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Nitric oxide, NO, which is generated by various components of the immune system, has been presumed to be cytotoxic. However, NO has been proposed to be protective against cellular damage resulting during ischemia reperfusion. Along with NO there is often concomitant formation of superoxide/hydrogen peroxide, and hence a synergistic relationship between the cytotoxic effects of nitric oxide and these active oxygen species is frequently assumed. To study more peroxide or to hypoxanthine/xanthine oxidase resulted in the degradation of the dopamine uptake and release mechanism. As was observed in the case of the V79 cells, the presence of NO essentially abrogated this peroxide-mediated cytotoxic effect on mesencephalic cells.Nitric oxide, NO, is one of the proposed cytotoxic species produced by the immune surveillance system (1, 2). Many reports also suggest that during the ischemia reperfusion event, NO mediates tissue injury (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). However, it has been suggested that NO can function as a protective agent on the basis of two lines of evidence: (i) NO synthase (NOS) inhibitors increase tissue damage during in vivo ischemia reperfusion within the cerebral cortex, and (ii) in more direct evidence, NO has been shown to prevent damage during ischemia reperfusion events in both brain and heart (13-18). Additional studies provide evidence that NO, though present, plays a minimal role in the pathological effects associated with ischemia reperfusion injury (19,20) or tumor necrosing factor-mediated cytotoxicity (21). Unfortunately, the exact role(s) NO plays in cytotoxicity in vivo is not clear because there are a number of different physiological functions NO or the NOS inhibitors could affect simultaneously. It has been proposed that NO or reactive nitrogen oxide species can directly cause cell death. However, primary neuronal cell cultures exposed to concentrations of NO as high as 1 mM show no adverse effects (22, 23). Many of the biological events in which NO has been proposed as a toxin occur concurrently with the production of reactive oxygen species-e.g., immune response and ischemia reperfusion injury.To clarify the relationship between the cytotoxic properties of the reactive oxygen species and NO, it is necessary to simplify the system. We now show that NO released from a series of compounds known as the "NONOates" (24) Abstract Service Registry Number 136587-13-8) were synthesized and assayed for NO production via chemiluminescence technique as described (24).Enzyme Analysis/Controls. The activity of XO was monitored in the absence and presence of 1 mM DEA/NO by two different assays. (i) Superoxide-induced reduction of ferricytochrome c to ferrocytochrome c was monitored spectrophotometrically at 550 nm (25). The reaction was carried out in a 1-ml volume in aerated 50 mM phosphate buffer (pH 7.8) containing 50 ,AM diethylenetriaminepentaacetic acid. HX was maintained at 2.5 mM and ferricytochrome c at 20 ,uM. The reactions were initiated with the addition of XO (final con...

BackgroundMicroRNAs (miRNAs) are small, highly conserved, non-coding RNA that alter protein expression and regulate multiple intracellular processes, including those involved in the response to cellular stress. Alterations in miRNA expression may occur following exposure to several stress-inducing anticancer agents including ionizing radiation, etoposide, and hydrogen peroxide (H2O2).Methodology/Principal FindingsNormal human fibroblasts were exposed to radiation, H2O2, or etoposide at doses determined by clonogenic cell survival curves. Total RNA was extracted and miRNA expression was determined by microarray. Time course and radiation dose responses were determined using RT-PCR for individual miRNA species. Changes in miRNA expression were observed for 17 miRNA species following exposure to radiation, 23 after H2O2 treatment, and 45 after etoposide treatment. Substantial overlap between the miRNA expression changes between agents was observed suggesting a signature miRNA response to cell stress. Changes in the expression of selected miRNA species varied in response to radiation dose and time. Finally, production of reactive oxygen species (ROS) increased with increasing doses of radiation and pre-treatment with the thiol antioxidant cysteine decreased both ROS production and the miRNA response to radiation.ConclusionsThese results demonstrate a common miRNA expression signature in response to exogenous genotoxic agents including radiation, H2O2, and etoposide. Additionally, pre-treatment with cysteine prevented radiation-induced alterations in miRNA expression which suggests that miRNAs are responsive to oxidative stress. Taken together, these results imply that miRNAs play a role in cellular defense against exogenous stress and are involved in the generalized cellular response to genotoxic oxidative stress.