Skin damage by tropospheric ozone.

Tropospheric (ground level) ozone (O3) is a secondary pollutant, emerging from other pollutants in the sunshine. Exposure to O3 correlates with higher pulmonary and cardiovascular mortality and affects reproductive health and the central nervous system acutely and chronically. Skin might be a potentially overlooked target organ of ambient O3. The experimental evidence suggests a positive correlation of O3 exposure with oxidative damage, impaired antioxidant defence and proinflammatory response in the skin. In time series studies it was observed that acute rises in O3 levels correlated with seeking medical help for skin conditions; however, whether these findings are specific to O3, is not yet clear. There is preliminary epidemiological evidence that long-term exposure to O3 is associated with premature skin aging. This finding was independent of co-exposure to other environmental factors affecting skin (e.g. ultraviolet radiation and air pollution). As concentrations of O3 are rising in many regions of the world, adverse cutaneous effects of O3 present a relevant public health concern.

[Skin damage by tropospheric ozone]. / Hautschäden durch troposphärisches Ozon.

Tropospheric (ground level) ozone (O3) is a secondary pollutant, emerging from other pollutants in the sunshine. Exposure to O3 correlates with higher pulmonary and cardiovascular mortality and affects reproductive health and the central nervous system acutely and chronically. Skin might be a potentially overlooked target organ of ambient O3. The experimental evidence suggests a positive correlation of O3 exposure with oxidative damage, impaired antioxidant defence and proinflammatory response in the skin. In time series studies it was observed that acute rises in O3 levels correlated with seeking medical help for skin conditions; however, whether these findings are specific to O3, is not yet clear. There is preliminary epidemiological evidence that long-term exposure to O3 is associated with premature skin aging. This finding was independent of co-exposure to other environmental factors affecting skin (e.g. ultraviolet radiation and air pollution). As concentrations of O3 are rising in many regions of the world, adverse cutaneous effects of O3 present a relevant public health concern.

Non-conventional rottlerin anticancer properties.

In the past few years, we focused the interest on rottlerin, an old/new natural substance that, over the time, has revealed a number of cellular and molecular targets, all potentially implicated in the fight against cancer. Past and recent literature well demonstrated that rottlerin is an inhibitor of enzymes, transcription factors and signaling molecules that control cancer cell life and death. Although the rottlerin anticancer activity has been mainly ascribed to apoptosis and/or autophagy induction, recent findings unveiled the existence of additional mechanisms of toxicity. The major novelties highlighted in this mini review are the ability to bind and inhibit key molecules, such as ERK and mTOR, directly, thus independently of upstream signaling cascades, and to cause a profound dysregulation of cap-dependent protein translation through the mTORC1/4EBP1/eIF4E axis and by inhibition of eIF2, an initiation factor of translation that is negatively regulated by endoplasmic reticulum (ER) stress. These last mechanisms, proved to be lethal in cancer cell lines derived from breast and skin, strongly enforce the potential of rottlerin as a promising natural lead compound for the development of novel therapeutic approaches.

NADPH oxidase activation and 4-hydroxy-2-nonenal/aquaporin-4 adducts as possible new players in oxidative neuronal damage presents in drug-resistant epilepsy.

A correlation between epilepsy and cellular redox imbalance has been suggested, although the mechanism by which oxidative stress (OS) can be implicated in this disorder is not clear. In the present study several oxidative stress markers and enzymes involved in OS have been determined. In particular, we examined the levels of 4-hydroxy-2-nonenal protein adducts (HNE-PA), a by-product of lipid peroxidation, and the activation of NADPH oxidase 2 (NOX2), as cellular source of superoxide (O(2)(-)), in surgically resected epileptic tissue from drug-resistant patients (N=50). In addition, we investigated whether oxidative-mediated protein damage can affect aquaporin-4 (AQP4), a water channel implicated in brain excitability and epilepsy. Results showed high levels of HNE-PA in epileptic hippocampus, in both neurons and glial cells and cytoplasmic positivity for p47(phox) and p67(phox) suggesting NOX2 activation. Interestingly, in epileptic tissue immunohistochemical localization of AQP4 was identified not only in perivascular astrocytic endfeet, but also in neurons. Nevertheless, negativity for AQP4 was observed in neurons in degeneration. Of note, HNE-mediated post-translational modifications of AQP4 were increased in epileptic tissues and double immunofluorescence clearly demonstrated co-localization of AQP4 and HNE-PA in epileptic hippocampal structures. The idea is that sudden, disorderly, and excessive neuronal discharges activates NOX2 with O(2)(-) production, leading to lipid peroxidation. The resulting generation of HNE targets AQP4, affecting water and ion balance. Therefore, we suggest that seizure induces oxidative damage as well as neuronal loss, thereby promoting neuronal hyperexcitability, also affecting water and ion balance by AQP4 modulation, and thus generating a vicious cycle.

Rottlerin and cancer: novel evidence and mechanisms.

Because cancers are caused by deregulation of hundreds of genes, an ideal anticancer agent should target multiple gene products or signaling pathways simultaneously. Recently, extensive research has addressed the chemotherapeutic potential of plant-derived compounds. Among the ever-increasing list of naturally occurring anticancer agents, Rottlerin appears to have great potentiality for being used in chemotherapy because it affects several cell machineries involved in survival, apoptosis, autophagy, and invasion. The underlying mechanisms that have been described are diverse, and the final, cell-specific, Rottlerin outcome appears to result from a combination of signaling pathways at multiple levels. This paper seeks to summarize the multifocal signal modulatory properties of Rottlerin, which merit to be further exploited for successful prevention and treatment of cancer.

Ozone and ozonated oils in skin diseases: a review.

Although orthodox medicine has provided a variety of topical anti-infective agents, some of them have become scarcely effective owing to antibiotic- and chemotherapeutic-resistant pathogens. For more than a century, ozone has been known to be an excellent disinfectant that nevertheless had to be used with caution for its oxidizing properties. Only during the last decade it has been learned how to tame its great reactivity by precisely dosing its concentration and permanently incorporating the gas into triglycerides where gaseous ozone chemically reacts with unsaturated substrates leading to therapeutically active ozonated derivatives. Today the stability and efficacy of the ozonated oils have been already demonstrated, but owing to a plethora of commercial products, the present paper aims to analyze these derivatives suggesting the strategy to obtain products with the best characteristics.

Nitroxide radical TEMPO reduces ozone-induced chemokine IL-8 production in lung epithelial cells.

Exposure to high levels of ozone (O(3)) damages respiratory tract epithelial cells. This research evaluated the ability of TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a stable nitroxide free radical, to decrease O(3)-mediated injury to a respiratory tract-derived cell line (A549 cells) by monitoring in this cell system the interleukine-8 (IL-8) production. TEMPO reduced O(3)-induced IL-8 production in A549 cells, as evidenced by PCR analysis, Western blot and ELISA assays. This behaviour is explainable on the basis of the reactivity between TEMPO with O(3) and/or O(3)-derived free radicals in biological systems. The study provides evidence that TEMPO reacts with O(3) and/or its cytotoxic products and may provide protections against O(3)-induced biotoxicities.

Antiproliferative and survival properties of PMA in MCF-7 breast cancer cell.

Although PKCs are assumed to be the main targets of phorbol esters (PMA), additional PMA effectors, such as chimaerins (a family of RacGTPase activating proteins) and RasGRP (exchange factor for Ras/Rap1), can counteract or strengthen the PKC pathways. In this study, we evaluated the proliferative behavior of PMA-treated MCF-7 breast cancer cell and found that: PMA induced growth arrest and inhibited cell death; PMA activated ERKs, which, in turn, induced p21; and inhibitors of ERK (PD98059) and PKC (GF109203X) prevented p21 induction and abolished the PMA survival effect. We conclude that PMA inhibits MCF-7 cell growth and simultaneously stimulates cell survival; both responses are linked to ERK-dependent and p53-independent p21 induction.

Rottlerin inhibits the nuclear factor kappaB/cyclin-D1 cascade in MCF-7 breast cancer cells.

In the course of a project aimed to clarify the molecular mechanisms by which phorbol 12-myristate 13-acetate (PMA)-activated forms of protein kinase C (PKC) promote growth arrest in an MCF-7 cell line, we found that the PKCdelta inhibitor Rottlerin was able by itself to block cell proliferation. In the current study, we investigated further the antiproliferative response to Rottlerin. Western blotting analysis of cytoplasmic/nuclear extracts showed that the drug did not prevent either extracellular signal-regulated kinase (ERK) activation by PMA or Akt phosphorylation, but did interfere with the NFkappaB activation process (both basal and PMA-stimulated), by lowering the levels of phospho-IkappaBalpha and preventing p65 nuclear migration. The growth arrest evoked by Rottlerin was not mediated by cell-cycle inhibitors p21 and p27 but was accompanied by a dramatic fall in the cyclin-D1 protein, the levels of which were not altered by the pan-PKC inhibitor GF 109203X, thus excluding a PKC-mediated mechanism in the Rottlerin effect. The parallel drop in cyclin-D1 mRNA suggested a down-regulation of the gene caused by the inhibition of nuclear factor-kappa B (NFkappaB), which occurs via a PKC-, Akt-, ERK- and mitochondrial uncoupling-independent mechanism. We provide preliminary evidence that the interference on the NFkappaB activation process likely occurs at the level of calcium/calmodulin-dependent protein kinase II (CaMKII), a known Rottlerin target. Indeed the drug prevented calcium-induced CaMKII autophosphorylation which, in turn, led to decreased NFkappaB activation.

Keratinocytes oxidative damage mechanisms related to airbone particle matter exposure.

Epidemiological evidences have correlated airbone particulate matter (PM) to adverse health effects, mainly linking to pulmonary and cardiovascular disease. Nevertheless, only recently, some studies reported detrimental effects of PM on other organs such as skin. In a recent work, we have reported increased oxidative and inflammatory responses in Reconstituted Human Epidermis (RHE) exposed to ambient particles (CAPs) and we also demonstrated the ability of CAPs to penetrate the skin tissue. The present study was aimed to better understand the cellular mechanisms beyond the oxidative changes induced by CAPs (5-10-25µg/mL) in human immortalized keratinocytes (HaCaT). After 24h of treatment, CAPs were able to enter the cells leading to a decrease in viability, increased levels of 4-hydroxinonenal products (4-HNE) and IL-1α release. Overall these data, suggest lipid and protein oxidative damage, as well as an increase of inflammatory response after being challenged with CAPs. In addition, 3h after CAPs exposure we found a significant increase in NF-kB and Nrf2 translocation into the nucleus. In contrast, no differences in gene expression and enzymatic activity of Nrf2 target genes were detected. This last finding could be explained by the ability of CAPs to possibly alter the binding of Nrf2 to the ARE DNA sequence.

Association between Siesta (Daytime Sleep), Dietary Patterns and the Presence of Metabolic Syndrome in Elderly Living in Mediterranean Area (Medis Study): The Moderating Effect of Gender.

OBJECTIVES: Several lifestyle parameters including diet, physical activity and sleep were associated in isolation with the presence of Metabolic Syndrome (MetS) in adults, to date there is a paucity of studies which evaluated their combined role aging populations and especially with respect to gender. Therefore, the aim of the present study was to provide a global consideration of the lifestyle factors associated with MetS among elderly individuals. DESIGN: Cross-sectional observational study. SETTING: 21 Mediterranean islands and the rural Mani region (Peloponnesus) of Greece. PARTICIPANTS: during 2005-2015, 2749 older (aged 65-100 years) from were voluntarily enrolled in the study. MEASUREMENTS: Dietary habits, energy intake, physical activity status, socio-demographic characteristics, lifestyle parameters (sleeping and smoking habits) and clinical profile aspects were derived through standard procedures. The presence of MetS was defined using the definition provided by NCEP ATP III (revised) and cluster analysis was used to identify overall dietary habit patterns. RESULTS: The overall prevalence of MetS in the study sample was 36.2%, but occurred more frequently in females (40.0% vs. 31.8%, respectively, p=0.03). Individuals with MetS were more likely to sleep during the day (89.4% vs. 76.8% respectively, p=0.039) and frequent 'siesta' was positively linked to the odds of MetS presence in females (Odds Ratio (OR) =3.43, 95% Confidence Intervals (CI): 1.08-10.9), but not for men (p=0.999). The lower carbohydrate (i.e., 45.2% of total daily energy, 120±16gr/day) dietary cluster was inversely associated with the odds for MetS presence, but only for men (OR=0.094, 95%CI: 0.010-0.883). CONCLUSIONS: Lifestyle parameters including sleep and diet quality are strongly associated with the presence of MetS in elderly cohort, but different their level of influence appears to be different, depending on gender. Further research is needed to better consider the role of lifestyle characteristics in the management of MetS in clinical practice.

Inhibitions of mTORC1 and 4EBP-1 are key events orchestrated by Rottlerin in SK-Mel-28 cell killing.

Earlier studies demonstrated that Rottlerin exerts a time- and dose-dependent antiproliferative effect on SK-Mel-28 melanoma cells during 24 h of treatment, but cytotoxicity due to cell death began only after a 48 h exposure. In the current study, in order to identify the type of cell death in this cell line, which is notoriously refractory to most anticancer therapies, and to clarify the underlying mechanisms of this delayed outcome, we searched for apoptotic, necrotic/necroptotic and autophagic traits in Rottlerin-exposed cells. Although SK-Mel-28 cells are both apoptosis and autophagy competent, Western blotting analysis, caspase activity assay, nuclear imaging and the effects of autophagy, apoptosis and necroptosis inhibitors, indicated that Rottlerin cytotoxicity was due to none of the aforementioned death mechanisms. Nevertheless, in growth arrested cells, the death did occur after a prolonged treatment and most likely ensued from the observed blockage of protein synthesis that reached levels expected to be incompatible with cell survival. From a mechanistic point of view, we ascribed this effect to the documented inhibition of mTORC1 activity; mTORC1 inhibition on the one hand led to a not deadly, rather protective autophagic response but, on the other hand caused a near complete arrest of protein synthesis. Interestingly, no cytotoxicity was found towards normal skin fibroblasts, which only resulted mildly growth arrested by the drug.

Ozone: A Multifaceted Molecule with Unexpected Therapeutic Activity.

A comprehensive outline for understanding and recommending the therapeutic use of ozone in combination with established therapy in diseases characterized by a chronic oxidative stress is currently available. The view of the absolute ozone toxicity is incorrect, because it has been based either on lung or on studies performed in artificial environments that do not correspond to the real antioxidant capacity of body compartments. In fact, ozone exerts either a potent toxic activity or it can stimulate biological responses of vital importance, analogously to gases with prospective therapeutic value such as NO, CO, H2S, H2, as well as O2 itself. Such a crucial difference has increasingly become evident during the last decade. The purpose of this review is to explain the aspects still poorly understood, highlighting the divergent activity of ozone on the various biological districts. It will be clarified that such a dual effect does not depend only upon the final gas concentration, but also on the particular biological system where ozone acts. The real significance of ozone as adjuvant therapeutic treatment concerns severe chronic pathologies among which are cardiovascular diseases, chronic obstructive pulmonary diseases, multiple sclerosis, and the dry form of age-related macular degeneration. It is time for a full insertion of ozone therapy within pharmaceutical sciences, responding to all the requirements of quality, efficacy and safety, rather than as either an alternative or an esoteric approach.

Phosphorylation-independent mTORC1 inhibition by the autophagy inducer Rottlerin.

We recently found that Rottlerin not only inhibits proliferation but also causes Bcl-2- and Beclin 1-independent autophagic death in apoptosis-resistant breast adenocarcinoma MCF-7 cells. Having excluded a role for canonical signaling pathways, the current study was aimed to investigate the contribution of the AMPK/mTOR axis in autophagy induction and to search for the upstream signaling molecules potentially targeted by Rottlerin. Using several enzyme inhibitors, Western blotting analysis, mTOR siRNA and pull down assay, we demonstrate that the Rottlerin-triggered autophagy is mediated by inhibition of mTORC1 activity through a novel AMPK and mTORC1 phosphorylation-independent mechanism, likely mediated by the direct interaction between Rottlerin and mTOR.

Activity of monomeric, dimeric, and trimeric flavonoids on NO production, TNF-alpha secretion, and NF-kappaB-dependent gene expression in RAW 264.7 macrophages.

Flavonoids are potent antioxidants and have been associated with lowering the risk of cardiovascular diseases. In this study, the effect of flavonoids (monomers, dimers and a trimer) as well as French maritime pine bark extract, Pycnogenol, on NO production, tumor necrosis factor-alpha (TNF-alpha) secretion and nuclear factor (NF)-kappaB activity was compared. Monomers and dimers repressed NO production, TNF-alpha secretion and NF-kappaB-dependent gene expression induced by interferon gamma, whereas the trimeric procyanidin C2 and Pycnogenol enhanced these parameters. In addition, in unstimulated RAW 264.7 macrophages, both procyanidin C2 and Pycnogenol increased TNF-alpha secretion in a concentration- and time-dependent manner. These results demonstrate that procyanidins act as modulators of the immune response in macrophages.

Ozone potentiates vitamin E depletion by ultraviolet radiation in the murine stratum corneum.

As the outermost layer of the skin, the stratum corneum is exposed to environmental oxidants. To investigate putative synergisms of environmental oxidative stressors in stratum corneum, hairless mice were exposed to ultraviolet radiation (UV) and ozone (O(3)) alone and in combination. Whereas a significant depletion of alpha-tocopherol was observed after individual exposure to either a 0.5 minimal erythemal dose of UV or 1 ppm O(3) for 2 h, the combination did not increase the effect of UV alone. However, a dose of 0.5 ppm O(3) x 2 h, which had no effect when used alone, significantly enhanced the UV-induced depletion of vitamin E. We conclude that concomitant exposure to low doses of UV and O(3) at levels near those that humans can be exposed to causes additive oxidative stress in the stratum corneum.

Effect of benzoyl peroxide on antioxidant status, NF-kappaB activity and interleukin-1alpha gene expression in human keratinocytes.

Benzoyl peroxide (BP) is used as a topical treatment for acne. Besides its anti-bacterial activity, the exact molecular mechanisms underlying its mode of action are not fully understood. In the current study, the effects of BP on cell viability, antioxidant status and, IL-1 and IL-8 gene expression were investigated in HaCaT keratinocytes. Keratinocytes incubated for 24 h with BP exhibited a dose-dependent cytotoxicity at concentrations above 250 microM. Furthermore, in the presence of 300 microM BP about 50% of the cellular vitamin E was depleted within the first 30 min. The intracellular ratio of oxidized to reduced glutathione (GSSG/GSH) was increased significantly starting 6 h after BP treatments indicating that BP reacts rapidly with targets in the cell membrane and more slowly with those in the cytosol. NF-kappaB transactivation was not significantly affected by BP. However, BP treatment of HaCaT keratinocytes resulted in a dose-dependent increase in IL-1alpha gene expression whereas no changes in IL-8 mRNA levels were observed. These results demonstrate that BP induces an inflammatory reaction mediated by oxidative stress by a pathway independent of the redox-sensitive transcription factor NF-kappaB.

Acrolein-induced cytotoxicity in cultured human bronchial epithelial cells. Modulation by alpha-tocopherol and ascorbic acid.

Acrolein is a highly reactive unsaturated hazardous air pollutant of human health concern, particularly as a component of cigarette smoke. In this study, the mechanisms of acrolein-induced cytotoxicity in human bronchial epithelial cells (HBE1) and the modulating effects of antioxidants were examined. Our results show that acrolein induces a cell death pathway in human bronchial epithelial cells, which retain key features of apoptosis, as indicated by phosphatidylserine (PS) externalization and DNA fragmentation. Acrolein-induced apoptosis was associated with depletion of cellular GSH and intracellular generation of oxidants. Supplementation of cells with either alpha-tocopherol or ascorbic acid was found to strongly inhibit acrolein-induced apoptosis and to prevent the increase in the generation of intracellular oxidants, although GSH depletion was unaffected. Moreover, recovery of cellular GSH levels after acrolein exposure was enhanced following either alpha-tocopherol or ascorbic acid supplementation. The intracellular generation of oxidants following acrolein exposure seems to be an important event triggering the apoptotic response in this model system.

Studies on the biological effects of ozone: 7. Generation of reactive oxygen species (ROS) after exposure of human blood to ozone.

The acceptance of any complementary medical approach is conditioned by the results obtained after the same scientific scrutiny applied in orthodox medicine. Otherwise any claim of efficacy remains in the realm of fiction. In the case of ozone therapy, the mechanisms of action have remained nebulous and in a series of publications we are trying to present the biochemical, immunological and morphological evidence in favour or against ozone therapy. We have now shown that ozone (O3) dissolved in the water of either plasma or serum or physiological saline generates reactive oxygen species (ROS), of which hydrogen peroxide (H2O2) can be unequivocally demonstrated by using specific methods for its detection. Lipids present in plasma preferentially those present in lipoproteins, undergo peroxidation that is somewhat O3-dose dependent and can be observed by the measurement of thiobarbituric acid reactive substances (TBARS). While the generation of H2O2 is crucial in activating both biochemical (hexose monophosphate shunt) and immunological (via the transcription factor NF-kB) mechanisms, the role of lipid oxidation products (LOP) remains to be investigated. We have shown here that there is a small but consistent induction of some cytokines (TNF-alpha, IFN-gamma and IL-2) when human blood is directly exposed to O3 concentrations up to 100 micrograms/ml per g of blood. On the other hand, isolated blood mononuclear cells (PBMC) in tissue culture medium are far more sensitive to the oxidant action of O3 as shown by a progressive reduction of the proliferation index with comparatively far lower O3, concentrations. On the whole, these results support the concept that much of the O3 toxicity is neutralized by the powerful antioxidant system of blood. The minimal hemolysis supports this idea but as far as platelets are concerned, we must mention that they tend to aggregate in heparinized blood, even when it is exposed to an O3 concentration of 40 micrograms/ml. In spite of the lack of side-effects after autohemotherapy, this drawback must be kept in mind and avoided in clinical practice.

Ozonation of blood during extracorporeal circulation. I. Rationale, methodology and preliminary studies.

We investigated whether exposure of blood ex-vivo to oxygen-ozone (O2-O3) through a gas exchanger is feasible and practical. We first evaluated the classical dialysis-type technique but we soon realized that semipermeable membranes are unsuitable because they are hydrophilic and vulnerable to O3. We therefore adopted a system with hydrophobic O3-resistant hollow fibers enclosed in a polycarbonate housing with a membrane area of about 0.5 m2. First we tested the system with normal saline, determining the production of hydrogen peroxide (H2O2) at O3 concentrations from 5 to 40 microg/ml. We then evaluated critical parameters by circulating swine blood in vitro; this revealed that heparin is not an ideal anticoagulant for this system. Finally, we performed several experiments in sheep and defined optimal anticoagulant dose (sodium citrate, ACD), priming solution, volume of blood flow per min, volume and concentration of O2-O3 mixture flowing countercurrent with respect to blood and the time necessary for perfusion in vivo. The biochemical parameters showed that an O3 concentration as low as 10 microg/ml is effective; this means that gas exchange and O3 reactivity are rapid and capable of inducing biological effects. The sheep showed no adverse effects even after 50 min of extracorporeal circulation at higher O3 concentrations (20 to 40 microg/ml) but the exchanger became less effective (low pO2 values) due to progressive clogging with cells.