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The aim of this work was to compare the genotoxic responses of mixtures of terpenes present in wastewaters of a citrus transformation factory with the genotoxicity of the individual compounds. Samplings of wastewater collected before (untreated sample) and past water purification by biological method (treated sample) were analyzed using Solid Phase Micro-extraction (SPME) followed by GC analyses. The chromatograms showed in all effluents the presence of four terpenes: pinene, -pinene, 3-carene, D-limonene. The concentrations of terpenes in the untreated sample were 1–3 orders of magnitude higher than in the treated sample. Genotoxicity was evaluated in the Salmonella reversion assay (Ames test) and in V79 cells by comet assay, by utilizing aqueous solutions the four terpenes at concentrations corresponding at those determined by SPME. In the Ames test, when tested individually, the four terpenes induced no or only a modest increase of genotoxic effects. On the contrary, the mixtures of terpenes present in untreated sample caused an increase highly significant of the revertants in TA100 strain, in presence of metabolic activation, in comparison to the control. The comet assay showed a significant increase in DNA migration in V79 cells after 1 or 6 h treatment with single or mixed terpenes. The possibility to photodegrade terpenes by using polycrystalline TiO2 irradiated with UV light was investigated. Photocatalytic tests carried out on both synthetic and actual aqueous effluents indicated that all terpenes were completely photodegraded, confirming the methodology effectiveness.

The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. In previous studies long-term progression of chromosomal instability was typified by increasing aneuploidy in Chinese hamster V79 and human keratinocyte cells treated with arsenite for a 24 hr exposure period followed by growth in arsenic-free medium for 40-120 cell generations. In the current study the role of progressive DNA methylation changes was evaluated in long-term cell cultures after brief arsenite treatments as above. We have found altered genomic methylation patterns in cells that were briefly exposed to arsenic with evidence for widespread dysregulation of CpG methylation that persists for many population doublings after the treatment. In V79 cell populations, progressive aneuploidy increases were notable by 50 cell generations after a 24 hr exposure to 1-10 uM arsenite. Dicentric chromosomes and/or telomeric associations, as well as complex chromosome rearrangements, occurred by 90 cells generations post treatment; and mutator and transformed phenotypes began to appear thereafter. This increasing genomic instability correlated with modifications of global DNA methylation patterns in V79 cells evaluated by 5-methylcytosine antibody binding and MeSAP-PCR. The results show that short-term exposure to arsenite induced an apparent genome hypomethylating effect within a short time after exposure. In identical protocols using human HaCaT keratinocytes exposed to low doses of arsenite (0.05-0.1 M) for 24 hr, genomewide methylation levels were measured by LINE1 pyrosequencing and gene-specific methylation status was assessed by Methylation-Specific-PCR for up to 40 generations post exposure. Global demethylation following treatment was supported by preliminary LINE-1 studies. Moreover, the study of gene-specific MSP and determination of expression levels by RT-PCR of several genes (p16, hMLH1, hMSH2, DNMT1, DNMT3a and DNMT3b) demonstrated that hMSH2 gene was epigenetically regulated by arsenite and that down regulation of DNMT3a and DNMT3b genes occurred in an arsenite dose-dependent manner. The results reported here demonstrate that acute 24 hr arsenic exposure promptly induces genome wide DNA hypomethylation, and support the hypothesis that the cells continue to undergo epigenetic reprogramming both at the gene and genomic levels in the absence of further arsenite treatment; thus likely contributing to long-lasting genomic instability that manifests as aberrant chromosomal, mutator and cell transformation effects.

Mitosis is regulated by checkpoints that delay mitotic progression when chromosome segregation errors occur. Inaccuracy in checkpoint processes can lead to chromosome instability both in number and structure (CIN). Arsenic is reported to induce CIN by perturbing mitotic spindles and checkpoints, however, its carcinogenic mechanisms are poorly understood. We previously studied the long-term progression of chromosomal instability in V79 cells treated acutely with arsenite (10 M, 24 hr) followed by growth in arsenic-free medium for 120 cell generations, and found time-dependent increase of aneuploid cells. Here, we treated V79-derived G12 cells with sub-lethal doses (0.1-1.0 µM) of arsenite for 10 days followed by growth in arsenite-free medium for 40 cell generations. Cytogenetic analysis at the end of treatment showed concentration-dependent increases in the frequency of aneuploid cells that was even higher after 40 cell generations. Furthermore, the mitotic index (MI) of arsenite-exposed cells was higher than untreated cells at the end of the 10-day treatment or after 40 cell generations without arsenite. Surprisingly, we found that submicromolar 10-day arsenic exposure induced a large amount of cells in anaphase with premature centromere division. These aberrant mitotic figures dramatically increased after 40 cell generations. Taken together, these results raise the possibility that chronic exposure to sub-lethal arsenic doses induces bypass of the spindle assembly checkpoints and may be mechanistically involved in the induction of cell transformation.

Numerosi studi confermano l’associazione tra fumo di sigaretta e insorgenza di patologie-polmonari, cardiovascolari e tumori. Il fumo, infatti, contiene diversi composti mutageni c cancerogeni. Premesso che non esiste una sigaretta sicura, è comunque possibile attuare strategie per caratterizzare e ridurre il rischio. In questo studio, abbiamo utilizzato test in vitro per investigare i possibili meccanismi d’azione del condensato di sigaretta (CSC), ovvero la frazione particolata del fumo, assorbita nei polmoni del fumatore e successivamente metabolizzata. Analisi precedenti avevano dimostrato che il CSC, dopo 24h di trattamento induce decremento della vitalità delle cellule Swiss3T3 (IC50 = 130 µg/ml), inibizione dell’efficienza di clonaggio (circa il 60% a`50 µg/ml) e attivazione delle caspasi già a partire dalla prima dose testata (25 µg/ml). La natura degli effetti genotossici del CSC è stata analizzata con il Comet assay che ha evidenziato la capacità del CSC di indurre rotture a singolo e doppio filamento del DNA, dopo trattamenti per 90min e 3 h, a partire dalla dose di 100 µg/ml (p

CSC is a complex chemical mixture containing about 4800 compounds, many of them have cytotoxic and mutagenic activities on mammalian cells. Most of these compounds are able to interact with DNA at different levels. Cells may respond to DNA damage by following different pathways, such as the DNA repair processes and the cell cycle and DNA damage checkpoint activation. To the aim to evaluate the biological effects of CSC on cells, alkaline comet assay and flow cytofluorimetry were used to examine DNA damage/repair and cell cycle progression. All experiments were performed by using CSC from standard cigarettes in the range of doses 30-180g/ml and Swiss 3T3 murine fibroblasts. Results obtained by comet assay showed that CSC induces DNA strand breaks, significantly higher after 90 min of treatment than 3hrs. This difference, particularly evident at 100 and 150g/ml, it is probably due to a fast repair that can be explained by the oxidative component of the DNA damage CSC-induced. To clarify these results, further investigations on the evaluation of the oxidative damage are in progress by applying two different methods. one is the detection of the oxidised bases on the DNA by using the modified protocol of Comet assay with FPG and endo III, the second is the analysis of the intracellular levels of ROS, measured as the ability of treated cells to oxidise a fluorogenic dye, is going to be carried out. Previous results of long-term survival showed that cells lose their ability to form colonies in dose-dependent manner, after 24hrs of CSC treatment and 168 hrs of culture. However, the cytofluorimetric analysis showed that a fraction of cells, blocked in G2/M immediately after 24hrs of treatment, are gradually granted to continue the cell cycle, after incubation for further 6hrs in medium CSC-free. Further investigations on the cell cycle alteration are on going.

E’ stato dimostrato che la diversa capacità di metabolizzare alcune sostanze è la conseguenza di differenze geneticamente determinate nelle attività di alcuni enzimi. In particolare, il citocromo P450 CYP2A6 ha diversi livelli di espressione interindividuali ed interetnici a causa di polimorfismi genetici; metabolizza circa il 70-80% della nicotina e ciò determina in alcune popolazioni una dipendenza interindividuale più o meno elevata dal fumo di sigaretta. Più in dettaglio, individui con alleli CYP2A6 con elevata attività metabolizzante (CYP2A6*1) avranno una maggiore dipendenza dalla nicotina, poiché questa, essendo metabolizzata velocemente, rimane nell’organismo per breve tempo: di conseguenza questi individui fumeranno molte sigarette per sopperire alla carenza di nicotina. Al contrario, individui con alleli CYP2A6 che codificano per un enzima con bassa attività metabolizzante (CYP2A6*2, ad esempio) avranno una minore dipendenza dalla nicotina in quanto questa, essendo metabolizzata più lentamente, rimane nell’organismo per più tempo e questa condizione indurrà il soggetto a fumare meno. Allo scopo di verificare che la correlazione fra genotipo CYP2A6 e dipendenza dal fumo di sigaretta sia valido anche nella popolazione della Sicilia Occidentale, abbiamo analizzato la distribuzione di alcuni alleli CYP2A6 in 39 soggetti donatori abituali di sangue ai quali, dopo consenso informato, è stato somministrato un questionario sullo stile di vita con particolare riferimento all’abitudine al fumo. Il DNA, estratto da linfociti di sangue periferico mediante il sistema FASST DNA Releaser, è stato utilizzato come stampo in una Multiplex-PCR per amplificare una regione da 1717 bp comprendente gli esoni 1-4 del gene CYP2A6. Successivamente il prodotto della Multiplex-PCR è stato utilizzato come stampo per reazioni Multiplex-Nested-PCR, nelle quali sono state separatamente utilizzate coppie di oligonucleotidi innesco specifiche per l’allele selvatico CYP2A6*1 e per l’allele mutato CYP2A6*2. In queste condizioni è stato amplificato un frammento da 414 bp compreso fra gli esoni 3 e 4 del gene CYP2A6 nel quale può ricadere la trasversione TA che caratterizza l’allele mutato CYP2A6*2. In tal modo è stato possibile determinare il genotipo CYP2A6 dei soggetti esaminati visualizzando i prodotti di amplificazione su gel di poliacrilammide 12% in condizioni non denaturanti. I nostri risultati indicano che il genotipo omozigote CYP2A6*1/*1 è presente soltanto in soggetti fumatori, che tutti i soggetti non fumatori presentano il genotipo eterozigote CYP2A6*1/*2 e che nessun soggetto presenta genotipo CYP2A6*2/*2; inoltre, genotipi con delezione totale o parziale del gene CYP2A6 sono presenti tanto in fumatori che in non fumatori. Ciò dimostra che polimorfismi del gene CYP2A6 sono in correlazione alla dipendenza dal fumo anche nella popolazione della Sicilia Occidentale e suggerisce che alla definizione di questo fenotipo concorrano fattori genetici ai quali si affiancano fattori di tipo comportamentale-sociale. Studi più estesi sono auspicabili per definire le frequenze genotipiche del gene CYP2A6 dal momento che diverse configurazioni alleliche di questo gene, modulando la dipendenza dal fumo, indirettamente definiscono la suscettibilità individuale a contrarre alcune malattie come il cancro.

Vinyl monomer acrylamide (AA), generally used in numerous industrial applications, has been classified by the International Agency for Research on Cancer (IARC) as “probably carcinogenic to humans” (group 2A), but the molecular mechanism underlying its genotoxicity has not fully known. Previously, we observed that Acrylamide (AA) was able to antagonize in vivo the citotoxicity of well know poison etoposide suggesting that topoisomerase II (Topo II) activity was affected by AA. In the current studies we investigated the inhibitory activity of acrylamide toward topoisomerase II by performing tests in vitro.

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 mitotic spindle microtubules. This affects both chromosome segregation and cytokinesis, resulting in aneuploidy that in turn can lead to cell death or genomic instability.

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 as endoreduplicated cells and chromosome breaks. Clastogenic damage was prevalent over aneuploidogenic damage as demonstrated by the higher proportion of kinetochore-negative micronuclei. Alkaline comet confirmed that monoterpene exposure caused DNA lesions in a concentration-dependent manner. This damage probably arose by increased reactive oxygen species (ROS) production. In order to assess the generation of ROS, the cells were incubated with CM-H(2)DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through ROS production.

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 caused, in the presence of metabolic activation, a highly significant increase of the revertants in TA100 strain in comparison to the control. The Comet assay showed a significant increase in DNA damage in V79 cells treated for 1 h with single or mixed terpenes. Moreover, the actual wastewater was found highly genotoxic in bacterial and mammalian cells. Photocatalytic tests completely photodegraded the pollutants present in aqueous wastewater and the initial high genotoxicity of samples of wastewater collected during the photocatalytic run, was completely lose in 3 h of irradiation.

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 as dicentric chromosomes and/or telomeric associations and other complex chromosome rearrangements that began to appear much later, at the 90th cell generation following arsenite exposure. The increasing genomic instability was further characterized by an increased frequency of spontaneous mutations. Furthermore, the long-lasting genomic instability was related to elevated levels of reactive oxygen species (ROS), which at the 50th cell generation appeared higher than in stable parental cells. To gain additional insight into the continuing genomic instability, we examined several individual clones isolated at different time points from the growing cell population. Chromosomally and morphologically unstable cell clones, the number of which increased with the expanded growth, were also present at early phases of growth without arsenite. All genomically unstable clones exhibited higher ROS levels than untreated cells suggesting that oxidative stress is an important factor for the progression of genomic instability induced by arsenite. Environ. Mol. Mutagen. 2011. © 2011 Wiley-Liss, Inc.

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. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears.

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 successfully achieved with the photocatalyst TiO 2 Degussa P25 in both the actual effluent and in synthetic solutions.

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 carcinoma living in same country, in order to found a correlation between CYP2E1 VNTR genotype and neoplasia. DNA was isolated from spit sample of 108 control subject and from the peripheral lymphocytes of 35 HCC patients. The 5’ flanking region of the CYP2E1 gene was amplified by polymerase chain reaction and examined for tandem repeat polymorphism using Nla IV restriction map. This study reports that only four of the ten possible genotype were found in all subjects. The modal genotype, found in both analyzed populations, is A2/A2. Interestingly, in a gender-based analysis of data this genotype was found more frequent in woman with disease that in control ones. These preliminary findings represent a first report of a gender-specific correlation between CYP2E1 VNTR polymorphism and hepatocarcinoma.

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 acrylamide pre-incubated with DTT converted kinetoplast DNA to the decatenated form, suggesting that acrylamide influences topoisomerase II activity through reaction with sulfhydryl groups on the enzyme. Furthermore, acrylamide did not induce the pBR322 DNA cleavage, as assessed by cleavage assay: thus, it cannot be regarded as a poison of topoisomerase II. As a catalytic inhibitor, acrylamide antagonizes the effect of etoposide, a topoisomerase II poison, as determined by clonogenic assay in V79 cells. This antagonism is confirmed by band depletion assay, from which it can be inferred that acrylamide reduces the level of catalytically active cellular topoisomerase II available for the action of etoposide. (C) 2009 Elsevier Ltd. All rights reserved. The vinyl monomer acrylamide is characterized by the presence of an a,b-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 acrylamide pre-incubated with DTT converted kinetoplast DNA to the decatenated form, suggesting that acrylamide influences topoisomerase II activity through reaction with sulfhydryl groups on the enzyme. Furthermore, acrylamide did not induce the pBR322 DNA cleavage, as assessed by cleavage assay; thus, it cannot be regarded as a poison of topoisomerase II. As a catalytic inhibitor, acrylamide antagonizes the effect of etoposide, a topoisomerase II poison, as determined by clonogenic assay in V79 cells. This antagonism is confirmed by band depletion assay, from which it can be inferred that acrylamide reduces the level of catalytically active cellular topoisomerase II available for the action of etoposide.

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 followed by growth in arsenite-free medium for 40 cell generations. Cytogenetic analysis at the end of treatment showed concentration-dependent increase in aneuploid cells frequency that was even higher after 40 cell generations. In addition to this finding a large amount of cells in anaphase and cells with chromosomes showing premature centromere division was observed. Western blotting analysis showed dose-dependent upregulation of Mad2 that returns to normal after 40 cell generations and persistent aberrant expression of the spindle assembly complex proteins p-BubR1 and Cenp-E. Taken together, these results demonstrate that arsenic induces aneuploidy independently of mode of treatment; in particular, results from chronic exposure to sub-lethal doses raise the possibility that arsenic induces bypass of the spindle assembly checkpoints which may be mechanistically involved in the induction of cell transformation.

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 stability by disturbing chromosome segregation. Cytogenetic analysis demonstrated that frequency of hypodiploid metaphases increased in a dose-dependent manner and, moreover, α-pinene induced endoreduplicated cells and double strand breaks. Alkaline comet confirmed that monoterpene exposure induced DNA lesions; in fact, OTM increased significantly in a dose-dependent manner. In order to assess whether the severe DNA damage evidenced by comet assay was originated through the ROS production, cells were incubated with CM-H2DCFDA and then analysed by flow cytometry. Results demonstrated an increase in fluorescence intensity after α-pinene treatment indicating an increased oxidative stress. On the whole, these findings strongly suggest that α-pinene is able to compromise genome stability preferentially through mitotic alterations and to damage DNA through reactive oxigen species production.

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 a direct-acting genotoxic agent, we investigated the possibility that its effects could depend on ROS formation. FACS analysis after CM-H2DCFDA staining evidenced an increase of ROS production at the lowest concentration (2.5 μM) of arsenite, while at higher doses (5 μM and 10 μM), ROS production decreased. An inverse correlation was found between ROS production and the expression of superoxide dismutases (SOD) 1 and 2. Finally, we found that PIPPin, an RNA-binding protein the concentration of which has been recently reported to change in response to stress induced by cadmium, undergoes a putative post-translational transition when cells are exposed to high doses of arsenicum.

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 intermediates produced during heterogeneous photocatalytic treatment, in vitro tests such as Ames test, with and without rat liver microsomal fractions (S9 mix), and micronucleus test, were used. Results obtained with Salmonella typhimurium (strain TA100) showed that paraquat and photocatalytic products were unable to induce gene mutations when photocatalysis was used in the presence of the optimum amount of TiO2, i.e. 0.4 g l−1, whereas an increase of revertants his+ per plate was observed after 300 min irradiation in the presence of very low amount of TiO2 (0.04 g l−1). The negative results from micronucleus test suggest that mutagenic, but non-clastogenic, late intermediates of paraquat photo-oxidation were formed when the photocatalytic runs of paraquat degradation were carried out by using 0.04 g l−1 of photocatalyst.

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 gene, TAS2R38 on chromosome 7, consists of a small, single coding exon 1002 bp. The non-tester allele differs from the tester one for three single nucleotide polymorphisms (SNPs) at position 49, 262 and 296 in the gene, determining two predominant haplotypes. The phenotypic assay to differentiate taster from non-taster is relatively easy and quick, as well as the genotyping of the known polymorphisms. Therefore, this phenotypic-genotypic assay results to be particularly suitable for molecular and population genetic studies on large populations and for teaching genetics at university and high school level by allowing students to assess the genotype-phenotype relationship on themselves. By providing students with paper samples soaked in different solutions of PTC and PROP it was been possible to assess the individual threshold of bitterness sensitivity. DNA was extracted from saliva by means of Qiagen Kit mini, and following PCR amplification, polymorphisms were evaluated by gel electrophoresis. We started to develop this assay involving hundreds of students of several Italian universities such as Caltanissetta, Palermo, Catania, Cosenza in the South of Italy, Pisa and Parma in the Centrum of Italy with the aim to describe the Italian population for this polymorphism. By considering the large number of samples, we expect to be able to assess the frequency of additional rare polymorphisms, possibly further characterizing defined populations. A detailed questionnaire concerning life style and diet was also administered to students for possible association studies with the genetic polymorphism. Moreover, investigations on isolated populations such as these leaving in mountains of Garfagnana (Lucca) was undertaken. The distribution frequency of TAS2R38 polymorphisms in different sub sets of Italian population and isolates, the possible relationship with food preferences and other life styles, such as alcohol drinking and smoking will be reported and discussed. Functional expression studies demonstrate that five different haplotypes from the hTAS2R38 gene, code for operatively distinct receptors. The responses of the three haplotypes we also tested in vivo correlate strongly with individuals' psychophysical bitter sensitivities to a family of compounds. These data provide a direct molecular link between heritable variability in bitter taste perception to functional that contain the N-C= moiety. The molecular mechanisms of perceived bitterness variability have therapeutic implications, such as helping patients to consume beneficial bitter-tasting compounds, for example, pharmaceuticals and selected phytochemicals (Bufe 2005). Our goal is to investigate correlations as a function of individual sensitivities to several bitter compounds representative of different chemical classes and, from these correlations, infer the number and variety of potential bitterness transduction system.

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytogenetic, "whole genome"/conventional PCR, EMSA and DNase footprinting techniques. The results obtained: (i) show that PTHrP(38-94)-amide gains access to the nuclear compartment of MDA-MB231 cell; (ii) demonstrate that PTHrP(38-94)-amide is a DNA-binding peptide; and, (iii) represent the first data to date on the potential molecular targets in both cellular chromatin and isolated oligonucleotides "in vitro".

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytogenetic, "whole genome"/conventional PCR, EMSA and DNase footprinting techniques. The results obtained: (i) show that PTHrP(38-94)-amide gains access to the nuclear compartment of MDA-MB231 cell; (ii) demonstrate that PTHrP(38-94)-amide is a DNA-binding peptide; and, (iii) represent the first data to date on the potential molecular targets in both cellular chromatin and isolated oligonucleotides "in vitro".

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 was affected by AA. The hypothesis was confirmed in in vitro tests: we observed, in fact, a strong inhibitory effect of AA against topoisomerase II in the kDNA assay and in topoisomerase II-mediated supercoiled DNA relaxation assay. In particular, this latter inhibition was not accompanied by stabilization of a covalent topoisomerase II-DNA intermediate. In order to characterize AA target(s), we pretreated pBR322 with AA and observed that this substrate became quickly incompetent in the topoisomerase II catalytic assay. These results suggest that DNA could be a target for Acrylamide-induced inhibition of topoisomerase II. Furthermore, preliminary results seem to indicate the possibility that another mode of action of acrylamide is related to its affinity for topoisomerase II sulphydryl groups, according to recent evidences reporting that thiol-reactive compounds can induce DNA damage through a nongenotoxic mechanism, such as the thiolation of the nuclear protein topoisomerase II.

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 multinucleated cells as well as aneuploid cells, seen just after treatment, reappeared since 60th cell generation. Metaphases with dicentric chromosomes or telomeric associations characterized cell population since 90th generation. To gain insight into the mechanism involved in perpetuating the unstable phenotype, groups of clones, stable and unstable, were analysed also for telomerase activity by TRAP assay and for levels of reactive oxygen species (ROS) measured by ability to oxidize fluorogenic dye. Some of the isolated unstable clones, also bearing chromosomal end-to-end fusions, maintained telomerase activity and were capable to proliferate accumulating genomic instability as well as transformed phenotype and spontaneous increased gene mutation oxidative stress associated. Furthermore these clones showed altered DNA methylation pattern. On the whole, these results raise the possibility that cell variants induced by the short-term exposure to arsenic probably gain a selective advantage when they are able to epigenetically reprogram their genome and proliferate in an error-prone mode.

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 induced by arsenic after treatment. In fact, cytogenetic, morphological and molecular investigations, performed at several time points during the expanded growth in drug-free medium, emphasized that genomic instability reappeared since 60th cell generation. Metaphases with dicentric chromosomes or telomeric associations characterized cell population since 90th cell generation. Some of the isolated clones also bearing chromosomal end-to-end fusions maintained telomerase activity and were capable to proliferate accumulating genomic instability as well as transformed phenotype and spontaneous increased gene mutation oxidative stress associated. On the whole, these results raise the possibility that the short-term exposure to arsenic induces altered DNA methylation pattern conferring a selective advantage to cell variants. Similarly to Karpinets and Foy hypothesis (Carcinogenesis, 2005) we believe these unstable cells epigenetically reprogram their genome and proliferate in an error-prone mode

Lo stato di metilazione del DNA genomico e del gene PTHrP è stato valutato con tecniche molecolari e citogenetiche in artrite reumatoide (AR), patologia autoimmune caratterizzata anche da alta incidenza di linfomi e da ipercalcemia per overespressione del gene PTHrP. La metilazione del DNA, infatti, ha un ruolo critico nello sviluppo delle malattie neoplastiche; il gene PTHrP avendo tre promotori uno dei quali contiene un’isola CpG è un buon candidato per la deregolazione da alterato pattern di metilazione locale. Le indagini sulla metilazione genomica, condotte su DNA estratto da sangue periferico di pazienti e di donatori e amplificato in reazioni di Methylation-Sensitive Arbitrarily Primed (MeS-AP)-PCR, hanno indicato che AR è fortemente associata a ipometilazione globale del DNA. Analogamente, l’analisi del pattern di metilazione del DNA cromosomico per mezzo della tecnica di immunofluorescenza indiretta con anticorpo anti-5 metilcitosina ha mostrato che le metafasi di linfociti periferici dei soggetti di controllo presentavano cromosomi quasi uniformemente decorati con grani brillanti; viceversa, i cromosomi dei pazienti AR erano debolmente fluorescenti senza grani discreti. Gli studi sulla metilazione del promotore 2 del gene PTHrP condotti su 5 siti all’interno dell’isola CpG utilizzando la tecnica Methylation-Sensitive Restriction Endonuclease Multiplex (MSREM)-PCR hanno mostrato che un sito tra i più vicini al sito di inizio della trascrizione è fortemente metilato in un gruppo significativamente grande di pazienti. Pertanto in AR ipometilazione genomica si accompagna a ipermetilazione gene-specifica, caratteristica comune a diversi tumori. Ciò indica che la suscettiblità ai linfomi può essere correlata a livelli alterati di metilazione e suggerisce l’opportunità di valutare lo stato di metilazione del DNA in pazienti sottoposti a terapie demetilanti, che insieme alla dieta e allo stile di vita, possono aumentare il rischio di evoluzione in patologie tumorali.

Early genetic instability induced in dividing V79-Cl3 Chinese hamster cells by inorganic arsenic, as demonstrated in our previous investigation, was evidenced by aneuploidy and nuclear abnormalities, but not by chromosomal rearrangements. Here we report the results of cytogenetic and morphological analyses performed on the progeny of cells dividing at the end of sodium arsenite treatment after they had been expanded through 120 generations (ASO cells) and then cloned. The acquired genetic instability persisted and was increased by highly unstable chromosomal rearrangements, namely dicentric chromosomes and telomeric associations, which were not seen following acute exposure. A peculiar finding was the preferential involvement of a particular chromosome in dicentric rearrangements observed in some isolated ASO clones. Interestingly, by immunostaining with anti-5-methylcytosine antibodies the genome-wide DNA hypomethylation, induced by arsenic immediately after the acute treatment, was found to affect those ASO clones characterized by aneuploidy and chromosomal rearrangements. These findings demonstrate that short-term exposure to arsenic has long-term effects and suggest that genome-wide DNA hypomethylation enhances genetic instability.