| Cancer is the second leading cause of death in developed countries, and exposure to environmental carcinogen is a major contributing factor for the cancer epidemic. Most of the known environmental carcinogens are genotoxins, meaning these agents cause cancer by damaging DNA. There are many types of DNA damage, including base modification which covers chemical changes to base altering their base pairing attributes and DNA adduction, single strand breaks (SSBs), double strand breaks (DSBs),intra- or inter-strand cross-links, among which DSBs are regarded as the most severe type of damage. A sensitive method to identify DNA damage would be helpful for us to identify potential environmental genotoxins, and thus be helpful for the prevention and therapy of cancer.Recently, the relationship between histone H2AX and DSBs has been gradually recognized. The histone H2AX is one of the variants of H2A. H2AX is phosphorylated (denoted as yH2AX) and forms "foci" which could be clearly viewed in the immunofluorescent microscope in response to DSBs induced by many stimuli. yH2AX plays an important role in the processing and repair of DSBs, it could recruit many DNA repair proteins including BRCA1,53BP1, Rad50 and NBS1 for the repair of DNA damage. The number of γH2AX foci detected by immunofluorescence is quantitatively the same as IR induced DSBs, and the disappearance of γH2AX foci is exactly consistent with the repair of DNA damages. Thus, γH2AX foci is proposedto be a potential biomarker for DSBs. And, it has been used for detecting many stimuli induced DSBs, including many chemicals, virus, and even heat shock.Comet assay, a classical method for detecting DNA damage, is frequently used to measure DNA strand breaks in single cells. Comet assay can be divided into two classes: one is neutral comet assay, which is used to detect DSBs in cells;and another one is alkaline comet assay used to detect SSBs and DSBs or even base modification.The monofunctional alkylating agent N-methyl-A^'-nitro-iV-nitrosoguanidine (MNNG) is a potent carcinogen and mutagen which can be found in tobacco smoke, and it is involved in the development of gastric and colorectal cancer in both animals and humans. It generally targets DNA and proteins to generate adducts, which can eventually lead to chromosomal aberrations, point mutations, and cell death. In addition, MNNG can also induce DNA strand breaks detected by using comet assay.Although yH2AX foci formation has been suggested as a sensitive way to detect DNA damages, it still needs to be carefully validated before being widely applied in research or other related areas. Therefore, using MNNG as the model chemical, both comet assay and immunofluorescent microscopy for yH2AX foci formation were conducted to evaluate MNNG-induced DNA damage, and the results were systematically compared for their consistency.MNNG induces the formation of yH2AX foci in FL cells in a time- and dose-dependent mannerAfter FL cells were treated with different concentrations of MNNG for different times, the yH2AX foci formation in FL cells were analyzed in the immunofluorescent microscopy.Results: MNNG induces the formation of yH2AX foci in FL cells in a time- and dose- dependent way. 0.1 \xg/ml and 1 ug/ml MNNG could dramatically induce the yH2AX foci in FL cells. However, from 10 min to 8 h time point, the percentage of cells with more than 30 foci/cell treated with 0.1 |ag/ml MNNG was increased and reached a peak;while at 24 h time point, the percentage of cells with more than 30foci/cell treated with 0.1 |ag/ml MNNG was decreased and ultimately was similar with the control at 48 time point. In contrast, 1 jig/ml MNNG showed its effect on yH2AX foci formation at 30 min, and the percentage of cells containing over 30 foci/cell increased steadily, and at the end of the 48 h time frame, almost all cells had at least 30 foci/cell. On the other hand, for 10 ug/ml MNNG, we observed a distinct whole nuclei staining pattern of yH2AX.Neutral comet assay demonstrates that MNNG induces DNA strand breaksAfter FL cells were treated with different concentrations of MNNG for different times, the DSBs formation in FL cells were analyzed by neutral comet assay.Results: For 0.1 ug/ml of MNNG, there were no significant changes for neither the percentage of cells with tail nor the length of tail at 10 and 30 min. However, 2 h later, the percentage of cells with tail jumped to 73%, with an average tail length of 10 um. This trend continued to 8 h, and then began to come down sometime after that. At 24 h, only 29% of cells still had tail, and the average tail length was decreased to about 5.49±3.98 um. At 48 h, both the percentage of cells with tail and the average tail length were comparable to those of controls. On the contrary, although 1 ug/ml MNNG also induced increases in both the percentage and tail length at 2 h, this trend continued and lasted to 48 h. In sharp contrast, neutral comet assay did not show any significant changes of 10 ug/ml MNNG treated cells in 8 h. Even more intriguing was that at 24 h, there was no cell with the comet tail, although at 48 h, about 33% cells showed the comet tail again.Alkaline comet assay reveals DNA strand breaks in whole nucleus staining cellsFL cells were treated with 10 jag/ml MNNG for 2 h, and then were further subjected to alkaline comet assay.Results: Alkaline comet assay revealed the presence of DNA damage induced by 10 |ig/ml MNNG, which indicates that there were other patterns of DNA damage other than DSBs.MNNG induces the formation of yH2AX foci in CHL cells in a time-dependent mannerAfter CHL cells were treated with 1 ng/ml of MNNG for different times, the yH2AX foci formation in CHL cells were analyzed in the immunofluorescent microscopy.Results: I ng/ml of MNNG induced yH2AX foci formation in CHL cells, which indicates that the yH2AX foci formation induced by MNNG is not cell type dependent.Wortmannin inhibits yH2AX foci formation in FL cells treated with MNNGFL cells were pre-incubated with wortmannin for 30 min, and then were subjected to the treatment with 1 ug/ml MNNG for 1.5 h and after then were further co-incubated with wortmannin for another 30 min, finally the yH2AX foci formation in FL cells were analyzed in the immunofluorescent microscopy.Results: 1 ng/ml of MNNG dramatically induced yH2AX foci formation in FL cells, which was inhibited by wortmannin, which demonstrated that PI-3K family kinases were responsible for the yH2AX foci formation induced by MNNGConclusion: Firstly, the results of yH2AX foci formation were highly consistent with the results of neutral comet assay although there existed some minor discrepancies. Thus, yH2AX foci formation could be used to detect DSBs and to some extent it could earlier detect the DNA damage than neutral comet assay. Second, 10 ug/ml MNNG induced distinct whole nuclei staining pattern might reflect other DNA damages than DSBs. Third, MNNG induced yH2AX foci formation was not cell type specific. Forth, MNNG induced yH2AX foci formation was dependent on PI-3K family kinases. |
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