A nutrigenomics approach for the study of anti-aging interventions: olive oil phenols and the modulation of gene and microRNA expression profiles in mouse brain.

PURPOSE: Middle-aged C57Bl/6J mice fed for 6 months with extra-virgin olive oil rich in phenols (H-EVOO, phenol dose/day: 6 mg/kg) showed cognitive and motor improvement compared to controls fed the same olive oil deprived of phenolics (L-EVOO). The aim of the present study was to evaluate whether these behavioral modifications were associated with changes in gene and miRNA expression in the brain. METHODS: Two brain areas involved in cognitive and motor processes were chosen: cortex and cerebellum. Gene and miRNA profiling were analyzed by microarray and correlated with performance in behavioral tests. RESULTS: After 6 months, most of the gene expression changes were restricted to the cerebral cortex. The genes modulated by aging were mainly down-regulated, and the treatment with H-EVOO was associated with a significant up-regulation of genes compared to L-EVOO. Among those, we found genes previously associated with synaptic plasticity and with motor and cognitive behavior, such as Notch1, BMPs, NGFR, GLP1R and CRTC3. The agrin pathway was also significantly modulated. miRNAs were mostly up-regulated in old L-EVOO animals compared to young. However, H-EVOO-fed mice cortex displayed miRNA expression profiles similar to those observed in young mice. Sixty-three miRNAs, out of 1203 analyzed, were significantly down-regulated compared to the L-EVOO group; among them, we found miRNAs whose predicted target genes were up-regulated by the treatment, such as mir-484, mir-27, mir-137, mir-30, mir-34 and mir-124. CONCLUSIONS: We are among the first to report that a dietary intervention starting from middle age with food rich in phenols can modulate at the central level the expression of genes and miRNAs involved in neuronal function and synaptic plasticity, along with cognitive, motor and emotional behavior.

Investigation of Aberrant Basaloid Cells in a Rat Model of Lung Fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (ILD) whose cause and pathogenesis are not yet well understood. Until now, no animal model of lung fibrosis succeeds in recapitulating all IPF features, thus the use of different rodent models is essential for the evaluation and development of new effective pharmacological treatments. Recently, the alveolar epithelial dysfunction has been emphasized in the etiopathogenesis context of IPF. Remarkably, the role of an aberrant basaloid cell type, primarily found in humans and confirmed in mice, seems to be crucial in the establishment and progression of the disease/model. Our work aimed to characterize for the first time this cell population in a rat model of lung fibrosis induced by a double bleomycin (BLM) administration, demonstrating the translational value of the model and its potential use in the testing of effective new drugs. METHODS: Rats received an intratracheal BLM administration at day 0 and 4. Animals were sacrificed 21 and 28 days post-BLM. The fibrosis evaluation was carried out through histological (Ashcroft score and automatic image analysis) and immunoenzymatic analysis. Immunofluorescence was used for the characterization of the aberrant basaloid cells markers. RESULTS: Lung histology revealed an increase in severe grades of Ashcroft scores and areas of fibrosis, resulting in a rise of collagen deposition at both the analyzed time-points. Immunofluorescence staining indicated the presence of KRT8+ cells in bronchial epithelial cells from both controls (saline, SAL) and BLM-treated animals. Interesting, KRT8+ cells were found exclusively in the fibrotic parenchyma (confirmed by the alpha-smooth muscle actin (α-SMA) staining for myofibroblasts) of BLM-treated animals. Moreover, KRT8+ cells co-expressed markers as Prosurfactant protein C (Pro-SPC) and Vimentin, suggesting their intermediate state potentially originating from alveolar type II (AT2) cells, and participating to the abnormal epithelial-mesenchymal crosstalk. CONCLUSION: Previous preclinical studies demonstrated the presence of KRT8+ aberrant basaloid-like cells in murine models of lung fibrosis. This work investigated the same cell population in a different rodent (the rat) model of lung fibrosis triggered by a double administration of BLM. Our results provided a further confirmation that, in rats, the intratracheal administration of BLM induced the appearance of a population of cells compatible with the KRT8+ alveolar differentiation intermediate (ADI) cells, as described previously in the mouse. This piece of work enforces previous evidence and further support the use of a rat model of BLM resembling the alveolar epithelial dysfunction to evaluate new clinical candidates for development in IPF.

Pyruvate metabolism dictates fibroblast sensitivity to GLS1 inhibition during fibrogenesis.

Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor-ß1 (TGF-ß1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-ß1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-ß1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-ß1-induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-ß1-induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-ß1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF. METHODS: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies. RESULTS: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies. CONCLUSIONS: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

Optimization of M3 Antagonist-PDE4 Inhibitor (MAPI) Dual Pharmacology Molecules for the Treatment of COPD.

Aiming at the inhaled treatment of pulmonary diseases, the optimization process of the previously reported MAPI compound 92a is herein described. The project was focused on overcoming the chemical stability issue and achieving a balanced bronchodilator/anti-inflammatory profile in rats in order to be confident in a clinical effect without having to overdose at one of the biological targets. The chemical strategy was based on fine-tuning of the substitution pattern in the muscarinic and PDE4 structural portions of the dual pharmacology compounds, also making use of the analysis of a proprietary crystal structure in the PDE4 catalytic site. Compound 10f was identified as a chemically stable, potent, and in vivo balanced MAPI lead compound, as assessed in bronchoconstriction and inflammation assays in rats after intratracheal administration. After the in-depth investigation of the pharmacological and solid-state profile, 10f proved to be safe and suitable for development.

Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis.

During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N'-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N'-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.

Effects of de-alcoholised wines with different polyphenol content on DNA oxidative damage, gene expression of peripheral lymphocytes, and haemorheology: an intervention study in post-menopausal women.

PURPOSE: Epidemiological studies suggest that a moderate consumption of wine is associated with a reduced risk of cardiovascular diseases and with a reduced mortality for all causes, possibly due to increased antioxidant defences. The present intervention study was undertaken to evaluate the in vivo effects of wine polyphenols on gene expression in humans, along with their supposed antioxidant activity. METHODS: Blood haemorheology and platelet function were also evaluated. In order to avoid interferences from alcohol, we used de-alcoholised wine (DAW) with different polyphenol content. A randomised cross-over trial of high-proanthocyanidin (PA) red DAW (500 mL/die, PA dose = 7 mg/kg b.w.) vs. low-PA rosé DAW (500 mL/die, PA dose = 0.45 mg/kg) was conducted in 21 post-menopausal women in Florence, Italy. Oxidative DNA damage by the comet assay and gene expression by microarray was measured in peripheral blood lymphocytes, collected during the study period. Blood samples were also collected for the evaluation of haematological, haemostatic, haemorheological, and inflammatory parameters. RESULTS: The results of the present study provide evidence that consumption of substantial amounts of de-alcoholised wine for 1 month does not exert a protective activity towards oxidative DNA damage, nor modifies significantly the gene expression profile of peripheral lymphocytes, whereas it shows blood-fluidifying actions, expressed as a significant decrease in blood viscosity. However, this effect does not correlate with the dosage of polyphenols of the de-alcoholised wine. CONCLUSIONS: More intervention studies are needed to provide further evidence of the health-protective effects of wine proanthocyanidins.

Discovery of M3 Antagonist-PDE4 Inhibitor Dual Pharmacology Molecules for the Treatment of Chronic Obstructive Pulmonary Disease.

In this paper, we report the discovery of dual M3 antagonist-PDE4 inhibitor (MAPI) compounds for the inhaled treatment of pulmonary diseases. The identification of dual compounds was enabled by the intuition that the fusion of a PDE4 scaffold derived from our CHF-6001 series with a muscarinic scaffold through a common linking ring could generate compounds active versus both the transmembrane M3 receptor and the intracellular PDE4 enzyme. Two chemical series characterized by two different muscarinic scaffolds were investigated. SAR optimization was aimed at obtaining M3 nanomolar affinity coupled with nanomolar PDE4 inhibition, which translated into anti-bronchospastic efficacy ex vivo (inhibition of rat trachea contraction) and into anti-inflammatory efficacy in vitro (inhibition of TNFα release). Among the best compounds, compound 92a achieved the goal of demonstrating in vivo efficacy and duration of action in both the bronchoconstriction and inflammation assays in rat after intratracheal administration.

Effects of dietary extra-virgin olive oil on behaviour and brain biochemical parameters in ageing rats.

The aim of the present study was to verify whether extra-virgin olive oil, a dietary component naturally containing phenolic antioxidants, has the potential to protect the brain from the deleterious effects of ageing. To accomplish this goal, we used male rats fed a high-energy diet containing either maize oil, or extra-virgin olive oil with high or low phenol content (720 or 10 mg total phenols/kg oil, corresponding to a daily dose of 4 or 0.05 mg total phenols/kg body weight, respectively) from age 12 months to senescence. The measured endpoints were biochemical parameters related to oxidative stress and functional tests to evaluate motor, cognitive and emotional behaviour. Olive oil phenols did not exert major protective actions on motor and cognitive function, as we observed only a tendency to improved motor coordination on the rotarod in the old animals treated with the oil rich in phenols (40 % average increase in the time to first fall; P = 0.18). However, an interesting finding of the present study was a reduced step-through latency in the light-dark box test, found in the older animals upon treatment with the oil rich in antioxidant phenols, possibly indicating an anxiety-lowering effect. This effect was associated with decreased glutathione reductase activity and expression in the brain, a phenomenon previously associated with decreased anxiety in rodents. These results indicate a previously undetected effect of a diet containing an olive oil rich in phenols. Further studies are warranted to verify whether specific food antioxidants might also have an effect on emotional behaviour.

Reduction of colonic inflammation in HLA-B27 transgenic rats by feeding Marie Ménard apples, rich in polyphenols.

Inflammatory bowel diseases (IBD) are immunomediated ailments affecting millions of individuals. Although diet is regarded as an important factor influencing IBD, there are no accepted dietary recommendations presently available. We administered 7.6 % lyophilised apples obtained from two cultivars (Golden Delicious and Marie Ménard, low and high in polyphenols, respectively) to HLA-B27 transgenic rats which develop spontaneous IBD. After 3 months feeding, rats fed Marie Ménard apples had reduced myeloperoxidase activity (3.6 (sem 0.3) v. 2.2 (sem 0.2) U/g tissue; P < 0.05) and reduced cyclo-oxygenase-2 (P < 0.05) and inducible NO synthase gene expression (P < 0.01) in the colon mucosa and significantly less diarrhoea (P < 0.05), compared with control rats. Cell proliferation in the colon mucosa was reduced significantly by feeding Golden Delicious apples, with a borderline effect of Marie Ménard apples. Gene expression profiling of the colon mucosa, analysed using the Whole Rat Genome 4 x 44 K Agilent Arrays, revealed a down-regulation of the pathways of PG synthesis, mitogen-activated protein kinase (MAPK) signalling and TNFalpha-NF-kappaB in Marie Ménard-fed rats. In the stools of the animals of this group we also measured a significant reduction of bacteria of the Bacteriodes fragilis group. In conclusion, the administration of Marie Ménard apples, rich in polyphenols and used at present only in the manufacturing of cider, ameliorates colon inflammation in transgenic rats developing spontaneous intestinal inflammation, suggesting the possible use of these and other apple varieties to control inflammation in IBD patients.

Oxidative DNA damage and plasma antioxidant capacity in type 2 diabetic patients with good and poor glycaemic control.

Diabetes mellitus is a complex metabolic disorder characterized by a disturbance in glucose metabolism. Recent evidence suggests that increased oxidative damage as well as reduction in antioxidant capacity could be related to the complications in patients with type 2 diabetes. The aim of this study was to measure plasma antioxidant status in type 2 diabetic patients with good and poor glycaemic control and its relationship with oxidative DNA damage. Thirty-nine type 2 diabetic patients and eighteen healthy subjects were recruited for this study. We found that diabetic patients had slightly, but not significantly lower antioxidant capacity, measured with the "ferric reducing ability of plasma" (FRAP) assay, than healthy subjects. On the contrary, oxidative DNA damage (measured by the Comet assay) in leukocytes obtained from diabetic patients was significantly higher compared to healthy subjects. Taking into account glucose control, we found that the FRAP level was significantly (p<0.05) lower in diabetic subjects with poor glycaemic control than healthy subjects, while patients with good glycaemic control had FRAP values similar to controls. We also observed an unexpected positive correlation between FRAP values and oxidative DNA damage in diabetic patients; moreover, a positive correlation was found between FRAP and glucose level or HbA(1c) in patients with poor glycaemic control. In conclusion, our results confirm that patients with type 2 diabetes have a higher oxidative DNA damage than healthy subjects and that plasma antioxidant capacity is significantly lower only in patients with poor glycaemic control, moreover, in these patients FRAP values are positively correlated with glycaemic levels and HbA(1c). These observations indicate that a compensatory increase of the antioxidant status is induced as a response to free radical overproduction in type 2 diabetes. Therefore, the addition of antioxidant supplements to the current pharmacological treatment could have potentially beneficial effects in diabetic patients with poor glycaemic control.

Liver and colon DNA oxidative damage and gene expression profiles of rats fed Arabidopsis thaliana mutant seeds containing contrasted flavonoids.

Plant polyphenols, such as flavonoids, comprise many compounds, ranging from simple phenolic molecules (i.e. flavonols, anthocyanins) to polymeric structures with high molecular weight (as proanthocyanidins, PAs). We investigated the effects of flavonoids by feeding Wistar rats Arabidopsis thaliana seeds carrying mutations in key enzymes of the flavonoid biosynthetic pathway (15% w/w seeds for 4 weeks). The seeds used were: Ws-2 wild-type containing flavonols and PAs, tt3-4 mutant containing flavonols only, ban-5 accumulating flavonols and anthocyanins, tt4-8 mutant, deprived of flavonoids. DNA oxidative damage was significantly reduced only in the liver of rats fed tt3-4 mutant seeds. Microarray analysis of the liver revealed down-regulation of genes associated with oxidative stress, Krebs cycle, electron transport and proteasome degradation in all experimental groups compared to the tt4-8-fed reference rats; therefore, these effects were due to the flavonol content and not to high molecular weight compounds. We observed a down-regulation of inflammatory response genes in the colon mucosa in ban-5- fed rats, probably due to anthocyanin content. In conclusion, flavonols exhibited antioxidant effects at systemic level, whereas high molecular weight flavonoids affected only the colon, probably due to their limited absorption.

Calibration of the comet assay for the measurement of DNA damage in mammalian cells.

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. Gamma-rays from 60Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with gamma- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10(9) Da, respectively, (0.50 and 0.52 breaks/10(6) bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.

Seasonal variations of DNA damage in human lymphocytes: correlation with different environmental variables.

Several types of DNA damage, including DNA breaks and DNA base oxidation, display a seasonal trend. In the present work, a sample of 79 healthy subjects living in the city of Florence, Italy, was used to analyse this effect. Three possible causative agents were taken into consideration: solar radiation, air temperature and air ozone level. DNA damage was measured in isolated human lymphocytes at different times during the year and the observed damage was correlated with the levels of these three agents in the days preceding blood sampling. Three time windows were chosen: 3, 7 and 30 days before blood sampling. DNA strand breaks and the oxidized purinic bases cleaved by the formamidopyrimidine glycosylase (FPG sites) were measured by means of the comet assay. The results of multivariate regression analysis showed a positive correlation between lymphocyte DNA damage and air temperature, and a less strong correlation with global solar radiation and air ozone levels.

Loss of tyrosinase activity confers increased skin tumor susceptibility in mice.

The tyrosinase (Tyr) gene encodes the enzyme tyrosinase that catalyses the conversion of L-tyrosine into DOPA (3,4-dihydroxyphenylalanine)-quinone. The albino mutation abrogates functional activity of tyrosinase resulting in deficiency of melanin pigment production in skin and retina. Tyr maps to a region in the central position of Chromosome 7 that contains a skin tumor-modifier locus. We rescued the albino mutation in transgenic mice to assess a possible role of Tyr gene in two-stage skin carcinogenesis. Transgenic expression of the functional Tyr(Cys) allele in albino mice (Tyr(Ser)) caused a reduction in skin papilloma multiplicity, in four independent experiments and at three dose levels of DMBA (9,10-dimethyl-1,2-benzanthracene). In vitro mechanistic studies demonstrated that transfection of the Tyr(Cys) allele in a human squamous cell carcinoma cell line (NCI-H520) increases tyrosinase enzyme activity and confers resistance to hydrogen peroxide-induced oxidative DNA damage. These results provide direct evidence that the Tyr gene can act as a skin cancer-modifier gene, whose mechanism of action may involve modulation of oxidative DNA damage.

Association between atmospheric ozone levels and damage to human nasal mucosa in Florence, Italy.

We evaluated the effects of urban air pollutants on human nasal mucosa over an 8-month period on 102 subjects living in Florence, Tuscany, Italy. A group of subjects living in a city with a lower level of pollution (Sassari, Sardinia, Italy) was also analyzed. Nasal mucosa cells were harvested by brushing, a noninvasive procedure. Half of the cells were used for genotoxicity studies using the alkaline comet assay, and half for morphological studies. The levels of DNA damage in the nasal mucosa were considerably higher (+73%) in the subjects living in Florence than in Sassari. High levels of atmospheric ozone in Florence air correlated with DNA damage, and to the prevalence of inflammatory pathologies of the upper respiratory tract, although the ozone concentrations were below the Italian recommended attention level. Furthermore, higher levels of DNA damage were correlated with a dysfunction in the ability to maintain a normal epithelial cell structure. These data suggest an association between ozone air levels and damage in the upper respiratory tract. It remains unclear whether ozone itself or other associated pollutants are responsible for the observed alterations.

Measurement of DNA breaks and oxidative damage in polymorphonuclear and mononuclear white blood cells: a novel approach using the comet assay.

DNA damage is thought to play a relevant role in degenerative diseases and aging. Therefore, measuring DNA damage in living cells without artifacts is a critical issue, especially with very sensitive methods, such as the comet assay, which can detect very low levels of DNA damage. We show here that the procedures of cell subtype isolation increase DNA damage measured in human white blood cells (WBC) with the comet assay. We describe a novel and simple method to measure DNA strand breaks and oxidative damage separately in polymorphonuclear and mononuclear leukocytes, using whole blood without previous cell isolation. This method can be useful for measuring DNA damage in different subtypes of human peripheral leukocytes, avoiding the artifacts and the time involved in the cell separation procedures.

Oxidative DNA damage in peripheral blood cells in type 2 diabetes mellitus: higher vulnerability of polymorphonuclear leukocytes.

Since oxidative stress is thought to play an important role in the pathogenesis and complications of diabetes, we used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as FPG (formamidopyrimidine DNA glycosylase)-sensitive sites, in peripheral blood cells (PBC) from type 2 diabetes patients and healthy controls. Oxidative DNA damage in leukocytes was increased in diabetic compared to normal subjects. However, no differences in the levels of DNA damage in isolated lymphocytes were found between the two groups. These data indicate a higher vulnerability to oxidative damage of polymorphonuclear as compared to mononuclear leukocytes in type 2 diabetes. Thus, the measurement of oxidative DNA damage in leukocytes by means of the comet assay is a suitable marker for the evaluation of systemic oxidative stress in diabetic patients.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo.

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

Chronic resveratrol treatment ameliorates cell adhesion and mitigates the inflammatory phenotype in senescent human fibroblasts.

We evaluated the effect of resveratrol on the senescence-associated secretory phenotype (SASP) and on adhesion-related processes in cultured human MRC5 fibroblasts. Presenescent cultures were chronically treated with or without 5 µM resveratrol. The development of SASP in MRC5 fibroblasts approaching senescence was significantly attenuated by resveratrol treatment, which reduced both gene expression and release of proinflammatory cytokines. Although to a lesser extent, 1 µM resveratrol proved to be effective on cytokine gene expression. Cell spreading capacity and plating efficiency were strikingly increased and accompanied by recovery of type I collagen expression to presenescent levels. As p16(INK4a) protein expression was not significantly modified, and based on our previous data, we propose that resveratrol does not affect fibroblast replicative senescence, but improves tissue maintenance and repair during normal cellular aging. Considering these low concentrations proved effective in vitro, translation of these data to human research on inflammation-related pathologies can be envisaged.