Marie MAM, et al: Extent of DNA Damage and Gastritis in Normal and Helicobacter pylori-Infected Patients 319 tensively to conditions where single cells are freely available in suspension, but less frequently to solid tissues. In order to per- form in vivo studies of epithelial cells, it is necessary to create a cell suspension from solid biopsy material. It is possible that this process could lead to spurious, ex vivo, DNA damage. For instance, both trypsinisation and scraping of cell cultures prior to performing the comet assay have been shown to cause DNA damage.6 We discovered that the high comet percentages in cells from normal specimens may reflect the true levels of DNA damage that occur during physiological processes. The theory supports our results which state that the cells die due to senescence in normal processes of cell turnover. Our results may imply that the normal surface gastric epithe- lial cells accumulate DNA damage for a longer time resulting in higher rate of DNA damage to be detected experimentally un- like H. pylori infected cells with rapid turnover. Another explanation is that the decreased repair mechanism of DNA damage by physiological cell death leads to a higher rate of DNA damage; the DNA repair mechanism does not take place in the normal cells and therefore DNA damaged cells per- centage is highly seen. It also could be the case that the gastric epithelial cells which became infected with H. pylori died more quickly which resulted in less cometed cells due to generation of younger cells with less DNA damage. Also, these H. pylori gastric infected cells might be develop a mutation which can result in cell phagocytosed and early gastric cells death. Our results are in contrary with results published by Ladeira et al .4 and Arabski et al .7 Both authors used comet assay to show that H. pylori infected patients have significant DNA dam- age than non infected patients. The above authors support their results with the theory of increased rates of cellular exfoliation8 and cell proliferation9 have been demonstrated in tissues infected with H. pylori. This implies that there is a higher rate of cell death than in normal tissue. Also they suggest that a normal cell dies quickly, and so it will be less likely that the DNA damage will be detected experimentally or possibly accumulated, thus showing a lower comet percentage which represents less DNA damage in the normal cell than what is actually occurring. Furthermore, the use of biopsy specimens for estimation of DNA damage may select different types of cell populations as- sociated with different histological diagnosis. Exfoliation of cell into the gastric lumen occurs at a faster rate in H. pylori infect- ed tissue than in normal tissue,10 so the majority of cells with DNA damage may not be included in the biopsy specimen. Only cells with single strand breaks caused by reactive oxygen spe- cies (ROS) which have not resulted in cell death would remain in the tissue.11 The H. pylori infected gastric cells are susceptible to mutation, also if they have a high degree of DNA damage they are more likely to die or be phagocytosed by inflammatory cells, resulting in a few cells with DNA damage to be detected. The mutation and a higher DNA damage in specimens from H. pylori infected tissue can be detected experimentally unless pro- gression to later stages of the preneoplastic sequence is occur- ring. When there is H. pylori infection in the gastric tissue there will be an accompanying inflammatory cell infiltrate, consisting of neutrophil polymorphs, monocytes, lymphocytes and plasma cells.10 When the infection is active neutrophil predominate and will be found locally at the site of inflammation,12 so they may be collected with the biopsy specimen. These additional cells would not be present in normal gastric tissue. Therefore, biop- sies taken from tissues with different histological profiles can be expected to have heterogonous cell population with varying proportion of different cells. In tissue infected with H. pylori the proportion of gastric epithelial cell present in the assay may have been less than in normal tissues, where they should be ac- counted for the majority of cells. The comet assay itself is not selective. Any cell with single strand breaks will form a comet. This will result in a distortion of the comet percentage from that which would have occurred if only gastric epithelial cells were present. This would reflect the average level of DNA damage in all of the cells present, instead of only that of the gastric epithelial cells. To overcome this disadvantage the cells in the specimen could be separated according to cell type before the assay is performed. This would be difficult to achieve and could cause artefactual DNA dam- age. Alternatively, extra biopsies of tissue from the same biopsy taken for the comet assay could be taken from each patient in order to investigate in detail the proportions of different cell types in each biopsy. Such an investigation was done at stan- dard incubation time of 40 minutes and no difference in cell types was found. The gradual reduction in DNA damage throughout the gastric layer cells in normal individuals may be due to the fact that the gastric epithelial cells are old and have accumulated damaged DNA with age. Also, the younger cells should be synthesized and are present at the crypts and muscular layer in a few days. The younger cells move towards the mucosa in a gradual man- ner. Although these younger cells are present at the crypts and muscular layer initially, after a few days these cells are present at the mucosa. The results of this study suggest that in normal individuals the highest degree of DNA damage was in the mucosa. The low- est degree of DNA damage was observed in the muscular layer and these finding may be due to the reasons mentioned above. In H. pylori patients the low degree of DNA damage through- out the gastric layer cells could be due to rapid turnover of the gastric cells as a result of inflammation and hence younger cells with low DNA damage are present in infected tissue. The younger cells in H. pylori patient are present through the gastric layer cells due to the processes of gastric cell turnover. These cells move towards the mucosa more rapidly in H. pylori pa-