H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[ a]pyrene and dibenzo[ a, l]pyrene diol epoxides

Abstract

Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AXγ). A549 cells were exposed to benzo[ a]pyrene diol epoxide [(+)- anti-BPDE] (a bay-region PAH) and dibenzo[ a, l]pyrene diol epoxide [(−)- anti-DBPDE] (a fjord-region PAH) and H2AXγ was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H 2O 2) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H 2O 2. Visualisation of H2AXγ formation demonstrated that the proportion of cells exhibiting H2AXγ staining at 1 h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H 2O 2 treatment, almost all cells demonstrated H2AXγ at 1 h. Western blot analysis of the H2AXγ formation also showed concentration and time-dependent response patterns. The kinetics of H2AXγ formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AXγ formation and persistence was related to both the number of adducts and their structural features.