DNA copy number alterations, gene expression changes and disease-free survival in patients with colorectal cancer: a 10 year follow-up.

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-ß 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.

Effects of Extreme Dilutions of Apis mellifica Preparations on Gene Expression Profiles of Human Cells.

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.

Effect of butyrate enemas on gene expression profiles and endoscopic/histopathological scores of diverted colorectal mucosa: A randomized trial.

BACKGROUND: 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. AIMS: 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. METHODS: 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. RESULTS: In group A endoscopic scores were significantly improved (p<0.01) 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. CONCLUSION: Butyrate enemas may prevent the atrophy of the diverted colon/rectum, thus improving the recovery of tissue integrity.

Long-term Neuroglial Cocultures as a Brain Aging Model: Hallmarks of Senescence, MicroRNA Expression Profiles, and Comparison With In Vivo Models.

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-ß deposition. Neuroglial cocultures displayed features of cellular senescence (increased senescence-associated-ß-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.

Occurrence, exposure, effects, recommended intake and possible dietary use of selected trace compounds (aluminium, bismuth, cobalt, gold, lithium, nickel, silver).

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.

Exploring the effects of homeopathic Apis mellifica preparations on human gene expression profiles.

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 (IL1ß, 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.

Expression of LGR-5, MSI-1 and DCAMKL-1, putative stem cell markers, in the early phases of 1,2-dimethylhydrazine-induced rat colon carcinogenesis: correlation with nuclear ß-catenin.

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 ß-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<0.01 compared to NM). DCAMKL-1 positive cells, distributed along the length of normal crypts, were reduced in MDF and tumours. Nuclear expression of MSI-1, located mainly at the base of normal crypts, was not observed in MDF or tumours. In both MDF and tumours, few cells co-expressed LGR-5 and NBC (LI: 1.0 ± 0.3 and 0.4 ± 0.2 in MDF and tumours, respectively). Notwithstanding the lower expression of DCAMKL-1 in tumours, the percentage of cells co-expressing DCAMKL-1 and NBC was higher than in NM (LI: 0.5 ± 0.1 and 0.04 ± 0.02 in tumours and NM, respectively). MSI-1 and NBC co-localization was not observed. Celecoxib did not reduce cells co-expressing LGR-5 and NBC. 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.

The frequency of granulocytes with spontaneous somatic mutations: a wide distribution in a normal human population.

Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10⁻6 to 37.5×10⁻6, with a median of 4.9×10⁻6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.

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

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

Long-term dietary extra-virgin olive oil rich in polyphenols reverses age-related dysfunctions in motor coordination and contextual memory in mice: role of oxidative stress.

The aim of this study was to evaluate the effects of olive oil phenols on brain aging in mice and to verify whether the antioxidant and antiinflammatory activities of these polyphenols were involved. C57Bl/6J mice were fed from middle age to senescence with extra-virgin olive oil (10% wt/wt dry diet) rich in phenols (total polyphenol dose/day, 6 mg/kg). Behavioral tests were employed to assess cognitive, motor, and emotional behavior after 6 or 12 months of treatment. Parameters of oxidative status and inflammation were measured in different brain areas at the same times and evaluated for correlation with behavioral changes. The treatment with olive oil phenols improved contextual memory in the step-down test to levels similar to young animals and prevented the age-related impairment in motor coordination in the rotarod test. This motor effect was correlated with reduced lipid peroxidation in the cerebellum (p<0.05), whereas the memory effect did not correlate with oxidation or inflammation parameters. In conclusion, this work points out that natural polyphenols contained in extra-virgin olive oil can improve some age-related dysfunctions by differentially affecting different brain areas. Such a modulation can be obtained with an olive oil intake that is normal in the Mediterranean area, provided that the oil has a sufficiently high content of polyphenols.

Antioxidant vitamins and mineral supplementation, life span expansion and cancer incidence: a critical commentary.

PURPOSE: 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. RESULTS: 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. ß-carotene and α-tocopherol, alone or in combination, increase cardiovascular and all-cause mortality or have no effect. In some studies, ß-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. CONCLUSIONS: 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.

Marie Ménard apples with high polyphenol content and a low-fat diet reduce 1,2-dimethylhydrazine-induced colon carcinogenesis in rats: effects on inflammation and apoptosis.

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-1ß, 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.

Mucin depleted foci, colonic preneoplastic lesions lacking Muc2, show up-regulation of Tlr2 but not bacterial infiltration.

Mucin depleted foci (MDF) are precancerous lesions of the colon in carcinogen-treated rodents and humans at high risk. Since MDF show signs of inflammation we hypothesized that the defective mucous production would expose them to the risk of being penetrated by intestinal bacteria, which can be sensed by Toll-like receptors (Tlrs) and activate inflammatory pathways. To verify this hypothesis we tested the expression of 84 genes coding for Tlrs and associated pathways using RT-qPCR in MDF (n = 7) from 1,2-dimethylhydrazine (DMH)-treated rats. Among the 84 tested genes, 26 were differentially expressed in MDF with 5 genes significantly up-regulated and 21 down-regulated when compared to the normal mucosa. Tlr2, as well as other downstream genes (Map4k4, Hspd1, Irak1, Ube2n), was significantly up-regulated. Among the genes regulating the NFkB pathway, only Map4k4 was significantly up-regulated, while 19 genes were not varied and 6 were down-regulated. Tlr2 protein was weakly expressed both in normal mucosa and MDF. To determine whether inflammation observed in MDF could be caused by bacteria contacting or infiltrating crypts, we performed fluorescence in situ hybridization (FISH) experiments with a rRNA universal bacterial probe. None of the 21 MDF tested, showed bacteria inside the crypts, while among the colonic tumors (n = 15), only one had very few bacteria on the surface and on the surrounding normal mucosa. In conclusion, the up-regulation of Tlr2 in MDF, suggests a link between this receptor and carcinogenesis, possibly related to a defective barrier function of these lesions. The data of FISH experiments do not support the hypothesis that inflammation in MDF and tumors is stimulated by bacterial infiltration.

Sustained proliferation and resistance to apoptosis after a cytotoxic insult are early alterations in rat colon carcinogenesis.

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.

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

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

Protective effects of resveratrol against senescence-associated changes in cultured human fibroblasts.

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.

Extremely low copper concentrations affect gene expression profiles of human prostate epithelial cell lines.

Although cellular copper metabolism is tightly regulated through a variety of copper transport proteins and chaperones, disturbances in copper homeostasis are involved in several pathological disorders. The aim of this study was to evaluate the effects of extremely low copper concentrations on gene expression profiles of a line of human prostate epithelial cells (RWPE) which grows in the absence of fetal calf serum, a source of variable and unpredictable copper. Cells were exposed to copper(II) sulfate for 24h at concentrations varying from 10(-6) to 10(-17)M and untreated reference cells were exposed to the same volume of copper-free water. Relative gene expression variations between copper-treated and control cells were studied with microarray technology using the Whole Human Genome Array from Agilent. Microarray data demonstrated that copper added to the medium varied gene expression at all concentrations tested. Many genes belonging to functional gene families were modulated by copper, some dose-dependently. Amongst these genes metallothioneins (MT1A and MT2A) were over-expressed at all copper concentrations, MT1M was up-regulated between 10(-6) and 10(-9)M, while MT1B, MT1E, MT1G and MT1H were up-regulated between 10(-6) and 10(-14)M. The heat shock protein (HSP) gene family showed similar behavior: some HSP genes were constantly up-regulated by copper (HSP90Ad, HSP90B1 and HSPD1) and others only at higher concentrations (HSP90AB1 and HSPA8). Reverse-transcription-PCR analysis, performed on four different genes on five biological replicates for selected genes, on each copper concentration tested, confirmed the trend observed in microarray results. In conclusion, we unexpectedly observed a modulation of gene expression even at extremely diluted copper concentrations, similar to that induced by toxic concentrations, possibly as a result of very tight control of free copper(II) levels inside the cells.

Prenatal exposure to carbon monoxide delays postnatal cardiac maturation.

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.

Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats.

BACKGROUND: Azoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats. METHODS: For gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 x 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 x 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent). RESULTS: Microarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFalpha/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc. CONCLUSION: The results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to note that one of the alterations concerned Apc, a key gene in colorectal carcinogenesis. The fact that many of the molecular alterations described in this study are documented in human colon tumours confirms the relevance of DMH-induced cancers as a powerful tool for the study of colon carcinogenesis and chemoprevention.

Air monitoring and assessment of occupational exposure to peracetic acid in a hospital environment.

A new automated method based upon solid phase micro-extraction (SPME)/fast gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative determination of airborne peracetic acid (PAA). The method is suitable for the quick assessment of brief acute exposure as well as for long-term environmental monitoring of PAA and can assist in improving safety and environmental quality in workplaces where disinfectants are used. During a monitoring campaign in the Regional Hospital of Florence, Italy, the 8-h average air concentration of PAA was 1/10 of the threshold limit value of time weighted average in 87% of the clinical units tested. However, the application of the new SPME method showed that short-term exposure to PAA could be relatively elevated in some hospital units with poor ventilation, allowing prompt intervention in order to reduce worker exposure to this potentially toxic compound.