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.

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.

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.

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.

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.

NF-kappaB-dependent anti-inflammatory activity of urolithins, gut microbiota ellagic acid-derived metabolites, in human colonic fibroblasts.

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 metabolised in vivo by 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-1beta treatment as an in vitro inflammation model. Uro-A and Uro-B (10 microm) inhibited PGE2 production (85 and 40 %, respectively) after IL-1beta 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-kappaB 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-kappaB and MAPK, down-regulation of COX-2 and mPGES-1 expressions, and consequently,via the reduction of PGE2 production. Taking into account that Uro did not enter the cells, a competitive binding for IL-1beta membrane receptor cannot be discarded.

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

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

Mucin-depleted foci show strong activation of inflammatory markers in 1,2-dimethylhydrazine-induced carcinogenesis and are promoted by the inflammatory agent sodium dextran sulfate.

Mucin-depleted foci (MDF), formed by dysplastic crypts devoid of mucins, have been identified in the colon of carcinogen-treated rodents and in humans at high risk for colon cancer. The lack of the protective layer of mucus may cause inflammation which has been linked to colon carcinogenesis, therefore, the expression of markers such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (i-NOS) and macrophage infiltration was studied with immunohistochemistry (IH) in MDF harvested from F344 rats treated with the colon carcinogen 1,2-dimethylhydrazine (DMH). The same determinations were performed in aberrant crypt foci (ACF) and, at a later time point, in tumours. A dramatic increase in COX-2, i-NOS and macrophage infiltration was observed in MDF but ACF showed a moderate increase compared with the paired normal mucosa. Tumours were positive for all the markers. RT-PCR experiments demonstrated that i-NOS RNA expression was increased in a set of MDF confirming the results obtained with immunohistochemistry. In an inflammation-cancer experimental model [mice treated with azoxymethane (AOM) and dextran sodium sulphate (DSS)], we observed that DSS-induced inflammation promoted MDF in a dose-dependent manner, whereas ACF were not affected. In conclusion, we report here for the first time a strong activation of the inflammatory process in MDF, which may contribute to the further progression of MDF to tumours.

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

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

Protective effect of 4-coumaric acid from UVB ray damage in the rabbit eye.

UV-induced oxidation damage seems to play a major role in a number of specific pathological conditions of intraocular tissues, such as cataract formation and retinal degeneration. Therefore, antioxidant and/or scavenger compounds might protect the eyes from UV-induced cellular damage. We previously reported that 4-coumaric acid (4-CA) is able to protect rabbit corneal-derived cells (SIRC) from UVB-induced oxidation damage. In this study we evaluated the protective effect of 4-CA against UVB-induced cell damage in rabbit cornea in vivo. Twelve male New Zealand albino rabbits were used; four rabbits were used as a control and received vehicle in one eye and 4-CA acid in the contralateral eye; eight rabbits were exposed to UVB rays (79.2mJ/cm(2)) and three days before to UV exposure each animal received 1 drop/day of vehicle in one eye and 1 drop/day of vehicle containing 4-CA (164ng) in the contralateral eye. Corneal and sclera tissues were removed and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) levels were measured. Superoxide dismutase (SOD) and xanthine oxidase (XO) activities were determined in aqueous humour. UVB-induced vessel hyper-reactivity was strongly reduced at 4 and 24h after UVB exposure after local treatment with 4-CA, 8-oxodGuo levels, a marker of oxidative DNA damage, were significantly increased (P<0.05) in sclera and cornea by UVB irradiation, but when 4-CA was administered to the conjunctiva in a buffered solution once a day for 3d before and 6d after UVB exposure, levels of 8-oxodGuo were similar to controls and significantly reduced (P<0.05) compared to UVB-treated corneas. XO activity in the aqueous humour was significantly increased. The administration of 4-CA for 3d before and 6d after UVB irradiation induced a small but significant (P<0.05) reduction of XO compared with control eyes. Our results indicate that the administration of 4-CA protects eye tissues, thus reducing the harmful effect of UVB radiation at low concentration, probably through its free radical scavenging and antioxidant properties. Therefore, 4-CA may be useful in protecting the eye from free radical damage following UVB exposure from sunlight, UV lamps and welding torches.

Frequent mutation of Apc gene in rat colon tumors and mucin-depleted foci, preneoplastic lesions in experimental colon carcinogenesis.

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 beta-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.

K-ras mutations and mucin profile in preneoplastic lesions and colon tumors induced in rats by 1,2-dimethylhydrazine.

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.

Eu.Gene Analyzer a tool for integrating gene expression data with pathway databases.

MOTIVATION: Eu.Gene Analyzer is an easy-to-use, stand-alone application that allows rapid and powerful microarray data analysis in the context of biological pathways. Its intuitive graphical user interface makes it an easy and flexible tool, even for the first-time user. Eu.Gene supports a variety of array platforms, organisms and pathway ontologies, transparently deals with multiple nomenclature systems and seamlessly integrates data from different sources. Two different statistical methods, the Fisher Exact Test and the Gene Set Enrichment Analysis (GSEA), are implemented to identify biological pathways transcriptionally affected under experimental conditions. A suite of tools is offered to define, visualize and share custom non-redundant pathway sets. In conclusion, Eu.Gene Analyzer is a new software application that takes advantage of information from multiple pathway databases to build a comprehensive interpretation of experimental results in a simple, intuitive environment.

Correspondence between flat aberrant crypt foci and mucin-depleted foci in rodent colon carcinogenesis.

BACKGROUND: Flat aberrant crypt foci (flat ACF) and mucin-depleted foci (MDF) are preneoplastic lesions identified in the colon of carcinogen-treated rodents stained with methylene blue (MB) and high iron diamine-alcian blue (HID-AB), respectively. The correspondence between flat ACF and MDF in the same colon of Min mice treated with azoxymethane (AOM) and of F344 rats treated with 1,2-dimethylhydrazine (DMH) was explored. MATERIALS AND METHODS: The position of each flat ACF was recorded on a digitally constructed photographic map of the MB-stained colon. The same colons were then stained with HID-AB and the position of each MDF was compared with that of flat ACF. RESULTS: The fraction of coincident lesions, identified as both flat ACF and MDF with the two staining methods, was 57% and 42%, in the Min mice and F344 rats, respectively. Flat ACF or MDF not coincident with the two staining methods were either undetectable or ACF with one of the two methods. CONCLUSION: Flat ACF and MDF show considerable, but not total, overlap.

No effects of olive oils with different phenolic content compared to corn oil on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

BACKGROUND: Some epidemiological and experimental studies suggest that olive oil, despite its elevated caloric content, may have protective activity against colon cancer, partially due to its phenolic content. However, little experimental evidence exists to support this claim in vivo. AIM OF THE STUDY: To test the effect of olive oils with different phenolic content in a well-characterized model of colon carcinogenesis, comparing them with corn oil (CO). METHODS: F344 rats were fed AIN-76 based diets for the entire experimental period; the diets contained 23% (w/w) of lipids from three different sources: extra-virgin olive oil rich in phenolic compounds (EV), rectified olive oil (ROO) with the same fatty acid composition but devoid of phenolic compounds and CO as a control diet. One week later, rats were induced with 1,2-dimethylhydrazine (DMH) (150 mg/kg b.w. x 2 times) to measure preneoplastic lesions (aberrant crypt foci (ACF) and mucin depleted foci (MDF)) and tumours in the intestine. RESULTS: Thirteen weeks after DMH, the numbers of ACF and MDF were similar in the different groups (ACF/colon were 344.9 +/- 27.0, 288.6 +/- 28.5 and 289.8 +/- 21.4 in CO, EV and ROO groups, respectively, means +/- SE; MDF/colon were 8.83 +/- 1.2, 8.41 +/- 1.5 and 8.75 +/- 1.6 in CO, EV and ROO groups, respectively, means +/- SE). Thirty-two weeks after DMH, the incidence of tumours (rats with tumours/rats in the group) did not differ among the different groups (20/21, 18/19 and 20/20 in the CO, EV, and ROO groups, respectively). Similarly, the number of tumours/ rat in the colorectum (both adenomas and cancers) was not different in the three different groups (2.33 +/- 0.26, 2.42 +/- 0.41 and 2.25 +/- 0.40 in CO, EV and ROO groups, respectively, means +/- SE). CONCLUSIONS: Olive oil, irrespective of its phenolic content, does not affect DMH-induced colon carcinogenesis in F344 rats compared with CO.

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

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