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Background: DNA copy number alterations (CNAs) and gene expression changes have amply been encountered in colorectal cancers (CRCs), but the extent at which CNAs affect gene expression, as well as their relevance for tumor development, are still poorly defined. Here we aimed at assessing the clinical relevance of these parameters in a 10 year follow-up study. Methods: Tumors and normal adjacent colon mucosa, obtained at primary surgery from 21 CRC patients, were subjected to (i) high-resolution array CGH (a-CGH) for the detection of CNAs and (ii) microarray-based transcriptome profiling for the detection of gene expression (GE) changes. Correlations between these genomic and transcriptomic changes and their associations with clinical and histopathological parameters were assessed with the aim to identify molecular signatures associated with disease-free survival of the CRC patients during a 10 year follow-up. Results: DNA copy number gains were frequently detected in chromosomes 7, 8q, 13, 19, 20q and X, whereas DNA copy number losses were frequently detected in chromosomes 1p, 4, 8p, 15, 17p, 18, 19 and 22q. None of these alterations were observed in all samples. In addition, we found that 2,498 genes were up- and that 1,094 genes were down-regulated in the tumor samples compared to their corresponding normal mucosa (p < 0.01). The expression of 65 genes was found to be significantly associated with prognosis (p < 0.01). Specifically, we found that up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, were strongly associated with a poor survival. Subsequent integrated analyses revealed that increased expression levels of the MMP9, BMP7, UBE2C, I-CAM, NOTCH3, NOTCH1, PTGES2, HMGB1 and ERBB3 genes were associated with copy number gains, whereas decreased expression levels of the MUC1, E2F2, HRAS and SIRT3 genes were associated with copy number losses. Pathways related to cell cycle progression, eicosanoid metabolism, and TGF-? and apoptosis signaling, were found to be most significantly affected. Conclusions: Our results suggest that CNAs in CRC tumor tissues are associated with concomitant changes in the expression of cancer-related genes. In other genes epigenetic mechanism may be at work. Up-regulation of the IL17RA, IGF2BP2 and ABCC2 genes, and of genes acting in the mTOR and cytokine receptor pathways, appear to be associated with a poor survival. These alterations may, in addition to Dukes' staging, be employed as new prognostic biomarkers for the prediction of clinical outcome in CRC patients.

Our purpose was to evaluate long-term neuroglial cocultures as a model for investigating senescence in the nervous system and to assess its similarities with in vivo models. To this aim, we maintained the cultures from 15 days in vitro (mature cultures) up to 27 days in vitro (senescent cultures), measuring senescence-associated, neuronal, dendritic, and astrocytic markers. Whole microRNA expression profiles were compared with those measured in the cortex of 18- and 24-month-old C57Bl/6J aged mice and of transgenic TgCRND8 mice, a model of amyloid-β deposition. Neuroglial cocultures displayed features of cellular senescence (increased senescence-associated-β-galactosidase activity, oxidative stress, γ-H2AX expression, IL-6 production, astrogliosis) that were concentration dependently counteracted by the antiaging compound resveratrol (1-5 µM). Among the 1,080 microRNAs analyzed, 335 were downregulated or absent in 27 compared with 15 days in vitro and resveratrol reversed this effect. A substantial overlapping was found between age-associated changes in microRNA expression profiles in vitro and in TgCRND8 mice but not in physiologically aged mice, indicating that this culture model displays more similarities with pathological than physiological brain aging. Our results demonstrate that neuroglial cocultures aged in vitro can be useful for investigating the cellular and molecular mechanisms of brain aging and for preliminary testing of protective compounds.

Background Diluted preparations obtained from Apis mellifica are reported in the homeopathic literature to have anti-inflammatory activity. The present study was designed to explore the effects on global gene expression profiles of human cells by means of microarrays, using Apis mellifica mother tincture (TM) and its 3C, 5C, 7C dynamized dilutions; the technique employed allowed us to study the changes in gene expression at concentrations much lower than those associated with pharmacological responses. Methods An RWPE-1 cell line (human immortalized prostate epithelial cells) was used to study the effects on global gene expression by transcriptomic analysis. Results Apis mellifica TM and its 3C, 5C, 7C dynamized dilutions modulated hundreds of genes; using cluster analysis we observed groups of genes up- or down-regulated with similar expression profiles among treatments; other genes showed opposite regulation profiles at low and high dilutions of Apis mellifica, suggesting a hormetic response. In particular, genes involved in cytokine expression, inflammatory processes, anti-oxidative responses and proteasome degradation were differentially, and sometimes divergently expressed by the TM or by Apis mellifica 3C, 5C and 7C dilutions. We confirmed these data by RT-PCR analyses on 5 selected candidate genes (IL1β, CD46, ATF1, UBE2Q2 and MT1X). Conclusions Apis mellifica TM modifies gene expression in human cells and has inhibitory effects on regulatory processes of inflammation; in addition, extremely diluted dynamized dilutions (3C, 5C and 7C) still exert significant effects on genes involved in inflammation and oxidative stress.

Gene expression analysis has been employed in the past to test the effects of high dilutions on cell systems. However, most of the previous studies were restricted to the investigation of few dilutions, making it difficult to explore underlying mechanisms of action. Using whole-genome transcriptomic analysis, we investigated the effects of a wide range of Apis mellifica dilutions on gene expression profiles of human cells. RWPE-1 cells, a nonneoplastic adult human epithelial prostate cell line, were exposed to Apis mellifica preparations (3C, 5C, 7C, 9C, 12C, 15C, and 30C) or to the reference solvent solutions for 24 hours; nonexposed cells were also checked for gene expression variations. Our results showed that even the most diluted solutions retained the ability to trigger significant variations in gene expression. Gene pathway analysis revealed consistent variations in gene expression induced by Apis mellifica when compared to nonexposed reference cells but not to reference solvent solutions. Since the effects of Apis Mellifica at extreme dilutions did not show dose–effect relationships, the biological or functional interpretation of these results remains uncertain.

Inflammation may increase cancer risk, therefore, we studied whether polyphenol-rich Marie Ménard (MM) apples with reported anti-inflammatory activity prevent 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats and, likewise whether high-fat (HF) diet promoting carcinogenesis, may affect inflammation. DMH-induced rats were fed for 15 weeks with: an HF diet (23% corn oil w/w); an HF diet containing 7.6% w/w lyophilized MM (apple diet (AD)); a low-fat (LF) diet and an HF diet containing piroxicam (PXC) (0.01% w/w) as control. Mucin depleted foci (MDF), precancerous lesions in the colon, were dramatically reduced in the AD, LF, and PXC groups compared with the HF. Peritoneal macrophage activation, an index of systemic inflammation, was significantly decreased in the AD, LF, and PXC groups. TNF-α, iNOS, IL-1β, IL-6 m-RNA expression in the colon, as well as CD68 cells and plasmatic PGE2 were lower in the AD, but not in the LF group. Apoptosis in the MDF of both the AD and LF-fed rats was significantly higher than in HF rats. In conclusion, AD has a strong chemopreventive effect, reducing inflammation, and increasing apoptosis, while the chemopreventive effect of the LF diet seems mediated mainly by increased apoptosis in MDF.

Experimental evidence indicates a strong connection between oxidative damage, cancer, and aging. Epidemiological observations suggest that a diet rich in fruits and vegetables is associated with lower incidence of some cancers and longer life expectancy; since fruits and vegetables contain natural antioxidants, a considerable effort has been dedicated to understanding their effects in experimental studies and in human trials.A: Effects of antioxidant-containing food and supplements on oxidation damage in humans. Intervention trials employing a variety of biomarkers have shown either a slight decrease in oxidation damage or no effect. B: Effects of selected antioxidants on mortality and cancer incidence. β-carotene and α-tocopherol, alone or in combination, increase cardiovascular and all-cause mortality or have no effect. In some studies, β-carotene and retinyl palmitate significantly increase the progression of lung cancer and aggressive prostate cancer. Protection against cardiovascular mortality or no effect of vitamin E has been reported, with an increase of all-cause mortality at dosages greater than 150 IU/day. Selenium showed beneficial effects on gastrointestinal cancer and reduced the risk of lung cancer in populations with lower selenium status. For multivitamin and mineral supplementation, no significant reduction of mortality or cancer incidence was observed, but some reports indicate a possible preventive effect in cervical cancer.The majority of supplementation studies indicate no variation of general mortality and of cancer incidence or a detrimental effect on both. Antioxidant supplements so far tested seem to offer no improvement over a well-balanced diet, possibly because of the choice of the substances tested or of an excessive dosage. However, new natural or synthetic compounds effective in vitro and in experimental studies might still be worth investigating in human trials.

To study the early alterations in carcinogenesis, we determined apoptosis and proliferation in rat mucin depleted foci (MDF), precancerous lesions in the colon under basal conditions and 24 h after treatment with 1,2-dimethylhydrazine (DMH), which induces apoptosis in the colon. Spontaneous apoptosis in MDF was higher than in normal mucosa (Apoptotic Index was 1.61 ± 0.30 and 0.21 ± 0.02 in MDF and normal mucosa, respectively, mean ± SE, p < 0.05). DMH (30 and 75 mg/kg) increased apoptosis in both normal mucosa and MDF (up to 20 times higher compared to basal levels in normal mucosa, but only two times in MDF). MDF had a higher and deregulated pattern of proliferation along the crypt compared to normal mucosa. After DMH, proliferation in normal mucosa was significantly depressed, but it did not vary in MDF. Survivin-Birc5 regulating apoptosis and proliferation was significantly over-expressed (RT-qPCR and immunohistochemistry experiments) in MDF vs. normal mucosa, but did not vary in response to DMH. The expression of the pro-apoptotic protein Bak did not vary in normal mucosa and MDF. Since inflammation is present in MDF, which may hamper apoptosis, we studied the effect of pre-treatment with aspirin (600 ppm in the diet for 10 days). No significant effects of aspirin were observed. In conclusion, MDF had a higher spontaneous apoptosis and proliferation coupled with a reduced response to apoptotic stimuli from cytotoxic compounds. Survivin over-expression in MDF indicates that this is an early event in colon carcinogenesis and suggests that down-regulation of Survivin may represent a strategy for cancer prevention.

Prenatal exposure to toxicants, such as maternal smoking, may impair cardiovascular autonomic maturation in infants. We recently showed that exposure of pregnant rats to a mild concentration of carbon monoxide (CO), a component of cigarette smoke, delays postnatal electrophysiological maturation of ventricular myocytes from newborns rats, likely predisposing to life-threatening arrhythmias. To get a comprehensive view of developmental molecular abnormalities induced, at cardiac level, by prenatal CO exposure, we used microarray analysis approach on the rat heart at 4, 7 and 20 days postnatal life. The relationship between molecular and functional alterations was investigated by assessing the ventricular expression of f-current, an electrophysiological marker of immature cardiac phenotype. Rats were prenatally exposed to 0 (CTR) or 150 p.p.m. CO and mRNA obtained from ventricular samples. Differential analysis and biological pathway analysis of microarray data were performed by using Newton's approach and the GENMAPP/MAPPFinder, respectively. The real-time RT-PCR reactions were performed by TaqMan probe-based chemistry. Freshly isolated patch-clamped ventricular cardiomyocytes were used to measure I(f). Genes and pathways controlling cell cycle and excitation-contraction coupling were significantly modified in CO-exposed rats. The higher effect was observed in cardiomyocytes harvested from 7-day-old rats, in which mRNA expression for crucial sarcomeric proteins (myosin and actin subunits, troponin I), transporters (Ca(2+) transporting ATPase) and enzymes (aldolase) were significantly downregulated. Accordingly, the molecular and functional expression of f-channels, which represents a marker of fetal ventricular phenotype, was transiently greater in CO-exposed rats (+200%) than in control ones. In conclusion, our study provides new insights into the molecular and functional mechanisms underlying cardiac maturation and its impairment by prenatal exposure to toxic components of smoking, such as CO.

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.

Previous studies have reported the anti-inflammatory properties of pomegranate extracts, suggesting that ellagitannins (ET) and ellagic acid (EA) are the main anti-inflammatory compounds. However, both ET and EA are metabolisedin vivoby the gut microbiota to yield urolithins (Uro) which can be found in the gut and in systemic bloodstream. The present study was carried out to evaluate the individual effect of EA and their microbiota-derived metabolites Uro on colon fibroblasts upon IL-1β treatment as anin vitroinflammation model. Uro-A and Uro-B (10 μm) inhibited PGE2production (85 and 40 %, respectively) after IL-1β stimulation, whereas EA did not show any effect. Uro-A, but not Uro-B, down-regulated cyclo-oxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) mRNA expression and protein levels. Both Uro inhibited NF-κB translocation to nucleus. Slight but significant effects were found in the activation of mitogen-activated protein kinase (MAPK) pathways. Uro-A lowered c-Jun N-terminal kinase phosphorylation state, and both Uro inhibited p38 activation. No metabolites derived from Uro or EA were found in the cell media upon incubation of EA or Uro with the cells, and only traces of the compounds were found inside the cells. The present results suggest that Uro, mainly Uro-A, are the main compounds that are responsible for the pomegranate anti-inflammatory properties. The mechanism of action implicated seems to be via the inhibition of activation of NF-κB and MAPK, down-regulation of COX-2 and mPGES-1 expressions, and consequently,via the reduction of PGE2production. Taking into account that Uro did not enter the cells, a competitive binding for IL-1β membrane receptor cannot be discarded.

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.

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.

The naturally occurring polyphenol resveratrol has been acknowledged with health-beneficial properties. Most of the studies dealing with its in vivo effects assay huge doses, not representative from a dietary point of view. Our aim was to ascertain whether resveratrol can exert anti-inflammatory activity in vivo at an attainable dietary dose. Rats were fed with 1 mg of resveratrol/kg/day (a human equivalent dose) for 25 days, and in the last 5 days, 5% dextran sulfate sodium (DSS) was administered to induce colitis. Effects on colon tissue damage, gut microbiota, reactive oxygen species, inflammatory markers and nitric oxide production as well as gene expression profile with microarrays were evaluated. Resveratrol increased lactobacilli and bifidobacteria as well as diminished the increase of enterobacteria upon DSS treatment. Resveratrol significantly protected the colonic mucosa architecture, reduced body weight loss, diminished the induced anemia and reduced systemic inflammation markers, colonic mucosa prostaglandin E(2), cycloxygenase-2, prostaglandin E synthase and nitric oxide levels. In addition, the expression of 2,655 genes in distal colon mucosa related to important pathways was varied. These results reinforce the concept of resveratrol as a dietary beneficial compound in intestinal inflammation at doses possibly attainable with resveratrol-enriched nutraceuticals.

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.

Tobacco consumption represents a major health hazard to humans and, despite anti-smoking campaigns, the number of smokers remains high; thus the reduction of toxic compounds from tobacco smoke may reduce the health hazards of smoking. In the last 25 years cigarette manufacturers have introduced a variety of filter designs to reduce toxic and carcinogenic substances in tobacco smoke (normal filters, NF). However, large quantities of harmful constituents are inefficiently retained by commonly used cigarette filters. Following a patented method we modified commercial cigarette filters (modified filter, MF) by injecting a DNA solution into the filter tips; we then evaluated the reduced polycyclic aromatic hydrocarbon (PAH) levels in mainstream tobacco smoke of MF relative to NF. The PAH measured were: fluoranthene (FLUO), pyrene (PY), benzo(a)anthracene (B(a)A), chrysene (CRY), benzo(a)pyrene (B(a)P), benzo(b)fluoranthene (B(b)F), benzo(k)fluoranthene (B(k)F), benzo(g,h,i)perylene (BGP), dibenzo(a,h)anthracene (DBA). The levels of PAH in cigarette smoke after MF were significantly reduced (P

Healthy volunteers (n = 50) were enrolled for studying the variation of gene expression induced by smoking in peripheral lymphocytes. RNAs from smokers (> 3 cigarettes/day, n = 20) and passive smokers (exposed to tobacco smoke > 3 h/day, n = 10) were hybridized versus a reference pool obtained by mixing equal amounts of RNA from 20 nonsmokers, and gene expression was analyzed using DNA microarrays containing 13,971 oligos. Principal component analysis showed that 99.7% of gene expression variability was related to plasma cotinine, age, and DNA oxidation damage. SAM and GenMAPP/MAPPFinder analyses showed that smokers, compared to nonsmokers, had 129 down-regulated and 87 up-regulated genes, whereas passive smokers, compared to nonsmokers, had 44 down-regulated and 159 up-regulated genes, mainly involved in pathways associated with the activation of defensive responses. Hierarchical cluster analysis identified two distinct clusters of smokers, characterized by different oxidative DNA damage: smokers with high DNA oxidation damage, compared to smokers with low DNA oxidation damage, had a large number (150) of down-regulated genes, mainly associated with xenobiotic metabolism, DNA damage and repair, inflammatory responses, lymphocyte activation, and cytokine activity, suggesting a reduced cellular response to toxic agents in this subset of smokers that could lead to an increased DNA oxidation damage.

Mucin-depleted foci (MDF) are microscopic dysplastic lesions induced in the colon of rodents by specific colon carcinogens. Most MDF show Wnt pathway activation, whereas only a subset shows mutations in the Ctnnb1 gene, coding for β-catenin. Because Apc is a member of the Wnt pathway and the most frequent mutated gene in human colon cancer, we tested whether MDF harbor Apc mutations. F344 rats were treated twice with 150 mg/kg of 1,2-dimethylhydrazine. After 15 or 28 weeks, MDF, aberrant crypt foci (ACF), and tumors were collected. We screened a segment of the Apc gene comprising the region homologous to the mutation cluster region (MCR) of human APC, which frequently shows mutations in experimental colon tumors. Mutations were identified by PCR amplification and sequencing in 6:24 MDF (25%), 7:23 tumors (30%), 0:24 ACF (0%). Most of the mutations (92%) in MDF and tumors were localized in a region upstream from the MCR. All mutations were single-base substitutions and mainly formed by G:C → A:T and C:G → T:A transitions. The pattern of nucleotide changes was similar in MDF and tumors, and, interestingly, the same mutation in codon 1047 was found in two MDF and in three tumors. Four out of the six mutations found in MDF were nonsense mutations, and two were missense. All mutations in tumors determined a protein truncation. These results show that Apc mutations are present in MDF with a frequency similar to that of tumors, strengthening the evidence that they are precancerous lesions in colon carcinogenesis. [Cancer Res 2007;67(2):445–9]

K-ras and mucin profile variations, associated with intestinal carcinogenesis, were studied in the preneoplastic lesions, mucin-depleted foci (MDF) and aberrant crypt foci (ACF), and in colonic tumors induced in rats by 1,2-dimethylhydrazine (DMH). The frequency of lesions with K-ras mutations was 23% (3/13), 5.5% (1/18) and 100% (14/14) in MDF, tumors and ACF, respectively. Two of three MDF mutated in K-ras also carried a missense mutation in Apc. We also tested the expression of MUC2, a mucin abundantly expressed in normal colon and M1/MUCA5C, up-regulated in colon carcinogenesis, using immunohistochemistry. MDF and tumors showed a dramatic reduction in the expression of MUC2, whereas ACF showed only a slight reduction. The expression of M1/MUC5AC was almost absent in normal mucosa, but was increased in all the lesions (MDF, tumors and ACF). The expression of the intestinal trefoil factor (ITF), a marker of goblet cell lineage, was reduced in MDF and tumors compared to normal mucosa but not in ACF. In conclusion, although K-ras mutations are present in all ACF, they are less frequent in MDF and tumors; M1/MUC5AC is a marker associated with all preneoplastic events while the reduction of MUC2 and ITF expression is selectively associated with more advanced lesions such as MDF and tumors. © 2007 Wiley-Liss, Inc.

A temporary stoma is often created to protect a distal anastomosis in colorectal surgery. Short-chain fatty acids, mainly butyrate, are the major fuel source for the epithelium and their absence in the diverted tract may produce mucosal atrophy and inflammation.To investigate whether the administration of sodium butyrate enemas (Naburen(©), Promefarm, Italy) could prevent mucosal inflammation and atrophy and affect gene expression profiles after ileo/colostomy.We performed a randomized, double-blind, placebo-controlled clinical trial, in patients with enterostomy performed for inflammatory bowel disease, colorectal cancer or diverticulitis. Twenty patients were randomly allocated to receive 30ml of sodium butyrate 600mmol/L (group A) or saline (group B), b.i.d. for 30 days.In group A endoscopic scores were significantly improved (p0.01) while mucosal atrophy was reduced or unchanged; in group B mucosal atrophy was increased in 42.8% of patients. Despite the high dose of butyrate used, no short-chain fatty acids were detectable by gas chromatography-mass spectrometry in colorectal biopsies. Group A patients showed up-regulation of genes associated with mucosal repair such as Wnt signalling, cytoskeleton regulation and bone morphogenetic protein-antagonists.Butyrate enemas may prevent the atrophy of the diverted colon/rectum, thus improving the recovery of tissue integrity.

Chronic inflammatory processes induce oxidative stress and lipid peroxidation (LPO), hereby generating DNA-reactive aldehydes such as trans-4-hydroxy-2-nonenal (HNE). Etheno-modified DNA bases are inter alia generated by reaction of DNA with HNE. Using an immunoaffinity-(32)P-postlabeling method, the authors have investigated etheno-DNA adduct levels 1,N (6)-ethenodeoxyadenosine (epsilondA) and of 3,N (4)-ethenodeoxycytidine (epsilondC) in the pancreas of chronic pancreatitis patients and in the colon of patients with inflammatory bowel disease. Both epsilondA and epsilondC levels were found to be significantly, 3 and 28 times, respectively, elevated in the inflamed pancreatic tissue. In contrast, only epsilondC was found to be increased in affected colonic mucosa of Crohn's disease (19 times) and of ulcerative colitis patients (4 times) when compared to asymptomatic tissues. In all three cancer-prone diseases, the mean epsilondC-levels in tissues were five- to ninefold higher than those of epsilondA. Differential or impaired DNA repair pathways of these adducts, known to occur by two different glycosylases are implicated. K-ras in pancreatic tumors and K-ras and p53 in colon mucosa in long-standing inflammatory bowel disease are known to be highly mutated. The conclusion is that promutagenic etheno-DNA adducts are generated as a consequence of chronic inflammation, acting as a driving force to malignancy in cancer-prone inflammatory diseases.

Tar and nicotine levels have been made to conform to EU standards as of 1 July 2004, but data on tobacco-derived carcinogenic compounds, such as PAH, in Chinese cigarettes are lacking in the literature. Levels of tar, nicotine, carbon monoxide and PAH were measured in 20 cigarette brands purchased in China between 2003 and 2004. Higher nicotine and tar levels were found in Chinese cigarettes than in European brands just 3 months before the above deadline; carcinogenic PAH levels were about 1.5 fold higher than in European cigarettes, but analysed singly, the mean value of benzo(a)pyrene (B(a)P) and dibenzo(a,h)anthracene (DBA), the most potent carcinogenic PAH yields, were 2.4 and 4.4 fold higher, respectively. Tar levels were well correlated with carcinogenic PAH (r = 0.53, P < 0.01), thus providing an easily measurable parameter for ranking various cigarette brands in developing countries where more sophisticated techniques might not be feasible for lack of funds and expertise.

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.

Benzo(a)pyrene [B(a)P] diolepoxide (BPDE)-DNA adducts were measured in the leukocytes of 41 healthy smokers using high-performance liquid chromatography coupled with a fluorimetric detector. The correlation between exposure to B(a)P through smoking and BPDE-DNA adduct levels was poor (r = 0.31), although subjects in the high exposure group [B(a)P > 50 ng/d] had a slightly higher level of adducts compared with the less exposed group (mean ± SE, 1.70 ± 0.3 versus 1.09 ± 0.1; P = 0.057). We studied the effect on BPDE-DNA adducts of individual variations in genes controlling B(a)P metabolism, classifying subjects in “low-risk” and “high-risk” genotypes for smoking-related B(a)P DNA damage. The high-risk group included subjects characterized by a combination of increased B(a)P activation [cytochrome P450 1A1 (CYP1A1) MspI and/or exon 7 Ile462Val allele variants and microsomal epoxide hydrolase (mEH) fast activity] and decreased deactivation ability [presence of glutathione S-transferase M1 (GSTM1) null allele and wild-type glutathione S-transferase P1 (GSTP1)]. The low-risk group included smokers with lower B(a)P activation (wild-type CYP1A1, low or intermediate mEH activity) and higher deactivation capacity (active GSTM1, GSTP1 Ile105Val allele). Subjects in the low-risk group had lower levels of BPDE-DNA adducts compared with subjects in the high-risk genotype group; this difference was significant using two markers (CYP1A1 and GSTM1, median ± SD, 0.77 ± 1.16 versus 1.89 ± 0.39; P = 0.03) or three markers (CYP1A1, GSTM1, and GSTP1, median ± SD, 0.66 ± 0.93 versus 1.43 ± 1.17; P = 0.013). The discrimination between groups was reduced when including mEH as an additional marker (P = 0.085). In conclusion, CYP1A1, GSTM1, and GSTP1 genotyping seems to be a risk predictor of BPDE-DNA adduct formation in leukocytes.

Crypt foci with absent or scant mucous production (mucin-depleted foci, MDF) were recently described by our group in the colon of azoxymethane (AOM)-treated rats. Since MDF are dysplastic and easy to quantify, we think that MDF are pre-neoplastic lesions that could be used as biomarkers for carcinogenesis. To test this hypothesis, we studied MDF in azoxymethane (AOM)-initiated rats treated with cholic acid (CHA), a promoter of colon carcinogenesis or with piroxicam (PXC), a colon cancer-inhibiting drug. Aberrant crypt foci (ACF) were determined as well. F344 male rats were treated with AOM (15 mg/kg x 2, s.c.) and then divided into: controls, which were fed AIN76 diet; CHA group, which was fed AIN76 diet containing CHA 0.5% w/w; PXC group, which was fed AIN76 diet containing PXC 0.02% w/w. Ten weeks after the first dose of AOM, the total number of MDF was significantly increased in rats treated with CHA (P

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.

Oxidative DNA damage was measured in the substantia nigra (SN), cortex, hippocampus, striatum and hypothalamus of 3- and 24-month-old rats, using single-cell gel electrophoresis (SCGE, 'comet' assay) which allows the detection of DNA breaks and oxidized bases. A significant increase in basal DNA damage was selectively found in the SN of aged rats. FPG-sensitive oxidative DNA damage was also significantly increased in the SN of aged rats and, to a lesser extent, in the cortex and hypothalamus. These data show a higher vulnerability of SN to oxidative damage with aging and indicate that the detection of DNA damage within discrete brain nuclei can provide a reliable tool for investigating oxidative damage in neurodegenerative processes.

The single-cell gel electrophoresis (comet assay) was used to evaluate the possibility of detecting single-strand breaks of brain DNA in the early phase of ischemia. Four hours after occlusion of the middle cerebral artery (MCAO) in rats, the percentage of DNA migrating into the comet tail (indicating the presence of breaks) increased from 11.4 ± 4.70 to 34.7 ± 9.2 (means ± SD) in the caudate and from 9.9 ± 4.3 to 42.8 ± 14.1 in the cortex. Interestingly, a subpopulation of cells exhibiting higher resistance to the ischemic insult was present in the caudate putamen, but not in the cortex. Administration of MK801, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, (1 mg/kg subcutaneously, 10 minutes before MCAO), reduced the ischemia-induced DNA breaks and the infarct volume, suggesting that excessive stimulation of NMDA receptors contributes to the formation of both DNA damage and infarct volume. In contrast, DPQ, an inhibitor of poly(ADP-ribose) polymerase (PARP) (10 mg/kg intraperitoneally, 2 hours before and 1 hour after MCAO), reduced the infarct volume but not DNA damage, suggesting that the neuroprotective actions of PARP inhibitors occur at a later step of the processes leading to postischemic neuronal death.

We studied the correlation between fecal levels of short-chain fatty acids (SCFA), bile acids (BA), and colonic mucosal proliferation in humans on a free diet. Subjects [n = 43: 27 men and 16 women; 61 +/- 7 and 59 +/- 6 (SE) yr old, respectively] were outpatients who previously underwent resection of at least two sporadic colon polyps. Mucosal proliferation was determined by [3H]thymidine incorporation in vitro in three colorectal biopsies obtained without cathartics and was expressed as labeling index (LI). BA were analyzed in feces by mass spectrometry and SCFA by gas chromatography. We found that increasing levels of BA in feces did not correlate with higher LI. On the contrary, higher levels of SCFA were significantly associated with lower LI in the colonic mucosa (P for trend = 0.02). In conclusion, in humans on a free diet, intestinal proliferation seems to be regulated by the levels of SCFA in feces and not by BA. Because a lower intestinal proliferation is associated with a decreased colon cancer risk, treatments or diets that increase colonic levels of SCFA might be beneficial for colonic mucosa.

Background: Minerals, metals, clays and rocks were widely used by physicians in the past. However, it was and it is well known that some inorganic elements at high dosage may have curative effects but also serious toxicity. The effects at low or ultra-low concentrations, on the contrary, are less documented, but the idea that low dosage supplementation might be beneficial to human health is widespread even in the present period. Methods: The main information about aluminium, bismuth, cobalt, gold, lithium, nickel and silver was selected and evaluated from a vast body of medical literature. Results: In modern times, most elements are proposed for human use at levels comparable with normal dietary intake, probably for precautionary considerations. Conclusion: Some inorganic trace compounds might have unexpected effects at extremely low dosages, but scientific demonstrations of beneficial effects of supplementation are mostly not available in the medical literature.

Background & Aims Red wine polyphenols inhibit chemically-induced oxidative DNA damage in vivo in experimental animals through a mechanism which is still unclear. On this basis, we tried to clarify the mechanisms of inhibition of DNA oxidation in vitro by wine extracts containing monomeric and polymeric phenols (WE) and monomer-free complex polyphenols and tannins (WCPT) from red wine. Methods Oxidative DNA damage was induced by incubating DNA with GSH/Fe3+ or cumene hydroperoxide (CumOOH) in vitro and using 8-OH–2-deoxyguanosine (8-OHdG) levels as a measure of DNA oxidation. Levels of 8-OHdG were determined by HPLC coupled with electrochemical detector (ESA). Results and conclusions WCPT and WE, at μM concentrations, reduced concentration-dependently oxidative DNA damage induced by GSH/Fe3+. WCPT and WE also reduced DNA oxidation by CumOOH. In conclusion, complex polyphenols and tannin extracts from red wine, with or without small molecular phenols, prevent oxidative DNA damage through a dual mechanism, iron binding and direct free radical scavenging.

The effect of black tea polyphenols on 1,2-dimethylhydrazine (DMH)-induced oxidative DNA damage in rat colon mucosa has been investigated. Fischer 344 rats were treated orally with thearubigin (TR) or theafulvin (TFu) for 10 days (40 mg/kg), injected ip with DMH (20 mg/kg) or saline and sacrificed 24 hr after DMH administration. The levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were measured in colonic mucosa DNA and expressed as a ratio relative to 2'-deoxyguanosine (2dG). Control rat mucosa had 8-OHdG values of 1.12 +/- 0.14/10(5) dG (mean +/- SEM, n=11), whereas DMH-treated rats significantly higher values (1.52 +/- 0.14/10(5) dG, n=26, P0.05). Pretreatment of rats with TR had significantly inhibited DMH-induced oxidative DNA damage 0.99 +/- 0.09/10(5) dG, n=10, P0.05) and a similar, although less marked, effect was observed with TFu (1.15 +/- 0.19/10(5), n=9, P=0.06). These findings confirm that DMH causes oxidative DNA damage in the colon mucosa of rats and demonstrate that this effect is prevented by the consumption of complex polyphenols from black tea.

We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 +/- 10.6 per 10(6) 2-deoxyguanosine (dG) [mean +/- SE], n = 16) compared with nonsmokers (15.3 +/- 1.8 per 10(6) dG, n = 31) and former smokers (17.8 +/- 1.5 per 10(6) dG, n = 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 +/- 17.1 per 10(6) dG, n = 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.

Benzo(a)pyrene diol-epoxide (BPDE)-DNA adducts and aryl hydrocarbon hydroxylase (AHH) activity were determined in white blood cells (WBCs) obtained from non-smokers, ex-smokers and smokers. DNA adduct levels ranged from non-detectable (ND) to 8.8 per 10 8 nucleosides in all samples analysed. The mean level in WBC from smokers (1.93±0.55 mean±SE) was similar to that from non-smokers (1.18±0.40) and ex-smokers (1.83±0.50). The distribution frequency of AHH activity in non-smokers was different from that in smokers, while 50% of non-smokers had AHH activity ranging from ND to 20 fmol mg -1 min -1 , This low AHH activity was not found in smokers; no correlation was found between AHH activity and tobacco consumption (r=0.097; P = 0.76; n=12). On the contrary, AHH activity was correlated with BPDE-DNA adduct levels in WBCs from the same subjects (r=0.457; P=0.019; n=26). Our findings indicate that there are limitations to the use of WBCs as surrogate cells for determining internal PAH exposure in target organs.

Background Glutathione S-transferases (GSTs)* are an important class of phase II, predominantly detoxifying, enzymes. The supergene family is composed of several isoenzymes, hetero- and homodimers, with tissue specific distribution and levels of expression. The hypothesis is that a higher expression of individual proteins within a specific tissue may be associated with a decreased burden of exposure to reactive carcinogens and ultimately with a decreased cancer risk in this tissue. Aims of the study Since nutrition is expected to contribute to the gene expression, it was the aim of this study to investigate the impact of dietary factors, especially resistant starch, and of the gut microflora, which may be influenced by diet, on the GSTs in colon cells of rats. Methods For this, a technique using high pressure liquid chromatography was established with which for the first time GST isoenzymes were analysed in colon cells and compared to the levels of the corresponding proteins in the liver of the same rat. Results It was found that colon cells contain mainly GST π and low amounts of μ but not GST α. In contrast, the predominant form of GSTs in the liver was α, then μ and hardly any π. Altogether, liver cells had approximately tenfold more total GSTs than colon cells. The feeding of “Crystalean”, a retrograded, high amylose starch which alters the fermentation profile and the composition of the microflora, led to higher levels of GST π in the colon. Furthermore, the comparison of GSTs in colon cells of germ-free rats revealed they were much lower than those observed in rats with conventional microflora. Conclusions These findings clearly demonstrate that the gut bacteria, or their metabolic products, enhance GST expression. The studies support the hypothesis that nutrition – by affecting the gut flora – may induce this potentially protective and important class of phase II enzymes in important tumor target cells.

An investigation was carried out to determine whether variations of dietary carbohydrates could modify the colonic flora in rats. Sprague-Dawley rats were fed with two equicaloric diets based on the AIN-76 diet (American Institute of Nutrition 1977) but differing from that diet in content of carbohydrates, i.e. high sucrose (64%) of high corn starch (64%). Feeding was continued for 9 months ad libitum and no variation in weight gain was recorded among the different diets. A prevalence of aerobes, and a significant reduction in the ratio anaerobes/aerobes in the faeces of rats on the high starch diet compared with the high sucrose diet, was observed. The anaerobe genera identified included Actinomyces, Bacteroides, Bifidobacterium, Clostridium, Eubacterium, Lactobacillus and Propionibacterium. Bacteroides was the most prevalent genus in both dietary groups (51.2 and 29.5% in the faeces of rats fed the sucrose and starch diets, respectively). In contrast, clostridia were prevalent in the starch-fed group (23.8%) and less so in the sucrose diet (11.5%), as propionibacteria were prevalent in faeces of rats fed the starch diet (15.5%), and low in the sucrose diet (3.9%). The remaining genera were scarce in faeces from rats on either diet. Total short-chain fatty acids (SCFA) were significantly higher in the faeces of animals fed the starch diet compared with those fed the sucrose diet. The relative concentrations of acetic, propionic and butyric acids were not significantly different between the two dietary groups. In conclusion, high starch diet can markedly modify the composition of faecal flora and alter considerably the faecal concentration of SCFAs, compound which might have a health-promoting effect.

We investigated in the rat the role of the Apc gene, which is mutated in familial adenomatous polyposis and sporadic colon cancer in the process leading from normal colonic mucosa to aberrant crypt foci (ACF) and finally to adenomas and adenocarcinomas. We analysed mutations in exon 15 of the rat Apc gene using in vitro synthesized protein assay in 66 ACF and in 28 colon tumours induced by azoxymethane. No Apc mutations were found in ACF, whereas five mutations were found in the tumours. The data suggest that mutations of the Apc gene are associated with the transition from ACF to adenoma and adenocarcinoma and not from normal mucosa to ACF.

The levels of 9 polycyclic aromatic hydrocarbons (PAHs) 6 of which carcinogenic were measured in the leaves of evergreen tree (Laurus nobilis) sampled in 13 locations in summer and winter in Tuscany, Italy. The carcinogenic PAH levels were correlated with the PAH air levels sampled at the same site. Samples from larger towns had higher PAH levels than those from medium and small towns. Leaves collected in the center of larger cities had higher carcinogenic PAH levels than samples from residential areas indicating that vehicular traffic is the main PAH source. Carcinogenic PAH levels in leaves collected in the winter in medium towns were considerably higher than expected, probably due to domestic heating. These findings demonstrate that air quality in terms of PAH contamination is not markedly different in towns of different size in Tuscany.

The ureic herbicide linuron [3-(3, 4-dichlorophenyl)-1-methoxy-1-methylurea] (CAS 330-55-2) was investigated for genotoxicity in a series of in vivo experiments. Since human exposure to herbicides is not only to the active principles, but also to all the chemicals present in the commercial formulation, we tested both pure and commercial linuron. Groups of rats were treated with gavage containing different doses of the herbicide (pure compound or commercial formulation) for 14 days. The doses were 150, 300 and 450 mg/kg b.wt. for the pure compound and 315.8, 631.6 and 947.4 mg/kg b.wt. for the commercial formulation (47.5% of linuron). Faeces and urine were collected at regular intervals. Urine specimens were analysed for their mutagenic metabolites, thioethers and D-glucaric acid content. Faeces extracts were tested for mutagenicity. Linuron's ability to cause DNA damage and cytogenetic effects was also investigated after treating groups of rats once with different doses of pure or commercial linuron. DNA single-strand breaks were assessed in rat liver using the alkaline elution technique and the single-cell microgel electrophoresis assay (SCGE: 'comet' assay), and in rat testes cells with the SCGE assay. Micronuclei induction was analysed in rat bone marrow erythrocytes. Results obtained were mainly negative when the excretion of mutagenic metabolites in urine and faeces of animals treated with the pure compound or with the linuron-based commercial formulation were monitored, whereas an increase in the urinary excretion of thioethers and D-glucaric acid was observed in rats treated with the commercial formulation. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the treated animals. However, linuron affected the viability of hepatocytes isolated from animals treated with higher doses. This cytotoxicity was accompanied by the induction of DNA single-strand breaks in the liver, as seen by the alkaline elution assay. The potential of pure linuron to induce in vivo DNA damage was confirmed with the microgel-electrophoresis technique ('comet' assay). Cytotoxicity was also seen in rat testes cells. However, no indication of DNA damage was visible.

Oxidative damage was quantified in the liver of rats by measuring the levels of 8-OH-2-deoxyguanosine (8-OH-2DG) relative to 2-deoxyguanosine in DNA after treating rats for 10 days at a total dose of 1 mg/kg/day with a mixture of the 15 pesticides most commonly found in Italian foods (comprised of dithiocarbamate, benomyl, procymidone, methidathion, chlorpyrifos-ethyl, parathion-methyl, chlorpropham, parathion, vinclozolin, chlorfenvinphos, pirimiphos ethyl, thiabendazole, fenarimol, diphenylamine and chlorothalonil). We fractionated this pesticide mixture into subgroups in order to determine which molecules, if any, induced DNA oxidative damage. The administration of diphenylamine (0.09-1.4 mg/kg/day) and chlorothalonil (0.13-1 mg/kg/day) induced a dose-dependent increase in 8-OH-2DG levels in liver DNA. The other 13 pesticides of the mixture on the contrary, did not produce oxidative liver DNA damage. These results indicate that the toxicity of low doses of pesticide mixtures present in food might be further reduced by eliminating diphenylamine and chlorothalonil.

We investigated the effects of smoking and exposure to environmental tobacco smoke (ETS) on oxidative DNA damage by measuring 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) levels in DNA of leukocytes of healthy donors (30 smokers, 29 nonsmokers, and 28 ETS-exposed subjects). Nonsmokers had lower 8-oxodGuo levels compared with smokers (5.94 ± 0.87 × 10−6 and 19.85 ± 4.75 × 10−6 2-deoxyguanosine, respectively, means ± SE, P = 0.00007). Subjects exposed to ETS had higher mean value of 8-oxodGuo compared with nonsmokers (9.18 ± 1.53 × 10−6 2-deoxyguanosine, mean ± SE), nonsignificant by univariate analysis (P = 0.074). Multiregression analysis indicated that the increase of 8-oxodGuo levels induced by ETS was significant (P = 0.045) and that coffee and tea consumption reduced DNA oxidation (P = 0.0053). Oxidative leukocyte DNA damage was positively correlated with plasma cotinine levels in ETS-exposed subjects (r = 0.47, P < 0.01, n = 28) and was increased by age in nonsmokers and ETS-exposed subjects (P = 0.049). The results seem to confirm that ETS exposure is capable of inducing some oxidative DNA damage in circulating leukocytes and that coffee and tea consumption might partially protect against smoking-induced oxidation damage. (Cancer Epidemiol Biomarkers Prev 2005;14(12):2975–7)

Background Colon cancer stem cells may drive carcinogenesis and account for chemotherapeutic failure. Although many markers for these cells have been proposed, there is no complete agreement regarding them, nor has their presence in the early phases of carcinogenesis been characterized in depth. Methods The expression of the putative markers LGR-5 (leucine-rich-repeat-containing G-protein-coupled receptor 5), MSI-1 (Musashi-1) and DCAMKL-1 (doublecortin and calcium/calmodulin-dependent protein kinase-like-1) was studied in normal colon mucosa (NM), in the precancerous lesions Mucin Depleted Foci (MDF) and in macroscopic tumours (adenomas) of 1,2-dimethylhydrazine-treated rats. Co-localization between these markers and nuclear β-catenin (NBC), an attributed feature of cancer stem cells, was also determined. Moreover, since PGE2 could increase NBC, we tested whether short-term treatment with celecoxib, a COX-2 inhibitor (2 weeks, 250 ppm in the diet) could reduce the expression of these markers. Results LGR-5 expression in NM was low (Labelling Index (LI): 0.22±0.03 (means±SE)) with positive cells located mainly at the base of the crypts. Compared to NM, LGR-5 was overexpressed in MDF and tumours (LI: 4.7±2.0 and 2.9±1.0 in MDF and tumours, respectively, P Conclusions Based on its prevalent localization at the base of normal crypts, as expected for stem cells, and on the overexpression in precancerous lesions and tumours, we support LGR-5, but not MSI-1 or DCAMKL-1, as putative neoplastic stem cell marker. In both MDF and tumours, we identified LGR-5-positive cells co-expressing NBC which could be a subpopulation with the highest stem cell features.

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.