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A number of risk factors have been linked epidemiologically with gastric cancer, but studies of DNA damage in gastric epithelial cells are limited. The comet assay is a simple technique for determining levels of DNA damage in individual cells. In this study, we have validated the comet assay for use in epithelial cells derived directly from human gastric biopsies, determined optimal conditions for biopsy digestion and investigated the effects of oxidative stress and digestion time on DNA damage. Biopsies taken at endoscopy were digested using combinations of pronase and collagenase, ethylenediaminetetra-acetic acid (EDTA) and vigorous shaking. The resultant cell suspension was assessed for cell concentration and epithelial cell and leukocyte content. A score for DNA damage, the comet %, was derived from the cell suspension, and the effect of various digestion conditions was studied. Cells were incubated with H(2)O(2) and DNA damage was assessed. Pronase and collagenase provided optimum digestion conditions, releasing 1. 12x10(5) cells per biopsy, predominantly epithelial. Of the 23 suspensions examined, all but three had leukocyte concentrations of less than 20%. The comet assay had high inter-observer (6.1%) and inter-assay (4.5%) reproducibility. Overnight storage of the biopsy at 4 degrees C had no significant effect on DNA migration. Comet % increased from a median of 46% in untreated cells to 88% in cells incubated for 45 min in H(2)O(2) (p=0.005). Serial 25-min digestions were performed on biopsies from 13 patients to release cells from successively deeper levels in the crypt. Levels of DNA migration were significantly lower with each digestion (r=-0.94, p<0.001), suggesting that DNA damage is lower in younger cells released from low in the gastric crypt. The comet assay is a reproducible measure of DNA damage in gastric epithelial cells. Damage accumulates in older, more superficial cells, and can be induced by oxidative stress.
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PMID:In vivo DNA damage in gastric epithelial cells. 1086 59

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm--around 300--and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for > 50% of the total cells in a typical adult worm. Therefore, isolating sperm from males is easier than from that of hermaphrodites. Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population. Males can be easily distinguished from hermaphrodites by observing the tail morphology. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures. Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids. Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm. Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes, and Nelson previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E. Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.
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PMID:Isolation and in vitro activation of Caenorhabditis elegans sperm. 2130 34