The Ability Of Benzo(a)pyrene And Azathioprine/cyclosporin A In The Induction Of γH2AX Foci Formation

Nowadays, the incidence of cancer is increasing significantly, in which many cases are associate with exposure to environmental carcinogens. It is an important task to develop a method which can accurately evaluate the carcinogenecity of chemicals. One type of environmental carcinogens is genotoxins, which cause cancer by damaging DNA. Another type of environmental carcinogens is epigenetic carcinogens, including some cytotoxins and immunosuppressants, which cause cancer by means other than DNA damage. Most of the known environmental carcinogens are genotoxins. There are many types of DNA damage induced by genotoxins, including single strand breaks (SSBs), double strand breaks (DSBs), DNA adduction, intra- or inter-strand cross-links and base modification, et al. DSBs are regarded as the most severe type of damage. Thus, a sensitive method to identify DSBs would be helpful for the prevention and therapy of cancer.Recently, researchers have found that histone H2AX is phosphorylated (denoted as γH2AX) by phosphatidylinositol 3-like kinase when responses to DSBs. γH2AX could recruit many other DNA repair proteins to DSBs and forms "foci", then repair the damaged DNA together. It has been shown that after exposure to ionizingradiation (IR), yH2AX foci form quickly, and the number of yH2AX foci detected by immunofluorescence is quantitatively the same as that of DSBs, suggesting that yH2AX is an indicator for DSBs induced by IR. Some other genotoxic agents, such as cisplatin, camptothecin, which do not induce DSBs directly but nonetheless cause DSBs indirectly have also been shown to induce yH2AX foci formation. Since both direct and indirect induction of DSBs can lead to yH2AX foci formation, we believe that yH2AX foci could be used in the detection of DSBs.Benzo(a)pyrene (B(a)P) is a type of Polycyclic aromatic hydrocarbon (PAH), which has strong carcinogenicity. It can be activated by cytochrome P450 to generate 7,8-dihydrodiol9,10-epoxidebenzo(a)pyrene (BPDE), which could be covalently linked to DNA. It can cause abnormal cell differentiation, and promote cancer incidence.Azathioprine (Aza) and cyclosporin A (CsA) are both immunosuppressants, which are mainly used for suppressing rejection after transplantation. Both are regarded as epigenetic carcinogens. They can promote necrosis of cells, and the cytotoxicity is not through DNA damage.To further examine the hypothesis that yH2AX foci formation is a specific marker of DSBs, we selected a group of physical/chemical stressors to test then-ability to induce yH2AX foci formation in human amnion FL cells. In this study, the ability of indirect-genotoxin B(a)P, and epigenetic carcinogens Aza/CsA in the induction yH2AX foci formation was evaluated.In MTT experiments, no significant cytotoxicity was observed in FL cells for the different concentrations of B(a)P (up to 100 jaM) tested during the 24 h period. On the other hand, both Aza (20 fig/ml) and CsA (20 |j.g/ml) had significant cytotoxic effects on FL cells.After B(a)P treatment for 10 min and 2 h, no significant induction of yH2AX foci was observed for all concentrations tested. But at 8 h and 24 h, all concentrationstested had induced yH2AX foci formation. B(a)P induced yH2AX foci formation also exhibited a dose-dependent manner, as the highest concentration (50 \iM) induced the most foci. Compared to 8 h, the percentage of cells containing yH2AX foci was also increased at 24h. Micronuclei formation was also observed by 5OjoM B(a)P treatment.Compared to control, Aza/CsA treatment did not induce yH2AX foci formation in FL cells.Neural comet assay revealed that B(a)P could induce DSBs, while Aza/CsA could not.Conclusions:1. Indirect-genotoxin B(a)P induces yH2AX foci formation in a time- and dose-dependent manner in FL cells.2. Epigenetic carcinogens Aza and CsA can not induce yH2AX foci formation in FL cells.3. Only those chemicals that can induce DSBs can induce yH2AX foci formation. yH2AX foci can be used to detect DSBs.