In the present study cells were exposed
In the present study, cells were exposed to a low level of arsenite and a low cytotoxic concentration of BaP. Thus, γ-H2AX loss correlated with repair of DNA damage in cells co-exposed to arsenite and BaP. These results show that arsenite inhibits repair of BaP-induced DNA damage. In a previous study, we showed that low levels of arsenite do not induce DNA damage. As a co-carcinogen, however, arsenite inhibits repair of DNA damage, allowing damage to accumulate. Genetic instability results from an accumulation of errors during DNA replication and repair (Wang et al., 2001). Also in the present study, we found that arsenite apparently induces genetic instability by affecting the repair of DNA damage. Our results are consistent with previous results, in which arsenic enhances BaP mutagenicity, including DNA adduct formation in mouse lung and skin (Evans et al., 2004) and chromosome damage in mouse bone-marrow cells (Lewinska et al., 2004). The maintenance of genomic integrity after DNA damage depends on the coordinated action of the DNA repair system and Apoptosis Inhibitor checkpoint controls. DNA damage from replication stress is commonly observed in hyperproliferative tumor cells and can trigger a signaling pathway including ATR, ATM, Chk-1, Chk-2, and γ-H2AX (Bartkova et al., 2005, Gorgoulis et al., 2005), potentially resulting in activation of cell-cycle checkpoints. In particular, ATR is primarily involved in the repair of single-strand DNA breaks (Hammond and Giaccia, 2004), and ATM mediates responses in the presence of DSBs (Kurz and Lees-Miller, 2004). BaP-induced carcinogenesis involves DSBs (Temviriyanukul et al., 2012). In the present study, which involved comparison of ATM, ATR, Chk-1, and Chk-2 in control and arsenite and BaP-coexposed cells, there was differential induction of these signal pathways. The ATM/Chk-2 pathway was induced by BaP, but the ATR/Chk-2 pathway was not appreciably altered. Therefore, we concluded that the ATM/Chk-2 pathway mediates responses in the presence of DSBs induced by BaP exposure. Depletion of a specific DNA repair gene would enhance genotoxicity and ultimately affect chemical-induced malignant transformation of cells. Inhibition of expression of DNA repair genes contributes to cancer development in humans (Bukhari et al., 2011, Negrini et al., 2010). ERCC1, ERCC2, ATM, and hMSH2 are components of human DNA repair pathways, and silencing of these genes renders cells more sensitive to chemical induced damages (Wijnhoven et al., 2007). Defects in ATM result in an insufficiency of homologous recombination repair, leading to an increasing incidence of hematological malignancies (Lavin, 2008). Long-term exposure to arsenite in drinking water results in decreases in expression of ERCC1, XPF, and XPB, which are involved in the NER repair system (Andrew et al., 2003). In the present study, we found that, in HBE cells, arsenite inhibits repair of BaP-induced DNA damage by blocking the ATM/Chk-2 pathway. Thus, it appears that arsenite induces deficiencies in DNA repair that shorten the latency of BaP-induced cell transformation. HIF-α and HIF-β are transcription factors involved in carcinogenesis and tumor progression (Gordan and Simon, 2007, Kuphal and Bosserhoff, 2011). Under nomoxic conditions, environmental chemicals, such as arsenite, nickel, and cobalt, induce the expression of HIFs (Kaczmarek et al., 2009), and their up-regulation is involved in the induction of cancers. We have reported that arsenite inhibits ubiquitin proteasome-dependent degradation of HIF-2α, which is involved in the transformation of cells (Xu et al., 2012). BaP is reported to have no effect on the expression of HIF-1α (Li et al., 2006), and our results are consistent with other reports that BaP does not induce HIFs. Co-treatment of cells with arsenite and BaP, however, led to induction of HIF-2α, which was involved in the process of arsenite-accelerated, BaP-induced neoplastic transformation of HBE cells. Hypoxia has been linked to genetic instability and tumor progression, and aberrant repair of DNA damage under hypoxia is a possible factor in hypoxia-mediated genetic instability. HIFs could down-regulate some genes involved in repair of DNA damage, including those for ATR, ATM, Chk-1, and Chk-2. HIF-2α expression, in the absence of HIF-1α, correlates with increased BRCA1 expression and efficient completion of the S phase, thereby permitting cell cycle progression in the face of replication stress while limiting p-Chk-2 accumulation (Gordan et al., 2008). Thus, it appears that the effect of HIF-2α on repair of DNA damage in HBE cells exposed to arsenite and BaP is involved in their malignant transformation. The present data show that inhibition of HIF-2α activation blocks the arsenite-accelerated, BaP-induced neoplastic transformation of HBE cells and that HIF-2α blocks the ATM/Chk-2 pathway in cells exposed to arsenite and BaP.