenance of proper chromatin structure and genomic stability (Dillon, 2004
; Jenuwein, 2006
; Martin and Zhang, 2005
). For instance, a greater extent of DNA methylation and histone H3K9, H3K27, and H4K20 trimethylation are associated with condensed chromatin structure and, consequently, gene silencing. In contrast, decreased DNA and histone H3K9, H3K27, and H4K20 methylation is associated with the formation of relaxed chromatin, increased transcription of the LINE1 and other repetitive DNA sequences, and a variety of genomic instability events. Additionally, it has been suggested that chromatin structure may influence the sensitivity of DNA to damage caused by various environmental agents (Falk et al., 2008
The results of our study support the hypothesis that interstrain differences in response to BD exposure may be caused by alterations in chromatin structure. A profound loss of DNA and histone H3K9, H3K27, and H4K20 methylation, a relaxation of chromatin structure, activation of expression of LINE1 and major and minor satellites, and liver toxicity in BD-exposed C57BL/6J mice support this suggestion. In contrast, in the livers of A/J mice exposed to BD, the hepatotoxic effect of BD was substantially lower, despite comparable amounts of THB-Gua adducts. This may be explained, in part, by a substantial upregulation of ABC-transporter genes and the ability of liver cells to retain a proper epigenetic status.
The most intriguing findings of our study are the substantially lower formation of THB-Gua adducts and negligible hepatotoxicity of BD exposure in CAST/EiJ mice. As mentioned above, CAST/EiJ was the only strain that upon BD exposure exhibited a substantial increase in histone H3K9, H3K4, H4K20, and especially H3K27 trimethylation. These observations were supported by an increase in expression of the Ezh2 histone methyltransferase. These changes resulted in formation of a compact heterochromatin structure that may decrease accessibility of DNA to BD-reactive intermediates yielding lower formation of THB-Gua adducts and less liver toxicity. In addition, increased capacity for detoxication/elimination or lower capacity of bioactivation of BD in CAST/EiJ strain, which may well be due to epigenetic modulation of gene expression, may also play a role.
The linkages between environmental agent-induced DNA damage, epigenetic effects, and subsequent risk of mutagenesis remain largely unexplored; however, there is increasing evidence that chromatin structure strongly influences DNA repair processes (Kinner et al., 2008
). Our data show that BD-induced DNA and histone hypomethylation effects that have been associated with carcinogenesis through spontaneous mutations, including loss of heterozygosity, chromosome translocation, and DNA deletion (Chen et al., 1998
), may lead to increased cancer susceptibility in some, but not all strains. In addition, strain-specific changes in chromatin structure may not only prevent chemical-induced DNA damage but also impact DNA repair because replication-independent endogenous DNA double-strand breaks are processed differently in methylated and unmethylated parts of chromatin (Kongruttanachok et al., 2010
). The relevance of the repair pathways to these strain-dependent differences remains to be elucidated because THB-Gua adducts are primarily lost through chemical depurination.
In conclusion, our findings demonstrate that significant epigenetic events occur following BD exposure and suggest that the differences in interindividual susceptibility to BD and other genotoxic chemicals may be determined by variations in epigenetic response. More importantly, the results of the present study suggest that assessment of carcinogen-induced epigenetic alterations, in addition to genetic changes, may facilitate identification of subpopulation susceptible to exposure.