Acrylamide: a probable catalytic topoisomerase II inhibitor

Rheumatoid arthritis-associated HLA-DRB1 genotypes in western Sicily

Acrilamide: un probabile inibitore della topoisomerasi II

Biological effects and photodegradation by TiO(2) of terpenes present in industrial wastewater.

Abstract The aim of this work was to study the biological effects of four monoterpenes, i.e. α-pinene, β-pinene, 3-carene and d -limonene present in the wastewater of a citrus transformation factory. The study was carried out by exposing V79 Chinese hamster cells to single terpene or to the mixture of four terpenes at concentrations corresponding to those in the wastewater evaluated by head space solid phase micro extraction and gas chromatography (HS-SPME-GC) analyses. Treatments with single or combined terpenes similarly affected cell vitality, but only the combined treatments induced the 6-thioguanine resistant mutants. Moreover the photocatalytic degradation of the four terpenes was suc…

Photocatalytic degradation of paraquat Combined with a genotoxicity Study of the Intermediate Products

TiO2 Supported over Hβ and HZSM-5 Photocatalysts for Salmonelle and Escherichia Coli Disinfection

Genomic instability induced by α-pinene in Chinese hamster cell line.

Here, we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene used in insecticides, solvents and perfumes. Morphological analysis, performed in V79-Cl3 cells exposed for 1 h to increasing concentrations (25 up to 50 μM) of α-pinene, indicated a statistically significant increase in micronucleated and multinucleated cell frequencies; apoptotic cells were seen at 40 and 50 μM. This monoterpene caused genomic instability by interfering with mitotic process; in fact, 50% of cells (versus 19% of control cells) showed irregular mitosis with multipolar or incorrectly localised spindles. Cytogenetic analysis demonstrated high-frequency hypodiploid metaphases as well…

Evaluation of Biological Effects and Heterogeneous Photodegradation of Terpenes Present in Wastewater From a Citrus Fruit Transformation Factory

Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme

Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associate…

Photodegradation of paraquat in aqueous suspension of polycrystalline TiO2 : genotoxicity by using ames test

The expression of PTHrP isoforms in differentiating human fat-derived mesenchymal stem cells

Biological effects of inorganic arsenic on primary cultures of rat astrocytes

It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed…

Antagonist effects of Acrylamide on clastogenity of VP16

We investigated on the Acrylamide (AA) capability of influencing the clastogenic effects of VP16, the topoisomerase II targeting drug, by performing sequential treatments in V79 Chinese hamster cells. The VP16 cytotoxicity resulted almost completely antagonized by preincubating cells with nontoxic concentrations of AA, as inferred by statistically significant differences versus response with VP16 alone. Moreover, the severe clastogenic effect of VP16, evidenced by the presence of complex structural chromosome aberrations and by high frequencies of micronulei and sister chromatid exchanges, was reduced by AA in a dose-dependent manner. For example, the frequency of micronucleated cells induc…

Genetic polymorphism of the bitter taste TAS2R38 gene in central Sicily

SNP variation in the bitter taste TAS2R38 gene evaluated in student populations of several italian universities and isolates

People vary widely in their sensitivities to bitter compounds, but the all intercorrelation of these sensitivities is unknown. The study of genetic influences on bitter taste perception originated from the discovery in the 1930s that some individuals had taste to phenylthiocarbamide(PTC), whereas others found it extremely bitter. Subsequently, many studies were carried out on PTC and the structurally related compound propylthiouracil (PROP) to assess this viability and to determine the root causes. Initial family studies strongly suggested that PTC no tasting was due to a recessive allele in a single gene and heritability was estimated at 0,5. 55-85% of variation in PTC detection. The PTC ge…

Photocatalytic Degradation of Paraquat and Genotoxicity of its Intermediate Products

Abstract The photocatalytic degradation of paraquat (1,1-dimethyl-4,4′-bipyridylium dichloride) aqueous solutions in the presence of polycrystalline TiO2 Degussa P25 irradiated by near-UV light was investigated. The substrate and total organic carbon concentrations were monitored by UV spectroscopy and TOC measurements, respectively: the complete photocatalytic mineralization of paraquat (20 ppm) was achieved after ca. 3 h of irradiation by using 0.4 g l−1 of catalyst amount at natural pH (ca 5.8). On the contrary no significant photodegradation of paraquat was observed in the absence of TiO2 under similar experimental conditions. To evaluate the genotoxicity of paraquat and its intermediat…

Persistent genomic instability by arsenic exposure in V79 Chinese hamster cells

Previously, we demonstrated that acute treatment with arsenic leads mammalian cells to exhibit persistent chromosomal instability and DNA hypomethylation, by performing investigations after about 2 months of subculturing. In order to evaluate quantitatively the continuing instability during the expanded growth, we carried out cytogenetic, morphologic and molecular analyses immediately after exposure and every week up to 112 cell generations. Briefly, V79 Chinese hamster cells were treated with 10 µM sodium arsenite (SA) for 24h; at the end of exposure, mitotic rounded-up cells were harvested by shake-off and allowed to grow in drug-free medium. The instability markers, micronucleated and mu…

Acrylamide catalytically inhibits topoisomerase II in V79 cells.

The vinyl monomer acrylamide is characterized by the presence of an alpha,beta-unsaturated carbonyl group that makes it reactive towards thiol, hydroxyl or amino groups and towards the nucleophilic centers in DNA. The ability of acrylamide to chemically modify protein thiols has prompted us to consider topoisomerase II as one possible target of acrylamide, since agents targeting protein sulfhydryl groups act as either catalytic inhibitors or poisons of topoisomerase II. Nuclear extracts from V79 Chinese hamster cells incubated with acrylamide reduced topoisomerase II activity as inferred by an inability to convert kinetoplast DNA to the decatenated form. Nuclear extracts incubated with acry…

Bitter taste genetics and food preference in italian population

Objective: To investigate the possible role of the polymorphic bitter taste gene, TAS2R38, known to be involved in the perception of the bitter synthetic chemical phenylthiocarbamide (PTC), in influencing food preference and body mass index(BMI). Methods: up to now more than 1500 university students (17-25 years old) at Catania, Cosenza, Rome, Palermo, Pisa, Parma, Chieti, Trento University have been enrolled in the study. DNA was extracted from saliva, and genotyped by TaqMan assay for the most frequent polymorphism (PAV/AVI) of TAS2R38 gene. A possible association between genotype and food preference was assessed by administering a detailed questionnaire for food preferences and life styl…

Biological effects of alpha-pinene in cultured mammalian cells

In this work we report the effects of exposure of mammalian cells to α-pinene, a bicyclic monoterpene founded in essential oils and used in insecticides, solvents, perfumes, etc.. Morphological analysis, performed in V79 cells exposed to increasing doses(25μM up to 50μM) of α-pinene, indicated a increase of dose-related nuclear abnormalities; apoptotic cells were seen at higher doses. Immunofluorescence with anti β- tubulin antibody showed that monoterpene induced genomic instability by interfering with mitotic process; in fact, 50% (vs 19% in the control cells) of irregular mitosis with multipolar or not correctly localized spindles were detected, suggesting that α-pinene affects cell stab…

Arsenite-induced aneuploidy following short and long-term exposure in mammalian cells

We studied the long-term progression of chromosomal instability in V79 cells treated acutely with arsenite (10mM, 24 hr) followed by growth in arsenic-free medium for 120 cell generations. Indirect immunostaining using anti-ß-tubulin antibody showed severe alterations in spindle morphology after only 6 h treatment and cytogenetic investigations carried out at the end of treatment revealed that the percentage of cells with 21 chromosomes (modal number of the cell line) decreased, making way for aneuploid cells. The acquired instability remained and propagated within the cell population. Moreover, we treated V79-derived G12 cells with sub-lethal doses (0.1-1.0 μM) of arsenite for 10 days foll…

Abnormal mitotic spindle assembly and cytokinesis induced by D-Limonene in cultured mammalian cells

D-Limonene is found widely in citrus and many other plant species; it is a major constituent of many essential oils and is used as a solvent for commercial purposes. With the discovery of its chemotherapeutic properties against cancer, it is important to investigate the biological effects of the exposure to D-Limonene and elucidate its, as yet unknown, mechanism of action. We reported here that D-Limonene is toxic in V79 Chinese hamster cells in a dose-dependent manner. Moreover, to determine the cellular target of D-Limonene, we performed morphological observations and immunocytochemical analysis and we showed that this drug has a direct effect on dividing cells preventing assembly of mito…

Determination of Monoterpens in Citrus Processing Factory’s Wastewater Using SPME/FAST-GC/MS

Cytochrome P450 2E1 variable number tandem repeat polymorphisms and health risks: A genotype-phenotype study in cancers associated with drinking and/or smoking

Cytochrome P450 2E1 (CYP2E1) is one of the main enzymes involved in the oxidation of ethanol and in the transformation of a number of potentially dangerous compounds. It has various polymorphic sites, one of which is a variable number tandem repeat (VNTR) polymorphism previously described in the 5'-flanking region. The aim of this study was to investigate the genotype-phenotype association between CYP2E1 VNTR polymorphisms and risky health habits in healthy subjects and to analyze the associations between these polymorphisms with drinking- and/or smoking-related cancers. We analyzed 166 healthy subjects by genotyping for the CYP2E1 VNTR polymorphism associated with drinking and/or smoking h…

POLIMORFISMI DEI GENI CYP2A6 E CYP2E1 IN RELAZIONE A STILI DI VITA IN UNA POPOLAZIONE DELLA SICILIA CENTRO-OCCIDENTALE

Le più recenti stime dell’OMS testimoniano che le patologie correlate all’abitudine al fumo e al consumo di alcool sono tra le principali cause di morte nei soggetti adulti ed è noto che la diversità interindividuale nell’adottare tali stili di vita è attribuibile, oltre che a fattori psico-sociali, anche a polimorfismi dei geni per i citocromi P450. Questo studio mira a verificare se esista una correlazione fra i genotipi CYP2A6 -CYP2E1 e abitudine al fumo e consumo d’alcool nella Sicilia centro-occidentale. I risultati ottenuti dall’analisi della distribuzione di alcuni alleli di entrambi i geni in 65 soggetti (41 donne e 24 uomini) hanno evidenziato che i genotipi CYP2A6*1/*4A e CYP2A6*1…

Early and late effects of arsenic exposure in mammalian cells

Previously we demonstrated that V79 Chinese hamster cells underwent either early genetic instability or apoptosis When exposed to sodium arsenite (SA). Genetic instability was evidenced by aneuploid and morphologically abnormal cells, but not by cells with chromosome aberrations. As dividing cells turned out to be the most sensitive to SA exposure, due to the arsenics direct action on the mitotic spindle assembly, we later ascertained the fate of genetically unstable cells escaping apoptosis, by harvesting mitotic rounded-up cells at the end of a 24 h treatment. The progeny of the exposed Chinese hamster cells showed an increased level of mutations related to genome DNA hypomethylation indu…

Long-Lasting Genomic Instability Following Arsenite Exposure inMammalian Cells: The Role of Reactive Oxygen Species

Previously, we reported that the progeny of mammalian cells, which has been exposed to sodium arsenite for two cell cycles, exhibited chromosomal instability and concurrent DNA hypomethylation, when they were subsequently investigated after two months of subculturing (about 120 cell generations) in arsenite-free medium. In this work, we continued our investigations of the long-lasting arsenite-induced genomic instability by analyzing additional endpoints at several time points during the cell expanded growth. In addition to the progressive increase of aneuploid cells, we also noted micronucleated and multinucleated cells that continued to accumulate up to the 50th cell generation, as well a…

CYP2E1 VNTR polymorphisms and hepatocarcinoma: a gender-specific correlation

Cytochrome P450 (CYP2E1) is often associate to susceptibility to alcohol-related diseases and various cancers, because of its role in the metabolism of multiple environmental xenobiotics. In the 5’- flanking region of the human CYP2E1 gene there are restriction fragment length polymorphism which are involved in the transcriptional regulation of the CYP2E1 gene. Recently a tandem repeat polymorphism (VNTR) in the 5’-flanking region of CYP2E1 was found. Because cytochrome P450 2E1 catalyzes the metabolic activation of pro-carcinogen and cytotoxic compound, we value the genetic distribution of this tandem repeat polymorphism in a healthy population, and in patients with hepatocellular carcinom…

PTHrP isoform expression in adipo- and osteo-differentiating human mesenchymal stem cells

Variable Number of Tandem Repeats (VNTR) gene polymorphism of CYP2E1 in patients with pancreatic adenocarcinoma

Context: The genetic polymorphism is considered a major source of variability, influencing the levels of gene expression. Cytochrome P450 2E1 (CYP2E1) is a mixed-function oxidase involved in the metabolism of the many endogenous and exogenous substances (ethanol, chemical carcinogens) in the hepatic and pancreatic tissue. CYP2E1 gene polymorphisms can cause various abilities of metabolize xenobiotic substances within a population with consequent increased susceptibility to various diseases,including cancer. One of the polymorphisms of the CYP2E1 gene is a VNTR (Variable Number Tandem Repeat) of some sequences in its "5 '- flanking region. Method : VNTR genotype CYP2E1 was determined by RFLP…

Genotoxicity of citrus wastewater in prokaryotic and eukaryotic cells and efficiency of heterogeneous photocatalysis by TiO(2).

Abstract The presence of (±)α-pinene, (+)β-pinene, (+)3-carene, and R-(+)limonene terpenes in wastewater of a citrus transformation factory was detected and analyzed, in a previous study, by using Solid Phase Micro-extraction (SPME) followed by GC analyses. Purpose of that research was to compare the genotoxic responses of mixtures of terpenes with the genotoxicity of the individual compounds, and the biological effects of actual wastewater. Genotoxicity was evaluated in the Salmonella reversion assay (Ames test) and in V79 cells by Comet assay. Ames tests indicated that the four single terpenes did not induce an increase of revertants frequency. On the contrary, the mixtures of terpenes ca…

Effect of inorganic arsenic on rat cortical astrocytes in culture

Although inorganic arsenic is a well known poisonous metalloid, the cellular and molecular mechanisms of its action remain elusive. The present study was aimed at analyzing the effects of NaAsO2 on primary cultures of rat astrocytes by determining DNA damage by comet assay, and by evaluating possible changes of the concentration of some conserved heat shock proteins. Cells treated with inorganic arsenic underwent induction of Hsp70, demonstrating a state of stress. Moreover, although micromolar NaAsO2 treatments (60 μM) only reduced cell viability to 60% respect to untreated cells, high DNA damage was already observed after 24h treatment with 10 μM arsenite. Since arsenic is known to be not…

In vivo and in vitro inhibitory effects of acrylamide on DNA topoisomerase II

Acrylamide (AA), a chemical produced in several foodstuffs when cooked at a high temperature, is considered a probable human carcinogen, but the molecular mechanism underlying its genotoxicity has not fully known. Numerous authors have reported the induction by AA of DNA double strand breaks and sister chromatid exchange (SCE); we here confirmed the acrylamide capability of damaging DNA by utilizing Comet assay, which showed a dose-dependent increase of tail lenght, in metabolically non competent V79 Chinese hamster cells. Moreover, we observed that Acrylamide (AA) was able to antagonize in vivo the citotoxicity of well know poison etoposide; this suggested that topoisomerase II activity wa…