Concetta Gardi

Ethanol-induced oxidative stress: basic knowledge

After a general introduction, the main pathways of ethanol metabolism (alcohol dehydrogenase, catalase, coupling of catalase with NADPH oxidase and microsomal ethanol-oxidizing system) are shortly reviewed. The cytochrome P450 isoform (CYP2E1) specifically involved in ethanol oxidation is discussed. The acetaldehyde metabolism and the shift of the NAD/NADH ratio in the cellular environment (reductive stress) are stressed. The toxic effects of acetaldehyde are mentioned. The ethanol-induced oxidative stress: the increased MDA formation by incubated liver preparations, the absorption of conjugated dienes in mitochondrial and microsomal lipids and the decrease in the most unsaturated fatty acids in liver cell membranes are discussed. The formation of carbon-centered (1-hydroxyethyl) and oxygen-centered (hydroxyl) radicals during the metabolism of ethanol is considered: the generation of hydroxyethyl radicals, which occurs likely during the process of univalent reduction of dioxygen, is highlighted and is carried out by ferric cytochrome P450 oxy-complex (P450–Fe3+O2·−) formed during the reduction of heme-oxygen. The ethanol-induced lipid peroxidation has been evaluated, and it has been shown that plasma F2-isoprostanes are increased in ethanol toxicity.

Cigarette smoke affects keratinocytes SRB1 expression and localization via H2O2 production and HNE protein adducts formation.

Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H2O2) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H2O2, induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.

Hydrocephalus, bronchiectasis, and ciliary aplasia.

A girl presented in the neonatal period with hydrocephalus, bronchiectasis, and ciliary aplasia. A common defect both in respiratory tract cilia and in ventricular ependyma cilia may explain the association of the two diseases.

Pulmonary vascular injury in pancreatitis: Evidence for a major role played by pancreatic elastase

Using an experimental model of pancreatitis in the rat, the role of trypsin and elastase in mediating lung vascular injury in this condition was examined. The induction of pancreatitis by injection of sodium cholate in the pancreas resulted in a significant decrease in serum trypsin inhibitory capacity, and in a complete saturation of serum elastase inhibitory capacity matched by the appearance of endothelial injury of pulmonary capillaries and edema formation. The complete lack of serum elastase inhibitory capacity was associated with the presence of elastase activity in serum and bronchoalveolar lavage (BAL) fluids. The pretreatment of animals with N-furoyl saccharin (a potent inhibitor of many serine proteinases) prevented lung capillary injury and the imbalance of serum proteinase-anti-proteinase activities as well as the appearance of any elastolytic activity in serum and BAL fluids. These findings which clearly demonstrate the protease dependence of the pulmonary vascular injury in our experimental model, strongly suggested a major role for elastase(s). The suppression, in the experimental model, of the serum elastase inhibitory capacity by using chloramine-T resulted in an earlier onset of lung vascular damage, a marked worsening of pulmonary lesions, and an increase of elastolytic levels in serum and BAL fluids. Furthermore the physical properties of the protein molecule with enzyme activity detected in BAL fluids were consistent with those of rat pancreatic elastase. The reported data strongly support the hypothesis that pancreatic elastase plays a major role in the development of pulmonary vascular injury after acute pancreatitis.

F2-isoprostanes stimulate collagen synthesis in activated hepatic stellate cells: a link with liver fibrosis?

Carbon tetrachloride (CCl4)-induced hepatic fibrosis has been considered to be linked to oxidative stress and mediated by aldehydic lipid peroxidation products. In the present study, we investigated whether collagen synthesis is induced by F2-isoprostanes, the most proximal products of lipid peroxidation and known mediators of important biological effects. By contrast with aldehydes, F2-isoprostanes act through receptors able to elicit definite signal transduction pathways. In a rat model of CCl4-induced hepatic fibrosis, plasma F2-isoprostanes were markedly elevated for the entire experimental period; hepatic collagen content also increased. When hepatic stellate cells (HSCs) from normal liver were cultured with F2-isoprostanes in the concentration range found in the in vivo studies (10−9–10−8 M), a striking increase in DNA synthesis (reversed by the thromboxane A2 antagonist SQ 29 548), in cell proliferation and in collagen synthesis was observed. Total collagen content was similarly increased. Moreover, F2-isoprostanes markedly increased the production of transforming growth factor-β1 by U937 cells, considered a model of liver macrophages. The data provide evidence for the possibility that F2-isoprostanes generated by lipid peroxidation in hepatocytes mediate HSC proliferation and collagen production seen in hepatic fibrosis.

Interaction between Neuroanatomical and Psychological Changes after Mindfulness-Based Training

Several cross-sectional studies have documented neuroanatomical changes in individuals with a long history of meditation, while a few evidences are available about the interaction between neuroanatomical and psychological changes even during brief exposure to meditation. Here we analyzed several morphometric indexes at both cortical and subcortical brain level, as well as multiple psychological dimensions, before and after a brief -8 weeks- Mindfulness Based Stress Reduction (MBSR) training program, in a group of 23 meditation naïve-subjects compared to age-gender matched subjects. We found a significant cortical thickness increase in the right insula and the somatosensory cortex of MBSR trainees, coupled with a significant reduction of several psychological indices related to worry, state anxiety, depression and alexithymia. Most importantly, an interesting correlation between the increase in right insula thickness and the decrease in alexithymia levels during the MBSR training were observed. Moreover, a multivariate pattern classification approach allowed to identify a cluster of regions more responsive to MBSR training across subjects. Taken together, these findings documented the significant impact of a brief MBSR training on brain structures, as well as stressing the idea of MBSR as a valuable tool for alexithymia modulation, also originally providing a plausible neurobiological evidence of a major role of right insula into mediating the observed psychological changes.

Iron overload enhances the development of experimental liver cirrhosis in mice

The role of iron in initiating liver fibrosis in iron overload diseases is not clearly established. Partly, this is due to the lack of suitable animal models that can produce the full liver pathology seen in genetic hemochromatosis. Recent advances in this field have demonstrated that iron may be interacting with other potential liver-damaging agents. The aim of this study was to investigate if feeding with carbonyl iron (CI) facilitates the development of carbon tetrachloride (CCl4)-induced liver fibrosis in the mouse. Mice were given a diet containing 3% CI and treated with CCl4 intraperitoneally twice weekly and 5% alcohol added to the drinking water for 12 weeks. Hepatic iron content increased 15- and 22-fold in animals receiving CI and CI + CCl4. At histological examination, iron-laden hepatocytes were found in CI treated animals, whereas these were absent in animals not exposed to CI. Mice receiving iron-enriched diet alone showed a mild fibrosis. Conversely, a marked collagen deposition was observed in CCl4 and CI + CCl4 groups. In particular, in this latter group, there was evidence of liver cirrhosis. Biochemical evaluation of collagen content substantiated histologic analysis. These results demonstrate that the addition of iron facilitates the development of cirrhosis in animals exposed to subtoxic doses of CCl4. This model may be useful in exploring the pathogenesis of liver cirrhosis. Moreover, its use in genetically altered mouse strains might provide new insight on the role of iron in fibrosis.

NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells.

During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O(3) per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H(2)O(2)) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis.

Reactivity of mouse alveolar macrophages to cigarette smoke is strain dependent

Cigarette smoke (CS) is a main risk factor in chronic obstructive pulmonary disease (COPD), but only 20% of smokers develop COPD, suggesting genetic predisposition. Animal studies have shown that C57BL/6J mice are sensitive to CS and develop emphysema, whereas Institute of Cancer Research (ICR) mice are not. To investigate the potential factors responsible for the different susceptibility of ICR and C57BL/6J mice to CS, we evaluated in alveolar macrophages (AMs) isolated from these strains of mice the possible mechanisms involved in the inflammatory and oxidative responses induced by CS. Lactate dehydrogenase (LDH) release revealed that C57BL/6J AMs were more susceptible to CS extract (CSE) toxicity than ICR. Differences were observed in inflammatory and oxidative response after CSE exposure. Proinflammatory cytokines and matrix metalloproteinases (MMPs) were increased in C57BL/6J but not ICR AMs. Control C57BL/6J AMs showed a higher baseline production of reactive oxygen species (ROS) and H(2)O(2) with lower baseline levels of GSH, nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), and glutathione peroxidase (GPX2). This was associated with reduced histone deacetylase-2 (HDAC2) expression, activation of NF-κB, and higher basal levels of TNF-α and IL-6. CSE induced a decrease in HDAC2 protein levels in both C57BL/6J and ICR AMs; however, the level of HDAC2 was significantly lower in C57BL/6 than in ICR AMs. Furthermore, CSE enhanced NF-κB-dependent cytokine release only in C57BL/6J AMs. We suggest that an imbalance in oxidative stress decreases HDAC2 levels and facilitates NF-κB binding, resulting in a proinflammatory response in C57BL/6J but not in ICR AMs. These results could contribute in understanding the different susceptibility to CS of these strains of mice.

A biochemical and morphological investigation of the early development of genetic emphysema in tight-skin mice

The tight-skin (Tsk) mouse has recently been proposed as a genetic model of emphysema. In the present study, the development of emphysema was investigated in these mice with histological, biochemical, and ultrastructural methods at 4 days and at 1 and 2 months of life. At 4 days after birth, histological examination of the lungs revealed only a mild enlargement of the primary sacculi. Neither biochemical nor ultrastructural changes were seen however at this time. At 1 month of age, the histological examination showed marked emphysema-like changes, characterized by enlargment of air spaces accompanied by destruction of alveolar walls. Biochemical analysis showed a marked decrease in insoluble elastin content and a significant increase in salt-extractable collagen. Ultrastructural investigation revealed edema fluid in the interstitium and broken and disorganized elastic fibers. All these findings strikingly resemble the changes which occur in the lungs early after an instillation of elastase. In the 2-month-old Tsk mice the histological lesion progressed in severity. The ultrastructural findings were similar to those observed at 1 month, and the biochemical changes showed no signs of recovery. Thus, in these mice, the emphysematous lesion develops very rapidly between 4 days and 1 month of life and shows the characteristics of an elastolytic process which is still ongoing at 2 months of age.