285 results on '"De Flora A"'
Search Results
2. Multi-drug resistant Pseudomonas aeruginosa nosocomial strains: Molecular epidemiology and evolution
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Etleva Dedej, Stefano Pascarella, Elisabetta Ferraro, Silvia Angeletti, Roberto Coppola, Giordano Dicuonzo, Fabio Francescato, Marta Fogolari, Eleonora Cella, Cecilia De Flora, Massimo Ciccozzi, Silvia Spoto, Lucia Florio, Mattia Prosperi, Raffaele Antonelli Incalzi, and Francesca Antonelli
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Models, Molecular ,0301 basic medicine ,Carbapenem ,medicine.medical_specialty ,medicine.drug_class ,Rome ,030106 microbiology ,Antibiotics ,Porins ,Microbial Sensitivity Tests ,Drug resistance ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Drug Resistance, Multiple, Bacterial ,Molecular genetics ,medicine ,Humans ,Infection control ,Pseudomonas Infections ,Phylogeny ,Cross Infection ,Molecular Epidemiology ,Base Sequence ,Molecular epidemiology ,Pseudomonas aeruginosa ,Hospitals ,Multiple drug resistance ,030104 developmental biology ,Infectious Diseases ,Carbapenems ,Sequence Alignment ,MDR P. aeruginosa ,nosocomial infection ,phylogenetic analysis ,medicine.drug - Abstract
Pseudomonas aeruginosa causes a wide variety of nosocomial infections. In the study, phylogenetic, selective pressure analysis and homology modelling were applied to oprD efflux pump gene with the aim to characterize multi-drug resistant strains circulating in the nosocomial setting, their transmission dynamics and ongoing evolution. One hundred ninety-three consecutive inpatients with Pseudomonas aeruginosa infection were enrolled at the University Campus Bio-Medico of Rome, between January 2015 and December 2016. oprD gene was sequenced in 20 nosocomial multi-drug resistant P. aeruginosa strains. Phylogeographic, selective pressure, residue conservation analysis and homology modelling were performed. Clinical epidemiological data were extracted from patient medical records. Multi-drug resistant strains accounted for the 36% of total strains and were responsible of 20 cases of nosocomial infections. P. aeruginosa infections occurred prevalently in the West area, especially at the location IIIW and in the Geriatric ward. The time of the most recent common ancestor indicated that strains could have been introduced in the hospital since the end of the year 2009 with the most probable location in general surgery ward. By selective pressure analysis, 29 positions under diversifying selection have been identified and mapped onto the OprD model. Most of the observed residue substitutions are predicted to be destabilizing and some of them occurred in the Loops 2 and 3 that are involved in solute selection and carbapenem susceptibility. The molecular and evolutionary analysis of Multi-drug resistant strains circulating in the nosocomial setting may provide useful insights into the epidemiology and the mechanisms leading to resistance, contributing to infection control improvement.
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- 2018
3. CD38 downregulation modulates NAD+ and NADP(H) levels in thermogenic adipose tissues
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Benzi, Andrea, primary, Sturla, Laura, additional, Heine, Markus, additional, Fischer, Alexander W., additional, Spinelli, Sonia, additional, Magnone, Mirko, additional, Sociali, Giovanna, additional, Parodi, Alessia, additional, Fenoglio, Daniela, additional, Emionite, Laura, additional, Koch-Nolte, Friedrich, additional, Mittrücker, Hans-Willi, additional, Guse, Andreas H., additional, De Flora, Antonio, additional, Zocchi, Elena, additional, Heeren, Joerg, additional, and Bruzzone, Santina, additional
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- 2021
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4. Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2
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Chiara Fresia, Hugo de Jonge, Denise Galante, Laura Sturla, Enrico Millo, Tiziana Vigliarolo, Antonio De Flora, Elena Cichero, Luisa Iamele, Lucrezia Guida, Valeria Booz, Claudia Scotti, Paola Fossa, and Elena Zocchi
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0301 basic medicine ,computational studies ,Mutant ,Cooperativity ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Humans ,Binding site ,Receptor ,Site-directed mutagenesis ,Abscisic acid ,Lanthionine ,Binding Sites ,organic chemicals ,fungi ,Mutagenesis ,Membrane Proteins ,Nuclear Proteins ,food and beverages ,Cell Biology ,Phosphate-Binding Proteins ,Surface Plasmon Resonance ,Abscisic acid (ABA) ,Binding affinity ,Human Lanthionine Synthetase Component C-Like Protein 2 (LANCL2) ,site-directed mutagenesis ,Recombinant Proteins ,030104 developmental biology ,Amino Acid Substitution ,chemistry ,Mutagenesis, Site-Directed ,Abscisic Acid - Abstract
Lanthionine synthetase component C-like protein 2 (LANCL2) has been identified as the mammalian receptor mediating the functional effects of the universal stress hormone abscisic acid (ABA) in mammals. ABA stimulates insulin independent glucose uptake in myocytes and adipocytes via LANCL2 binding in vitro, improves glucose tolerance in vivo and induces brown fat activity in vitro and in vivo. The emerging role of the ABA/LANCL2 system in glucose and lipid metabolism makes it an attractive target for pharmacological interventions in diabetes mellitus and the metabolic syndrome. The aim of this study was to investigate the presence of ABA binding site(s) on LANCL2 and identify the amino acid residues involved in ABA binding. Equilibrium binding assays ([3H]-ABA saturation binding and surface plasmon resonance analysis) suggested multiple ABA-binding sites, prompting us to perform a computational study that indicated one putative high-affinity and two low-affinity binding sites. Site-directed mutagenesis (single mutant R118I, triple mutants R118I/R22I/K362I and R118I/S41A/E46I) and equilibrium binding experiments on the mutated LANCL2 proteins identified a high-affinity ABA-binding site involving R118, with a KD of 2.6 nM ± 1.2 nM, as determined by surface plasmon resonance. Scatchard plot analysis of binding curves from both types of equilibrium binding assays revealed a Hill coefficient >1, suggesting cooperativity of ABA binding to LANCL2. Identification of the high-affinity ABA-binding site is expected to allow the design of ABA agonists/antagonists, which will help to understand the role of the ABA/LANCL2 system in human physiology and disease.
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- 2018
5. Inactivation kinetics of antibiotic resistant Escherichia coli in secondary wastewater effluents by peracetic and performic acids
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Campo, Neus, primary, De Flora, Cecilia, additional, Maffettone, Roberta, additional, Manoli, Kyriakos, additional, Sarathy, Siva, additional, Santoro, Domenico, additional, Gonzalez-Olmos, Rafael, additional, and Auset, Maria, additional
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- 2020
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6. Interactions between ethanol and cigarette smoke in a mouse lung carcinogenesis model
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Rosanna T. Micale, Roumen Balansky, Manasi Nikolov, Silvio De Flora, Marietta Iltcheva, S La Maestra, Gancho Ganchev, and Vernon E. Steele
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Lung Diseases ,Male ,0301 basic medicine ,Pathology ,medicine.medical_specialty ,Lung Neoplasms ,Cigarette smoke ,Ethanol ,Lung tumors ,Histopathological alterations ,Cytogenetic damage ,Carcinogenesis ,Pulmonary toxicity ,Physiology ,Toxicology ,medicine.disease_cause ,Mice ,03 medical and health sciences ,Clastogen ,0302 clinical medicine ,Pregnancy ,Smoke ,Tobacco ,medicine ,Animals ,Drug Interactions ,Carcinogen ,Lung ,Neovascularization, Pathologic ,Chemistry ,Body Weight ,Central Nervous System Depressants ,CYP2E1 ,Survival Analysis ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Female ,Tobacco Smoke Pollution ,Chemical and Drug Induced Liver Injury ,Mutagens ,Respiratory tract - Abstract
Both ethanol and cigarette smoke are classified as human carcinogens. They can synergize, especially in tissues of the upper aerodigestive tract that are targeted by both agents. The main objective of the present study was to evaluate the individual and combined effects of ethanol and smoke in the respiratory tract, either following transplacental exposure and/or postnatal exposure. We designed two consecutive studies in mouse models by exposing Swiss H mice to oral ethanol and/or inhaled mainstream cigarette smoke for up to 4 months, at various prenatal and postnatal life stages. Clastogenic effects and histopathological alterations were evaluated after 4 and 8 months, respectively. Ethanol was per se devoid of clastogenic effects in mouse peripheral blood erythrocytes. However, especially in mice exposed both transplacentally throughout pregnancy and in the postnatal life, ethanol administration was associated not only with liver damage but also with pro-angiogenetic effects in the lung by stimulating the proliferation of blood vessels. In addition, these mice developed pulmonary emphysema, alveolar epithelial hyperplasias, microadenomas, and benign tumors. On the other hand, ethanol interfered in the lung carcinogenesis process resulting from the concomitant exposure of mice to smoke. In fact, ethanol significantly attenuated some smoke-related preneoplastic and neoplastic lesions in the respiratory tract, such as alveolar epithelial hyperplasia, microadenomas, and even malignant tumors. In addition, ethanol attenuated cigarette smoke clastogenicity. In conclusion, preclinical studies provide evidence that, in spite of its pulmonary toxicity, ethanol may mitigate some noxious effects of cigarette smoke in the respiratory tract.
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- 2016
7. CD38 downregulation modulates NAD+ and NADP(H) levels in thermogenic adipose tissues
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Sonia Spinelli, Laura Emionite, Andrea Benzi, Joerg Heeren, Markus Heine, Friedrich Koch-Nolte, Elena Zocchi, Alessia Parodi, Laura Sturla, Santina Bruzzone, Alexander W. Fischer, Hans Willi Mittrücker, Mirko Magnone, Daniela Fenoglio, Giovanna Sociali, Andreas H. Guse, and Antonio De Flora
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0301 basic medicine ,medicine.medical_specialty ,Adipose tissue ,Dehydrogenase ,White adipose tissue ,CD38 ,03 medical and health sciences ,0302 clinical medicine ,Downregulation and upregulation ,Internal medicine ,Brown adipose tissue ,medicine ,Molecular Biology ,NAD kinase ,Chemistry ,Thermogenesis ,Cell Biology ,NAD(P)(H) ,030104 developmental biology ,medicine.anatomical_structure ,Endocrinology ,030220 oncology & carcinogenesis ,Browning ,NAD+ kinase ,Brown and white adipose tissue - Abstract
Different strategies to boost NAD+ levels are considered promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme involved in the regulation of different cell functions. In the context of systemic energy metabolism, it has been demonstrated that brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis. In this study we aim to understand the functional relevance of CD38 for NAD+ and energy metabolism in BAT and WAT, also using a CD38−/− mouse model. During cold exposure, an increase in NAD+ levels occurred in BAT of wild type mice, together with a marked downregulation of CD38, as detected at the mRNA and protein level. CD38 downregulation was observed also in WAT of cold-exposed mice, where it was accompanied by a strong increase in NADP(H) levels. Accordingly, NAD kinase and glucose-6-phosphate dehydrogenase activities were enhanced in WAT (but not in BAT). Increased NAD+ levels were observed in BAT/WAT from CD38−/− compared with wild type mice, in line with CD38 being a major NAD+-consumer in AT. CD38−/− mice kept at 6 °C had higher levels of Ucp1 and Pgc-1α in BAT and WAT, and increased levels of phosphorylated hormone-sensitive lipase in BAT, compared with wild type mice. These results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT.
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- 2021
8. Pharmacological Modulation of Lung Carcinogenesis in Smokers: Preclinical and Clinical Evidence
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Rosanna T. Micale, Roumen Balansky, Gancho Ganchev, Marietta Iltcheva, Vernon E. Steele, Silvio De Flora, and Sebastiano La Maestra
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0301 basic medicine ,Lung Neoplasms ,Phenethyl isothiocyanate ,Carcinogenesis ,anti-inflammatory drugs ,Smoking Prevention ,antidiabetic drugs ,Pharmacology ,Toxicology ,Lapatinib ,Article ,antineoplastic drugs ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Animals ,Anticarcinogenic Agents ,Humans ,pharmacological prevention ,Vorinostat ,Bexarotene ,Aspirin ,Animal ,business.industry ,cigarette smoke ,Smoking ,Ascorbic acid ,Disease Models, Animal ,lung cancer ,030104 developmental biology ,chemistry ,Smoking Cessation ,030220 oncology & carcinogenesis ,Disease Models ,business ,Licofelone ,medicine.drug - Abstract
Many drugs in common use possess pleiotropic properties that make them capable of interfering with carcinogenesis mechanisms. We discuss here the ability of pharmacological agents to mitigate the pulmonary carcinogenicity of mainstream cigarette smoke. The evaluated agents include anti-inflammatory drugs (budesonide, celecoxib, aspirin, naproxen, licofelone), antidiabetic drugs (metformin, pioglitazone), antineoplastic agents (lapatinib, bexarotene, vorinostat), and other drugs and supplements (phenethyl isothiocyanate, myo-inositol, N-acetylcysteine, ascorbic acid, berry extracts). These drugs have been evaluated in mouse models mimicking interventions either in current smokers or in ex-smokers, or in prenatal chemoprevention. They display a broad spectrum of activities by attenuating either smoke-induced preneoplastic lesions or benign tumors and/or malignant tumors. Together with epidemiological data, these findings provide useful information to predict the potential effects of pharmacological agents in smokers.
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- 2016
9. Exposing native cyprinid (Barbus plebejus) juveniles to river sediments leads to gonadal alterations, genotoxic effects and thyroid disruption
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Luigi Viganò, Silvio De Flora, Marco Gobbi, Giuseppe Mascolo, Claudio Roscioli, Alberto Izzotti, Giovanna Guiso, and Alberta Mandich
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Male ,Barbus juvenile ,Intersex ,DNA adducts ,Thyroid hormones ,Bile ,River sediment ,Geologic Sediments ,Health, Toxicology and Mutagenesis ,Cyprinidae ,Thyroid Gland ,Drainage basin ,Zoology ,Endocrine System ,Aquatic Science ,Vitellogenin ,Rivers ,Barbus plebejus ,Tributary ,Animals ,Gonads ,geography ,Barbel ,geography.geographical_feature_category ,biology ,Ecology ,Aquatic animal ,Environmental Exposure ,Environmental exposure ,biology.organism_classification ,Gene Expression Regulation ,Italy ,Liver ,biology.protein ,Water Pollutants, Chemical - Abstract
Juveniles (50 days post hatch) of a native cyprinid fish (Barbus plebejus) were exposed for 7 months to sediments from the River Lambro, a polluted tributary impairing the quality of the River Po for tens of kilometers from their confluence. Sediments were collected upstream of the city of Milan and downstream at the closure of the drainage basin of the River Lambro. Chemical analyses revealed the presence of a complex mixture of bioavailable endocrine-active chemicals, with higher exposure levels in the downstream section of the tributary. Mainly characterized by brominated flame retardants, alkylphenols, polychlorinated biphenyls, and minor co-occurring personal care products and natural hormones, the sediment contamination induced reproductive disorders, as well as other forms of endocrine disruption and toxicity. In particular, exposed male barbel exhibited higher biliary PAH-like metabolites, overexpression of the cyp1a gene, vitellogenin production in all specimens, the presence of oocytes (up to 22% intersex), degenerative alterations in their testis, liver fat vacuolization, a marked depression of total thyroxine (T4) and triiodothyronine (T3) plasma levels, and genotoxic damages determined as hepatic DNA adducts. These results clearly demonstrate that Lambro sediments alone are responsible for recognizable changes in the structure and function of the reproductive and, in general, the endocrine system of a native fish species. In the real environment, exposure to waterborne and food-web sources of chemicals are responsible for additional toxic loads, and the present findings thus provide evidence for a causal role of this tributary in the severe decline observed in barbel in recent decades and raise concern that the fish community of the River Po is exposed to endocrine-mediated health effects along tens of kilometres of its course.
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- 2015
10. Multi-drug resistant Pseudomonas aeruginosa nosocomial strains: Molecular epidemiology and evolution
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Angeletti, Silvia, primary, Cella, Eleonora, additional, Prosperi, Mattia, additional, Spoto, Silvia, additional, Fogolari, Marta, additional, De Florio, Lucia, additional, Antonelli, Francesca, additional, Dedej, Etleva, additional, De Flora, Cecilia, additional, Ferraro, Elisabetta, additional, Incalzi, Raffaele Antonelli, additional, Coppola, Roberto, additional, Dicuonzo, Giordano, additional, Francescato, Fabio, additional, Pascarella, Stefano, additional, and Ciccozzi, Massimo, additional
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- 2018
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11. Abscisic Acid Transport in Human Erythrocytes
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Emilia Turco, Lucrezia Guida, Tiziana Vigliarolo, Elena Zocchi, Chiara Fresia, Antonio De Flora, and Enrico Millo
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2'-Disulfonic Acid ,Erythrocytes ,Peripheral membranes ,Environmental stress ,4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ,Biochemistry ,RBC ,Cell membrane ,chemistry.chemical_compound ,Plant Growth Regulators ,Anion Exchange Protein 1, Erythrocyte ,Abscisic acid ,Cells, Cultured ,Chromatography, High Pressure Liquid ,Mammals ,Chromatography ,Cultured ,Blotting ,food and beverages ,Biological membranes ,Cell membranes ,Cells ,Chromatographic analysis ,Cytology ,Stem cells Chromatographic methods ,Functional activities ,Human erythrocytes ,Human red blood cell ,Intracellular side ,Pancreatic beta cells ,Cell biology ,Erythrocyte ,medicine.anatomical_structure ,High Pressure Liquid ,Abscisic acid transport ,liposome ,abscisic acid (ABA) ,Signal transduction ,Western ,Intracellular ,Signal Transduction ,membrane transporter reconstitution ,Blotting, Western ,Adenylate kinase ,Biology ,Cyclase ,Chlorides ,Membrane Biology ,ATP-release ,medicine ,Humans ,Molecular Biology ,Band 3 ,cyclic AMP (cAMP) ,Anion Exchange Protein 1 ,organic chemicals ,Cell Membrane ,fungi ,Biological Transport ,Cell Biology ,ATP ,chemistry ,Abscisic Acid ,biology.protein ,4'-Diisothiocyanostilbene-2 - Abstract
Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [3H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [3H]ABA and [35S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone. Background: The plant stress hormone abscisic acid (ABA) is present and active in mammalian cells. Results: Band 3 protein is required for ABA influx into red blood cells (RBC); intracellular ABA activates adenylate cyclase resulting in [cAMP]i increase and subsequent ATP release. Conclusion: ABA influx through Band 3 activates ATP release from RBC. Significance: Paracrine ABA may regulate the ATP-mediated vasodilator response to inflammation.
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- 2015
12. Identification of a high affinity binding site for abscisic acid on human lanthionine synthetase component C-like protein 2
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Cichero, Elena, primary, Fresia, Chiara, additional, Guida, Lucrezia, additional, Booz, Valeria, additional, Millo, Enrico, additional, Scotti, Claudia, additional, Iamele, Luisa, additional, de Jonge, Hugo, additional, Galante, Denise, additional, De Flora, Antonio, additional, Sturla, Laura, additional, Vigliarolo, Tiziana, additional, Zocchi, Elena, additional, and Fossa, Paola, additional
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- 2018
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13. CD73 Protein as a Source of Extracellular Precursors for Sustained NAD+ Biosynthesis in FK866-treated Tumor Cells
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Annalisa Salis, Alessia Grozio, Alessio Nencioni, Santina Bruzzone, Laura Sturla, Irene Caffa, Nadia Raffaelli, Debora Soncini, Giovanna Sociali, and Antonio De Flora
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Nicotinamide phosphoribosyltransferase ,Down-Regulation ,Biology ,CD38 ,GPI-Linked Proteins ,Biochemistry ,Gene Expression Regulation, Enzymologic ,chemistry.chemical_compound ,Piperidines ,Cell Line, Tumor ,Neoplasms ,Extracellular ,Humans ,Gene Silencing ,Nicotinamide Phosphoribosyltransferase ,5'-Nucleotidase ,Molecular Biology ,Nicotinamide Mononucleotide ,Nicotinamide mononucleotide ,Acrylamides ,Membrane Glycoproteins ,Cell Death ,Nicotinamide ,Cell Biology ,NAD ,ADP-ribosyl Cyclase 1 ,Neoplasm Proteins ,Gene Expression Regulation, Neoplastic ,chemistry ,Nicotinamide riboside ,Cytokines ,NAD+ kinase ,Intracellular ,Signal Transduction - Abstract
NAD(+) is mainly synthesized in human cells via the "salvage" pathways starting from nicotinamide, nicotinic acid, or nicotinamide riboside (NR). The inhibition with FK866 of the enzyme nicotinamide phosphoribosyltransferase (NAMPT), catalyzing the first reaction in the "salvage" pathway from nicotinamide, showed potent antitumor activity in several preclinical models of solid and hematologic cancers. In the clinical studies performed with FK866, however, no tumor remission was observed. Here we demonstrate that low micromolar concentrations of extracellular NAD(+) or NAD(+) precursors, nicotinamide mononucleotide (NMN) and NR, can reverse the FK866-induced cell death, this representing a plausible explanation for the failure of NAMPT inhibition as an anti-cancer therapy. NMN is a substrate of both ectoenzymes CD38 and CD73, with generation of NAM and NR, respectively. In this study, we investigated the roles of CD38 and CD73 in providing ectocellular NAD(+) precursors for NAD(+) biosynthesis and in modulating cell susceptibility to FK866. By specifically silencing or overexpressing CD38 and CD73, we demonstrated that endogenous CD73 enables, whereas CD38 impairs, the conversion of extracellular NMN to NR as a precursor for intracellular NAD(+) biosynthesis in human cells. Moreover, cell viability in FK866-treated cells supplemented with extracellular NMN was strongly reduced in tumor cells, upon pharmacological inhibition or specific down-regulation of CD73. Thus, our study suggests that genetic or pharmacologic interventions interfering with CD73 activity may prove useful to increase cancer cell sensitivity to NAMPT inhibitors.
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- 2013
14. Dose-responsiveness and persistence of microRNA expression alterations induced by cigarette smoke in mouse lung
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Mariagrazia Longobardi, Silvio De Flora, Alberto Izzotti, Anna Camoirano, Vernon E. Steele, Patrizia Larghero, and Cristina Cartiglia
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medicine.medical_specialty ,Microarray ,Health, Toxicology and Mutagenesis ,medicine.disease_cause ,DNA Adducts ,Mice ,Downregulation and upregulation ,Pregnancy ,Smoke ,Internal medicine ,Tobacco ,microRNA ,Sense (molecular biology) ,Genetics ,medicine ,Animals ,Cluster Analysis ,Lung ,Molecular Biology ,Principal Component Analysis ,Dose-Response Relationship, Drug ,Microarray analysis techniques ,business.industry ,Smoking ,Deoxyguanosine ,Microarray Analysis ,Rats ,MicroRNAs ,Dose–response relationship ,Endocrinology ,medicine.anatomical_structure ,Animals, Newborn ,Gene Expression Regulation ,8-Hydroxy-2'-Deoxyguanosine ,Female ,Smoking Cessation ,Tobacco Smoke Pollution ,business ,Carcinogenesis ,Biomarkers - Abstract
Our previous studies demonstrated that exposure to cigarette smoke (CS), either mainstream or environmental, results in a remarkable downregulation of microRNA expression in the lung of both mice and rats. The goals of the present study were to evaluate the dose responsiveness to CS and the persistence of microRNA alterations after smoking cessation. ICR (CD-1) neonatal mice were exposed whole-body to mainstream CS, at the doses of 119, 292, 438, and 631mg/m(3) of total particulate matter. Exposure started within 12h after birth and continued daily for 4 weeks. The levels of bulky DNA adducts and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) were measured by (32)P postlabeling procedures, and the expression of 697 mouse microRNAs was analyzed by microarray. The highest CS dose was lethal. Exposure to CS caused a dose-dependent increase of DNA alterations. DNA adducts and, even more sharply, 8-oxodGuo were reverted 1 and 4 weeks after smoking cessation. Exposure to CS resulted in an evident dysregulation of microRNA expression profiles, mainly in the sense of downregulation. The two lowest doses were not particularly effective, while the highest nonlethal dose produced extensive microRNA alterations. The expression of most downregulated microRNAs, including among others 7 members of the let-7 family, was restored one week after smoking cessation. However, the recovery was incomplete for a limited array of microRNAs, including mir-34b, mir-345, mir-421, mir-450b, mir-466, and mir-469. Thus, it appears that microRNAs mainly behave as biomarkers of effect and that exposure to high-dose, lasting for an adequate period of time, is needed to trigger the CS-related carcinogenesis process in the experimental animal model used.
- Published
- 2011
15. Functional characterization of a synthetic abscisic acid analog with anti-inflammatory activity on human granulocytes and monocytes
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Chiara Fresia, Andrea Galatini, Alessia Grozio, Annalisa Salis, Laura Sturla, Elena Zocchi, Luca Bagnasco, Gianluca Damonte, Marta Bellotti, Antonio De Flora, Mirko Magnone, Tiziana Vigliarolo, Enrico Millo, Santina Bruzzone, and Lucrezia Guida
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medicine.medical_treatment ,Biophysics ,Inflammation ,Biology ,Granulocyte ,Binding, Competitive ,Biochemistry ,Monocytes ,Structure-Activity Relationship ,chemistry.chemical_compound ,Phagocytosis ,medicine ,Humans ,Receptor ,Molecular Biology ,Abscisic acid ,Cells, Cultured ,Innate immune system ,Chemotaxis ,Monocyte ,Anti-Inflammatory Agents, Non-Steroidal ,Cell Membrane ,fungi ,Membrane Proteins ,Nuclear Proteins ,food and beverages ,Cell Biology ,Phosphate-Binding Proteins ,Recombinant Proteins ,medicine.anatomical_structure ,chemistry ,medicine.symptom ,Abscisic Acid ,Granulocytes ,Prostaglandin E - Abstract
The phytohormone abscisic acid (ABA), in addition to regulating several important physiological functions in plants, is also produced and released by human granulocytes and monocytes where it stimulates cell activities involved in the innate immune response. Here we describe the properties of an ABA synthetic analog that competes with the hormone for binding to human granulocyte membranes and to purified recombinant LANCL2 (the human ABA receptor) and inhibits several ABA-triggered inflammatory functions of granulocytes and monocytes in vitro: chemotaxis, phagocytosis, reactive oxygen species production and release of prostaglandin E(2) (PGE(2)) by human granulocytes, release of PGE(2) and of monocyte chemoattractant protein-1 by human monocytes. This observation provides a proof of principle that ABA antagonists may represent a new class of anti-inflammatory agents.
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- 2011
16. Influence of laser hardening on the tribological properties of forged steel for hot rolls
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M.G. De Flora and Massimo Pellizzari
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Materials science ,Carbon steel ,Metallurgy ,Oxide ,Surfaces and Interfaces ,Tribology ,engineering.material ,Condensed Matter Physics ,Microstructure ,Hardness ,Surfaces, Coatings and Films ,chemistry.chemical_compound ,chemistry ,Mechanics of Materials ,Martensite ,Materials Chemistry ,Hardening (metallurgy) ,engineering ,Adhesive ,Composite material - Abstract
The influence of laser hardening on the tribological behaviour of a forged steel for hot profiled rolls is evaluated using a customary test rig which provides the rolling–sliding contact between the roll and a C40 plain carbon steel counterpart. Samples treated with different lasers and processing parameters were considered. Starting from a base value of 300 HV the surface hardness was increased up to 800 HV and the total case depth ranged from 1.2 to 2 mm. The benefits of the laser treatment could be clearly observed at low test temperature, due to the positive influence of a hard martensite microstructure in preventing severe metallic wear by adhesive fracture. The influence of laser hardening was less evident at high temperature, where the tendency of different microstructures towards the formation of wear protective oxide layers could result in a better or worse resistance compared to the base steel, respectively. The capability of the roll material to produce wear particles and their entrapment within the contact interface resulted of fundamental importance for the formation of oxide glazes.
- Published
- 2011
17. P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy
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Santina Bruzzone, Giovanna Basile, Elena Zocchi, Antonio De Flora, Laura Sturla, Federica Benvenuto, Iliana Moreschi, Cesare Usai, Angelo Schenone, Fulvia Fiorese, and Lucilla Nobbio
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congenital, hereditary, and neonatal diseases and abnormalities ,Small interfering RNA ,Blotting, Western ,Ciliary neurotrophic factor ,Biochemistry ,Calcium in biology ,Animals, Genetically Modified ,Rats, Sprague-Dawley ,Basal (phylogenetics) ,Dorsal root ganglion ,Charcot-Marie-Tooth Disease ,1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine ,Gene duplication ,Purinergic P2 Receptor Antagonists ,medicine ,Extracellular ,Animals ,Enzyme Inhibitors ,RNA, Small Interfering ,Molecular Biology ,Gene ,Cells, Cultured ,Membrane Potential, Mitochondrial ,Microscopy ,biology ,Receptors, Purinergic P2 ,Reverse Transcriptase Polymerase Chain Reaction ,Mechanisms of Signal Transduction ,Cell Biology ,Immunohistochemistry ,Molecular biology ,Rats ,Cell biology ,medicine.anatomical_structure ,Gene Expression Regulation ,biology.protein ,Calcium ,Receptors, Purinergic P2X7 ,Schwann Cells ,Myelin Proteins ,Demyelinating Diseases - Abstract
Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca(2+)](i)) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca(2+)](i) is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca(2+) into CMT1A SC. Correction of the altered [Ca(2+)](i) in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca(2+)](i) and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.
- Published
- 2009
18. Abscisic Acid Released by Human Monocytes Activates Monocytes and Vascular Smooth Muscle Cell Responses Involved in Atherogenesis
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Mirko Magnone, Antonio De Flora, Domenico Palombo, Lucrezia Guida, Elena Zocchi, Santina Bruzzone, Gianluca Damonte, Enrico Millo, Cesare Usai, Sonia Scarfì, and Laura Sturla
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Arterial tissue ,Vascular smooth muscle ,Second Messenger Systems ,Biochemistry ,Hemostatics ,Monocytes ,Muscle, Smooth, Vascular ,chemistry.chemical_compound ,Plant Growth Regulators ,Cell Movement ,Prostaglandin E2 ,Abscisic acid ,Aorta ,Cells, Cultured ,Chemokine CCL2 ,Reverse Transcriptase Polymerase Chain Reaction ,Mechanisms of Signal Transduction ,NF-kappa B ,Thrombin ,food and beverages ,Atherogenesis ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,Second messenger system ,Abscisic acid, Activated platelets, ADP-ribose, Arterial tissue, Atherogenesis ,ADP-ribose ,medicine.drug ,Blotting, Western ,Biology ,Dinoprostone ,Paracrine signalling ,medicine ,Humans ,RNA, Messenger ,Platelet activation ,Autocrine signalling ,Molecular Biology ,Cell Proliferation ,Monocyte ,fungi ,Activated platelets ,Cell Biology ,Atherosclerosis ,Platelet Activation ,chemistry ,Cyclooxygenase 2 ,Calcium - Abstract
Abscisic acid (ABA) is a phytohormone recently identified as a new endogenous pro-inflammatory hormone in human granulocytes. Here we report the functional activation of human monocytes and vascular smooth muscle cells by ABA. Incubation of monocytes with ABA evokes an intracellular Ca2+ rise through the second messenger cyclic ADP-ribose, leading to NF-kappaB activation and consequent increase of cyclooxygenase-2 expression and prostaglandin E2 production and enhanced release of MCP-1 (monocyte chemoattractant protein-1) and of metalloprotease-9, all events reportedly involved in atherogenesis. Moreover, monocytes release ABA when exposed to thrombin-activated platelets, a condition occurring at the injured vascular endothelium; monocyte-derived ABA behaves as an autocrine and paracrine pro-inflammatory hormone-stimulating monocyte migration and MCP-1 release, as well as vascular smooth muscle cells migration and proliferation. These results, and the presence of ABA in human arterial plaques at a 10-fold higher concentration compared with normal arterial tissue, identify ABA as a new signal molecule involved in the development of atherosclerosis and suggest a possible new target for anti-atherosclerotic therapy.
- Published
- 2009
19. Hot friction and wear behaviour of high speed steel and high chromium iron for rolls
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M.G. De Flora, Massimo Pellizzari, and D. Cescato
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Materials science ,Metallurgy ,Surfaces and Interfaces ,Tribology ,Condensed Matter Physics ,Microstructure ,Surfaces, Coatings and Films ,Abrasion (geology) ,Carbide ,Mechanics of Materials ,Martensite ,Materials Chemistry ,Tempering ,High-speed steel ,Eutectic system - Abstract
The wear resistance at high temperature of different high speed steels (HSS) for rolls is evaluated on the basis of a tribological test aimed at reproducing the damage mechanisms occur during hot rolling. The test is positively used to highlight the different wear and friction behaviour of high speed steels and high chromium irons to be used in the early finishing stands of hot strip mill. The microstructure of these materials is given by a tempered martensitic matrix surrounded by an interconnected network of primary and eutectic carbides. In this paper a closer look into the properties of high speed steels is proposed. A wide range of microstructural conditions was obtained by changing the chemical composition and even by tempering the steels at different temperatures, in order to obtain different matrix microhardnesses. The wear behaviour can be explained on the basis of the operating mechanism, given by the combination of abrasion, triboxidation and by adhesion. While abrasion rules the tribological behaviour of HSS, triboxidation becomes more and more decisive in controlling the behaviour of HiCr irons. In these alloys the friction coefficient shows a transition (not observed in HSS), which was ascribed to the transition form a metal–oxide to an oxide–oxide contact, i.e., to the formation of wear protective layers. The tribological behaviour was correlated to the microstructure.
- Published
- 2009
20. The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes
- Author
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Malara, Alessandro, primary, Fresia, Chiara, additional, Di Buduo, Christian Andrea, additional, Soprano, Paolo Maria, additional, Moccia, Francesco, additional, Balduini, Cesare, additional, Zocchi, Elena, additional, De Flora, Antonio, additional, and Balduini, Alessandra, additional
- Published
- 2017
- Full Text
- View/download PDF
21. Abscisic acid enhances glucose disposal and induces brown fat activity in adipocytes in vitro and in vivo
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Sturla, Laura, primary, Mannino, Elena, additional, Scarfì, Sonia, additional, Bruzzone, Santina, additional, Magnone, Mirko, additional, Sociali, Giovanna, additional, Booz, Valeria, additional, Guida, Lucrezia, additional, Vigliarolo, Tiziana, additional, Fresia, Chiara, additional, Emionite, Laura, additional, Buschiazzo, Ambra, additional, Marini, Cecilia, additional, Sambuceti, Gianmario, additional, De Flora, Antonio, additional, and Zocchi, Elena, additional
- Published
- 2017
- Full Text
- View/download PDF
22. Mutagenesis and cardiovascular diseases
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Alberto Izzotti and Silvio De Flora
- Subjects
Genetics ,Mutation ,education.field_of_study ,Molecular epidemiology ,DNA damage ,Health, Toxicology and Mutagenesis ,Population ,Cancer ,Inflammation ,Biology ,Bioinformatics ,medicine.disease_cause ,medicine.disease ,Germline mutation ,medicine ,medicine.symptom ,education ,Molecular Biology ,Oxidative stress - Abstract
Although no generalization can be made, it is of interest that cancer, cardiovascular diseases, and other chronic conditions often share common risk factors and common protective factors as well as common pathogenetic determinants, such as DNA damage, oxidative stress, and chronic inflammation. Atherosclerosis is the most important cause of vascular forms representing the major cause of death in the population of many geographical areas. A great deal of studies support the "response-to-injury" theory. A variety of experimental and epidemiological findings are also in favor of the somatic mutation theory, which maintains that the earliest event in the atherogenic process is represented by mutations in arterial smooth muscle cells, akin to formation of a benign tumor. These two theories can be harmonized, also taking into account the highly diversified nature of atherosclerotic lesions. Molecular epidemiology studies performed in our laboratory and other laboratories have shown that DNA adducts are systematically present in arterial smooth muscle cells, and their levels are correlated with atherogenic risk factors known from traditional epidemiology. Oxidative DNA damage was also consistently detected in these cells. The role of glutathione S-transferase polymorphisms on the frequency of the above molecular alterations and of arterial diseases is rather controversial. Prevention of both cancer and atherosclerosis is based on avoidance of exposure to risk factors and on fortification of the host defense mechanisms by means of dietary principles and chemopreventive drugs.
- Published
- 2007
23. Lipid peroxidation-derived etheno-DNA adducts in human atherosclerotic lesions
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Jagadeesan Nair, Silvio De Flora, Alberto Izzotti, and Helmut Bartsch
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Male ,medicine.medical_specialty ,DNA damage ,Health, Toxicology and Mutagenesis ,Inflammation ,medicine.disease_cause ,Deoxycytidine ,Lipid peroxidation ,DNA Adducts ,chemistry.chemical_compound ,medicine.artery ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Aorta, Abdominal ,Molecular Biology ,Aged ,Aged, 80 and over ,chemistry.chemical_classification ,Aorta ,Reactive oxygen species ,Deoxyadenosines ,Molecular Structure ,Cell growth ,Smoking ,Middle Aged ,Atherosclerosis ,Endocrinology ,chemistry ,Biochemistry ,Lipid Peroxidation ,medicine.symptom ,DNA ,Oxidative stress - Abstract
Atherosclerosis and cancer are characterized by uncontrolled cell proliferation and share common risk factors, such as cigarette smoking, dietary habits and ageing. Growth of smooth muscle cells (SMCs) in atherosclerotic plaques may result from DNA damage, caused either by exogenous mutagens or by agents endogenously generated due to oxidative stress and lipid peroxidation (LPO). Hydroxy-2-nonenal (HNE), a major LPO product, binds covalently to cellular DNA to form the exocyclic etheno-DNA-base adducts, 1,N(6)-ethenodeoxyadenine (varepsilondA) and 3,N(4)-ethenodeoxycytosine (varepsilondC). By applying an ultrasensitive (32)P-postlabeling-immunoaffinity method, varepsilondA and varepsilondC were quantified in abdominal aorta SMCs from 13 atherosclerotic patients and 3 non-smoking subjects without atherosclerotic lesions. The levels of etheno-adducts ranged for varepsilondA from 2.3 to 39.6/10(8)dA and for varepsilondC from 10.7 to 157.7/10(8)dC, with a high correlation between varepsilondA and varepsilondC (r=0.84, P=0.0001). Etheno-adduct levels were higher in atherosclerotic smokers than in ex-smokers for both varepsilondA (means 15.2 versus 7.3, P=0.06) and varepsilondC (71.9 versus 51.6, not significant). varepsilondC levels were higher in either ex-smokers (P=0.03) or smokers (P=0.07) than in non-smokers. There was a poor correlation between either varepsilondA or varepsilondC and 8-hydroxy-2'-deoxyguanosine, whereas significant positive correlations were detected with the levels of several postlabeled bulky aromatic DNA adducts. In conclusion, two different types of DNA damage may be involved in atherosclerotic plaque formation and progression: (i) bulky aromatic compounds, to which aorta SMCs are chronically exposed in smokers, can either covalently bind to DNA, induce redox-cycling via quinone intermediates and/or activate local chronic inflammatory processes in the arterial wall; ii) this in turn leads to a self perpetuating generation of reactive oxygen species, LPO-products and increasing DNA-damage, as documented by the presence of high levels of miscoding etheno-DNA adducts in human aorta SMCs.
- Published
- 2007
24. Survival of atherosclerotic patients as related to oxidative stress and gene polymorphisms
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Lucia Perrone, Marina Vercelli, Giuseppe Minniti, Silvio De Flora, Alberto Izzotti, and A. Piana
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medicine.medical_specialty ,Mitochondrial DNA ,Homocysteine ,DNA damage ,Health, Toxicology and Mutagenesis ,Kaplan-Meier Estimate ,Biology ,medicine.disease_cause ,chemistry.chemical_compound ,Internal medicine ,Genetics ,medicine ,Humans ,Prospective Studies ,Molecular Biology ,Gene ,Atherosclerosis ,Malondialdehyde ,Glutathione ,Molecular biology ,Oxidative Stress ,Endocrinology ,chemistry ,Methylenetetrahydrofolate reductase ,biology.protein ,Restriction fragment length polymorphism ,Biomarkers ,Polymorphism, Restriction Fragment Length ,Oxidative stress ,DNA Damage ,Follow-Up Studies - Abstract
A prospective molecular epidemiology study was implemented in a cohort of 98 subjects suffering from severe atherosclerotic lesions requiring removal of an abdominal aorta fragment. We previously published the results relative to detection, in the aorta medium layer, of bulky DNA adducts and fluorescent polycyclic aromatic hydrocarbon-related DNA adducts, oxidative DNA damage, and mitochondrial DNA 4977 common deletion, as well as GSTM1 and GSTT1 gene polymorphisms. We report herein new data, relative to oxidative stress biomarkers, including oxidative DNA damage in both inner and medium aorta layers, malondialdehyde in the medium layer, homocysteine and reduced glutathione in plasma, and those relative to additional gene polymorphisms, including NAT1, NAT2, OGG1, MTHFR, Leiden factor V, and prothrombin. The results of biochemical and molecular analyses were related to survival of the patients, whose average age was 70 at the start of the follow up. During the following 14 years, 71.4% of them died. The results obtained provide evidence for the crucial impact of oxidative stress and certain gene polymorphisms on clinical and biochemical patterns as well as on survival of patients. Survival was significantly affected not only by traditional risk factors for atherosclerosis but also by molecular end-points and adverse gene polymorphisms, and by their combinations.
- Published
- 2007
25. Chemoprevention of smoke-induced alopecia in mice by oral administration of l-cystine and vitamin B6
- Author
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Francesco D'Agostini, Tanya M. Pennisi, Paolo Fiallo, and Silvio De Flora
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Drug ,medicine.medical_specialty ,media_common.quotation_subject ,Cystine ,Administration, Oral ,Apoptosis ,Dermatology ,Chemoprevention ,Biochemistry ,Mice ,chemistry.chemical_compound ,Oral administration ,Internal medicine ,medicine ,Animals ,Molecular Biology ,media_common ,Smoke ,Dose-Response Relationship, Drug ,integumentary system ,business.industry ,Body Weight ,Alopecia ,Glutathione ,Vitamin B 6 ,Acetylcysteine ,Mice, Inbred C57BL ,Dose–response relationship ,Endocrinology ,chemistry ,Female ,Tobacco Smoke Pollution ,business ,Hair Follicle ,Cysteine - Abstract
Summary Background We previously demonstrated that high doses of environmental cigarette smoke (ECS) induce alopecia in mice. This effect was prevented by the oral administration of N-acetylcysteine (NAC), an analogue and precursor of l -cysteine and reduced glutathione. Objectives The present study aimed at assessing whether l -cystine, the oxidized form of l -cysteine, which is a key hair component, may behave like NAC in inhibiting ECS-induced alopecia and modulating the mechanisms responsible for this condition. Methods C57BL/6 mice were exposed whole-body to ECS in a smoking machine. Groups of mice received in the diet, at three dose levels, a mixture of l -cystine with vitamin B6, which plays a role in l -cystine incorporation in hair cells. Occurrence of alopecia areas and apoptosis of hair bulb cells were evaluated for up to 6 months of exposure, and the time course induction of micronucleated erythrocytes in peripheral blood was investigated. Results The frequency of micronucleated erythrocytes was increased by ECS, irrespective of treatment with l -cystine/vitamin B6. ECS-induced alopecia and apoptosis of hair bulb cells in all exposed mice. l -Cystine/vitamin B6 inhibited alopecia in a dose-dependent fashion. Conclusions High-dose ECS induces apoptosis-related alopecia in mice, and oral administration of l -cystine/vitamin B6 is an effective preventive treatment.
- Published
- 2007
26. Interactions between ethanol and cigarette smoke in a mouse lung carcinogenesis model
- Author
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Balansky, Roumen, primary, Ganchev, Gancho, additional, Iltcheva, Marietta, additional, Nikolov, Manasi, additional, La Maestra, S., additional, Micale, Rosanna T., additional, Steele, Vernon E., additional, and De Flora, Silvio, additional
- Published
- 2016
- Full Text
- View/download PDF
27. Oral chromium(VI) does not affect the frequency of micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses
- Author
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Marietta Iltcheva, Silvio De Flora, and Roumen Balansky
- Subjects
Chromium ,Male ,Hematopoietic System ,Health, Toxicology and Mutagenesis ,medicine.medical_treatment ,Intraperitoneal injection ,Administration, Oral ,Physiology ,medicine.disease_cause ,Mice ,chemistry.chemical_compound ,Fetus ,Chromates ,Genetics ,medicine ,Animals ,Potassium dichromate ,Micronuclei, Chromosome-Defective ,Carcinogen ,chemistry ,Toxicity ,Sodium dichromate ,Micronucleus test ,Immunology ,Female ,Potassium Dichromate ,Micronucleus ,Genotoxicity - Abstract
Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500 mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7 mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.
- Published
- 2006
28. Development of novel cancer chemopreventive agents in Europe – Neglected Cinderella or rising phoenix?
- Author
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James A. Crowell, Maurizio D'Incalci, Silvio De Flora, Enrico Mihich, Helmut Bartsch, Norbert Frank, Andreas J. Gescher, Clarissa Gerhäuser, Christian Dittrich, Giampaolo Tortora, and Christian Steffen
- Subjects
Cancer Research ,medicine.medical_specialty ,Pathology ,business.industry ,Cancer chemoprevention ,Alternative medicine ,Cancer ,medicine.disease ,Human health ,Clinical work ,Oncology ,Novel agents ,Family medicine ,Potential biomarkers ,medicine ,business ,Pharmaceutical industry - Abstract
Agents that prevent cancer, delay its onset, or revert premalignant conditions could have dramatic beneficial impacts on human health. Although there is an urgent need to develop cancer chemopreventive agents, researchers in the field suspect that this area of scientific endeavour in Europe leads a Cinderella existence, both in terms of perception of importance and research funding. In order to review current activities in this prevention field and to seek a consensus position, an exploratory workshop was held in September 2005 at the German Cancer Research Center (DKFZ) in Heidelberg, Germany, sponsored mainly by the European Science Foundation (ESF), and also supported by the European Association for Cancer Research (EACR) and the German Cancer Society (DKG). The 35 experts from European countries and the United States of America assessed state-of-the-art cancer chemoprevention research in Europe. The aims that the workshop organizers had pre-defined were: i) assessment of the usefulness of animal models for agent identification; ii) review of ongoing preclinical and clinical work on novel agents; iii) discussion of potential biomarkers predictive for cancer preventive efficacy; and finally iv) the potential role that European pharmaceutical industries could play in furthering chemopreventive agent development. Overall the workshop aimed at raising awareness among European clinical and laboratory researchers of the importance of the development of novel, efficacious and safe cancer preventive agents.
- Published
- 2006
29. Mechanistic approaches to chemoprevention of mutation and cancer
- Author
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Silvio De Flora, Lynnette R. Ferguson, and Giorgio Bronzetti
- Subjects
Glutathione S-transferases ,Tumor angiogenesis ,Antimutagen ,Health, Toxicology and Mutagenesis ,Apoptosis ,Biology ,Anticarcinogen ,Neoplasms ,Genetics ,Screening method ,medicine ,Animals ,Anticarcinogenic Agents ,Humans ,Molecular Biology ,Cancer ,Antimutagenic Agents ,medicine.disease ,DNA repair enzymes ,Mutagenesis ,Selective estrogen receptor modulator ,Mutation (genetic algorithm) ,Cancer research ,Extended time - Abstract
This is the eighth special issue of Mutation Research to focus on antimutagenesis and anticarcinogenesis. It covers a wide range of mechanisms from prevention of cancer initiation by antimutagens through to inhibition of tumour angiogenesis and selective estrogen receptor modulators. New screening methods and new biomarkers are also elucidated. There is increasing reason to believe that the long-term use of a combination of anticarcinogens, over an extended time span, may provide a realistic prospect of reducing the current burden of human cancers.
- Published
- 2005
30. Modulation of apoptosis by cancer chemopreventive agents
- Author
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Carlo Bennicelli, Alberto Izzotti, Francesco D'Agostini, Silvio De Flora, and Roumen Balansky
- Subjects
Male ,Phenethyl isothiocyanate ,Light ,Health, Toxicology and Mutagenesis ,Nude ,Respiratory System ,Apoptosis ,Stimulation ,drug effects/genetics/physiology ,medicine.disease_cause ,Epithelium ,Rats, Sprague-Dawley ,Mice ,chemistry.chemical_compound ,Sulindac ,Pregnancy ,Neoplasms ,Smoke ,Oligonucleotide Array Sequence Analysis ,Animals, Anticarcinogenic Agents ,pharmacology, Antineoplastic Agents ,pharmacology, Apoptosis ,drug effects/genetics/physiology, Epithelium ,drug effects/physiology, Female, Gene Expression Profiling, Humans, In Situ Nick-End Labeling, Light, Male, Mice, Mice ,Nude, Molecular Sequence Data, Neoplasms ,pathology/prevention /&/ control, Oligonucleotide Array Sequence Analysis, Pregnancy, Rats, Rats ,Sprague-Dawley, Respiratory System ,drug effects/metabolism/pathology, Smoke, Sulindac ,pharmacology, Tobacco ,drug effects/metabolism/pathology ,Female ,medicine.drug ,Programmed cell death ,Molecular Sequence Data ,Mice, Nude ,Antineoplastic Agents ,pathology/prevention /&/ control ,Biology ,Tobacco ,Oltipraz ,In Situ Nick-End Labeling ,Genetics ,medicine ,Animals ,Anticarcinogenic Agents ,Humans ,Molecular Biology ,Gene Expression Profiling ,Ascorbic acid ,Rats ,drug effects/physiology ,chemistry ,Immunology ,Cancer research ,Sprague-Dawley ,pharmacology ,Carcinogenesis - Abstract
A review of almost 2000 studies showed that the large majority of 39 putative cancer chemopreventive agents induced "spontaneous" apoptosis. Inhibition of the programmed cell death triggered by a variety of stimuli was consistently reported only with ascorbic acid, alpha-tocopherol, and N-acetylcysteine (NAC). We performed experimental studies in rodents exposed to cigarette smoke, either mainstream (MCS) or environmental (ECS), and UV-A/B-containing light. The nonsteroidal anti-inflammatory drug sulindac did not affect the apoptotic process in the skin of light-exposed mice and in the lungs of ECS-exposed mice. Likewise, 5,6-benzoflavone, indole-3-carbinol, 1,2-dithiole-3-thione and oltipraz failed to modulate apoptosis in the respiratory tract of ECS-exposed rats. Phenethyl isothiocyanate further enhanced the frequency of apoptosis in pulmonary alveolar macrophages and bronchial epithelial cells, and upregulated several genes in the lung of ECS-exposed rats. Both individually and in combination with oltipraz, NAC inhibited apoptosis in the respiratory tract of rats exposed either to MCS or ECS. Moreover, NAC attenuated the ECS-related overexpression of proapoptotic genes and normalized the levels of proapoptotic proteins in rat lung. The transplacental administration of NAC to mice considerably attenuated gene overexpression in the liver of fetuses exposed to ECS throughout pregnancy. Inhibition of apoptosis by chemopreventive agents reflects their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress that trigger apoptosis. On the other hand, enhancement of apoptosis is a double-edged sword, since it represents a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other degenerative diseases. We suggest that stimulation of apoptosis by so many chemopreventive agents, as reported in the literature, may often reflect the occurrence of toxic effects at high doses.
- Published
- 2005
31. Multiple drug resistance, antimutagenesis and anticarcinogenesis
- Author
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Silvio De Flora and Lynnette R. Ferguson
- Subjects
Abcg2 ,biology ,Health, Toxicology and Mutagenesis ,Antimutagenic Agents ,Mutagen ,ATP-binding cassette transporter ,Endogeny ,Pharmacology ,medicine.disease_cause ,Drug Resistance, Multiple ,Multiple drug resistance ,Biochemistry ,Neoplasms ,Genetics ,biology.protein ,medicine ,Animals ,Anticarcinogenic Agents ,Humans ,ATP Binding Cassette Transporter, Subfamily B, Member 1 ,Carcinogenesis ,Molecular Biology ,Gene ,Carcinogen - Abstract
Many cells are protected from excess levels of exogenous chemicals, including mutagens and carcinogens as well as pharmaceutical agents, by being actively extruded through the action of one or more of a series of ATP-binding cassette drug transporter proteins. Those known to be important in humans are the multidrug resistance proteins (P-glycoproteins, encoded by the mdr 1 and 3 genes), multidrug-resistance-associated proteins (MRP1–7) and the breast cancer resistance protein (BCRP). These proteins have overlapping but distinct cellular locations and substrate specificities, and jointly govern the likelihood of penetration or distribution of a given mutagen or carcinogen into various tissues including the brain, testis, ovaries and fetus. Thus, they can affect the absorption, distribution and excretion of mutagens and carcinogens, as well as of their metabolites and conjugates, in most cases acting to prevent or reduce mutagenesis or carcinogenesis. However, because ABC transporters may limit the success of chemotherapy, there has been a considerable effort by the pharmaceutical industry to develop inhibitors of this transport process, and these are increasing in use. In general, the mutagenicity of many chemicals may be increased at the cellular levels by the action of these inhibitors, while the altered absorption characteristics favour greater uptake into the body. Thus, in many cases, such inhibitors may counter the antimutagenic and anticarcinogenic effect of the multidrug resistance mechanisms. There are exceptions, however. An increasing number of single nucleotide polymorphisms in multidrug resistance genes are being identified in humans, and may account for many of the significant differences in inter-individual susceptibility to exogenous and endogenous mutagenic and carcinogenic insults.
- Published
- 2005
32. Birth-related genomic and transcriptional changes in mouse lung
- Author
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Mariagrazia Longobardi, Elena Tampa, Anna Camoirano, Roumen Balansky, Cristina Cartiglia, Alberto Izzotti, and Silvio De Flora
- Subjects
Fetus ,Lung ,DNA repair ,Health, Toxicology and Mutagenesis ,Transplacental ,Biology ,medicine.disease_cause ,Molecular biology ,Andrology ,Acetylcysteine ,Pulmonary respiration ,medicine.anatomical_structure ,Gene expression ,Genetics ,medicine ,Oxidative stress ,medicine.drug - Abstract
Birth is characterized by a sudden transition from the maternal-mediated respiration to the autonomous pulmonary respiration. Notwithstanding the importance of the involved functional and metabolic changes, little is known about possible DNA alterations occurring in the lung during the perinatal period. We comparatively evaluated genomic and transcriptional changes in the lung of fetuses and newborn Swiss albino mice, whose dams had either been untreated or treated with oral N-acetyl- l -cysteine (NAC) throughout the pregnancy period. In the less than 24 h period elapsing between the end of fetal life and the start of post-natal life, nucleotide alterations occurred in mouse lung, as shown by a significant increase of both bulky DNA adducts and 8-hydroxy-2′-deoxyguanosine levels, detected by 32 P post-labeling procedures. The frequency of micronuclei in peripheral blood erythrocytes was not significantly increased after birth. Multigene expression analysis of 746 selected genes, by cDNA arrays, showed that 33 of them (4.4%) were upregulated in the lung of newborn mice, as compared with fetuses. The overexpressed genes were mainly involved in protective mechanism as a response to oxidative changes, alterations of glutathione metabolism, cellular stress, and damage to DNA and proteins. The transplacental treatment with NAC totally prevented birth-related genomic alterations in lung DNA. NAC did not change the basal gene expression in mouse fetal lung, but attenuated the upregulation of most genes involved in oxidative stress, stress response, and DNA repair in the lung of newborn mice. In fact, only 13 genes (1.7%) were overexpressed in newborns from NAC-treated dams. It therefore appears that administration of NAC during pregnancy is beneficial not only to counteract the adverse effects of toxic agents, as supported by previous studies, but also to attenuate birth-related DNA alterations.
- Published
- 2003
33. Modulation of cigarette smoke-related end-points in mutagenesis and carcinogenesis
- Author
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Roumen Balansky, Maria Bagnasco, Carlo Bennicelli, Silvio De Flora, Anna Camoirano, Elena Tampa, Francesco D'Agostini, Alberto Izzotti, Ronald A. Lubet, Cristina Cartiglia, and Maria Grazia Longobardi
- Subjects
Lung Neoplasms ,Phenethyl isothiocyanate ,Health, Toxicology and Mutagenesis ,medicine.disease_cause ,Toxicology ,chemistry.chemical_compound ,In vivo ,Smoke ,Oltipraz ,Genetics ,Animals ,Humans ,Medicine ,Lung cancer ,Molecular Biology ,Carcinogen ,business.industry ,Smoking ,Cancer ,medicine.disease ,Gene Expression Regulation ,chemistry ,Models, Animal ,Carcinogens ,Cancer research ,Animal studies ,business ,Carcinogenesis ,Mutagens - Abstract
The epidemic of lung cancer and the increase of other tumours and chronic degenerative diseases associated with tobacco smoking have represented one of the most dramatic catastrophes of the 20th century. The control of this plague is one of the major challenges of preventive medicine for the next decades. The imperative goal is to refrain from smoking. However, chemoprevention by dietary and/or pharmacological agents provides a complementary strategy, which can be targeted not only to current smokers but also to former smokers and passive smokers. This article summarises the results of studies performed in our laboratories during the last 10 years, and provides new data generated in vitro, in experimental animals and in humans. We compared the ability of 63 putative chemopreventive agents to inhibit the bacterial mutagenicity of mainstream cigarette smoke. Modulation by ethanol and the mechanisms involved were also investigated both in vitro and in vivo. Several studies evaluated the effects of dietary chemopreventive agents towards smoke-related intermediate biomarkers in various cells, tissues and organs of rodents. The investigated end-points included metabolic parameters, adducts to haemoglobin, bulky adducts to nuclear DNA, oxidative DNA damage, adducts to mitochondrial DNA, apoptosis, cytogenetic damage in alveolar macrophages, bone marrow and peripheral blood erytrocytes, proliferation markers, and histopathological alterations. The agents tested in vivo included N-acetyl-L-cysteine, 1,2-dithiole-3-thione, oltipraz, phenethyl isothiocyanate, 5,6-benzoflavone, and sulindac. We started applying multigene expression analysis to chemoprevention research, and postulated that an optimal agent should not excessively alter per se the physiological background of gene expression but should be able to attenuate the alterations produced by cigarette smoke or other carcinogens. We are working to develop an animal model for the induction of lung tumours following exposure to cigarette smoke. The most encouraging results were so far obtained in models using A/J mice and Swiss albino mice. The same smoke-related biomarkers used in animal studies can conveniently be applied to human chemoprevention studies. We participated in trials evaluating the effects of N-acetyl-L-cysteine and oltipraz in smokers from Italy, The Netherlands, and the People's Republic of China. We are trying to develop a pharmacogenomic approach, e.g. based on genetic metabolic polymorphisms, aimed at predicting not only the risk of developing cancer but also the individual responsiveness to chemopreventive agents.
- Published
- 2003
34. Formation of DNA adducts in the aorta of smoke-exposed rats, and modulation by chemopreventive agents
- Author
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Anna Camoirano, Cristina Cartiglia, Silvio De Flora, Elena Tampa, and Alberto Izzotti
- Subjects
Male ,Phenethyl isothiocyanate ,Arteriosclerosis ,Health, Toxicology and Mutagenesis ,Aorta, Thoracic ,Thiophenes ,Pharmacology ,Weight Gain ,Chemoprevention ,Tobacco smoke ,Rats, Sprague-Dawley ,DNA Adducts ,Eating ,chemistry.chemical_compound ,medicine.artery ,Oltipraz ,Genotype ,Genetics ,medicine ,Animals ,Thoracic aorta ,Anticarcinogen ,Aorta ,Chemistry ,Smoking ,Thiones ,Acetylcysteine ,Rats ,Biochemistry ,Pyrazines ,Toxicity - Abstract
Our previous studies showed that nucleotide alterations, evaluated by (32)P postlabeling, are systematically detected in smooth muscle cells of atherosclerotic lesions localized in the aorta of surgical patients. The level of these molecular lesions was correlated with the occurrence of known atherogenic risk factors, among which the number of currently smoked cigarettes, and was significantly enhanced in individuals having a null GSTM1 genotype as compared to individuals carrying the GSTM1 genotype. The present study had the dual objective of evaluating the formation of DNA adducts in the whole thoracic aorta of Sprague-Dawley rats, exposed whole-body to cigarette smoke for 28 consecutive days, and of investigating the effects of chemopreventive agents given orally during the same period. High levels of (32)P postlabeled DNA adducts were formed in the aorta of smoke-exposed rats, with an overall 11 times increase over the total levels observed in sham-exposed rats, and with increases ranging between three and 63 times for seven individual DNA adducts. Supplement of the diet with either 1,2-dithiole-3-thione, phenethyl isothiocyanate or 5,6-benzoflavone had no or poor effects on the smoke-related formation of nucleotide alterations in the aorta. In contrast, oltipraz, given with the diet, N-acetyl-L-cysteine, given with drinking water and, even more potently, their combination exerted remarkable protective effects. The results of this experimental study, together with the previous findings in humans, suggest that DNA alterations may contribute to the atherogenic process, clarify a possible mechanism of cigarette smoke, a well known atherogen, and show the potential protective effects of certain drugs towards these alterations.
- Published
- 2001
35. Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase by kinetic and crystallographic characterization of site-specific mutants
- Author
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Antonio De Flora, Michela Tonetti, Camillo Rosano, Gaetano Izzo, Martino Bolognesi, Angela Bisso, and Laura Sturla
- Subjects
Guanosine Diphosphate Mannose ,Models, Molecular ,Protein Conformation ,Stereochemistry ,Mannose ,Isomerase ,Reductase ,Crystallography, X-Ray ,Catalysis ,Fucose ,Substrate Specificity ,Structure-Activity Relationship ,chemistry.chemical_compound ,Multienzyme Complexes ,Structural Biology ,Deoxy Sugars ,Enzyme Stability ,Escherichia coli ,Coenzyme binding ,Phosphofructokinase 2 ,Molecular Biology ,Short-chain dehydrogenase ,Binding Sites ,Escherichia coli Proteins ,Hydrogen Bonding ,Ketone Oxidoreductases ,Enzyme structure ,Kinetics ,Crystallography ,Amino Acid Substitution ,chemistry ,Biochemistry ,Mutation ,Mutagenesis, Site-Directed ,Chromatography, Thin Layer ,Protons ,Carbohydrate Epimerases ,Holoenzymes ,NADP ,Sugar Alcohol Dehydrogenases - Abstract
GDP-4-keto-6-deoxy- d -mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP- l -fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP- l -fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy- d -mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy- d -mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1.45–1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy- d -mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.
- Published
- 2000
36. Age-related increases of 8-hydroxy-2′-deoxyguanosine and DNA–protein crosslinks in mouse organs
- Author
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Cristina Cartiglia, Roumen Balansky, Maurizio Taningher, Silvio De Flora, and Alberto Izzotti
- Subjects
Aging ,medicine.medical_specialty ,Ratón ,DNA damage ,Health, Toxicology and Mutagenesis ,Inbred Strains ,Mice, Inbred Strains ,Oxidative phosphorylation ,Lesion ,DNA Adducts ,Mice ,chemistry.chemical_compound ,Internal medicine ,Genetics ,medicine ,Animals ,Trifluoroacetic Acid ,Deoxyguanosine ,Lung ,Mutagenesis ,Age Factors ,Brain ,Proteins ,8-Hydroxy-2'-deoxyguanosine ,Heart ,Cross-Linking Reagents ,Endocrinology ,Biomarkers ,DNA Damage ,Female ,Isotope Labeling ,Liver ,Phosphorus Radioisotopes ,chemistry ,Biochemistry ,8-Hydroxy-2'-Deoxyguanosine ,Ageing ,medicine.symptom - Abstract
Experimental data suggest a possible role of DNA damage in aging, mainly related to oxidative lesions. With the objective of evaluating DNA lesions as molecular biomarkers of aging, we measured 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and DNA-protein crosslinks (DPXL) levels in different organs of mice aged 12 and 24 months. 8-OH-dG was detected by 32P postlabelling after removing unmodified dG by trifluoracetic acid, which prevented the artificial formation of 8-OH-dG during 32P labelling procedures. Appreciable 8-OH-dG amounts were detected in 12-month-old mice in liver (1.8 +/- 0.7 8-OH-dG/10(5) normal nucleotides), brain (1.6 +/- 0.5) and heart (2.3 +/- 0.5). In 24-month-old mice these values were higher in all examined organs (liver, 2.7 +/- 0.4; brain, 3.6 +/- 1.1; heart, 6.8 +/- 2.2 8-OH-dG/10(5) normal nucleotides). This accounted for a 1.5-fold increase in liver (not significant), 2.3-fold increase in brain (P < 0.01), and 3.0-fold increase in heart (P < 0.001). A similar trend was observed for DPXL levels, which were the 1.8 +/- 0.3%, 1.2 +/- 0.2%, and 2.2 +/- 0.3% of total DNA in liver, brain, and heart of 12-month-old mice and 1.9 +/- 0.4%, 2.0 +/- 0.4%, and 3.4 +/- 0.5% in 24-month-old mice, with ratios of 1.0, 1.7 (P < 0.01), and 1.5 (P < 0.001), respectively. Highly significant correlations between 8-OH-dG and DPXL levels were recorded in brain (r = 0.619, P < 0.001) and heart (r = 0.800, P < 0.0001), but not in liver (r = 0.201, not significant). These data suggest that brain and heart are more severely affected by the monitored age-related DNA lesions than liver, which can be ascribed to certain characteristics of these postmitotic organs, including the low detoxifying capacities, the high oxygen consumption, and the impossibility to replace damaged cells by mitosis. The strong correlation between 8-OH-dG and DPXL supports a possible contribution of oxidative mechanisms to formation of DPXL in those organs, such as brain and heart, which play a primary role in the aging of the whole organism.
- Published
- 1999
37. The metabolism of 6-deoxyhexoses in bacterial and animal cells
- Author
-
Angela Bisso, Antonio De Flora, Laura Sturla, Davide Zanardi, Michela Tonetti, and Umberto Benatti
- Subjects
Glycoconjugate ,Biology ,Reductase ,Rhamnose ,Biochemistry ,Cell membrane ,chemistry.chemical_compound ,Biosynthesis ,Guanosine Diphosphate Fucose ,Glycosyltransferase ,medicine ,Animals ,Humans ,Hydro-Lyases ,Fucose ,chemistry.chemical_classification ,General Medicine ,Metabolism ,medicine.anatomical_structure ,Enzyme ,Models, Chemical ,chemistry ,Dehydratase ,biology.protein ,Carbohydrate Epimerases - Abstract
L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.
- Published
- 1998
38. Reduction of hexavalent chromium by fasted and fed human gastric fluid. I. Chemical reduction and mitigation of mutagenicity
- Author
-
De Flora, Silvio, primary, Camoirano, Anna, additional, Micale, Rosanna T., additional, La Maestra, Sebastiano, additional, Savarino, Vincenzo, additional, Zentilin, Patrizia, additional, Marabotto, Elisa, additional, Suh, Mina, additional, and Proctor, Deborah M., additional
- Published
- 2016
- Full Text
- View/download PDF
39. Reduction of hexavalent chromium by fasted and fed human gastric fluid. II. Ex vivo gastric reduction modeling
- Author
-
Kirman, Christopher R., primary, Suh, Mina, additional, Hays, Sean M., additional, Gürleyük, Hakan, additional, Gerads, Russ, additional, De Flora, Silvio, additional, Parker, William, additional, Lin, Shu, additional, Haws, Laurie C., additional, Harris, Mark A., additional, and Proctor, Deborah M., additional
- Published
- 2016
- Full Text
- View/download PDF
40. Pharmacological Modulation of Lung Carcinogenesis in Smokers: Preclinical and Clinical Evidence
- Author
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De Flora, Silvio, primary, Ganchev, Gancho, additional, Iltcheva, Marietta, additional, La Maestra, Sebastiano, additional, Micale, Rosanna T., additional, Steele, Vernon E., additional, and Balansky, Roumen, additional
- Published
- 2016
- Full Text
- View/download PDF
41. Exposing native cyprinid (Barbus plebejus) juveniles to river sediments leads to gonadal alterations, genotoxic effects and thyroid disruption
- Author
-
Viganò, Luigi, primary, De Flora, Silvio, additional, Gobbi, Marco, additional, Guiso, Giovanna, additional, Izzotti, Alberto, additional, Mandich, Alberta, additional, Mascolo, Giuseppe, additional, and Roscioli, Claudio, additional
- Published
- 2015
- Full Text
- View/download PDF
42. Mechanisms of inhibitors of mutagenesis and carcinogenesis
- Author
-
Silvio De Flora
- Subjects
Mechanism (biology) ,Health, Toxicology and Mutagenesis ,Animals, Anticarcinogenic Agents ,pharmacology, Antimutagenic Agents ,pharmacology, Humans ,Mutagenesis (molecular biology technique) ,Antimutagenic Agents ,Computational biology ,Biology ,medicine.disease_cause ,Protective Agents ,Genetics ,medicine ,Animals ,Anticarcinogenic Agents ,Humans ,pharmacology ,Carcinogenesis ,Molecular Biology - Abstract
For a rational implementation of chemoprevention strategies it is essential not only to assess the efficacy and safety of putative inhibitors by using a variety of test systems but also to understand the mechanisms involved. This article proposes a detailed classification of mechanisms along with pertinent examples of agents which may be potentially exploited in the host-addressed prevention of cancer and other mutation-related diseases. The classification, presented in tabulated form, covers a variety of mechanisms interfering with different phases of mutagenesis and carcinogenesis. However, the reported sequence of events is not meant to follow a rigid scheme, and several mechanisms are reiterated throughout evolution of these processes. Similarly, a number of protective agents are shown to work through multiple and often interconnected mechanisms.
- Published
- 1998
43. DNA fragmentation, DNA-protein crosslinks, 32P postlabeled nucleotidic modifications, and 8-hydroxy-2′-deoxyguanosine in the lung but not in the liver of rats receiving intratracheal instillations of chromium(VI). Chemoprevention by oral N-acetylcysteine
- Author
-
Anna Camoirano, Michele Orlando, Silvio De Flora, Maria Bagnasco, and Alberto Izzotti
- Subjects
DNA damage ,Health, Toxicology and Mutagenesis ,8-Hydroxy-2'-deoxyguanosine ,Molecular biology ,DNA extraction ,Acetylcysteine ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Sodium dichromate ,Genetics ,medicine ,DNA fragmentation ,Toxicokinetics ,Molecular Biology ,DNA ,medicine.drug - Abstract
An in vivo study was carried out with the objectives of evaluating ( a ) the localization of DNA lesions resulting from exposure to chromium(VI) by the respiratory route, ( b ) the molecular nature of DNA alterations, and ( c ) modulation of DNA damage by a known chemopreventive agent. To this purpose, Sprague-Dawley rats received intratracheal instillations of sodium dichromate (0.25 mg/kg body weight) for three consecutive days, and the day after the last treatment lung and liver were removed for DNA purification. The results showed a selective localization of DNA lesions in the lung but not in the liver, which can be ascribed to toxicokinetics and metabolic characteristics of chromium(VI). DNA alterations included DNA-protein crosslinks, DNA fragmentation, nucleotidic modifications, and 8-hydroxy-2′-deoxyguanosine. The last two endpoints were evaluated, for the first time in chromium toxicology, by means of 32 P postlabeling procedures. This methodology was adapted to the detection of the DNA damage produced by those reactive oxygen species which result from the intracellular reduction of chromium(VI). The oral administration of the thiol N -acetylcysteine completely prevented any induction of DNA lesions in lung cells.
- Published
- 1998
44. Expression of CD38 Increases Intracellular Calcium Concentration and Reduces Doubling Time in HeLa and 3T3 Cells
- Author
-
Aurora Costa, Carla Marchetti, Santina Bruzzone, Antonio Daga, Lucrezia Guida, Luisa Franco, Elena Zocchi, Antonio De Flora, and Cesare Usai
- Subjects
ADP-ribosyl Cyclase ,Cell Membrane Permeability ,chemistry.chemical_element ,CD38 ,Calcium ,Biology ,Biochemistry ,Cyclase ,Calcium in biology ,Mice ,NAD+ Nucleosidase ,Antigens, CD ,Animals ,Humans ,Molecular Biology ,Calcium metabolism ,Membrane Glycoproteins ,Cell Cycle ,Biological Transport ,3T3 Cells ,DNA ,Cell Biology ,ADP-ribosyl Cyclase 1 ,Antigens, Differentiation ,Cell biology ,chemistry ,Cyclase activity ,Intracellular ,HeLa Cells - Abstract
CD38 is a bifunctional ectoenzyme, predominantly expressed on hematopoietic cells during differentiation, that catalyzes the synthesis (cyclase) and the degradation (hydrolase) of cyclic ADP-ribose (cADPR), a powerful calcium mobilizer from intracellular stores. Due to the well established role of calcium levels in the regulation of apoptosis, proliferation, and differentiation, the CD38/cADPR system seems to be a likely candidate involved in the control of these fundamental processes. The ectocellular localization of the cyclase activity, however, contrasts with the intracellular site of action of cADPR. Here we demonstrate that ectocellular expression of human CD38 in CD38(-) HeLa and 3T3 cells results in intracellular CD38 substrate (NAD+ + NADH) consumption and product (cADPR) accumulation. Furthermore, a causal relationship is established between presence of intracellular cADPR, partial depletion of thapsigargin-sensitive calcium stores, increase in basal free cytoplasmic calcium concentration, and decrease of cell doubling time. The significant shortening of the S phase in CD38(+) HeLa cells, as compared with controls, demonstrates an effect of intracellular cADPR on the mammalian cell cycle.
- Published
- 1998
45. Cyclic GMP-dependent and -independent Effects on the Synthesis of the Calcium Messengers Cyclic ADP-ribose and Nicotinic Acid Adenine Dinucleotide Phosphate
- Author
-
Hon Cheung Lee, Richard M. Graeff, Luisa Franco, and Antonio De Flora
- Subjects
ADP-ribosyl Cyclase ,Biochemistry ,Cyclase ,Cyclic ADP-ribose ,chemistry.chemical_compound ,Adenosine Triphosphate ,NAD+ Nucleosidase ,Antigens, CD ,Animals ,Kinase activity ,Cyclic GMP ,Molecular Biology ,Ovum ,Adenosine Diphosphate Ribose ,Cyclic ADP-Ribose ,Nicotinic acid adenine dinucleotide phosphate ,Nicotinamide ,Chemistry ,Cell Biology ,ADP-ribosyl Cyclase 1 ,Antigens, Differentiation ,Kinetics ,Sea Urchins ,Calcium ,NAD+ kinase ,Cyclase activity ,NADP - Abstract
Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) have been shown to mobilize intracellular Ca2+ stores by totally independent mechanisms, which are pharmacologically distinct from that activated by inositol trisphosphate. Although cADPR and NAADP are structurally and functionally different, they can be synthesized by a single enzyme having ADP-ribosyl cyclase activity. In this study, three different assays were used to measure the metabolism of cADPR in sea urchin egg homogenates including a radioimmunoassay, a Ca2+ release assay, and a thin layer chromatographic assay. Soluble and membrane-bound ADP-ribosyl cyclases were identified and both cyclized NAD to produce cADPR. The soluble cyclase was half-maximally stimulated by 5.3 microM cGMP, but not by cAMP, while the membrane-bound form was independent of cGMP. The two forms of the cyclase were also different in the pH dependence of utilizing nicotinamide guanine dinucleotide (NGD), a guanine analog of NAD, as substrate, indicating they are two separate enzymes. The stimulatory effect of cGMP required ATP or ATPgammaS (adenosine 5'-O-(3-thiotriphosphate)) and a cGMP-dependent kinase activity was shown to be present in the soluble fraction. The degradation of cADPR to ADP-ribose was catalyzed by cADPR hydrolase, which was found to be predominantly associated with membranes. Similar to the membrane-bound cyclase, the cADPR hydrolase activity was also independent of cGMP. Both the soluble and membrane fractions also catalyzed the synthesis of NAADP through exchanging the nicotinamide group of NADP with nicotinic acid (NA). The base-exchange activity was independent of cGMP and the half-maximal concentrations of NADP and NA needed were about 0.2 mM and 10 mM, respectively. The exchange reaction showed a preference for acidic pH, contrasting with the neutral pH optimum of the cyclase activities. The complex metabolic pathways characterized in this study indicate that there may be a multitude of regulatory mechanisms for controlling the endogenous concentrations of cADPR and NAADP.
- Published
- 1998
46. The CD38/cyclic ADP-ribose system: A topological paradox
- Author
-
Antonio De Flora, Luisa Franco, Lucrezia Guida, and Elena Zocchi
- Subjects
ADP-ribosyl Cyclase ,T-Lymphocytes ,media_common.quotation_subject ,CD38 ,Biology ,Topology ,Biochemistry ,Cyclic ADP-ribose ,Gene Expression Regulation, Enzymologic ,chemistry.chemical_compound ,NAD+ Nucleosidase ,Antigens, CD ,Multienzyme Complexes ,Aplysia ,Animals ,Homeostasis ,Humans ,Internalization ,Ovum ,media_common ,Orphan receptor ,Adenosine Diphosphate Ribose ,Cyclic ADP-Ribose ,Membrane Glycoproteins ,Cell Membrane ,Cell Biology ,ADP-ribosyl Cyclase 1 ,Antigens, Differentiation ,Cell biology ,chemistry ,Sea Urchins ,Second messenger system ,Calcium ,Signal transduction ,Intracellular ,Granulocytes - Abstract
CD38 was first identified as a lymphocyte differentiation antigen that showed typical properties of an orphan receptor involved in many programs of cell proliferation and activation. However, CD38 proved also to be a bifunctional ectoenzyme that catalyzes the transient formation of cyclic ADP-ribose (cADPR) in a variety of cell types. This property raises many intriguing and so far unanswered questions, since cADPR is a new second messenger molecule directly involved in the control of calcium homeostasis by means of receptor-mediated release of calcium from ryanodine-sensitive intracellular stores. The relationship between receptor-like and enzymatic properties of CD38 is still unknown. The apparent topological paradox of ectocellular synthesis and intracellular activity of cADPR might be explained by: (a) influx of cADPR across the plasma membrane to reach its target stores, as suggested by experiments on cerebellar granule cells; and (b) NAD(+)-induced internalization, following membrane oligomerization, of CD38 with consequent partial import of cADPR metabolism to an intracellular compartment, as recently observed in lymphoid B cells. These two distinct mechanisms and other potential ones (e.g. binding of ectocellularly formed cADPR to cell surface receptors and initiation of signal-transducing pathways across the plasmamembrane) seem to be paradigmatic of processes affecting different types of cells. Although in some biological systems, such as Aplysia and sea urchin egg, cADPR metabolism is restricted to the intracellular environment, in mammalian cells the CD38/cADPR system provides new challenges in terms of subcellular compartmentation and qualifies as an unusual example of "ectobiochemistry" with potential, still unrecognized, properties of cellular regulation.
- Published
- 1997
47. Effects of the Murine L929 and L1210 Cell Lines on Nitric Oxide and TNF-α Production by RAW 264.7 Murine Macrophages
- Author
-
Michela Tonetti, Enrico Millo, A. De Flora, Laura Sturla, and Angela Bisso
- Subjects
Cell type ,Cell signaling ,Fibrosarcoma ,Biophysics ,Cell Communication ,Nitric Oxide ,Biochemistry ,Nitric oxide ,law.invention ,Mice ,chemistry.chemical_compound ,law ,Tumor Cells, Cultured ,medicine ,Animals ,Leukemia L1210 ,Autocrine signalling ,Molecular Biology ,biology ,Tumor Necrosis Factor-alpha ,Chemistry ,Macrophages ,Cell Biology ,medicine.disease ,Coculture Techniques ,Cell biology ,Cell culture ,Immunology ,biology.protein ,Recombinant DNA ,Antibody - Abstract
Co-cultures of the murine macrophagic cell line RAW 264.7 with the L929 fibrosarcoma cell line, but not with the leukemia L1210 cell line, showed enhanced NO production over control RAW 264.7 cells. This potentiating effect, which was observed in detectably mycoplasma-free conditions and required low concentrations of recombinant murine IFN-gamma, was due to soluble factors released from L929 cells and not to physical contact between the two cell types. The soluble factors were able to induce TNF-alpha in the macrophages and to potentiate the TNF-alpha release induced by IFN-gamma. Increased generation of NO in RAW 264.7 cells co-cultured with L929 cells was prevented by a neutralizing anti-TNF-alpha antibody, suggesting that TNF-alpha is an autocrine factor for iNOS expression in these conditions. Also the L929 cell line showed a 4- to 5-fold enhanced NO production following co-culture with RAW 264.7 cells, thus indicating that exposure of tumor cells to macrophages can lead to an increased iNOS expression in tumor cells themselves.
- Published
- 1997
48. Extracellular ATP Enhances mRNA Levels of Nitric Oxide Synthase and TNF-α in Lipopolysaccharide-Treated Raw 264.7 Murine Macrophages
- Author
-
Umberto Benatti, Laura Sturla, Marco Giovine, Michela Tonetti, and A. De Flora
- Subjects
Lipopolysaccharides ,Lipopolysaccharide ,Molecular Sequence Data ,Biophysics ,Nitric Oxide ,Biochemistry ,Cell Line ,Mice ,chemistry.chemical_compound ,Adenosine Triphosphate ,Extracellular ,Animals ,RNA, Messenger ,Autocrine signalling ,Molecular Biology ,DNA Primers ,chemistry.chemical_classification ,Base Sequence ,biology ,Tumor Necrosis Factor-alpha ,Macrophages ,Purinergic receptor ,Cell Biology ,Molecular biology ,Nitric oxide synthase ,Enzyme ,chemistry ,Cell culture ,biology.protein ,Tumor necrosis factor alpha ,Amino Acid Oxidoreductases ,Nitric Oxide Synthase - Abstract
Extracellular ATP potentiates, by activation of P2y-type purinergic receptors, the production of NO induced by low doses of lipopolysaccharide (LPS) in the murine macrophagic cell line RAW 264.7 (Tonetti et al. (1994) Biochem. Biophys. Res. Commun. 203, 430-434). Release of TNF-alpha, known to be an autocrine factor for iNOS expression, was enhanced, too, following exposure of either LPS-induced or uninduced cells to externally added micromolar ATP. Reverse transcription-PCR experiments showed that extracellular ATP increases mRNA levels of both inducible NO synthase (iNOS) and of TNF-alpha to extents comparable to those of enzymatic and biological activities, respectively. These data demonstrate that activation of purinergic receptors by extracellular ATP results in an enhanced expression of the iNOS and TNF-alpha genes.
- Published
- 1995
49. Enhanced levels of DNA adducts in the liver of woodchucks infected with hepatitis virus
- Author
-
Alberto Izzotti, L Scatolini, Debra Walsh, Silvio De Flora, and Joellen Lewtas
- Subjects
Male ,isolation /&/ purification ,Mutagen ,Toxicology ,medicine.disease_cause ,DNA Adducts ,DNA adduct ,medicine ,metabolism/pathology/virology ,Animals ,Hepatitis B Virus, Woodchuck ,Metabolic Detoxication ,Biotransformation ,Carcinogen ,Hepatitis B virus ,biology ,Chemistry ,Woodchuck hepatitis virus ,General Medicine ,Hepatitis B ,biology.organism_classification ,medicine.disease ,Glutathione ,Molecular biology ,4-Nitroquinoline-1-oxide ,metabolism, Animals, Biotransformation, Carcinogens ,metabolism, DNA Adducts ,metabolism, Female, Glutathione ,metabolism, Hepatitis B Virus ,Woodchuck ,isolation /&/ purification, Hepatitis B ,metabolism/pathology/virology, Liver ,metabolism/pathology/virology, Male, Marmota, Metabolic Detoxication ,Drug ,Liver ,Hepadnaviridae ,Biochemistry ,Marmota ,Hepatocellular carcinoma ,Inactivation, Metabolic ,Carcinogens ,Female ,Hepatitis B Virus ,metabolism - Abstract
Liver DNA specimens from woodchucks kept in captivity, 10 naturally infected with hepatitis virus (WHV) and five WHV-free, were examined for the presence of carcinogen-DNA adducts by 32 P-postlabeling. The number of adducts was significantly higher in WHV carriers than in uninfected animals, and the total amounts of adducts per 10 9 nucleotides were also considerably enhanced by WHV infection, when using both butanol extraction (22.2 ± 7.1 vs, 12.6 ± 2.8, means ± S.D.) and nuclease P 1 enrichment (8.5 ± 5.9 vs. 2.8 ± 1.7). Two individual adducts were also significantly higher in WHV carriers. No significant variation occurred as related to age, sex or time length of captivity. These findings are consistent with our previous studies supporting an enhanced metabolism of chemical hepatocarcinogens in both human and woodchuck hepadnavirus infections. Several significant and remarkable correlations were pointed out by relating DNA adduct data to more than 30 virological, histopathological and metabolic parameters which had been previously evaluated in the same animals. For instance, numbers and/or levels of adducts were positively related to the amounts of virus present in hepatocytes, to cell damage (γ-glutamyltranspeptidase activity), to the severity of the liver histopathological picture, and to monooxygenase activities, while they were inversely related to cellular glutathione concentrations and to detoxification of the directacting mutagen 4-nitroquinoline 1-oxide. The major adduct significantly correlated with the metabolic activation of the aromatic amine 2-aminofluorene and of the heterocyclic amines 3-amino-1- methyl -5 H -pyrido(4,3)indole (Trp-P-2) and 2-amino-3,4-dimethylimidazo(4,5-f) quinoline (MeIQ), whereas another adduct significantly correlated with the metabolic activation of the mycotoxin aflatoxin B 1 . Thus, the enhanced metabolism of chemical hepatocarcinogens and the increased formation of carcinogen-DNA adducts in the liver of WHV carriers appear to represent one of the mechanisms contributing to the association between chronic hepadnavirus infection and development of primary hepatocellular carcinoma.
- Published
- 1995
50. Hepatic and biliary biomarkers in rainbow trout injected with sediment extracts from the River Po (Italy)
- Author
-
Silvio De Flora, Maria Bagnasco, F. Melodia, Luigi Viganò, Carlo Bennicelli, and Attilio Arillo
- Subjects
chemistry.chemical_classification ,geography ,Environmental Engineering ,geography.geographical_feature_category ,Ecology ,Health, Toxicology and Mutagenesis ,Glutathione peroxidase ,Glutathione reductase ,Public Health, Environmental and Occupational Health ,Sediment ,General Medicine ,General Chemistry ,Glutathione ,Biology ,biology.organism_classification ,Pollution ,chemistry.chemical_compound ,Trout ,chemistry ,Environmental chemistry ,Tributary ,Environmental Chemistry ,Rainbow trout ,Salmonidae - Abstract
Immature rainbow trout (Oncorhyncus mykiss) were injected i.p. with varying doses of extracts of sediments collected from the River Po (Northern Italy), upstream and downstream the immission of a heavily contaminated tributary (River Lambro). Six days after treatment, metabolic biomarkers were monitored in fish liver and bile. Microsomal enzyme activities, including arylhydrocarbon hydroxylase, ethoxyresorufin O-deethylase, aminopyrine N-demethylase and uridine-5′-diphosphoglucuronyltransferase, were induced by exposure to the polluted sediment to a moderate yet statistically significant extent, with dose-related effects. The cytosolic enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase were not affected by injection of sediment extracts, whereas both upstream and downstream specimens produced a depletion of nonprotein sulfhydryl groups. Elimination of fluorescent metabolites occured in the bile of trout injected with the polluted sediment extract, whereas organic extracts of bile were devoid of mutagenic activity in strain TA98 of S. typhimurium. In spite of some positive responses, the method used appears to be less sensitive in revealing the toxicological impact of pollution than either exposure of rainbow trout larvae to river sediment or in situ exposure of the same fish species in river water.
- Published
- 1995
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