Biomedical subjects

F Marcon

Publications and source records attributed to F Marcon.

At least 19 recordsLinked to original sources

PubMed2006

Influence of DNA repair polymorphisms on biomarkers of genotoxic damage in peripheral lymphocytes of healthy subjects.

DNA repair polymorphisms may represent susceptibility factors affecting DNA integrity, and possibly cancer risk, in human population. In order to elucidate the influence of a few widely studied DNA repair polymorphisms on individual levels of DNA damage and their possible interaction with lifestyle and environmental exposures, 171 subjects from a well-characterized human population enrolled in a previous study on genetic effects of air pollution were genotyped for the XRCC1 Arg280His and Arg399Glu, XRCC3 Thr241Met and ERCC2 Lys751Gln polymorphisms. The association between DNA repair genotype, alone or in combination with metabolic genotype, on the levels of SCE, micronuclei and tail moment values in peripheral lymphocytes was evaluated. A significant influence of the ERCC2 genotype on SCE frequency was observed. Subjects with ERCC2 751 Gln/Gln genotype had significantly higher risk of high (above the median) SCE/cell with respect to Lys/Lys referents (OR 4.55, 95% CI 1.48-13.99). A non-significantly elevated OR was also observed in Gln/Lys heterozygotes, suggesting a gene dosage effect. When subjects were categorized by smoking habits and professional exposure, the variant ERCC2 751 Gln/Gln genotype was associated with elevated SCE rates in non-smokers and in exposed subjects, but not in smokers. The results of this study support the hypothesis that some DNA repair polymorphisms exert a modifying effect on individual levels of DNA damage in healthy subjects, possibly also modulating cancer risk.

Adult↗

PubMed2005

[Feasibility and significance of a transoesophageal electrophysiological investigation in children and adolescents with Wolff-Parkinson-White syndrome].

UNLABELLED: An electrophysiological investigation is the most reliable means of detecting malignant forms of Wolff-Parkinson-White syndrome (WPW). However, an endocavity investigation is an invasive procedure, especially in young subjects with few symptoms. The aim of this study was to examine the feasibility and results of an electrophysiological study performed by the transoesophageal route in children with WPW. The study was performed in 70 children aged between 11 and 19 years (mean 15 +/- 3) with an obvious ECG appearance of WPW: 13 had dizziness or syncope (group I), 25 had tachycardia (group II) and 32 were asymptomatic (group III). The ages were similar in all three groups. The transoesophageal electrophysiological investigation without premedication consisted of atrial stimulation at increasing frequencies and programmed atrial stimulation using one and two extra stimuli delivered in the basal state and after infusion of 2 to 5 microg of isoproterenol. RESULTS: The investigation was completed in all the children except one in group II. A paroxysmal junctional tachycardia was induced in 7 group I children (54%), 22 in group II (92%) and 4 in group III (12.5%). Atrial fibrillation lasting more than one minute was induced in 7 group I children (54%), 6 in group II (25%) and 6 in group III (19%). The percentage of malignant forms combining rapid conduction in the bundle of Kent at a rate of more than 240/min in the basal state or more than 300/min with isoproterenol, and atrial fibrillation was 54% in group I, 21% in group II, and 22% in group III. In conclusion, a transoesophageal electrophysiological investigation was possible as an outpatient procedure in children older than 10 years, and allowed the detection of potentially serious forms whatever the indication for the investigation, with nevertheless a significantly higher incidence in those presenting with dizziness or syncope. The incidence of 22% for potentially malignant forms in asymptomatic children provides an incentive to recommend an ECG in all children older than 10 years participating in an active sport in order to detect WPW and to propose oesophageal investigation.

Adolescent↗

PubMed2004

Human-hamster hybrid cells used as models to investigate species-specific factors modulating the efficiency of repair of UV-induced DNA damage.

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species.

Animals↗

PubMed2003

Folate status, metabolic genotype, and biomarkers of genotoxicity in healthy subjects.

Gene-environment interactions play an important role in folate metabolism, with a potential impact on human health. Deficiencies in the uptake of key micronutrients and variant genotypes can affect the folic acid cycle, modulating methyl group transfer in key processes and leading to increased cancer risk and Down syndrome incidence. So far, the significance of folate status and metabolic genotypes on baseline levels of DNA damage in normal individuals has not been fully elucidated. In this study, the possible modulation of SCE, micronuclei and tail moment values in peripheral lymphocytes by plasma levels of folic acid, homocysteine and vitamin B12, and by the methylenetetrahydrofolate reductase (MTHFR) C677T and methionine synthase reductase (MTRR) A66G polymorphisms was investigated in 191 healthy subjects. The results obtained show a highly significant (P = 0.001) positive association between plasma levels of vitamin B12 and frequencies of both SCE and high frequency cells (HFC, above 90 degrees percentile) in smokers. No significant effect was observed in non-smokers. Moreover, after correction for age, gender and GSTM1 genotype, a significant association (P = 0.026) between the MTRR 66GG variant genotype and higher micronucleus rates was observed. Tail moment values were not affected by any of the independent variables considered. Overall, the results obtained suggest that both folate status and relevant metabolic genotype can influence background levels of DNA damage in normal subjects. The significant association observed in smokers between plasma vitamin B12 and SCE frequencies may highlight the effect of methylation status on DNA damage and repair, although the role of other, unidentified dietary factors cannot be ruled out. At the same time, micronucleus data indicate that the MTRR 66GG variant may represent another individual trait of relative genomic instability, thus supporting epidemiological data on increased risk of Down syndrome conception in MTRR 66GG subjects.

Adult↗

PubMed2003

Analysis of micronuclei in peripheral blood lymphocytes of traffic wardens: effects of exposure, metabolic genotypes, and inhibition of excision repair in vitro by ARA-C.

The cytokinesis-block micronucleus (MN) assay in peripheral lymphocytes was used to assess the genetic effects of the occupational exposure to traffic fumes in policemen from the Municipality of Rome. The study population consisted of 192 subjects engaged in traffic control (exposed, 134 subjects), or in office work (controls, 58 subjects). Groups were balanced for age, gender, and smoking habits. The average benzene exposure during the workshift was 9.5 and 3.8 microg/m(3) in exposed individuals and controls, respectively. All subjects were genotyped for CYP1A1, CYP2E1, GSTM1, GSTT1, and DT-diaphorase polymorphisms. The incidence of micronuclei and micronucleated cells was recorded in 1,000 binucleated cells harvested 66 hr after mitogen stimulation. Regression analysis of data showed that MN frequency was mainly modulated by the age (P = 0.001) and gender (P = 0.001) of the study subjects (relatively higher in the elderly and females), whereas it was unaffected by the occupational exposure to traffic fumes and smoking habits. A weak (P = 0.02) association between lower MN frequency and the GSTM1 null genotype was also observed. In order to improve the sensitivity of the method to excision-repairable lesions, a modified protocol, with exposure of cells to the repair inhibitor cytosine arabinoside (Ara-C) during the first 16 hr of growth, was applied to 78 subjects (46 exposed and 32 controls). The results confirmed the higher MN frequency in females (P < 0.05), but failed to demonstrate any significant effect of chemical exposure (occupational or related to smoking habits). When the frequency of MN induced by Ara-C (i.e., spontaneous values subtracted) was considered, a significant inverse correlation with age was observed (P = 0.005), possibly related to the age-dependent decrease in repair proficiency.

Adult↗

PubMed2002

Biomonitoring of exposure to urban air pollutants: analysis of sister chromatid exchanges and DNA lesions in peripheral lymphocytes of traffic policemen.

In order to elucidate the health effects of occupational exposure to traffic fumes, a few biomarkers of early genetic effect were investigated in Rome traffic policemen. One hundred and ninety healthy subjects engaged in traffic control (133 subjects) or in office work (57 subjects) participated the study. For all subjects, detailed information on smoking habits and other potential confounders were recorded by questionnaires. Average exposure of the study groups to benzene and other aromatic hydrocarbons was evaluated in a parallel exposure survey. All workers were genotyped for the following metabolic polymorphisms: CYP1A1 (m1, m2, and m4 variants), CYP2E1 (PstI and RsaI), NQO1 (Hinf1), GSTM1 and GSTT1 (null variants). In this paper, the results of the analysis of sister chromatid exchanges (SCE) in peripheral lymphocytes, and DNA damage by alkaline (pH 13) comet assay in mononuclear blood cells are reported. No statistically significant difference in the frequency of SCE or high frequency cells (HFC) was observed between traffic wardens and office workers (controls), despite the significantly higher exposure to benzene of the former (average group exposure 9.5 versus 3.8microg/m(3), 7h TWA). Conversely, both SCE per cell and HFC were highly significantly (P<0.001) increased in smokers compared to nonsmokers, showing a significant correlation (P<0.001) with the number of cigarettes per day. Multiple regression analyses of data, with metabolic polymorphisms, smoking habits, alcohol consumption, age, gender, and family history of cancer as independent variables, showed that smoking habits, and possibly the CYP2E1 variant genotypes, were the main factors explaining the variance of both SCE and HFC. Within smokers, an association of borderline significance between the CYP1A1 variant genotypes and increased SCE (P=0.050) and HFC (P=0.090) was found. This effect was mainly observed in light smokers (<15 cigarettes per day). The analysis of DNA damage by comet assay did not highlight any statistically significant difference between the exposed and control workers. Moreover, no significant model explaining tail moment variance was obtained by multiple regression analysis using the independent variables shown above. On the whole, these results indicate that exposure to moderate air pollution levels does not result in a detectable increase of genetic damage in blood cells. This evidence does not rule out any possibility of adverse effects, but strongly suggests that in urban residents life-style related factors, such as tobacco smoking, give the prevailing contribution to individual genotoxic burden.

Adult↗

PubMed2002

Detection of 1cen--1q12 lesions in different phases of the cell cycle: dual colour FISH analysis of peripheral lymphocytes from subjects with occupational exposure to petroleum fuels.

Dual colour FISH was used to assess the genotoxic effects of exposure to petroleum fuels and low benzene levels in peripheral lymphocytes of 12 gasoline station attendants. Labelled DNA probes were used for hybridization of the 1cen and 1q12 contiguous regions of chromosome 1, allowing simultaneous detection of hyperploidy and breakages in both interphase and metaphase cells. The analysis of interphase cells (either unstimulated or mitogen stimulated) showed a prevalence of cells with signal separation in exposed workers compared to matched controls. This difference was highly significant (P < 0.001) in stimulated lymphocytes (9.9 +/- 3.3 and 6.5 +/- 1.5 per thousand in exposed and controls, respectively). Far lower incidences of breaks, with no relation to chemical exposure, were detected in metaphase cells (0.3 +/- 0.8 versus 0.7 +/- 1.0 per thousand, respectively). The analysis of post-mitotic, cytokinesis-blocked cells again showed a relatively high incidence of nuclei with displacement of fluorescent signals (7.2 +/- 2.4 and 5.6 +/- 1.7 per thousand, respectively), suggesting that chromatin decondensation, rather than alteration of DNA strand integrity, led to signal separation in interphase nuclei. Even though the mechanism leading to the separation of alpha and classical satellites in interphase nuclei has not been elucidated, the significant association between cytogenetic findings and intensity of benzene exposure (as shown by the analysis of internal exposure biomarkers) suggests that signal displacement in 1cen-1q12 may be a marker of chemical exposure.

Cell Cycle↗

PubMed2001

Environmental and biological monitoring of traffic wardens from the city of Rome.

A molecular epidemiological study on Roman policemen is ongoing. The results of a first assessment of the occupational exposure to aromatic compounds of 66 subjects engaged in traffic control and of 33 office workers are presented in this paper. Passive personal samplers and urinary biomarkers were used to assess exposure to benzene and polycyclic hydrocarbons during work shifts. The results obtained indicate that benzene exposure in outdoor workers is about twice as high as in office workers (geometric mean 7.5 and 3.4 micrograms/m3, respectively). The distribution of individual exposure values was asymmetrical and skewed toward higher values, especially among traffic wardens. Environmental benzene levels recorded by municipal monitoring stations during work shifts (geometric mean 11.2 micrograms/m3) were in the first instance comparable to or greater than individual exposure values. However, several outlier values were observed among personal data that greatly exceeded average environmental benzene concentrations. Among the exposure biomarkers investigated, only blood benzene correlated to some extent with previous exposure to benzene, while a seasonal variation in the excretion of 1-hydroxypyrene and trans-muconic acid was observed in both study groups. In conclusion, these results suggest that outdoor work gives a greater contribution than indoor activities to benzene exposure of Roman citizens. Moreover, relatively high-level exposures can be experienced by outdoor workers, even in the absence of large-scale pollution episodes.

Adult↗

PubMed2000

Recombination between homologous chromosomes does not play a dominant role in the formation of radiation-induced chromosomal aberrations.

PURPOSE: In mammalian cells, the relevance of homologous recombination in radiation-induced double-strand break (DSB) repair is not yet well understood. In the present work, the role of recombination between homologous chromosomes and homology-directed repair of DSB were studied, using X-ray-induced chromosomal aberrations as an end-point. MATERIALS AND METHODS: Human-hamster hybrid cells containing one or two copies of human chromosome 8 were used. If recombination between homologous chromosomes plays a dominant role in DSB repair, it is expected that X-irradiation of cells with two copies of chromosome 8 would result in a lower frequency of aberrations involving this chromosome compared with cells with only one copy of chromosome 8. The aberrations involving human chromosome 8 were detected by fluorescence in situ hybridization (FISH). Furthermore, a comparison between the hamster cell line XR-C1 (defective in non-homologous repair), CHO-9 (the wild-type cells) and the cell line XR-C1#8 (in which the defect of XR-C1 is complemented by human chromosome 8) was made to determine, indirectly, the involvement of homology-directed recombination in DSB repair. RESULTS: The observed frequencies of aberrations per human chromosome 8 were not significantly different between cells containing one or two copies of this chromosome. The frequency of chromatid-type aberrations was doubled in XR-C1 cells compared with CHO-9 and XR-C1#8 cells. CONCLUSIONS: In hamster cells, recombination between homologous chromosomes appears not to have a major role in the formation of radiation-induced chromosomal aberrations, while nonhomologous repair seems to be important in both the G and G2 phases of the cell cycle.

Animals↗

PubMed1999

Chromosome damage and aneuploidy detected by interphase multicolour FISH in benzene-exposed shale oil workers.

A multicolour tandem-labelling fluorescence in situ hybridization (FISH) procedure was used to detect chromosome alterations in peripheral blood cells of a group of Estonian petrochemistry workers. Twelve workers employed in benzene production and five cokery workers, together with eight unexposed rural controls, were enrolled in the study. The methodology employed, based on the in situ hybridization of adjacent centromeric and pericentromeric regions, allowed the simultaneous detection of both chromosome breakage, involving damage-prone pericentromeric regions, and hyperploidy in interphase cells. Blood smears from all subjects were hybridized with chromosome 1 specific probes, in order to detect genotoxic damage in circulating lymphocytes and granulocytes. Moreover, lymphocyte cultures were established, harvested 48 h following mitogen stimulation and hybridized with the tandem chromosomes 1 and 9 probes. No significant difference in the incidence of breakage was detected in the nucleated cells of blood smears of exposed vs. control subjects. In contrast, modest but significantly increased frequencies of breakage affecting both chromosomes 1 and 9 were observed in the cultured lymphocytes of the benzene-exposed workers compared to the unexposed controls, suggesting an expression of premutagenic lesions during the S-phase in vitro. Across the entire study group, the frequencies of breakage affecting chromosomes 1 and 9 in the stimulated lymphocytes were highly intercorrelated (p < 0.001). No significant difference was found in the incidence of hyperploidy among the study groups, although a tendency to higher values was observed in benzene-exposed workers. Although the relatively small size of the study groups does not allow firm conclusions on the role of occupational exposure, the observed patterns are suggestive of effects in the benzene-exposed workers. This work also shows that tandem labelling FISH can be usefully applied in human biomonitoring, allowing the simultaneous detection of both hyperploidy and chromosome breakage at interphase in different cell types.

Adult↗

PubMed1999

Follow-up results of transvenous occlusion of patent ductus arteriosus with the buttoned device.

OBJECTIVES: The purpose of this presentation is to document results of buttoned device (BD) occlusion of patent ductus arteriosus (PDA) in a large number of patients with particular emphasis on long-term follow-up in an attempt to provide evidence for feasibility, safety and effectiveness of this method of PDA closure. BACKGROUND: Immediate and short-term results of BD occlusion of PDA have been documented in a limited number of children. METHODS: During a six-year period ending August 1996, transcatheter BD closure of PDA was attempted in 284 patients, ages 0.3 to 92 years (median 7) under a protocol approved by the local institutional review boards and FDA with an investigational device exemption in U.S. cases. RESULTS: The PDAs measured 1 to 15 mm (median 4) at the narrowest diameter; 20 were larger than 8 mm and 10 larger than 10 mm. They were occluded with devices measuring from 15 to 35 mm delivered via 7F (N = 140) or 8F (N = 144) sheaths. Successful implantation of the device was accomplished in 278 (98%) of 284 patients. The Qp:Qs decreased from 1.8+/-0.6 (mean+/-SD) to 1.09+/-0.19 (p < 0.001). Effective occlusion defined as no (N = 167 [60%]) or trivial (N = 79 [28%]) residual shunt was achieved in 246 (88%) patients. All types of PDAs, irrespective of the shape (conical, tubular or short), size (small or large) or length (short or long) of the PDA and previously implanted Rashkind devices, could be occluded. Follow-up data, 1 to 60 months (median 24) after device implantation, were available in 234 (84%) patients. Seven (3%) patients required reintervention to treat residual shunt with (N = 2) or without (N = 5) hemolysis. Actuarial reintervention-free rates were 95% at 1 and 5 years. There was gradual reduction of actuarial residual shunts and were 40%, 28%, 21%, 14%, 11%, 10%, 6% and 0% respectively at 1 day, 1, 6, 12, 24, 36, 48 and 60 months after device implantation. Incorporation of folding plug over the button loop in 10 additional patients produced immediate and complete occlusion of PDA. CONCLUSIONS: This large multiinstitutional experience confirms the feasibility, safety and effectiveness of buttoned device closure of PDAs. All types of PDAs irrespective of the shape, length and diameter can be effectively occluded. Incorporation of folding plug over the button loop produces complete PDA occlusion at the time of device implantation.

Adolescent↗

PubMed1999

Analysis of chromosome loss and non-disjunction in cytokinesis-blocked lymphocytes of 24 male subjects.

Chromosome malsegregation in peripheral blood lymphocytes of 24 healthy male subjects was analysed by means of fluorescence in situ hybridization with centromeric probes of chromosomes 7, 11, 18 and X. On the basis of the distribution of centromeric signals in cytokinesis-blocked cells, both loss (leading to centromere-positive micronuclei) and non-disjunction (resulting in an unbalanced distribution of signals in the main nuclei) of the hybridized chromosomes in vitro were identified. In addition, the incidence of binucleated cells with two hyperploid nuclei, possibly arising from mitotic division of trisomic types, was determined. In this way, the incidence of chromosome malsegregation in vivo and in vitro could be compared in the same cell samples. The results obtained show that ageing is positively correlated with the incidence of malsegregation of chromosome X in peripheral lymphocytes of male subjects and confirm the higher susceptibility of chromosome X to malsegregation in comparison with autosomes. A positive correlation between in vitro and in vivo malsegregation rates was observed for both chromosome X and for autosomes. Finally, relatively high frequencies of multiple malsegregation events, greater than expected for independent events, were recorded for both chromosome X and for autosomes, indicating that the abnormal segregation of chromosomes may be connected to a general dysfunction of the mitotic apparatus. The correlation observed between in vitro and in vivo malsegregation frequencies and the association of both parameters with ageing suggest that analysis of chromosome malsegregation in binucleated cells is a useful tool in the study of genomic instability in human populations.

Adult↗

PubMed1998

Genetic effects of petroleum fuels: II. Analysis of chromosome loss and hyperploidy in peripheral lymphocytes of gasoline station attendants.

Molecular cytogenetic methods were applied to investigate the effect of the occupational exposure to low concentrations of benzene and petroleum fuels on genomic stability. Twelve male gasoline station attendants (average benzene exposure of 0.32 mg/m3 as 8h TWA) and 12 age- and smoking-matched unexposed controls were selected for the study. The incidence of hyperploidy and polyploidy in peripheral lymphocytes was evaluated through in situ hybridization of interphase cells, harvested 24 hr after stimulation, with centromeric probes of chromosomes 7, 11, 18, and X. For half of the subjects, metaphases harvested 24 hr later were analyzed. The incidence of chromosome loss in vitro was determined in cytokinesis-blocked cells, harvested at 66 hr, through the hybridization of micronuclei with a pancentromeric probe. Ten thousand chromosomes (more than 200 metaphases equivalent) and 2,000 binucleated cells/person were scored for hyperploidy and micronucleus analysis, respectively. The results obtained did not show any exposure-related excess of hyperploidy or micronucleus formation. Conversely, the age of the subjects was significantly correlated with several markers of genomic instability, such as the incidence of chromosome X and chromosome 18 hyperploidy, total hyperploidy and polyploidy, and close to statistical significance with chromosome loss. Smoking habits did not appear to contribute significantly to the effects measured. The parallel analysis of hyperploidy and polyploidy in interphase nuclei in 24-hr cultures and in metaphase cells harvested 24 hr later showed basically similar incidences of aneuploid cells, indicating that no significant selection against hyperploid and polyploid types occurred during the first cell cycle in vitro.

Air Pollutants, Occupational↗

PubMed1997

[Tilt-table test. Value in children and adolescents].

The tilt test is a non-invasive investigation which reproduces malaises or vagal syncope. This is a common condition in children and adolescents and the value of this investigation in this population is therefore self-evident. The tilt test was performed from 4 years of age in 105 children aged 4 to 18 years (mean 10 +/- 4 years) who had suffered one or several malaises with syncope. This test, using a resting period of 20 minutes, then a tilt of 70-80 degrees for 40 minutes, was considered to be positive if the symptoms could be reproduced or if the heart rate slowed to less than 50/min or if the blood pressure fell by more than 30 mmHg. The test was positive in 46 children (44%). Ten remained asymptomatic but had significant reductions in blood pressure: 36 children reproduced their symptoms. The positivity was related to a cardio-inhibitory response in 4 cases, vasodepressive in 20 cases, mixed in 20 cases and "psychiatric" in 2 cases: sinus tachycardia preceded positivity in 43 of the 46 cases. Only 21 children were treated either by disopyramide (12 cases) or betablockers (9 cases). All but one of the children became asymptomatic but the tilt test remained positive in 7 of the 9 patients treated with betablockers and in 3 children treated with disopyramide. The essential value of this investigation was to stop investigations in positive cases, to reassure parents and the children and to allow the pursuit of sporting activities. The authors conclude that the tilt test is an investigation easy to carry out in the young child and which spares complementary investigation in cases of positivity and guides medical treatment.

Adolescent↗

PubMed1996

Sex chromosome loss and non-disjunction in women: analysis of chromosomal segregation in binucleated lymphocytes.

Chromosomal lagging and non-disjunction are the main mechanisms of chromosomal malsegregation at mitosis. To date, the relative importance of these two events in the genesis of spontaneous or induced aneuploidy has not been fully elucidated. A methodology based on in situ hybridization with centromeric probes in binucleated lymphocytes was previously developed to provide some insight into this matter. With this method, both chromosomal loss and non-disjunction can be simultaneously detected by following the distribution of specific chromosomes in the nuclei and micronuclei of binucleated cells. In this study, this approach was used for studying the role of chromosomal loss and non-disjunction in the age-related malsegregation of sex chromosomes in females. For this purpose, cultures of cytokinesis-blocked lymphocytes were established from 12 healthy women ranging in age from 25 to 56. The occurrence of malsegregation of X chromosomes in vitro was estimated in binucleated cells that contained four signals, which originates from the division of normal disomic cells. In this cell population, the frequencies of X chromosome loss and non-disjunction ranged from 0% to 1.69% (mean 0.75%), and from 0.20% to 1.33% (mean 0.57%), respectively. This indicates that both events contribute to malsegregation of X chromosomes in vitro. Moreover, a small but not negligible fraction of binucleated cells with two or six copies of the X chromosome was noticed in all donors. These cells, which are thought to arise from parental monosomic and trisomic types, may indicate the malsegregation of X chromosomes in vivo. The frequency of X chromosome aneuploidy both in vivo and in vitro significantly correlated with the age of donors. Analysis of chromosomal distribution in unbalanced cells demonstrated that both X homologues were frequently involved. The frequency of such multiple events (0.17%) was far greater than that expected by mere chance, indicating a tendency to multiple malsegregation events in the cell population investigated. Finally, parallel analysis of the segregation of chromosomes X and 1 in five of the donors confirmed the greater (about tenfold) susceptibility of X chromosomes to malsegregate compared with autosomes.

Adult↗

PubMed1996

Analysis of chromosome segregation by means of fluorescence in situ hybridization: application to cytokinesis-blocked human lymphocytes.

The application of methods based on in situ hybridization to centromeric regions to cytokinesis-blocked cells provides a convenient way for the analysis of chromosome segregation in interphase cells. In this way, the reciprocal segregation patterns in daughter nuclei can be visualized and most of the problems related to the artefactual loss or gain of chromosomes which flaw other methods are avoided. In this work, the methodology has been applied to human lymphocytes to investigate the influence of donor age on spontaneous malsegregation rates, the occurrence of multiple malsegregation events, and the effect of the cytokinesis-blocking agent cytochalasin B (Cyt B) on spontaneous and induced chromosome malsegregation. The results obtained with 14 male donors, aged 22-57 years, demonstrated a significant (p < 0.001) increase in the frequency of micronuclei and X chromosome missegregation (both non-disjunction and chromosome loss) with the increasing age of the donors. Moreover, a similar association was observed with cultures hybridized with either chromosome 8 or 18 centromere probes, suggesting that the age-related loss of fidelity in chromosome segregation in vitro may be a general trait. The investigation of the distribution of multiple malsegregation events in cultured lymphocytes of eight male and nine female donors, with the simultaneous hybridization with pairs of centromeric probes (for chromosomes X and 8 or X and 18), demonstrated a large excess of multiple events with respect to that expected by random segregation. This fact may highlight the existence of cellular subpopulation(s) prone to malsegregate, or indicate that the malsegregation of one chromosome is able to affect the fidelity of segregation of the other chromosomes. Finally, the possible influence of Cyt B on chemically induced malsegregation has been investigated with the analysis of chromosomes X and 8 signals in nuclei of lymphocyte cultures treated with vinblastine (2.5-20 ng/ml) in the presence and absence of 6 micrograms/ml Cyt B. Vinblastine induced a small increase in hyperploidy of either chromosome X or 8 at 10 ng/ml in cultures treated with Cyt B. Without Cyt B, a significant increase of hyperploidy was only observed at the highest dose assayed (20 ng/ml). This vinblastine dosage had a severe inhibitory effect on cultures treated with Cyt B, where no binucleated cells were detected. At all doses, a relatively greater mitotic index was observed in cultures with Cyt B, suggesting a synergistic effect of this drug with vinblastine. Most notably, at the two highest vinblastine dosages (10 and 20 ng/ml), a large incidence of polyploid nuclei was observed in cytokinesis-blocked cultures, whereas none or far lower increases of polyploidy were found in the absence or Cyt. B. This results provides direct evidence of the potential of Cyt B to indirectly interfere with chromosome misdistribution induced by a spindle poison, to be considered before drawing firm conclusions from kinesis-blocked systems.

Adult↗

PubMed1996

Induction of humoral immunity toward 2-acetylaminofluorene in mice: modulation of DNA binding after 4 weeks dietary exposure to the carcinogen.

In order to investigate the modulatory effect of the immune response induced by recurrent carcinogen exposure, anti-2-acetylaminofluorene (anti-2-AAF) IgG were elicited in Swiss mice before subsequent carcinogen administration. The immunization schedule consisted of three weekly i.p. injections of 2-acetylaminofluorene (2-AAF)-gelatin conjugate, followed by a final immunogen injection 14 days later. At the end of treatment, the presence of specific anti-2-AAF antibodies in blood serum of all immunized animals was demonstrated. The immunization procedure did not affect liver metabolic activities, as evaluated using liver homogenates for the exogenous activation of 2-AAF to mutagen. After immunization, mice were fed 2-AAF pelleted in the diet at 50 and 150 p.p.m. for 4 weeks and killed at the end of treatment. The determination of DNA adducts by ELISA in liver and spleen of treated animals revealed significantly (P < 0.01-0.001) lower 2-AAF adduct levels in both tissues of immunized mice with respect to non-immunized animals (both naive and pretreated with the adjuvant alone). This result suggests that the specific humoral immunity elicited by repeated carcinogen exposure may be able to modulate the genotoxic effect induced by subsequent carcinogen administration.

2-Acetylaminofluorene↗

PubMed1996

Analysis of chromosome segregation in cytokinesis-blocked human lymphocytes: non-disjunction is the prevalent damage resulting from low dose exposure to spindle poisons.

The chromosome malsegregation pattern produced by the spindle poisons vinblastine (VBL) and colchicine (COL) in human lymphocytes was investigated. For this purpose, the fluorescence in situ hybridization with centromeric DNA probes for chromosomes X and 1 was applied to cell cultures treated with cytochalasin B, a cytokinesis-blocking agent. With this method, chromosome segregation in daughter nuclei - retained in the same envelope - can be easily analysed, simultaneously determining the loss and non-disjunction of specific chromosomes. Preliminary experiments demonstrated that the aneugenic effects elicited by low dose exposure to spindle poisons were effectively detected with treatments from the S/G2 phase (43 h) to harvest of cell cultures (60 or 72 h), with no drug-free medium recovery. This exposure protocol was used in subsequent experiments, where COL and VBL were applied at concentrations which had no effect on the cell cycle ranging to producing marked mitotic block. To account for sex differences in chromosome X instability, lymphocyte cultures from both male and female donors were used to study X chromosome malsegregation. Chromosome 1 malsegregation, however, was analysed in female lymphocytes only. VBL induced reproducible, significant increases of micronuclei in cytokinesis-blocked cells at 5 ng/ml and over. In female lymphocytes, chromosome X loss was observed at 5 ng/ml whereas chromosome 1 loss was only observed at 10 ng/ml. In male lymphocytes, no significant chromosome loss was observed. On the other hand, non-disjunction of both chromosomes X and 1 was effectively induced in female lymphocytes even at 1.25 ng/ml, the lowest dose tested. In male lymphocytes, non-disjunction of chromosome X was observed at 5 ng/ml. Treatments with COL produced a significant increase of micronucleated cells only at the highest dose tested (20 ng/ml). No significant increase in the incidence of either chromosome X or chromosome 1 loss was observed. With cell cultures from donors of both genders, a significant increase in non-disjunction of chromosome X was observed at 5 ng/ml. At the same dose, chromosome 1 non-disjunction significantly increased. These results suggest that in cytokinesis-blocked human lymphocytes, non-disjunction is the prevalent error in chromosome segregation induced by low dose exposure to spindle poisons. Interestingly, non-disjunction was effectively induced even at doses which did not produce detectable detrimental effects on the cell cycle.

Aneuploidy↗