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Biomedical subjects

A Botta

Publications and source records attributed to A Botta.

At least 19 recordsLinked to original sources
PubMed2007

Exposure to genotoxic agents, host factors, and lifestyle influence the number of centromeric signals in micronuclei: a pooled re-analysis.

We pooled data from three biomonitoring studies using the cytokinesis-block micronucleus assay in peripheral blood lymphocytes in combination with fluorescence in situ hybridization. Centromere-positive micronuclei (C+MN) were classified in two groups: those containing one centromere (C1+MN) and those with two or more (Cx+MN). The three studies evaluated untreated cancer patients, welders, and pathologists/anatomists exposed to formaldehyde. The total number of subjects included in the pooled re-analysis was 113. A higher frequency of C+MN was observed in cancer patients and exposed workers, who showed significant differences from controls in all studies. C1+MN were particularly increased in the group of pathologists/anatomists, who showed a 3.29 times higher frequency than controls (95% CI: 2.04-5.30). A borderline increase in Cx+MN was observed in welders when compared to the corresponding control group (FR: 1.31; 95% CI: 0.99-1.74). An evident effect of gender was found, with significantly increased frequencies of all endpoints measuring aneuploidy in females (C+MN, C1+MN, and Cx+MN). Alcohol consumption had a significant effect on total MN frequency and particularly on C+MN and C1+MN. In conclusion, scoring the number of centromeric signals in the micronucleus assay provides additional information about the mechanism of action of various genotoxic agents, and the role of confounding factors may be more specifically accounted for. Indeed, C+MN could be efficiently used in biomonitoring studies as an independent biomarker of exposure and early biological effect. The use of centromeric signals allows the identification of two further endpoints, representing two alternative pathways of chromosome loss, i.e., impaired chromosome migration, leading to increased C1+MN frequency, and centrosome amplification, possibly leading to Cx+MN with two or more centromeric signals.

Case-Control Studies↗
PubMed2006

Evolution of DNA strand-breaks in cultured spermatocytes: the Comet Assay reveals differences in normal and gamma-irradiated germ cells.

In reproductive toxicity assessment, in vitro systems can be used to determine mechanisms of action of toxicants. However, they generally investigate the immediate effects of toxicants, on isolated germ cells or spermatozoa. We report here the usefulness of in vitro cultures of rat spermatocytes and Sertoli cells, in conjunction with the Comet Assay to analyze the evolution of DNA strand-breaks and thus to determine DNA damage in germ cells. We compared cultures of normal and gamma-irradiated germ cells. In non-irradiated spermatocytes, the Comet Assay revealed the presence of DNA strand-breaks, which numbers decreased with the duration of the culture, suggesting the involvement of DNA repair mechanisms related to the meiotic recombination. In irradiated cells, the evolution of DNA strand-breaks was strongly modified. Thus our model is able to detect genotoxic lesions and/or DNA repair impairment in cultured spermatocytes. We propose this model as an in vitro tool for the study of genotoxic injuries on spermatocytes.

Animals↗
PubMed2006

Number of centromeric signals in micronuclei and mechanisms of aneuploidy.

Genome instability or changes in chromosome structure and number are important facets of oncogenesis. Aneuploidy is a major cause of human reproductive failure and plays a large role in cancer. It is therefore important that any increase in its frequency due to occupational exposure to mutagens and carcinogens should be recognized and controlled. In recent years, the cytokinesis-block micronucleus assay has emerged as a biomarker of chromosome/genome damage relevant to cancer. Fluorescent in situ hybridisation using human pancentromeric DNA probes discriminates between the presence of acentric chromosomal fragments and whole chromosomes in binucleated micronucleated lymphocytes. The separated analysis of centromeric micronuclei may improve the sensitivity of the micronucleus assay in detecting genotoxic effects and chromosome instability. Our previous findings suggest that aneugenic events leading to micronuclei (MN) containing a single centromere (C1+MN) and two or more centromeres (Cx+MN) may arise through different pathways. Chromosome migration impairment would lead to increased C1+MN frequency whereas centrosome amplification would induce Cx+MN with three or more centromeric signals. Additional studies that target cellular defects on the centrosome (microtubule nucleation, organization of the spindle poles, cell cycle progression) are required to better understand aneuploid cell production.

Aneuploidy↗
PubMed2006

Genotoxic risk assessment of pathology and anatomy laboratory workers exposed to formaldehyde by use of personal air sampling and analysis of DNA damage in peripheral lymphocytes.

A study was conducted to evaluate the genotoxic effect of occupational exposure to formaldehyde on pathology and anatomy laboratory workers. The level of exposure to formaldehyde was determined by use of passive air-monitoring badges clipped near the breathing zone of 59 workers for a total sampling time of 15 min or 8 h. To estimate DNA damage, a chemiluminescence microplate assay was performed on 57 workers before and after a 1-day exposure. Assessment of chromosomal damage was carried out by use of the cytokinesis-blocked micronucleus assay (CBMN) in peripheral lymphocytes of 59 exposed subjects in comparison with 37 controls matched for gender, age, and smoking habits. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 18 exposed subjects and 18 control subjects randomized from the initial populations. Mean concentrations of formaldehyde were 2.0 (range <0.1-20.4 ppm) and 0.1 ppm (range <0.1-0.7 ppm) for the sampling times of 15 min and 8 h, respectively. No increase in DNA damage was detected in lymphocytes after a one-workday exposure. However, the frequency of binucleated micronucleated cells was significantly higher in pathologists/anatomists than in controls (16.9‰±9.3 versus 11.1‰±6.0, P=0.001). The frequency of centromeric micronuclei was higher in exposed subjects than in controls (17.3‰±11.5 versus 10.3‰±7.1) but the difference was not significant. The frequency of monocentromeric micronuclei was significantly higher in exposed subjects than in controls (11.0‰±6.2 versus 3.1‰±2.4, P<0.001), while that of the acentromeric micronuclei was similar in exposed subjects and controls (3.7‰±4.2 and 4.1‰±2.7, respectively). The enhanced chromosomal damage (particularly chromosome loss) in peripheral lymphocytes of pathologists/anatomists emphasizes the need to develop safety programs.

Adult↗
PubMed2006

A combined analysis of XRCC1, XRCC3, GSTM1 and GSTT1 polymorphisms and centromere content of micronuclei in welders.

The aims of the present study were to assess clastogenic and aneugenic properties of welding fumes using fluorescent in situ hybridization (FISH) with a human pancentromeric DNA probe. The involvement of genetic polymorphisms in DNA repair genes (p.Arg399Gln of XRCC1 and p.Thr241Met of XRCC3) and in detoxification genes (GSTM1 and GSTT1) on the centromere content of micronuclei (MN) was also evaluated. This study included 27 male welders working without any collective protection device and a control group (n = 30). The welders showed significantly higher levels of chromosome/genome damage compared to the controls. The frequencies of MN and centromere-positive MN (C+MN) per 1,000 binucleated cells were significantly higher in the exposed group than in the control group (7.1 per thousand +/- 3.7 versus 4.9 per thousand +/- 1.8; P = 0.012 and 3.5 per thousand +/- 1.8 versus 2.4 per thousand +/- 1.2; P = 0.018, respectively, Mann-Whitney U-test). The centromere-negative MN (C-MN) frequency was higher in the exposed subjects than in the controls (3.6 per thousand +/- 3.4 versus 2.5 per thousand +/- 1.4), but the Mann-Whitney U-test did not yield a significant result. In the total population, the GSTM1 and GSTT1 polymorphisms significantly affected the frequencies of C-MN and C+MN defined by FISH. GSTM1 positive subjects showed an increased C-MN frequency and GSTT1 null subjects showed an elevated C+MN frequency. When GSTM1 and GSTT1 genotypes were included in multiple regression analysis, the effect of the occupational exposure could better be demonstrated; both C+MN and C-MN were significantly increased in the welders. Our results suggest that the combined analysis of genetic polymorphisms and centromeres in MN may improve the sensitivity of the micronucleus assay in detecting genotoxic effects.

Adult↗
PubMed2006

Italian guidelines for molecular analysis in myotonic dystrophies.

Myotonic dystrophies, the most common form of adult muscular dystrophy, comprise at least two forms, clinically and genetically heterogeneous. Myotonic dystrophy type 1 and type 2 are both caused by unstable repetitions in untranslated gene regions: a [CTG]n expansion in the 3' region of the DMPK gene on chromosome 19q13 (DM1) and [CCTG]n tetranucleotide repeat located in the first intron of the ZNF9 gene on chromosome 3q21 (DM2). DM clinical features are caused by a gain of functions RNA mechanism in which the CUG and CCUG repeats alter nuclear functions, including alternative splicing of shared genes. Southern blot and/or polymerase chain reaction PCR-based approaches allow the detection of DM mutations in almost 100% of cases, however, the expansion size and the elevated grade of somatic instability make molecular testing for DM a diagnostic challenge. The increased use of DNA testing for DM generates many questions regarding the indications and interpretations of the test which require standardized methods, routinely available in molecular genetic laboratories. Here, we propose Guidelines for the molecular diagnosis of DM1 and DM2 approved by the Italian Ministry of Health in 2005 (Piano Nazionale Linee Guida, PNLG). Best practice for DM molecular analysis in diagnostic application, presymptomatic and prenatal testing, using direct and indirect approaches are described, with particular attention focused on ethical, legal and social issues. Overviews of materials used in the molecular diagnosis, as well as internet resources, are also included.

Genetic Testing↗
PubMed2005

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons.

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance. The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model. First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively. Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel. There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.

Aneugens↗
PubMed2005

Transmission ratio distortion in the spinal muscular atrophy locus: data from 314 prenatal tests.

BACKGROUND: Spinal muscular atrophy (SMA) is a recessive neurodegenerative disorder characterized by the loss of alpha-motor neurons in the spinal cord and subsequent death of motor neuron cells. SMA occurs with a frequency of 1 in 6,000 live births, with a carrier frequency of 1 in 40, and is a leading genetic cause of infant mortality. SMA is caused by loss or mutation of the telomeric survival motor neuron gene (SMN1), which is deleted in almost 94% of SMA patients OBJECTIVE: To analyze the transmission ratio at the SMA locus, examining the segregation of the SMN1-deleted alleles in 314 fetuses from carrier parents who requested prenatal testing for the disease. METHODS: Prenatal diagnosis of SMA in families at 25% risk of the disease has been performed on chorionic villous sampling specimens, through direct detection of the SMN1 gene mutation and linkage analysis using microsatellite markers from the 5q13 region. Analysis of the genotypic/allelic frequencies of the SMN1 gene was performed using the chi2 test, assuming a recessive mendelian inheritance. RESULTS: Of 314 fetuses analyzed, 95 were homozygous for the wild-type allele (30.3%), 154 were carriers (49.0%), and the remaining 65 were homozygous for the mutated allele (20.7%). Statistical analysis demonstrated that proportion of fetuses predicted with SMA is lower than 25% expected for a recessive disorder, resulting in a transmission rate of the SMN1-deleted allele deviant from the 50% expected in a random the segregation of a mendelian tract (p = 0.016) CONCLUSIONS: This is the first study to evaluate the genotypic frequencies at the spinal muscular atrophy (SMA) locus based on data derived from prenatal analysis, which are not subject to ascertainment bias. The analysis showed a transmission ratio distortion at the SMA locus in favor of the SMN1 wild-type alleles.

Chorionic Villi Sampling↗
PubMed2005

Risk assessment of welders using analysis of eight metals by ICP-MS in blood and urine and DNA damage evaluation by the comet and micronucleus assays; influence of XRCC1 and XRCC3 polymorphisms.

The aims of the present study were to assess the occupational risk of welders using analysis of metals in biological fluids, DNA damage evaluation by complementary genotoxic endpoints and the incidence of polymorphisms in DNA repair genes. A biomonitoring study was conducted that included biometrology (blood and urinary concentrations of aluminium, cadmium, chromium, cobalt, lead, manganese, nickel, zinc by ICP-MS), comet and cytokinesis-block micronucleus assays in peripheral lymphocytes and genetic polymorphisms of XRCC1 (p.Arg399Gln) and XRCC3 (p.Thr241Met). This study included 60 male welders divided into two groups: group 1 working without any collective protection device and group 2 equipped with smoke extraction systems. A control group (n = 30) was also included in the study. Higher chromium, lead and nickel blood and urinary concentrations were detected in the two groups of welders compared to controls. Statistically differences between welders of group 1 and group 2 were found for blood concentration of cobalt and urinary concentrations of aluminium, chromium, lead and nickel. The alkaline comet assay revealed that welders had a significant increase of OTMchi2 distribution at the end of a work week compared to the beginning; a significant induction of DNA strand breaks at the end of the week was observed in 20 welders out of 30. The cytokinesis-block micronucleus assay showed that welders of group 1 had a higher frequency of chromosomal damage than controls. The XRCC1 variant allele coding Gln amino acid at position 399 was found to be associated with a higher number of DNA breaks as revealed by the comet assay. Increased metal concentrations in biological fluids, DNA breaks and chromosomal damage in lymphocytes emphasized the need to develop safety programmes for welders.

Adult↗
PubMed2004

Characterization of a single nucleotide polymorphism in the ZNF9 gene and analysis of association with myotonic dystrophy type II (DM2) in the Italian population.

Myotonic dystrophy type 2 (DM2) is a dominant inherited disorder clinically similar to myotonic dystrophy type 1 (DM1) with a peculiar pattern of multisystemic phenotypic features. The mutation responsible for DM1 is a CTG repeat in the 3' UTR of the dystrophia myotonica protein kinase gene (DMPK) on chromosome 19q13.3, while DM2 is caused by an unstable CCTG expansion in intron 1 of the zinc finger protein 9 gene (ZNF9) on chromosome 3q21.3. Southern blotting analysis is the conventional test used to determinate the size of the repeats in the molecular diagnosis of DM2. However, the large number of CCTG repeats and their somatic instability complicates this diagnostic protocol. In order to improve the DM2 test, we have recently characterised a single nucleotide polymorphism located in the first intron of the ZNF9 gene. This SNP consists in a C to A nucleotide change, which creates or disrupts and ApaI enzyme restriction site, easily detectable by PCR amplification followed by restriction analysis. We genotyped this SNP in 30 unrelated DM2 patients and 70 unrelated Italians healthy individuals. Our results show that this polymorphism is in linkage disequilibrium with the DM2 mutation.

Case-Control Studies↗
PubMed2004

Low-level arsenite activates the transcription of genes involved in adipose differentiation.

In this study we analyzed gene expression in 3T3-F442A pre-adipocyte cells that differentiate in the presence of micro-molar arsenate concentration. Two concentrations of arsenite (As2O3, 0.25 micromol/L and 0.5 micromol/L) were applied for three days with and without insulin (170 nmol/L) and gene expressions were evaluated by quantitative RT-PCR. The genes included genes of oxidative-stress responses: heme-oxygenase-1 (HO1) and the hypoxia inducible factor 1a (HIF1alpha), genes of cell-cycle: c-jun and Kruppel like factor 5 (KLF5), and genes that play important roles in adipose determination: a peroxisome proliferator-activated receptor (PPARgamma) and a CCAAT/ enhancer binding protein (C/EBPalpha). Arsenite induced the expression of HO1, HIF1alpha, KLF5, PPARgamma and C/EBPalpha. These results suggest that under condition of oxidative stress arsenite induces genes that are required for adipose differentiation.

3T3 Cells↗
PubMed2004

Celiac disease and inflammatory bowel disease in pregnancy.

An adverse influence on reproductive life and obstetric complications are known to occur in women with celiac disease (clinical and subclinical disease) or inflammatory bowel diseases. Treatment can improve the pregnancy outcome; therefore, it is advisable that a clinical evaluation is performed by a joint team of obstetricians, internists and surgeons. The preconception clinical evaluation of the affected women is useful to focus on the different clinical aspects of the disease and to indicate specific therapeutic strategies. In this study a review of the literature regard to celiac disease and inflammatory bowel disease in pregnancy is presented.

Celiac Disease↗
PubMed2002

Cytogenetic monitoring of industrial radiographers using the micronucleus assay.

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years+/-11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv+/-49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7 per thousand +/-5.2 versus 6.6 per thousand +/-3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C-MN) in exposed subjects than in controls (8.5 per thousand +/-4.9 versus 2.2 per thousand +/-1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C-MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.

Adult↗
PubMed2002

Acentromeric micronuclei are increased in peripheral blood lymphocytes of untreated cancer patients.

Increased micronucleated cell rates, dicentric chromosomes, and other chromosomal damages have been reported in lymphocytes of cancer patients prior to the initiation of chemotherapy, and/or radiotherapy. The cause of these chromosomal damages in these lymphocytes remains unclear. In the present work, we investigated whether these micronuclei mainly reflect structural or numerical chromosomal aberrations by applying the cytokinesis-blocked micronucleus (CBMN) assay in combination with fluorescent in situ hybridization (FISH) of a DNA centromeric probe on blood samples of 10 untreated cancer patients (UCPs), and 10 healthy subjects (HSs). Micronucleated binucleated lymphocyte rate was significantly increased in patients (mean+/-S.D.: 19.0 per thousand +/-14.1 versus 9.2 per thousand +/-4.6 in controls). Trinucleated cytokinesis-blocked cells were not significantly higher in patients than in controls. Acentromeric, centromeric, and multicentromeric micronucleus levels were two-fold higher in patients than in controls, but the difference was significant only with acentromeric micronuclei. The percentage of micronuclei containing one or more centromeres averaged 69.2, and 71.5% in patients, and controls, respectively. The percentage of micronuclei containing several centromeres was 44.7% in patients, and 54.6% in controls. Among centromere-positive micronuclei, the percentage of micronuclei containing several centromeres averaged 59.7% in patients, and 75.4% in controls. These results indicate that genetic instability in peripheral blood lymphocytes of UCPs occurs because of enhanced chromosome breakage. However, a substantial proportion of this genetic instability occurs because of defects in chromosome segregation.

Adult↗
PubMed2001

The expression of genes induced in melanocytes by exposure to 365-nm UVA: study by cDNA arrays and real-time quantitative RT-PCR.

Ultraviolet A radiation (UVA; 320-400 nm) constitutes more than 90% of the terrestrial UV solar energy. This type of radiation generates reactive oxygen species and consequently induces DNA damage. UVA irradiation is now considered to be an important carcinogen agent especially in the development of melanoma. UVA radiation is known to activate several pathways in mammalian cells. We have used cDNA arrays to analyze differential gene expression in primary cultures of human melanocytes in response to 365-nm UVA. Among 588 genes studied, 11 were overexpressed. These genes included genes involved in cell cycle regulation (GADD45, CIP1/WAF1), in stress response (HSP70, HSP40, HSP86), in apoptosis (GADD153, tristetraproline) and genes encoding transcription factors (EGR-1, ETR-101, c-JUN, ATF4). This coordinate gene regulation was confirmed by real-time quantitative RT-PCR.

Cell Differentiation↗
PubMed2001

Cloning and characterization of the gene encoding human NPL4, a protein interacting with the ubiquitin fusion-degradation protein (UFD1L).

The ubiquitin fusion-degradation gene (UFD1L) encodes the human homologue of the yeast ubiquitin fusion-degradation 1 protein, an essential component of the ubiquitin-dependent proteolytic turnover and mRNA processing. Although the UFD1L gene has been mapped in the region commonly deleted in patients with DiGeorge syndrome (DGS)/velocardiofacial syndrome (VCFS), correlation between its haploinsufficiency and the phenotype has not yet been established. The only functional data available about mammalian Ufd1p is the ability to form a complex with the rat Npl4 protein, a component of the nuclear pore complex. In this paper we report the cloning and molecular characterization of the human NPL4 gene. This gene encodes for a protein 96% homologous to the rat Npl4, and 44 and 34% homologous to the C. elegans and S. cerevisiae Npl4 gene products, respectively. Fluorescence in situ hybridization experiments on human metaphases localized the NPL4 gene on the most telomeric region of chromosome 17q. Northern blots analysis on foetal and adult human tissues revealed a major approximately 4.5 kb transcript most abundant in heart, brain, kidney and skeletal muscle. In order to test a potential relationship between nuclear transport defects and some aspect of the DGS/VCFS phenotype, we also exclude the presence of mutations in the NPL4 coding sequence in a subset of patients with DGS/VCFS and no detectable 22q11 deletion or mutations at the UFD1L locus.

Abnormalities, Multiple↗
PubMed2001

Association study of a promoter polymorphism of UFD1L gene with schizophrenia.

Schizophrenia or schizoaffective disorders are often found in patients affected by DiGeorge/velo-cardio-facial syndrome (DGS/VCFS) as a result of hemizygosity of chromosome 22q11.2. We evaluated the UFD1L gene, mapping within the DGS/VCFS region, as a potential candidate for schizophrenia susceptibility. UFD1L encodes for the ubiquitin fusion degradation 1 protein, which is expressed in the medial telencephalon during mouse development. Using case control, simplex families (trios), and functional studies, we provided evidence for association between schizophrenia and a single nucleotide functional polymorphism, -277A/G, located within the noncoding region upstream the first exon of the UFD1L gene. The results are supportive of UFD1L involvement in the neurodevelopmental origin of schizophrenia and contribute in delineating etiological and pathogenetic mechanism of the schizophrenia subtype related to 22q11.2 deletion syndrome.

Adaptor Proteins, Vesicular Transport↗