Hydrogen peroxide (H 2 O 2 ) is widely regarded as a cytotoxic agent whose levels must be minimized by the action of antioxidant defence enzymes. In fact, H 2 O 2 is poorly reactive in the absence of transition metal ions. Exposure of certain human tissues to H 2 O 2 may be greater than is commonly supposed: substantial amounts of H 2 O 2 can be present in beverages commonly drunk (especially instant coffee), in freshly voided human urine, and in exhaled air. Levels of H 2 O 2 in the human body may be controlled not only by catabolism but also by excretion, and H 2 O 2 could play a role in the regulation of renal function and as an antibacterial agent in the urine. Urinary H 2 O 2 levels are influenced by diet, but under certain conditions might be a valuable biomarker of`oxidative stress'. ß

Reactive species such as free radicals are constantly generated in vivo and DNA is the most important target of oxidative stress. Oxidative DNA damage is used as a predictive biomarker to monitor the risk of development of many diseases. The comet assay is widely used for measuring oxidative DNA damage at a single cell level. The analysis of comet assay output images, however, poses considerable challenges. Commercial software is costly and restrictive, while free software generally requires laborious manual tagging of cells. This paper presents OpenComet, an open-source software tool providing automated analysis of comet assay images. It uses a novel and robust method for finding comets based on geometric shape attributes and segmenting the comet heads through image intensity profile analysis. Due to automation, OpenComet is more accurate, less prone to human bias, and faster than manual analysis. A live analysis functionality also allows users to analyze images captured directly from a microscope. We have validated OpenComet on both alkaline and neutral comet assay images as well as sample images from existing software packages. Our results show that OpenComet achieves high accuracy with significantly reduced analysis time.

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Resveratrol, a constituent of grapes and other food products, has been shown to prevent carcinogenesis in murine models. We report here that resveratrol induces apoptotic cell death in HL60 human leukemia cell line. Resveratrol-treated tumor cells exhibit a dose-dependent increase in externalization of inner membrane phosphatidylserine and in cellular content of subdiploid DNA, indicating loss of membrane phospholipid asymmetry and DNA fragmentation. Resveratrol-induced cell death is mediated by intracellular caspases as observed by the dose-dependent increase in proteolytic cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and the ability of caspase inhibitors to block resveratrol cytotoxicity. We also show that resveratrol treatment enhances CD95L expression on HL60 cells, as well as T47D breast carcinoma cells, and that resveratrol-mediated cell death is specifically CD95-signaling dependent. On the contrary, resveratrol treatment of normal human peripheral blood lymphocytes (PBLs) does not affect cell survival for up to 72 hours, which correlates with the absence of a significant change in either CD95 or CD95L expression on treated PBLs. These data show specific involvement of the CD95-CD95L system in the anti-cancer activity of resveratrol and highlight the chemotherapeutic potential of this natural product, in addition to its recently reported chemopreventive activity. © 1998 by The American Society of Hematology.

Reports about the effects of ascorbate (vitamin C) on cultured cells are confusing and conflicting. Some authors show inhibition of cell death by ascorbate, whereas others demonstrate that ascorbate is cytotoxic. In this report, using three different cell types and two different culture media (Dulbecco's modified Eagle's medium and RPMI 1640), we show that the toxicity of ascorbate is due to ascorbate-mediated production of H2O2, to an extent that varies with the medium used to culture the cells. For example, 1 mM ascorbate generates 161 +/- 39 microM H2O2 in Dulbecco's modified Eagle's medium and induces apoptosis in 50% of HL60 cells, whereas in RPMI 1640 only 83 +/- 17 microM H2O2 is produced and no apoptosis is detected. Apoptosis is prevented by catalase, and direct addition of H2O2 at the above concentration to the cells has similar effects to ascorbate. These results show that ascorbate itself is not toxic to the cell lines used and that effects of ascorbate in vivo cannot be predicted from studies on cultured cells. The ability of ascorbate to interact with different cell culture media to produce H2O2 at different rates could account for many or all of the conflicting results obtained using ascorbate in cultured cell assays.

The cell surface receptor Fas is a major trigger of apoptosis. However, expression of the Fas receptor in many tumor cell types does not correlate with sensitivity to Fas‐mediated cell death. Because a prooxidant state is a common feature of tumor cells, we examined the role of intracellular reactive oxygen intermediates in the regulation of Fas‐mediated cytotoxicity. Our results show that an oxidative stress induced by increasing the intracellular superoxide anion (O2‐) concentration can abrogate Fas‐mediated apoptosis in cells which are constitutively sensitive to Fas. Conversely, an O2‐ concentration decrease is observed to sensitize cells which are naturally resistant to Fas signals. These observations suggest that intracellular O2‐ may play a key role in regulating cell sensitivity to a potentially lethal signal and provide tumor cells with a natural, inducible mechanism of resistance to Fas‐mediated apoptosis.

Hydrogen peroxide (H P O P ) is considered to be a mediator of apoptotic cell death but the mechanism by which it induces apoptosis is unclear. Here, we show that cells undergoing apoptosis from exposure to H P O P display a significant decrease in intracellular concentration of superoxide (y 3 2 ) which is associated with a reduction of the intracellular milieu, as measured by an increase in the GSH/GSSG ratio and a decrease in intracellular pH. The notion that a decrease in intracellular y 3 2 concentration triggers apoptosis is supported by the observation that H P O P -mediated apoptosis could be retarded in cells in which the intracellular y 3 2 concentration is maintained at or above the cellular baseline level by inhibition of the major y 3 2 scavenger superoxide dismutase (Cu/Zn SOD). Taken together, our observations indicate that a decrease in the intracellular y 3 2 concentration, reduction and acidification of the intracellular milieu constitute a signal for H P O P -mediated apoptosis, thereby inducing a reductive as opposed to an oxidative stress.z 1998 Federation of European Biochemical Societies.

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