AimsHyperoxic breathing might lead to redox imbalance and signaling changes that affect cerebral function. Paradoxically, hypoxic breathing is also believed to cause oxidative stress. Our aim is to dissect the cerebral tissue responses to altered O2 fractions in breathed air by assessing the redox imbalance and the recruitment of the hypoxia signaling pathways.ResultsMice were exposed to mild hypoxia (10%O2), normoxia (21%O2) or mild hyperoxia (30%O2) for 28 days, sacrificed and brain tissue excised and analyzed. Although one might expect linear responses to %O2, only few of the examined variables exhibited this pattern, including neuroprotective phospho- protein kinase B and the erythropoietin receptor. The major reactive oxygen species (ROS) source in brain, NADPH oxidase subunit 4 increased in hypoxia but not in hyperoxia, whereas neither affected nuclear factor (erythroid-derived 2)-like 2, a transcription factor that regulates the expression of antioxidant proteins. As a result of the delicate equilibrium between ROS generation and antioxidant defense, neuron apoptosis and cerebral tissue hydroperoxides increased in both 10%O2 and 30%O2, as compared with 21%O2. Remarkably, the expression level of hypoxia-inducible factor (HIF)−2α (but not HIF-1α) was higher in both 10%O2 and 30%O2 with respect to 21%O2InnovationComparing the in vivo effects driven by mild hypoxia with those driven by mild hyperoxia helps addressing whether clinically relevant situations of O2 excess and scarcity are toxic for the organism.ConclusionProlonged mild hyperoxia leads to persistent cerebral damage, comparable to that inferred by prolonged mild hypoxia. The underlying mechanism appears related to a model whereby the imbalance between ROS generation and anti-ROS defense is similar, but occurs at higher levels in hypoxia than in hyperoxia.

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Melatonin is known to exert antitumour activity in several types of human cancers, but the underlying mechanisms as well as the efficacy of different doses of melatonin are not well defined. Here, we test the hypothesis whether melatonin in the nanomolar range is effective in exerting antitumour activity in vivo and examine the correlation with the hypoxia signalling mechanism, which may be a major molecular mechanism by which melatonin antagonizes cancer. To test this hypothesis, LNCaP human prostate cancer cells were xenografted into seven-wk-old Foxn1nu/nu male mice that were treated with melatonin (18 i.p. injections of 1 mg/kg in 41 days). Saline-treated mice served as control. We found that the melatonin levels in plasma and xenografted tissue were 4× and 60× higher, respectively, than in control samples. Melatonin tended to restore the redox imbalance by increasing expression of Nrf2. As part of the phenotypic response to these perturbations, xenograft microvessel density was less in melatonin-treated animals, indicative of lower angiogenesis, and the xenograft growth rate was slower (P < 0.0001). These changes were accompanied by a reduced expression of Ki67, elevated expression of HIF-1α and increased phosphorylation of Akt in melatonin than saline-treated mice. We conclude that the beneficial effect of melatonin in reducing cancer growth in vivo was evident at melatonin plasma levels as low as 4 nm and was associated with decreased angiogenesis. Higher HIF-1α expression in xenograft tissue indicates that the antitumour effect cannot be due to a postulated antihypoxic effect, but may stem from lower angiogenesis potential.

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Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O 2, hypoxia-inducible factor 1␣ (HIF-1␣) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O 2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1␣ production by improved tissue O 2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1␣ expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1␣ in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O 2 for 10 days) with or without CEpo administered by daily s.c. injection (10 g͞kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1␣ and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1␣ expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.apoptosis ͉ brain ͉ hypoxia-inducible factor 1␣ ͉ heart ͉ Epo receptor

In vivo systemic hypoxia promotes prostate cancer growth regardless of HIF-1alpha expression level and neovascularization, suggesting an important role for hypoxia-dependent pathways that do not involve HIF-1alpha, as the phosphatidyl inositol-3-phosphate signaling cascade.

Exposure to hypoxia triggers a variety of adverse effects in the brain that arise from metabolic stress and induce neuron apoptosis. Overexpression of the hypoxia-inducible factor-1alpha (HIF-1alpha) is believed to be a major candidate in orchestrating the cell's defense against stress. To test the impact of HIF-1alpha on apoptosis during chronic hypoxia in vivo, we examined the protective effect of modulating the nitric oxide (NO)/cGMP pathway by sildenafil, a selective inhibitor of phosphodiesterase-5 (PDE-5). Male ICR/CD-1 mice were divided into 3 groups (n = 6/group): normoxic (21% O(2)), hypoxic (9.5% O(2)), and hypoxic with sildenafil (1.4-mg/kg intraperitoneal injections daily). At the end of the 8-day treatment period, the mice were euthanized and cerebral cortex biopsies were harvested for analyses. We found that sildenafil: (1) did not significantly alter the hypoxia-induced weight loss and hemoglobin increase, but did augment plasma nitrates+nitrites and the tissue content of cGMP and phosphorylated (P) NO synthase III; (2) reversed the hypoxia-induced neuron apoptosis (terminal deoxynucleotidyl transferase positivity and double-staining immunofluorescence, P = 0.009), presumably through increased bcl-2/Bax (P = 0.0005); and (3) did not affect HIF-1alpha, but rather blunted the hypoxia-induced increase in P-ERK1/2 (P = 0.0002) and P-p38 (P = 0.004). We conclude that upregulating the NO/cGMP pathway by PDE-5 inhibition during hypoxia reduces neuron apoptosis, regardless of HIF-1alpha, through an interaction involving ERK1/2 and p38.

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