H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides.
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 (H2AXgamma). 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 H2AXgamma was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H(2)O(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(2)O(2). Visualisation of H2AXgamma formation demonstrated that the proportion of cells exhibiting H2AXgamma staining at 1h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H(2)O(2) treatment, almost all cells demonstrated H2AXgamma at 1h. Western blot analysis of the H2AXgamma formation also showed concentration and time-dependent response patterns. The kinetics of H2AXgamma formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AXgamma formation and persistence was related to both the number of adducts and their structural features.
Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, S-171 77 Stockholm, Sweden.
Ase
A
Mattsson
Bengt
B
Jernström
Ian A
IA
Cotgreave
Edyta
E
Bajak
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000494:pharmacology*; D018383:Bay-Region, Polycyclic Aromatic Hydrocarbon; D001564:Benzo(a)pyrene / Q000494:pharmacology*; D001580:Benzopyrenes / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D020552:Comet Assay; D018736:DNA Adducts / Q000494:pharmacology*; D004249:DNA Damage; D053938:DNA Fragmentation / Q000187:drug effects; D004852:Epoxy Compounds / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D010766:Phosphorylation / Q000187:drug effects; D013997:Time Factors
D001580:Benzopyrenes; D018736:DNA Adducts; D004852:Epoxy Compounds; C468783:H2AX protein, human; D006657:Histones; C121930:dibenzo(a,l)pyrene diol epoxide; C087596:11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo(a,l)pyrene; D001564:Benzo(a)pyrene; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide