unscheduled DNA synthesis and genotoxicity

Results from a recent, new assay suggest that omeprazole, a potent inhibitor of gastric acid secretion, is genotoxic. The principle of this assay is that the non-proliferating zone of surface gastric epithelial cells can be selectively removed by controlied digestion so that any incorporation of tritiated thymidine into these cells represents unscheduled DNA synthesis. Parietal cells (which are located below the uppermost proliferating cells) and proliferating cells in semiconservative, regular DNA synthesis could always be shown in the digested fraction, and as regular DNA synthesis takes up a thousand fold more thymidine than unscheduled DNA synthesis, any signal from unscheduled synthesis would therefore be swamped. The digestion process was also uneven, as histological analysis showed denuded patches ofmucosa, and gland like structures were seen in the digest. Quantification of the number of silver grains over the nuclei showed no increase in low level labelling after omeprazole administration, indicating that there was no unscheduled DNA synthesis. The labelling index of undigested gastric tissue from omeprazole treated rats was not significantly different from that ofthe control group, despite an increase in the plasma gastrin value. (Gut 1993; 34: 235-241) Imperial Cancer Research Fund, Histopathology Unit, Lincoln's Inn Fields, London R A Goodlad C Y Lee N A Wright Histopathology Department and Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London M R Alison C E Sarraf M A Ghatei S R Bloom C6rrespondence to: Dr R A Goodlad, Imperial Cancer Research Fund, Histopathology Unit, 35-43 Lincoln's Inn Fields, London WC2A 3PN Accepted for publication 27 July 1992 Carcinogenesis is a multistage process involving permanent changes in DNA.' DNA repair can occur after genotoxic damage as 'unscheduled DNA synthesis', and misor incomplete DNA repair may be related to carcinogenesis. While there are several well established tests for genotoxicity,2 3 these are either in vitro assays or use non-gastrointestinal tissue systems, thus a new in vivo test could have considerable potential. Such genotoxicity assay for the rat stomach has recently been described by Burlinson,4 who reported that the powerful proton pump inhibitor, omeprazole,5' stimulates unscheduled DNA synthesis.7 Other anti-acid drugs, namely loxtidine, cimetidine, and ranitidine did not have this effect.8 These findings have considerable implications9 but are at variance with the data generated from the extensive application of established genotoxicity methods. "' The site specific mode of action of omeprazole, in which the active form (sulphenamide) is generated only in the cannuliculi of the parietal cells has led to the value of these data being questioned. ' The basis of the Burlinson technique is that non-proliferating gastric surface epithelium cells are selectively removed by enzymatic digestion in vitro, the test agents having been previously administered in vivo. Detection of tritiated thymidine in the cell digests should then be proportional to the amount of unscheduled DNA synthesis. The validation of such a technique is a matter of some importance. If unscheduled DNA synthesis activity is used as a means of detecting genotoxic events, it is vitally important that it is distinguished from semiconservative, schedule, regular DNA synthesis as the uptake of tritiated thymidine during regular DNA synthesis is much greater than that during unscheduled DNA synthesis. Differentiation of unscheduled DNA synthesis from regular DNA synthesis accompanying cell division in the gastric mucosa is technically difficult. While regular DNA synthesis is maximal around the isthmus neck junction of the gastric glands, it is detectable up to five cell positions from the surface. Any method that relies on selective separation of non-dividing surface cells to identify unscheduled DNA synthesis requires stringent controls to exclude contamination with deeper dividing cells. This is particularly important in situations where there is a stimulus to cell proliferation, when cells undergoing regular DNA synthesis may be seen closer to the surface.