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Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress-induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence-associated secretory phenotype (SASP), which contributes to generate a pro-inflammatory and pro-tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti-inflammatory and anti-tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation-induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ-irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence-associated (SA)-β-Gal-staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP-AMP Synthase (cGAS) activation. IL-6, IL-8, MCP-1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation-induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB-mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy.

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers., Here, members of the hCOMET COST Action program provide a consensus statement on the Minimum Information for Reporting Comet Assays (MIRCA).

The nutraceutical effects ofWe aimed to evaluate the effects of a specific by-product of olive oil called "pâté" (OlP) administered as tablets, on cardiovascular and metabolic risk factors.The study was a crossover trial with 2 intervention periods. Nineteen participants (mean age: 38 years) took 4 tablets/day of either olive pâté (corresponding to 30 mg/day of hydroxytyrosol) or placebo for 2 months followed by a 2-month washout and another 2 months of crossed over treatment.After the intervention with pâté, participants showed a statistically significant reduction in plasma levels of total cholesterol (-10.8 mg/dL), LDL cholesterol (-10.8 mg/dL) and urea (-4.1 mg/dL), and a significant increase in calcium levels (+0.3 mg/dL). Leukocyte response to exogenous oxidative stress was significantly reduced (-12.8%) and levels of the antioxidant transcription factor Nrf-2 increased by 88.9%. Plasma levels of the pro-inflammatory protein MCP-1 were significantly reduced (-9.0 pg/mL).In conclusion, the intake of OlP showed positive effects on several cardiovascular risk factors, demonstrating the nutraceutical potential of a widely available but, to date, underestimated olive oil by-product.

This short narrative review describes the use of the comet assay to evaluate the formation of genotoxic compounds in the gut lumen in human studies. The fecal water genotoxicity assay is based on ability of the gut content to induce genotoxicity in a cellular model, employing the aqueous component of the feces (fecal water) as this is supposed to contain most of the reactive species and to convey them to the intestinal epithelium. This non-invasive and low-cost assay has been demonstrated to be associated with colon cancer risk in animal models, and although the final validation against human tumors is lacking, it is widely used as a colo-rectal cancer risk biomarker in human nutritional intervention studies. The contribution given to the field of nutrition and cancer by the FW genotoxicity assay is highlighted, particularly in conjunction with other risk biomarkers, to shed light on the complex relationship among diet, microbiota, individual subject characteristics and the formation of genotoxic compounds in the gut.

Objective Emerging evidence demonstrates that excessive accumulation of senescent cells is associated with some chronic diseases and suggests a pathogenic role of cellular senescence in fibrotic processes, such as that occurring in ageing or in SSc. Recently we demonstrated that parvovirus B19 (B19V) activates normal human dermal fibroblasts and induces expression of different profibrotic/pro-inflammatory genes. This observation prompted us to investigate whether it is also able to induce fibroblast senescence as a potential pathogenetic mechanism in B19V-induced fibrosis. Methods Primary cultures of fibroblasts were infected with B19V and analysed for the acquisition of senescence markers, such as morphological modifications, senescence-associated β-galactosidase (SA-β-gal) activity, DNA damage response and expression of senescence-associated secretory phenotype (SASP)-related factors. Results We demonstrated that B19V-infected fibroblasts develop typical senescence features such as enlarged and flat-shaped morphology and SA-β-gal activity similar to that observed in SSc skin fibroblasts. They also developed an SASP-like phenotype characterized by mRNA expression and release of some pro-inflammatory cytokines, along with activation of the transcription factor nuclear factor κB. Moreover, we observed B19V-induced DNA damage with the comet assay: a subpopulation of fibroblasts from B19V-infected cultures showed a significantly higher level of DNA strand breaks and oxidative damage compared with mock-infected cells. An increased level and nuclear localization of γH2AX, a hallmark of DNA damage response, were also found. Conclusions B19V-induced senescence and production of SASP-like factors in normal dermal fibroblasts could represent a new pathogenic mechanism of non-productive B19V infection, which may have a role in the fibrotic process.

Chemotherapy is still widely used as a coadjutant in gastric cancer when surgery is not possible or in presence of metastasis. During tumor evolution, gatekeeper mutations provide a selective growth advantage to a subpopulation of cancer cells that become resistant to chemotherapy. When this phenomenon happens, patients experience tumor recurrence and treatment failure. Even if many chemoresistance mechanisms are known, such as expression of ATP-binding cassette (ABC) transporters, aldehyde dehydrogenase (ALDH1) activity and activation of peculiar intracellular signaling pathways, a common and universal marker for chemoresistant cancer cells has not been identified yet. In this study we subjected the gastric cancer cell line AGS to chronic exposure of 5-fluorouracil, cisplatin or paclitaxel, thus selecting cell subpopulations showing resistance to the different drugs. Such cells showed biological changes, among them, we observed that the acquired chemoresistance to 5-fluorouracil induced an endothelial-like phenotype and increased the capacity to form vessel-like structures. We identified the upregulation of thymidine phosphorylase (TYMP), which is one of the most commonly reported mutated genes leading to 5-fluorouracil resistance, as the cause of such enhanced vasculogenic ability.

Background MDR in bacteria is threatening to public health. Overexpression of efflux pumps is an important cause of MDR. The co-administration of antimicrobial drugs and efflux pump inhibitors (EPIs) is a promising approach to address the problem of MDR. Objectives To identify new putative EPIs and to characterize their mechanisms of action. Methods The effects of four selected piperazine derivatives on resistance-nodulation-cell division (RND) pumps was evaluated in Escherichia coli strains overexpressing or not expressing RND pumps by assays aimed at evaluating antibiotic potentiation, membrane functionality, ethidium bromide accumulation and AcrB expression. The cytotoxicity of selected piperazines towards primary cultures of human dermal fibroblasts was also investigated. Results Four molecules enhanced levofloxacin activity against strains overexpressing RND efflux pumps (AcrAB-TolC and AcrEF-TolC), but not against RND pump-deficient strains. They had little effects on membrane potential. Molecule 4 decreased, whereas the other three increased, membrane permeability compared with untreated control cells. The four molecules showed differences in the specificity of interaction with RND efflux pumps, by inactivating the transport of one or more antibiotics, and in the levels of ethidium bromide accumulation and of acrB expression inhibition. Conclusions Piperazine derivatives are good candidates as inhibitors of RND efflux pumps. They decreased the activity of RND pumps by mixed mechanisms of action. Small structural differences among the molecules can be critical in defining their behaviour.

This study was designed within the frame of the COST Action hCOMET 15132 (Working Group 6), with the aim of comparing different peripheral blood cell preparations for their feasibility in human biomonitoring studies, using the comet assay for the evaluation of DNA damage. Basal levels of strand breaks/ALS and formamidopyrimidine DNA glycosylase (Fpg) - sites, and H2O2 (500 μM)-induced strand breaks, were measured in whole blood, peripheral blood mononuclear cells - lymphocytes and monocytes - and buffy coat; in fresh and 1, 4 and 12 weeks-frozen samples. The comparison among the fresh preparations showed that the basal levels of DNA damage were all very low and similar in the three samples. Frozen whole blood samples stored in cryostraws without cryoprotection showed similar basal levels of DNA damage as fresh samples, indicating that this preparation, often chosen for biobanks, resists efficiently freezing/thawing artifacts. However, long-term storage of frozen buffy coat samples in cryostraws and with no cryopreservative did not appear feasible. Storage up to 3 months of frozen cryoprotected peripheral blood mononuclear cells induced small increases in basal strand breaks and no other statistically significant modification. Altogether, this study suggests that whole blood could be the most suitable sample to be used to perform comet assay in human epidemiological biomonitoring for genotoxicity assessment in frozen samples, such as those stored in biobanks.

DNA damage and repair activity are often assessed in blood samples from humans in different types of molecular epidemiology studies. However, it is not always feasible to analyse the samples on the day of collection without any type of storage. For instance, certain studies use repeated sampling of cells from the same subject or samples from different subjects collected at different time-points, and it is desirable to analyse all these samples in the same comet assay experiment. In addition, flawless comet assay analyses on frozen samples open up the possibility of using this technique on biobank material. In this article we discuss the use of cryopreserved peripheral blood mononuclear cells (PBMCs), buffy coat (BC) and whole blood (WB) for analysis of DNA damage and repair using the comet assay. The published literature and the authors’ experiences indicate that various types of blood samples can be cryopreserved with only a minor effect on the basal level of DNA damage. There is evidence to suggest that WB and PBMCs can be cryopreserved for several years without much effect on the level of DNA damage. However, care should be taken when cryopreserving WB and BCs. It is possible to use either fresh or frozen samples of blood cells, but results from fresh and frozen cells should not be used in the same dataset. The article outlines detailed protocols for the cryopreservation of PBMCs, BCs and WB samples.

[Abstract] The alkaline comet assay, or single cell gel electrophoresis, is one of the most popular methods for assessing DNA damage in human population. One of the open issues concerning this assay is the identification of those factors that can explain the large inter-individual and inter-laboratory variation. International collaborative initiatives such as the hCOMET project - a COST Action launched in 2016 - represent a valuable tool to meet this challenge. The aims of hCOMET were to establish reference values for the level of DNA damage in humans, to investigate the effect of host factors, lifestyle and exposure to genotoxic agents, and to compare different sources of assay variability. A database of 19,320 subjects was generated, pooling data from 105 studies run by 44 laboratories in 26 countries between 1999 and 2019. A mixed random effect log-linear model, in parallel with a classic meta-analysis, was applied to take into account the extensive heterogeneity of data, due to descriptor, specimen and protocol variability. As a result of this analysis interquartile intervals of DNA strand breaks (which includes alkali-labile sites) were reported for tail intensity, tail length, and tail moment (comet assay descriptors). A small variation by age was reported in some datasets, suggesting higher DNA damage in oldest age-classes, while no effect could be shown for sex or smoking habit, although the lack of data on heavy smokers has still to be considered. Finally, highly significant differences in DNA damage were found for most exposures investigated in specific studies. In conclusion, these data, which confirm that DNA damage measured by the comet assay is an excellent biomarker of exposure in several conditions, may contribute to improving the quality of study design and to the standardization of results of the comet assay in human populations. This article is based upon work from COST Action hCOMET CA15132, supported by COST (European Cooperation in Science and Technology www.cost.eu) - STSM fellowships for Mirta Milić (IMROH, EU 19); IMROH, Zagreb, Croatia, Institute for Medical Research and Occupational Health (IMROH), Zagreb, Croatia, and the Ministry of Science, Education and Sports of the Republic of Croatia (Grant No. 022-0222148-2125) (EU4); Cancer Plan for PestiBG; Grant number: no ENV201401(EU 8, EU9); Italian Ministry of Education, University and Research PRIN 2005, prot. 2005058197 and Cariplo Foundation (Milan, Italy), Rif. Pratica 2007-5810 and Rif. Pratica 2010.2303 (EU 18); Associazione Italiana per la Ricerca sul Cancro (AIRC) (IG 2015/17564). (EU19); European Union Integrated Projects New Generis, 6th Framework Programme, Priority 5: Food Quality and Safety; Newborns and Genotoxic Exposure Risks, FOOD-CT-2005-016320 (EU22); ACT project No. 036APy/09 and No. 005DBB/12 (EU 24); FCT-SFRH/BPD/96196/2013, SFRH/BPD/100948/2014, Portugal (EU 26); MZ 2012/8-UKBA-8; VEGA 1/0703/13, APVV 15-0063 (EU30); Xunta de Galicia (XUGA 10605B98; INCITE08PXIB106155PR; ED481B2016/190-0; Grants ED431B2019/02), Spain (EU 32); Grant 01 173034, Ministry of Education, Science and Technological Development of the Republic of Serbia (EU 42); The Centre for Industrial and Technological Development within National Strategic Consortia for Techical Research (Industrial Research diets and food with specific characteristics for elderly, SENIFOOD); University of Navarra LE/97; Physiopathology of Obesity and Nutrition (CIBER Obn); Carlos III Health Research Institute (CB12/03/30002); Ministerio de Economia y Compatitividad (‘Ramón y Cajal’ Programme, RYC-2013-14370) of the Spanish Government for personal support (EU 45); the Ministry of Education, Youth and Sports of the Czech Republic project Healthy Aging in Industrial Environment HAIE (CZ.02.1.01/0.0/0.0/16_019/0000798) which is co-financed by the European Union (European Structural and Investment funds; Operation Programme Research, Development and Education); MYES LO 1508 (EU 46); MICRODIAB Study; ClinicalTrials.org (#NCT02231736) (EU 52); The study was funded by the Italian Ministry for Education, University and Scientific Research (MIUR) - Research No. 2005-062547 (EU14, EU53); Projects financed from Serbian Ministry of Education, Science and Technological Development #11146002, #175035, #173034 (EU 54); Mehr foundation organisation, UK (EU 55); MCTI/CNPQ No. 01/2016-Universal; FAPESC No. 09/2015; MEC/MCTI/CAPES/CNPQ/FAPS/ No. 09/2014, Brazil (CSA 6); the National Nuclear Energy Agency of Indonesia (Badan Tenaga Nuklir Nasional) with contract number 080.01.06 3447.001 001.052.A (AS4); Slovak Grant Agency (APVT-21 013202, APVT-21- 017704); Ministry of Health, Slovak Republic (2005/43-SZU-21, 2006/07- SZU-02 MZ SR, 2005/42-SZU-20

Differently substituted β-hydroxy- and β-amino dialkyl and alkyl-aryl tellurides and selenides have been prepared through ring-opening reactions of epoxides and aziridines with selenium- or tellurium-centered nucleophiles. The antioxidant properties and the cytotoxicity of such compounds have been investigated on normal human dermal fibroblasts. Most of the studied compounds exhibited a low cytotoxicity and a number of them proved to be non-toxic, not showing any effect on cell viability even at the highest concentration used (100 μM). The obtained results showed a significant antioxidant potential of the selected organotellurium compounds, particularly evident under conditions of exogenously induced oxidative stress. The antioxidant activity of selenium-containing analogues of active tellurides has also been evaluated on cells, highlighting that the replacement of Se with Te brought about a significant increase in the peroxidase activity.

The comet assay is a well-accepted biomonitoring tool to examine the effect of dietary, lifestyle, environmental and occupational exposure on levels of DNA damage in human cells. With such a wide range of determinants for DNA damage levels, it becomes challenging to deal with confounding and certain factors are inter-related (e.g. poor nutritional intake may correlate with smoking status). This review describes the effect of intrinsic (i.e. sex, age, tobacco smoking, occupational exposure and obesity) and extrinsic (season, environmental exposures, diet, physical activity and alcohol consumption) factors on the level of DNA damage measured by the standard or enzyme-modified comet assay. Although each factor influences at least one comet assay endpoint, the collective evidence does not indicate single factors have a large impact. Thus, controlling for confounding may be necessary in a biomonitoring study, but none of the factors is strong enough to be regarded a priori as a confounder. Controlling for confounding in the comet assay requires a case-by-case approach. Inter-laboratory variation in levels of DNA damage and to some extent also reproducibility in biomonitoring studies are issues that have haunted the users of the comet assay for years. Procedures to collect specimens, and their storage, are not standardized. Likewise, statistical issues related to both sample-size calculation (before sampling of specimens) and statistical analysis of the results vary between studies. This review gives guidance to statistical analysis of the typically complex exposure, co-variate, and effect relationships in human biomonitoring studies.

Chemoresistance is the primary cause of chemotherapy failure. Compelling evidence shows that micro RNAs (miRNAs) contribute to reprogram cancer cells toward a resistant phenotype. We investigate the role of miRNAs in the response to acute treatment with 5-FU in colon cancer-resistant cells. We performed a global gene expression profile for the entire miRNA genome and found a change in the expression of four miRNAs following acute treatment with 5-FU. Among them, we focused on miR-210-3p, previously described as a key regulator of DNA damage repair mechanisms and mitochondrial metabolism. We show that miR-210-3p downregulation enables resistant cells to counteract the toxic effect of the drug increasing the expression of RAD-52 protein, responsible for DNA damage repair. Moreover, miR-210-3p downregulation enhances oxidative phosphorylation (OXPHOS), increasing the expression levels of succinate dehydrogenase subunits D, decreasing intracellular succinate levels and inhibiting HIF-1α expression. Altogether, these adaptations lead to increased cells survival following drug exposure. These evidence suggest that miR-210-3p downregulation following 5-FU sustains DNA damage repair and metabolic adaptation to counteract drug treatment.

Background Genetic polymorphisms in genes involved in pain modulation have been reported to be associated to opioid efficacy and safety in different clinical settings. Methods The association between COMT Val158Met polymorphism (rs4680) and the inter-individual differences in the response to opioid analgesic therapy was investigated in a cohort of 87 Italian paediatric patients receiving opioids for cancer pain (STOP Pain study). Furthermore, a systematic review of the association between opioid response in cancer patients and the COMT polymorphism was performed in accordance with the Cochrane Handbook and the Prisma Statement. Results In the 87 paediatric patients, pain intensity (total time needed to reach the lowest possible level) was significantly higher for G/G than A/G and A/A carriers (p-value = 0.042). In the 60 patients treated only with morphine, the mean of total dose to reach the same pain intensity was significantly higher for G/G than A/G and A/A carriers (p-value = 0.010). Systematic review identified five studies on adults, reporting that opioid dose (mg after 24 h of treatment from the first pain measurement) was higher for G/G compared to A/G and A/A carriers. Conclusions Present research suggests that the A allele in COMT polymorphism could be a marker of opioid sensitivity in paediatric cancer patients (STOP Pain), as well as in adults (Systematic Review), indicating that the polymorphism impact could be not age-dependent in the cancer pain context. Trial registration Registration number: CRD42017057831. Electronic supplementary material The online version of this article (10.1186/s12885-019-5310-4) contains supplementary material, which is available to authorized users.

Background Convincing evidence suggests that the risk of colorectal cancer (CRC) is increased by the typical Western diet characterized by high consumption of red and processed meat. In addition, some epidemiological studies suggest a reduction in the risk of CRC associated with fish consumption. The role of the gut microbiome in this diet-associated risk is not well understood. Methods/design This is a randomized parallel open clinical trial comprising a total of 150 clinically healthy subjects randomly assigned to three groups: a meat-based diet of which 4 portions per week are red meat (1 portion = 150 g), 3 portions per week are processed meat (1 portion = 50 g), and 1 portion per week is poultry (1 portion = 150 g), for a total amount of 900 g per week of meat and derivatives; a meat-based diet supplemented with alpha-tocopherol; and a pesco-vegetarian diet excluding fresh and processed meat and poultry, but which includes 3 portions per week of fish for a total amount of 450 g per week. Each intervention will last 3 months. The three diets will be isocaloric and of three different sizes according to specific energy requirements. Anthropometric measurements, body composition, and blood and fecal samples will be obtained from each participant at the beginning and end of each intervention phase. The measure of the primary outcome will be the change from baseline in DNA damage induced by fecal water using the comet assay in a cellular model. Secondary outcome measures will be changes in the profile of fecal microbiomes, global fecal and urinary peroxidation markers, and neoplastic biomarkers. Discussion Although epidemiological data support the promoting role of meat and the possible protective role of fish in colon carcinogenesis, no study has directly compared dietary profiles characterized by the presence of these two food groups and the role of the gut microbiome in these diet-associated CRC risks. This study will test the effect of these dietary profiles on validated CRC risk biomarkers. Trial registration ClinicalTrials.gov, NCT03416777. Registered on 3 May 2018.

Intake of folate (vitamin B9) is strongly inversely linked with human cancer risk, particularly colon cancer. In general, people with the highest dietary intake of folate or with high blood folate levels are at a reduced risk (approx. 25%) of developing colon cancer. Folate acts in normal cellular metabolism to maintain genomic stability through the provision of nucleotides for DNA replication and DNA repair and by regulating DNA methylation and gene expression. Folate deficiency can accelerate carcinogenesis by inducing misincorporation of uracil into DNA, by increasing DNA strand breakage, by inhibiting DNA base excision repair capacity and by inducing DNA hypomethylation and consequently aberrant gene and protein expression. Conversely, increasing folate intake may improve genomic stability. This review describes key applications of single cell gel electrophoresis (the comet assay) in assessing genomic instability (misincorporated uracil, DNA single strand breakage and DNA repair capacity) in response to folate status (deficient or supplemented) in human cells in vitro, in rodent models and in human case-control and intervention studies. It highlights an adaptation of the SCGE comet assay for measuring genome-wide and gene-specific DNA methylation in human cells and colon tissue.

Background To describe frequency, preventability and seriousness of adverse drug reactions (ADRs) in children as cause of emergency department (ED) admission and to evaluate the association between specific factors and the reporting of ADRs. Methods A retrospective analysis based on reports of suspected ADRs collected between January 1st, 2012 and December 31st, 2016 in the ED of Meyer Children’s Hospital (Italy). Demographics, clinical status, suspected drugs, ADR description, and its degree of seriousness were collected. Logistic regression was used to estimate the reporting odds ratios (RORs) with 95% confidence intervals (CIs) of potential predictors of ADR seriousness. Results Within 5 years, we observed 834 ADRs (1100 drug-ADR pairs), of whom 239 were serious; of them, 224 led to hospitalization. Patients were mostly treated with one drug. Among patients treated with more than one drug, 78 ADRs presented a potential interaction. The most frequently reported ADRs involved gastrointestinal system. The most frequently reported medication class was antinfectives. Risk of serious ADR was significantly lower in children and infants compared to adolescents (ROR 0.41 [95% CI: 0.27–0.61] and 0.47 [0.32–0.71], respectively), and it was significantly increased in subjects exposed to more than one drug (ROR 1.87 [1.33–2.62] and 3.01 [2.07–4.37] for subjects exposed to 2 and 3 or more drugs, respectively). Gender, interactions and off-label drug use did not influence the risk of serious ADRs. Conclusion Active surveillance in pharmacovigilance might represent the best strategy to estimate and characterize the clinical burden of ADRs in children. Electronic supplementary material The online version of this article (10.1186/s40360-018-0207-4) contains supplementary material, which is available to authorized users.

Senescent cells are characterized by an increased secretion of inflammatory and growth factors, known as the "senescence-associated secretory phenotype" (SASP), producing a pro-tumoral and pro-angiogenic microenvironment. This work proposes chronic resveratrol treatment (5 µM for 5 weeks, termed R5) of senescent MRC5 fibroblasts as a mean to mimic and target the angiogenic trait of stromal fibroblast SASP. Senescent fibroblast conditioned medium (CM sen) was effective in enhancing the angiogenic properties of endothelial colony-forming cells (ECFCs), that is, invasive activity and capillary morphogenesis capability in vitro, that were significantly reduced when conditioned media were collected after resveratrol pretreatment (CM senR5). The attenuation of ECFC angiogenic phenotype induced by CM senR5 was accompanied by reduced protein levels of epidermal growth factor and urokinase plasminogen activator receptors (EGFR, uPAR), and by a related decreased activation of receptor-tyrosine-kinase signaling pathways. IL8 levels were found reduced in CM senR5 compared to CM sen, with the associated reduction of IL8-CXCR2 binding in ECFCs. IL8-subtraction mitigated the pro-angiogenic features of CM sen and the associated intracellular signaling in ECFCs, indicating a prominent role of IL8 in the pro-angiogenic effects of CM sen. IL8 modulation is an important mechanism underlying the antiangiogenic activity of resveratrol on MRC5 SASP.

The pharmacological modulation of collagen turnover is a strategy potentially useful in different skin conditions. The serine protease inhibitor Serpin A1 and portions of its C-terminal region have been investigated as collagen modulators. To clarify the mechanisms by which the C-terminal 409-418 peptide SA1-III increases extracellular type I collagen levels, to compare its activities range with that of the originator molecule Serpin A1, and to evaluate its efficacy in primary cultures from adult and aged human subjects. The different forms of type I collagen were analyzed by means of western blot in cell lysates and cell-conditioned media of primary human dermal fibroblasts obtained from subjects of different ages. Gelatin zymography was used to investigate the degrading enzymes. Cell viability and in vitro wound healing tests were used to evaluate cell proliferation. The SA1-III peptide increased extracellular collagen levels by reducing degradation, with no effect on cellular biosynthesis or cell proliferation mechanisms. A reduced level of MMP-2 and MMP-9 was also found in cell media upon peptide treatment. No peptide effect was detected on inflammatory mediators gene expression in resting and LPS-stimulated fibroblasts, or in the wound healing test. The SA1-III peptide is a good collagen modulator candidate, protecting collagen against degradation without detectable actions on biosynthesis, acting at reasonably low concentrations, and non-interfering with cell proliferation. It is effective in primary fibroblasts from young and aged subjects. These effects can prove useful in pathological and physiological skin conditions in which collagen degradation is excessive compared to the synthetic capacity.

Senescent cells display an increase in the secretion of growth factors, inflammatory cytokines and proteolytic enzymes, termed the “senescence-associated-secretory-phenotype” (SASP), playing a major role in many age-related diseases. The phenolic compounds present in extra-virgin olive oil are inhibitors of oxidative damage and have been reported to play a protective role in inflammation-related diseases. Particularly, hydroxytyrosol and oleuropein are the most abundant and more extensively studied. Pre-senescent human lung (MRC5) and neonatal human dermal (NHDF) fibroblasts were used as cellular model to evaluate the effect of chronic (4–6 weeks) treatment with 1 μM hydroxytyrosol (HT) or 10 μM oleuropein aglycone (OLE) on senescence/inflammation markers. Both phenols were effective in reducing β-galactosidase-positive cell number and p16 protein expression. In addition, senescence/inflammation markers such as IL-6 and metalloprotease secretion, and Ciclooxigenase type 2 (COX-2) and α-smooth-actin levels were reduced by phenol treatments. In NHDF, COX-2 expression, Nuclear Factor κ-light-chain-enhancer of activated B cells (NFκB) protein level and nuclear localization were augmented with culture senescence and decreased by OLE and HT treatment. Furthermore, the inflammatory effect of Tumor Necrosis Factor α (TNFα) exposure was almost completely abolished in OLE- and HT-pre-treated NHDF. Thus, the modulation of the senescence-associated inflammatory phenotype might be an important mechanism underlying the beneficial effects of olive oil phenols.

Cellular senescence is related to organismal aging and is observed after DNA damaging cancer therapies, that induce tumor-suppressive modifications, but it is characterized by a strong increase in secreted factors, termed the "senescence-associated secretory phenotype" (SASP). Particularly, SASP from stroma senescent fibroblasts creates a cancer-favoring microenvironment, providing targets for anti-cancer interventions. In the present article, chronic treatment (5 weeks) with 5 µM resveratrol has been used to modulate senescence-related protumoral features of MRC5 fibroblasts, reducing SASP-related interleukins IL1α, IL1β, IL6, and IL8; transforming-growth-factor-β (TGFβ); matrix metallo-proteinases MMP3 and MMP2; urokinase plasminogen activator (uPA); receptor proteins uPAR, IL6R, insulin growth factor receptor-1 (IGF-1R), TGFβ-R2, and CXCR4. The cellular nuclear-factor-kB (NF-kB) protein level was also reduced, confirming its role in the induction of SASP. Resveratrol pretreated MRC5 fibroblasts were resistant to activation by TGFβ. Resveratrol treatment of senescent MRC5 induced the production of conditioned media (CM) which counteracted the protumoral effect of senescent CM on A375 and A375-M6 melanoma cell proliferation and invasiveness, and reduced the expression of epithelial-to-mesenchymal transition markers related to malignant features. This experimental approach proposes a treatment that targets the senescent stromal cell phenotype to induce an anti-tumor hosting microenvironment, which is suitable for both preventive and therapeutic purposes.

There are no clinical tools to functionally assess degree of DNA damage in breast cancer. The comet assay is an accepted research tool for assessing DNA damage, however, most cancer studies have assessed lymphocytes as surrogate cells. The aim of this pilot study was to use the comet assay in early breast cancer directly in tumor tissue to compare DNA damage between and within traditionally defined subgroups, and to explore intra-tumoral heterogeneity. Scrapings of tumor and healthy breast tissue were obtained at primary surgery from 104 women. Comet assay was applied to quantitatively assess DNA damage, revealing substantial inter- and intra-subgroup variation. Marked intra-tumoral heterogeneity was evident across all subgroups. The degree of DNA damage for an individual could not be predicted by breast cancer subgroup. Comet assay warrants further study as a potential clinical tool for identification of tumoral DNA damage and ultimately, individualised use of DNA damaging therapy.

The modulation of collagen turnover can be a relevant pharmacological target in the context of treating either pathological or pathophysiological conditions, such as collagen-related diseases and skin aging. Our recent work has focused on the search for short-chain peptides as lead compounds for further development of compounds that enhance the production of type I collagen. In this study we selected and synthesized overlapping peptides of the C-terminal portion of serpin A1 (residues 393-418), the impact of which on collagen production has been reported previously, in order to identify shorter and still active fragments and to provide insight on the mechanisms involved. The biological activity of each fragment was evaluated with cultured normal human dermal fibroblasts, and changes in the amounts of collagen were monitored in collected culture media by a sandwich ELISA technique developed in house. Interestingly, we identified a decapeptide, termed SA1-III (Ac-MGKVVNPTQK-NH2 ), as a promising candidate for our purposes; it is able to induce a significant increase in type I collagen levels in the culture medium of treated cells at micromolar concentrations.

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.

Recent research has focused on natural compounds possibly endowed with antiaging effects. Resveratrol is a stilbene compound produced by different plants with many biologic activities, including an antiaging effect, which has been demonstrated both in vitro in eukaryotic cells and in vivo in mice. We studied the effect of resveratrol on cultured human MRC5 fibroblasts, a widely used in vitro model in aging studies. The chronic treatment of MRC5 cells until senescence with 5 μM resveratrol induced a small increase in the total number of replications completed by the cultures at senescence, showed protective effects against DNA oxidative damage, and reduced senescence-associated increases in nuclear size and DNA content. A reduction in the levels of acetylated forms of H3 and H4 histones and p53 protein was also found.

cope To investigate the effect of pomegranate mesocarp, a polyphenol-rich by-product of juice production, in colorectal cancer (CRC) chemoprevention. Methods and results A mesocarp decoction (PMD) was administered for 6 weeks in the diet to Pirc rats, mutated in Apc, a key-gene in CRC. Mucin Depleted Foci (MDFs), as CRC biomarkers, were reduced in PMD-fed rats compared to Controls (MDF/colon: 34±4 vs 47±3, P = 0.02). Apoptosis in MDFs from PMD-treated rats was increased compared to Controls (2.5±0.2 vs 1.6±0.2, P

In 71 adults residing in Florence, Italy, enrolled in a prospective study, we investigated the correlation between individual levels of oxidative DNA damage detected by the Comet assay in circulating lymphocytes, and a specific ozone exposure score calculated in 10 different time-windows (0-5 to 0-90 days) before blood drawing, based on daily measurements provided by the local environmental monitoring system. Overall, statistically significant positive correlations between average ozone concentrations and DNA damage emerged in almost all time-windows considered; correlations were more evident among males, non-smokers, and traffic-exposed workers. Multivariate regression analyses taking into account selected individual characteristics, showed an independent effect on DNA damage of average ozone concentrations in the last 60-90 days before blood drawing. Local residents showed a divergent pattern with correlations restricted to shorter time-windows. Our results suggest that ozone concentrations at ground levels modulate oxidative DNA damage in circulating lymphocytes of residents of polluted areas.

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 Menard, low and high in polyphenols, respectively) to HLA-B27 transgenic rats which develop spontaneous IBD. After 3 months feeding, rats fed Marie Menard 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 Menard 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 Menard-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 Menard 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.

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

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.

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.

The comet assay methodology was used to monitor nuclear changes occurring in MRC5 human fibroblasts during transition from young to senescent cultures and to study heterogeneity of senescent populations. Nuclear morphology and size, DNA content per nucleus, and DNA damage (basal strand break, total damage, and oxidized base levels) were evaluated; moreover, visually identified large and small nuclei were analyzed separately and arranged in classes of increasing DNA damage. Oxidized base levels were definitely lower in young versus senescent fibroblasts of which, however, a significant proportion showed negligible DNA damage. Nuclear size enlargement accompanying senescence was almost equally influenced by cell ploidy increase and also by a chromatin decondensation process involving diploid cells. It is noteworthy that DNA damage in senescent fibroblasts correlated significantly to nuclear size, but not to DNA content. The comet assay allowed us to identify different senescent phenotypes and to investigate changes in nuclear features and/or DNA damage irrespective of time elapsed in culture.

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.

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.

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve problems in the measurement of DNA oxidation that have resulted in varying estimates of the extent of this damage in humans. HeLa cells, sent to members for analysis, were either untreated, or treated with light in the presence of a photosensitizer to induce different amounts of 8-oxo-7,8-dihydroguanine (8-oxoGua) in DNA. Laboratories employing HPLC with electrochemical detection were able to measure the induced damage with similar efficiency; dose response gradients for seven of the eight sets of results were almost identical. GC-MS and HPLC-MS/MS, employed in three laboratories, did not convincingly detect the dose response. An alternative approach to measuring base oxidation employs the enzyme formamidopyrimidine DNA N-glycosylase (FPG) to convert 8-oxoGua to strand breaks, which are then measured by alkaline unwinding, alkaline elution, or the comet assay. Ten laboratories used this approach; five were able to detect the dose response in cells treated with photosensitizer plus light (at lower doses than for chromatographic methods, because the enzymic methods are more sensitive and less prone to spurious oxidation). Median values for 8-oxoGua (or FPG-sensitive sites) in untreated cells were 4.01 per 10(6) guanines for chromatographic methods, and 0.53 per 10(6) guanines for techniques based on FPG. (C) 2003 Elsevier Inc.

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up to resolve the problems associated with the measurement of background levels of oxidative DNA damage (in particular 8-oxo-7,8-dihydroguanine, or 8-oxoGua) in human cells. A tendency for DNA oxidation to occur during sample preparation prior to chromatography has been recognized as the source of a very substantial artefact. To assess the success of attempts to eliminate the artefact, ESCODD has distributed to its members standard samples of pig liver and HeLa cells for analysis. Estimates of 8-oxoGua in pig liver, using chromatographic techniques, ranged from 2.23 to 441 per 10(6) guanines, with a median of 10.47 per 10(6) guanines. Chromatographic analysis of HeLa cell DNA gave a range of 1.84 to 214 per 10(6) guanines with a median of 5.23 per 10(6) guanines. HeLa cell DNA was also analysed by an enzymic approach, in which whole cell DNA was treated with formamidopyrimidine DNA glycosylase, which nicks DNA at sites of 8-oxoGua, and the breaks measured with the comet assay, alkaline unwinding or alkaline elution. Values with these methods ranged from 0.06 to 4.988-oxoGua per 10(6) guanines, with a median of 0.79 per 10(6) guanines. Although there are clearly still serious discrepancies between methods and laboratories, the lowest estimates by chromatography (arguably those in which the artefact was best controlled) are only 2.5 times higher than the median value obtained with the enzymic approach.

The aim of this work was to evaluate the effect of stilbenes from different cultivars of Vitis vinifera on tumour proliferation. Extracts were obtained from elicited V. vinifera cell cultures and characterised by HPLC/DAD/MS. Cell growth was evaluated in four human cancer cell lines and in normal human fibroblasts. The cells were exposed to the extracts or to trans-resveratrol, used as reference molecule, for 48 h, at 1–10 μM concentrations of total stilbenoids. All the extracts exhibited antiproliferative activity, mediated by modulation of the cell cycle and induction of cytotoxicity in cancer but not in normal cell lines, and positively correlated with the content in dimeric stilbenoids. The Alphonse Lavallée extract was the most active, and the obtained stilbenoid fraction resulted 8–10 times more active than trans-resveratrol. Extracts from V. vinifera cell cultures could represent new sources of active stilbenoid compounds to be further assayed in in vivo studies for their antitumoural properties.

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.

International audience; 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. 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 ros, 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. 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. More intervention studies are needed to provide further evidence of the health-protective effects of wine proanthocyanidins.

The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo-or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.

Extra-virgin olive oils (EVOO), high in phenolic compounds with antioxidant properties, could be partly responsible for the lower mortality and incidence of cancer and CVD in the Mediterranean region. The present study aims to measure oxidative DNA damage in healthy human subjects consuming olive oils with different concentrations of natural phenols. A randomised cross-over trial of high-phenol EVOO (high-EVOO; 592mg total phenols/kg)Vlow-phenol EVOO (low-EVOO; 147mg/kg) was conducted in ten postmenopausal women in Florence. Subjects were asked to substitute all types of fat and oils habitually consumed with the study oil (50g/d) for 8 weeks in each period. Oxidative DNA damage was measured by the comet assay in peripheral blood lymphocytes, collected at each visit during the study period. Urine samples over 24h were collected to measure the excretion of the olive oil phenols. The average of the four measurements of oxidative DNA damage during treatment with high-EVOO was 30% lower than the average during the low-EVOO treatment (p=0·02). Urinary excretion of hydroxytyrosol and its metabolite homovanillyl alcohol were significantly increased in subjects consuming high-EVOO. Despite the small sample size, the present study showed a reduction of DNA damage by consumption of an EVOO rich in phenols, particularly hydroxytyrosol.

We studied the effects of extremely low frequency (50 Hz) (ELF) electromagnetic fields (EMF) on peripheral human blood lymphocytes and DBY747 Saccharomyces cerevisiae strain as models of eukaryotic cells. Graded exposure to 50 Hz magnetic flux density (MFD) was obtained with a Helmholtz coil system set at 1, 10 or 100 µT for 18 h. EMF effects on DNA were studied with the single gel cell electrophoresis assay (comet assay) on lymphocytes. Gene expression profiles of EMF-exposed human and yeast cells were evaluated with DNA microarrays containing 13,971 and 6,212 oligonucleotides, respectively. After exposure to EMF we did not observe an increase in the amount of strand breaks or DNA oxidated bases relative to controls, or variation in gene expression profiles. The results suggest that ELF-EMF do not induce DNA damage nor affect gene expression in these two different eukaryotic cell systems.

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.

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.

Lung macrophages may play a relevant role in oxidative processes producing both superoxide anion (O(2)(-)) and NO. In this view, an antioxidant therapy can be useful in the treatment of systemic sclerosis (SSc) patients. N-Acetylcysteine (NAC) is able to expand natural antioxidant defenses by increasing intracellular gluthatione concentration and it has been proposed as an antioxidant therapy in respiratory distress syndromes. The aim of our study was to determine whether lung macrophages obtained from SSc patient bronchoalveolar lavage (BAL) express the inducible form of nitric oxide synthase (iNOS) and whether NAC can reduce the peroxynitrite (ONOO(-)) and O(2)(-) production of these cells. Alveolar macrophages were isolated from BAL of 32 patients and used for the immunocytochemical determination of iNOS, and the production of ONOO(-) and O(2)(-) was measured by fluorimetric or spectrophotometric methods, respectively. Lung macrophages obtained from SSc patients expressed a higher level of iNOS compared to healthy subject cells. NAC preincubation (5 x 10(-5)M, 24h) significantly reduced (-21%) the ONOO(-) production in formyl Met-Leu-Phe (fMLP)-activated cells and slightly reduced it under resting conditions, whereas NAC preincubation was unable to modify the release of O(2)(-) both in basal condition and in fMLP-stimulated cells. We conclude that since SSc lung macrophages express high levels of iNOS and produce a significant quantity of ONOO(-), NAC administration reduces ONOO(-) production and can be an useful treatment to alleviate SSc symptoms.

Polyphenols from tea and other beverages such as red wine have been regarded with interest as possible chemopreventive agents against cancer. Here we report that red wine polyphenols (50 mg/kg) administered with the diet to F344 rats for 16 weeks inhibited colon carcinogenesis induced by azoxymethane (AOM, 7.4 mg/kg, total dose 74 mg/kg) or dimethylhydrazine (DMH, 30 mg/kg, total dose, 300 mg/kg). Polyphenol-treated animals had a consistently lower tumour yield compared to controls. In polyphenol-treated rats, the main bacterial strains in the faeces at sacrifice were Bacteroides, Lactobacillus and Bifidobacterium spp., whereas microorganisms predominantly identified in control-fed rats were Bacteroides, Clostridium and Propionibacterium spp. Wine polyphenols (57 mg/kg for 10 days, by gavage), administered to rats not treated with carcinogens, produced a significant decrease in the basal level of DNA oxidative damage of the colon mucosa as measured with the comet assay (average pyrimidine oxidation was reduced by 62% and purine oxidation by 57%, p

In order to evaluate dietary and lifestyle determinants of oxidative DNA damage we used a modification of the 'comet assay' (single cell alkaline gel electrophoresis), with the fpg enzyme (formamidopyrimidine DNA glycosilase), to measure the basal level of DNA oxidation in peripheral lymphocytes donated by 71 healthy adults living in Florence, Italy. Detailed information about dietary and lifestyle habits was collected by two validated and standardized questionnaires; we also measured plasma concentrations of selected micro-nutrients (six carotenoids, retinol, alpha- and gamma-tocopherol). DNA damage, measured as percent DNA migrated in the comet tail (mean 4.67%, interquartile range 2.36-6.62%), was not associated with gender, age, weight, body mass index, physical activity or smoking history. A positive correlation with height and period of blood sampling emerged: DNA damage tended to be higher among taller subjects (P = 0.02) and in samples obtained in summer months (P = 0.02). Multivariate analyses showed a positive association with coffee (P = 0.01) and tomato consumption (P = 0.05). Instead, the consumption of cruciferous vegetables tended to be negatively associated with oxidative damage (P = 0.09). Furthermore, a positive non-significant association between the consumption of total vegetables and fresh fruit and DNA damage emerged (P = 0.08 and P = 0.10, respectively). The estimated intake of simple sugars showed a strong positive association with oxidative DNA damage (P = 0.01), while vitamin E showed a borderline positive association (P = 0.06). The plasma levels of several micro-nutrients did not appear to influence DNA damage. Our results, although based on a relatively small group of subjects, indicate that individual dietary and lifestyle habits only modestly affect the levels of lymphocyte DNA oxidation and suggest that specific dietary patterns, rich in fresh fruit and vegetables, are not clearly related to decreased oxidative damage in peripheral lymphocytes in a Mediterranean population.