[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.

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.

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.

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.

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.

Comparative effects between electronic and cigarette smoke in human keratinocytes and epithelial lung cells.

Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the toxicity of the commercial e-CIGs. Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke (CS), were exposed to e-CIG vapor and CS using an in vitro system. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured by the Bio-Plex assay. The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine although their effects were less harmful to that of CS. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators. Based on our results, we can state that exposure to e-CIG vapors results in far less toxic than exposure to CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate cytokines release. This study will hopefully promote the development of safer e-CIGs to help people quit smoking.

[Oxidative stress and Rett syndrome]. / La sindrome di Rett: ruolo dello stress ossidativo.

The oxidative stress (OS) hypothesis is able to explain several features of Rett syndrome (RTT), a pervasive development disorder almost exclusively affecting females mainly caused by a mutation in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. In particular, the generation of an OS imbalance is related to MeCP2 gene mutation type, as well as natural history, clinical heterogeneity of the disease, and is compatible with the potential reversibility of the disease observed in the RTT animal models. In addition, our findings indicate the importance of blood as a suitable biological fluid for detecting markers of central nervous system oxidative damage in RTT and underline the key role of interaction between organic chemists, OS biochemists, and clinicians in revealing potential new markers of the disease and identifying potential new targets and interventional strategies aimed at improving the quality of life of these patients, affected by a so far incurable disease. Further efforts in the near future are needed in order to dissect the "black box" of the molecular events likely linking the MeCP2 gene mutation to OS derangement and subsequent disease expression.

Resveratrol protects SR-B1 levels in keratinocytes exposed to cigarette smoke.

Cigarette smoking (CS) has been strongly linked to several health conditions including heart disease, lung cancer, and other respiratory and circulatory ailments. Deleterious effects of cigarette smoking on skin have also been well documented, but unlike effects on other organs, damage does not depend upon inhalation. The upper layer of the skin, the stratum corneum (rich in cholesterol fatty acids and ceramide), is very susceptible to damage induced by exposure to environmental stressors that can modify its lipid composition and thereby affect its function of protecting skin from dehydration. Scavenger receptor B1 (SR-B1) is involved in the uptake of cholesterol in several tissues including skin. We previously demonstrated that CS exposure induces formation of aldehyde (HNE) adducts that decrease SR-B1 expression. As topical resveratrol, a well-known polyphenolic stilbene, has been demonstrated to show benefits against skin disorders, we investigated its possible role as a protective agent against CS-induced reduction of SR-B1 expression in cutaneous tissue. In this study, we demonstrate that resveratrol at doses ranging from 0.5 to 10 µM is not toxic and is able to increase SR-B1 protein levels in a dose-dependent manner in human keratinocytes. Moreover, when the cells that were pretreated with various doses of resveratrol were exposed to CS, the loss of SR-B1 was prevented in a dose-dependent manner. In addition, in keratinocytes, resveratrol was also able to prevent an increase in HNE-protein adducts induced by CS. In particular resveratrol was able to prevent HNE-SR-B1 adduct formation. Thus, resveratrol seems to be a natural compound that could provide skin with a defense against exogenous stressors by protecting the essential cholesterol receptor, SR-B1.

Hypoxia induces cell damage via oxidative stress in retinal epithelial cells.

Retinal diseases (RD), including diabetic retinopathy, are among the most important eye diseases in industrialized countries. RD is characterized by abnormal angiogenesis associated with an increase in cell proliferation and apoptosis. Hypoxia could be one of the triggers of the pathogenic mechanism of this disease. A key regulatory component of the cell's hypoxia response system is hypoxia-inducible factor 1 alpha (HIF-1α). It has been demonstrated that the induction of HIF-1α expression can be also achieved in vitro by exposure with cobalt chloride (CoCl2), leading to an intracellular hypoxia-like state. In this study we have investigated the effects of CoCl2 on human retinal epithelium cells (hRPE), which are an integral part of the blood-retinal barrier, with the aim to determine the possible role of oxidative stress in chemical hypoxia-induced damage in retinal epithelial cells. Our data showed that CoCl2 treatment is able to induce HIF-1α expression, that parallels with the formation of reactive oxygen species (ROS) and the increase of lipid 8-isoprostanes and 4-hydroxynonenal (4-HNE) protein adducts levels. In addition we observed the activation of the redox-sensitive transcription factor nuclear factor-kappaB (NFkB) by CoCl2 which can explain the increased levels of vascular endothelial growth factor (VEGF). The increased number of dead cells seems to be related to an apoptotic process. Taken together these evidences suggest that oxidative stress induced by hypoxia might be involved in RD development through the stimulation of two key-events of RD such as neo-angiogenesis and apoptosis.

Ozonated oils as functional dermatological matrices: effects on the wound healing process using SKH1 mice.

Wound tissue repair is a complex and dynamic process of restoring cellular structures and tissue layers. Improvement of this process is crucial for several pathologies characterized by chronic delayed wound closure such as diabetes, and the investigation of new approaches aimed to ameliorate the wound healing process is under continuous evolution. Recently, the usage of vegetable matrices in the form of ozonated oils has been proposed and several researchers have shown a positive effect in the wound, based on their bactericidal, antiviral, and antifungal properties. The present study was undertaken to compare the effect that different ozonated oils (olive, sesame and linseed) with the same level of ozonation have on wound healing rate in SKH1 mice. Several histological parameters and the level of key proteins such as VEGF and PCNA have been analyzed. Only treatment with ozonated sesame oil shows a faster wound closure in the first 7 days. This effect paralleled with the increased VEGF and PCNA levels, NFκB nuclear translocation and 4-HNE formation. The present study shows that not only the ozonation grade is of importance for the improvement of wound healing process but also the typical composition of the oil.

Free radicals and antioxidants: how to reestablish redox homeostasis in chronic diseases?

This review examines the biological role of oxidants and antioxidants continuously produced by all living cells. Physiologically, human beings, who have inherited good genes, used to eat moderately a healthy diet and exercised daily, both systems are equally important and essential in maintaining a normal long life. However, the aging process slowly leads to a disequilibrium that is accentuated in pathologies such as diabetes, cardiovascular, degenerative, pulmonary, infective diseases, and cancer. All of these diseases shorten the life span in about 80% of individuals and represent a huge social-economic problem for health authorities. Several factors such as excessive feeding, smoking, alcoholism, and a poor life-style conjure up to their realization. Their progress, initially promoted by some pathogens and a wrong life-style, is deeply accentuated by an excessive and deranged production of deadly oxidants no longer tameable by an inhibited control of the antioxidant defences. Effective orthodox drugs are able to slow down these ailments but they impoverish the quality of life because they cannot reactivate the innate ability to restore the complexity of the antioxidant system. Several potential approaches to renew this system have been discussed and their possible roles to reactivate a valid protection in at least some of the outlined pathologies. It is hoped to evaluate this integrated medical approach because it represents a sheet anchor for many patients.

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.

Beta-carotene prevents ozone-induced proinflammatory markers in murine skin.

Beta-carotene has been thought to protect against oxidative stress generated by ultraviolet radiation and thus prevents skin cancer and skin aging (Biesalski and Obermueller-Jevic, 2001). However, nothing is known about its potential effects against other environmental sources of oxidative stress such as ozone (O3) in skin. Intake of oral beta-carotene supplements before exposure to sunlight (and thus inevitably also to (O3) has been recommended on a population-wide basis. However, although some studies have shown beta-carotene as providing skin protection as an antioxidant, other studies using skin cells in culture have shown that beta-carotene may have unexpected prooxidant properties (Obermüller-Jevic, et al., 2001). Given this, there is an ongoing debate regarding the protective or potentially harmful role(s) of beta-carotene in human skin. In this study, the effect of beta-carotene on ozone's effects on the skin of hairless mice was assessed. After feeding a diet supplemented with 0.5% beta-carotene for 1 month, mice were subjected to O3 exposure (0.8 ppm 6 h/day; 7 days) and the induction of proinflammatory markers such as tumor necrosis factor-alpha (TNFalpha), macrophage inflammatory protein 2 (MIP2), and inducible nitric oxide synthase (iNOS), and markers of oxidative stress, heme-oxygenase-1 (HO-1), were quantitated. The data showed that beta-carotene downregulated the induction of TNFalpha, MIP2, iNOS, and HO-1 in response to O3. We conclude that beta-carotene provides protection against O3-induced skin oxidative stress in vivo, which is consistent with a protective role for beta-carotene in the skin.

Endothelin receptor A expression in human inflammatory cells.

Most inflammatory diseases show elevated levels of endothelin-1 (ET-1) probably due to an alteration in vascular structure and function with activation/accumulation of inflammatory cells. The ET receptors (ET(A), ET(B)) are widely expressed in all human vessels, consistent with the main role of ET-1 in maintaining physiological vascular tone. Previous findings have shown the expression on inflammatory cells such as neutrophils (PMNs) and macrophages (MØs) of ET-1 and endothelin-converting enzyme-1 (ECE-1) (the key enzyme in the biosynthesis of ET-1). Therefore the role of ET-1 cannot be related only to the vasoactivity. Our study was aimed to determine the expression and the cellular location of ET receptors in both human PMNs and MØs by the use of RT-PCR assay, Western blot analysis and immunocytological methods. Our results showed for the first time that PMNs and MØs clearly expressed ET(A) (mRNA and protein). Considering that the overproduction of ET-1 following endothelial dysfunction and inflammation, contributes to pathophysiological processes such as vascular hypertrophy, cell proliferation and fibrosis, our results suggest that PMNs and MØs can also play a key role in vascular dysfunctions via the possible formation of an autocrine loop between ET-1 and ET(A).