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Abstract: Transmission electron microscopy was used to study the ultrastructural changes induced by exposure to Cd or Zn in three species of ciliated protozoa: Colpoda steinii, Cyrtolophosis elongata and Drepanomonas revoluta. The main cytoplasmic alterations were partial mitochondrial degeneration, cytoplasmic vacuolisation, accumulation of membranous debris and autophagosome formation. At the nuclear level we detected nucleolar fusion in the macronucleus, and micronuclear membrane modifications. We compared these modifications with those coinciding with ciliate encystment (a differentiation process induced by environmental nutritional stress) and with changes in eukaryotic cells treated with staurosporine, a potent protein kinase inhibitor considered to be an apoptosis inducer. Exposure to heavy metals also coincided with the appearance of electron-dense accumulations in the cytoplasm, which might be related to metallothionein-mediated detoxification. The results are compared with previously reported data from ciliates and microalgae treated with heavy metals. [Copyright &y& Elsevier]

Background: Salivary nitrate is reduced by oral bacteria to form nitrite, which is further reduced to NO in the acidic gastric lumen. Dietary nitrate increases gastric NO which has profound effects on the gastric mucosa but it is unknown if the lower physiological levels of salivary nitrite, derived mainly from NO synthase, also affects gastric function. In an attempt to study this, we examined the gastric mucus layer in germ free (GF) and conventional mice given different doses of nitrate and nitrite. Since GF animals completely lack oral bacteria they cannot convert salivary nitrate to nitrite, and consequently they cannot form gastric NO. Methods: Mice were given either nitrate (140mg/kg day) or nitrite (14mg/kg day) in the drinking water for one week. Some GF mice were also given a very low dose of nitrite (1mg/kg day) corresponding to the levels provided by swallowing of fasting saliva. The firmly adherent gastric mucus layer was measured in vivo in anestetized mice using micropipettes. Results: Surprisingly, under basal conditions GF animals were almost completely devoid of the firmly adherent mucus layer (9±1μm) compared to conventional mice (34±2μm). Nitrate supplementation further increased the mucus thickness in conventional animals (46±1μm) but had no effect in GF mice (9±1μm). In contrast, a high dose of nitrite increased the mucus thickness in GF mice (41±1μm) to the same levels as in conventional animals (45±1μm). Remarkably, even physiological levels of nitrite given to GF animals increased the mucus thickness to the same extent (42±2μm). Conclusion: These results suggest that oral symbiotic bacteria play a role in physiological regulation of gastric mucus generation via bioactivation of salivary nitrate. Interestingly, in the absence of a dietary nitrate intake, salivary nitrate and nitrite mainly originates from the l-arginine/NOS pathway. This indicates the existence of a salvage pathway where oxidized NO from the endothelium and elsewhere is physiologically recycled via NOS-independent pathways. Disclosure: Nothing to disclose. [ABSTRACT FROM AUTHOR]

Background: Inorganic nitrate and nitrite have emerged as alternative substrates for nitric oxide (NO) generation in the gastrointestinal tract, and have shown to be protective against drug-induced gastric injury. The aim of this study was to investigate the preventive and therapeutic effects of nitrate and nitrite in a model of experimental colitis., Methods: Colitis was induced in mice by administrating dextran sulfate sodium (DSS) with concurrent administration of nitrite (1 mM) or nitrate (10 mM) in the drinking water for 7 days. A therapeutic approach was also investigated by initiating nitrite treatment 3 days after DSS-induced colitis. Clinical and inflammatory markers were assessed and the colonic mucus thickness was measured in vivo. The effect of nitrite on wound healing was evaluated using colon epithelial cells., Results: Concurrent administration of DSS and nitrite (1 mM) alleviated inflammation as determined by reduced disease activity index score (DAI) and increased colon length, while nitrate (10 mM) only reduced the DAI-score. Nitrite also displayed therapeutic effects by ameliorating established colonic inflammation with reduced colonic expression of iNOS and improving histopathology. DSS-induced decrease in colonic mucus thickness was completely prevented by nitrite administration. In addition, goblet cell abundance was lower by DSS treatment, but was increased by addition of nitrite. Further studies using colon epithelial cells revealed an NO-dependent improvement in wound healing with nitrite administration., Conclusion: Nitrite exerts both preventive and therapeutic effects in colonic inflammation. The protective effects involve preservation of an intact adherent mucus layer and regulation of epithelial cell restitution.

In damaged or proliferating endothelium, production of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS) is associated with elevated levels of reactive oxygen species (ROS), which are necessary for endothelial migration. We aimed to elucidate the mechanism that mediates NO induction of endothelial migration. NO downregulates expression of peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1 alpha), which positively modulates several genes involved in ROS detoxification. We tested whether NO-induced cell migration requires PGC-1 alpha downregulation and investigated the regulatory pathway involved. PGC-1 alpha negatively regulated NO-dependent endothelial cell migration in vitro, and inactivation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway, which is activated by NO, reduced NO-mediated downregulation of PGC-1 alpha. Expression of constitutively active Foxo3a, a target for Akt-mediated inactivation, reduced NO-dependent PGC-1 alpha downregulation. Foxo3a is also a direct transcriptional regulator of PGC-1 alpha, and we found that a functional FoxO binding site in the PGC-1 alpha promoter is also a NO response element. These results show that NO-mediated downregulation of PGC-1 alpha is necessary for NO-induced endothelial migration and that NO/protein kinase G (PKG)-dependent downregulation of PGC-1 alpha and the ROS detoxification system in endothelial cells are mediated by the PI3K/Akt signaling pathway and subsequent inactivation of the FoxO transcription factor Foxo3a.

The integrity of mitochondrial function is fundamental to cell life. The cell demands for mitochondria and their complex integration into cell biology, extends far beyond the provision of ATP. It follows that disturbances of mitochondrial function lead to disruption of cell function, expressed as disease or even death. Mitochondria are major producers of free radical species and also possibly of nitric oxide, and are, at the same time, major targets for oxidative damage. In this review we consider recent developments in our knowledge of how the mitochondrial production of reactive oxygen species (ROS) plays a critical role in several major human pathologies. We will also consider recent advances in our understanding of the molecular mechanisms involved in mitochondrial ROS detoxification.