Apolipoprotein B is a new target of the GDNF/RET and ET-3/EDNRB signalling pathways.

BACKGROUND: GDNF/RET and Endothelin-3 (ET-3)/EDNRB regulate survival, differentiation, migration, and proliferation of neural crest-derived cells. Although several RET and EDNRB signalling mediators have been characterized, most of the genes targeted by these two pathways are still largely unknown. We focused our study on apolipoprotein B (APOB) as a novel target gene of the RET and EDNRB pathways, based on previous data obtained using a Caenorhabditis elegans strain mutant for the homologue of mammalian ECE1. METHODS: Molecular and cellular studies of Apob were performed in the murine Neuro2a cells, an in vitro model for studying neural crest-derived cell development, along with a mouse knock-in for the Hirschsprung-associated mutation Ret(C620R). Silencing for Apob and Ret has been performed via shRNA. KEY RESULTS: GDNF/RET and ET-3/EDNRB cooperated in inducing neuronal differentiation resulting in Apob activation in Neuro2a cell line. Apob expression was downregulated in mouse embryos homozygous for the Ret(C620R) mutation and presenting a severe Hirschsprung phenotype. Ret silencing prevented Apob expression increase. MAPK P38 kinase activation evoked Apob expression via GDNF/RET signalling in Neuro2a cells. A p53-dependent repressor element in Apob promoter resulted in a reduced Apob expression. Silencing of Apob reduced HuD protein expression. CONCLUSIONS & INFERENCES: Apob is a novel downstream target of the RET/EDNRB pathways with a role in neuronal survival and maintenance, as indicated by its effect on HuD expression. Our data provide a conceptual framework to investigate and establish the role of APOB gene in severe gut dysmotility.

Spontaneous and bleomycin-induced chromosome damage in non cancer thyroid patients.

BACKGROUND: Presence of chromosome damage in lymphocytes of patients affected by several diseases, including cancer, was detected by the micronucleus (MN) assay. Individual susceptibility to DNA damage, considered as a risk factor for cancer, can be also evaluated using the bleomycin (BLM) sensitivity test. MATERIALS AND METHODS: We aimed to evaluate spontaneous or BLM-induced MN frequencies in autoimmune (AI, n = 19) and non autoimmune (NAI, n = 11) thyroid patients, not receiving (131)I radiometabolic therapy with respect to a control group of 18 healthy subjects. According to thyroid function, patients were also divided into hypothyroid (n = 10), euthyroid (n = 13) or hyperthyroid (n = 7) subjects. RESULTS: Spontaneous MN frequencies of AI and NAI patients did not differ from those of controls. Hypothyroid patients had more elevated MN basal levels (9.00 + or - 1.71 per thousand) than hyperthyroid (3.75 + or - 1.17 per thousand, P < 0.05) and euthyroid (5.38 + or - 0.97 per thousand, P < 0.01) patients or healthy subjects (4.17 + or - 0.63 per thousand, P < 0.01). In particular, the hypothyroid AI group showed the highest value (9.79 + or - 2.26 per thousand, P < 0.01). All thyroid patients responded differently to BLM than controls (39.90 + or - 2.48 per thousand vs. 31.08 + or - 2.51 per thousand, P = 0.0377). The NAI group had BLM-induced MN levels (45.00 + or - 2.56 per thousand) significantly higher (P = 0.0215) than AI patients (36.95 + or - 3.49 per thousand) or healthy subjects (31.08 + or - 2.51 per thousand). No significant difference was seen when patients were stratified according to autoimmunity. CONCLUSIONS: We report that hypothyroid patients exhibit a moderate increase in the level of spontaneous genome damage, and that AI thyroid patients resulted to be less sensitive than NAI patients to the mutagen sensitivity test. In prospective, it may be of interest to reinvestigate hypothyroid patients when correction of their dysfunction is achieved.

Susceptibility to chromosome malsegregation in lymphocytes of women who had a Down syndrome child in young age.

Recent findings seem to converge towards a unified hypothesis trying to relate Down's syndrome (DS), trisomy 21 and Alzheimer's disease (AD). The majority of DS individuals develop neuropathological characteristics of AD by the age of 40. Previous cytogenetic studies performed by us showed an increased frequency of aneuploidy in peripheral lymphocytes and fibroblasts of AD patients and a preferential occurrence of chromosome 21 in malsegregation events. An increased frequency of AD among young mothers of individuals with DS (MDS) is reported. This study investigates the cytogenetic characteristics and the predisposition to chromosome malsegregation of peripheral blood lymphocytes in a group of women (n = 35) who had a Down syndrome child in young age (<35 years) and in a control group (n = 30). We applied the micronucleus assay and the dual-color FISH in order to assess the susceptibility to malsegregation events. The results indicate a higher frequency of binucleated micronucleated cells in MDS in respect to the control group (16.1+/-9.1 per thousand versus 8.7+/-5.4 per thousand). Moreover, our data reveal that peripheral lymphocytes of MDS are more prone to chromosome non-disjunction with both chromosomes, 13 and 21, equally involved.

Oxidative and genotoxic damage after radio-iodine therapy of Graves' hyperthyroidism.

PURPOSE: To evaluate genetic damage and oxidative stress following a single therapeutic dose of 131I in Graves' disease patients monitored up to 180 days after treatment. MATERIALS AND METHODS: Genetic damage induction was estimated as the increase in micronuclei in peripheral lymphocytes of patients. As indicators of radiogenic oxidative stress, vitamin E and lipoperoxide levels were assessed in the plasma of patients, as well as the release of plasmic clastogenic factors measured by the induction of micronuclei in vitro in peripheral lymphocytes of a healthy donor. RESULTS: Vitamin E depletion lasted at least 3 days and the basal level was restored within 7 days. No statistically significant variations were observed in lipoperoxide plasma levels. A sharp increase of micronuclei in the peripheral lymphocytes of patients was correlated (p < 0.001) with the release of clastogenic factor in the plasma. The highest micronucleus value was negatively correlated (p < 0.03) with the lowest vitamin E level observed in each patient. CONCLUSIONS: Micronuclei induction was the direct consequence not only of the energy deposition of 131I on the genetic material, but also of oxidative stress, likely via the release of clastogenic factor.

Spontaneous sister chromatid exchange and chromosome aberration frequency in humans: the familial effect.

The possible effects of environmental and genetic factors on spontaneous frequencies of sister chromatid exchanges (SCEs) and cells with chromosome aberrations (CAs) in human lymphocytes were investigated by analysing 177 completed families (mother, father and at least one child). After removing the effects of methodological, biological and life-style factors by the use of multifactor analysis of variance (MANOVA), SCEs and CAs residuals were analysed by simple correlation analysis and principal component analysis. SCEs and CAs inter-familiar variability was higher than that found within families. A significant correlation was found between the average SCE frequencies shared by parents (the so-called 'midpoint parents', or 'midparent') and offspring (linear slope b=0.26+/-0.07, p<0.05), but also between mother and father (b=0.23+/-0.11, p<0.05) suggesting the presence of an effective environmental factor. The midparent-offspring correlation was found to be sustained by the mother-offspring relationship (b=0.28+/-0.08, p<0.05), being the father-offspring correlation not significant (b=0.16+/-0.11, p0.05). Concerning CAs, no statistically significant correlation between parents was found, but the strong relationship between mother and offspring was confirmed (b=0.468+/-0.11, p<0.001). The SCEs correlation between mother vs. offspring disappeared for older offspring (over 23 years old). The obtained findings strongly showed that the genetic make-up is barely detectable in the presence of domestic environment factors which are shown to play the major role in determining the interfamilial variability of SCE and CA in a general population. These results strengthen the suitability of the use of SCEs and CAs analysis in human cytogenetic surveillance for the detection of effective environmental factors.

Primed in situ labeling (PRINS): a method for rapid identification and quantification of human chromosomes in both lymphocytes and sperm nuclei.

In this work, a specific primer for X alphoid satellite DNA was used to detect chromosome X through primed in situ labeling (PRINS). The method allows the rapid identification of chromosome X in metaphase and its quantification in interphase. PRINS is equally applicable to both lymphocytes and sperm nuclei.

Common fragile sites on human chromosomes represent transcriptionally active regions: evidence from camptothecin.

Cellular processes involved in the expression of fragile sites (FS) have been investigated by studying the possible modulation of their induction by camptothecin, a specific topoisomerase I inhibitor. Expression of FS was induced by aphidicolin and then camptothecin was administered to cultures during G2 phase. Under these conditions, a very high number of chromosome aberrations were obtained: R-bands carrying FS were specifically involved in breakage and, in particular, the common FS (cFS) bands already expressed in aphidicolin-treated cultures were the most affected. These data show that the expressed FS are preferential targets of camptothecin, that is, regions where topoisomerase I-cleavable complexes are formed. This allows us to hypothesize that cFS could represent the cytogenetic expression of transcriptionally active regions. These treatments were able to induce, besides the known FS, four new FS, namely 1p34, 6p21, 6q25, and 15q15.

Induction of aneuploidy by the antineoplastic drug estramustine in human lymphocytes.

Estramustine (EM) is an antineoplastic drug used in the therapy of human prostatic carcinoma. The aim of our work was to evaluate the potential aneuploidogenic activity of estramustine, by analysing its cytogenetic effects induced in human lymphocytes. To estimate the ability of EM to induce mitotic spindle disturbances, two parameters were used: the presence of c-mitoses (according to the degree of chromatid spreading and contraction) and mitotic index evaluation (increase after exposure indicating the accumulation of mitoses). EM induced c-mitoses and mitotic index increases starting from the 4 microM dose: statistically significant increases were observed up to the highest dose (40 microM). A strong correlation between c-mitoses and mitotic index increase was found. The micronucleus (MN) assay combined with the fluorescence in situ hybridization technique with a pancentromeric DNA probe was also carried out. Compared to the control, EM induced significant MN increases in binucleated lymphocytes at two doses (8-16 microM). Moreover, we found that estramustine induced significant percentages of MN with positive hybridization signal at the same doses, confirming the presence of entire chromosomes in micronuclei. Additional experiments included induction of numerical and structural chromosome aberrations, and evaluation of sister chromatid exchanges (SCE) and satellited (D- and G-group chromosomes) chromosome associations. The results of numerical chromosome aberration analysis indicated that EM was positive in inducing a statistically significant increase in aneuploid cells and/or polyploid cells at all doses tested. On the basis of these observations, EM may be defined as a typical aneuploidy inducer, whereas it was not found to increase the frequency of structural chromosome aberrations and SCE frequency.

Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations, and micronuclei in human lymphocytes.

Intra- and interindividual variations of baseline frequencies of cytogenetic end points in lymphocytes of human populations have been reported by various authors. Personal characteristics seem to account for a significant proportion of this variability. Several studies investigating the role of age as a confounding factor in cytogenetic biomonitoring found an age-related increase of micronucleus (MN) frequency, whereas contradictory results were reported for chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs). We have quantitatively evaluated the effect of age on SCE, CA, and MN through the analysis of a population sample that included data from several biomonitoring studies performed over the last few decades in 12 Italian laboratories. The large size of the data set, i.e., more than 2000 tests for each end point, allowed us to estimate the independent effect of age, taking into account other covariates, such as sex, smoking habits, occupational exposure, and inter- and intralaboratory variability. A greater frequency of the mean standardized values by increasing of age was observed for all of the end points. A leveling off was evident in the last age classes in the trend of MN frequencies. Frequency ratios (FRs), which express the increase of the cytogenetic damage with respect to the first age classes, i.e., 1-19 years, were estimated using Poisson regression analysis after adjustment for the potential confounding factors and confirmed the increasing trend by age class for all three end points. The most dramatic increase was observed for MN, with a FR that approaches the value of 2 at the age class 50-59 (FR, 1.97; 95% confidence interval, 1.43-2.71) and remains substantially unchanged thereafter. The trend of FRs for CA is more homogeneous, with a constant rise even in the older classes, whereas the frequency of SCE increases with age to a lesser extent, reaching a plateau in the age class 40-49 and the maximum value of FR in the age class over 70 (FR, 1.14; 95% confidence interval, 1.07-1.23). In conclusion, our results point to an age-related increase of the chromosome damage in lymphocytes and emphasize the need to take into account the potential confounding effect of this variable in the design of biomonitoring studies based on chromosome damage.

Structure-activity relationship in the induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells by regioisomeric phenanthrene quinones.

Four regioisomeric phenanthrene (PH) quinones (Q) were investigated for their ability to induce chromosomal damage and spindle disturbances. PH 1,4-Q and PH 1,2-Q induced structural as well as numerical chromosomal aberrations, whereas the isomers PH 9,10-Q and PH 3,4-Q were virtually inactive in this respect, However, all four compounds enhanced the frequency of c-mitoses.

Atomic force microscope imaging of chromosome structure during G-banding treatments.

Surface topography of human chromosomes was examined by atomic force microscopy during treatments for G-banding. Trypsin treatment resulted in a structural modification in the chromatin. Subsequent Giemsa staining caused a general swelling of the chromosomal surface that was greater in the areas of G-band positive regions. By means of a quantitative evaluation method we showed that the G-banding process produces a 10-fold enhancement of a pre-existing pattern of chromatin between G-band positive and G-band negative regions on mitotic chromosomes.

Aphidicolin-sensitive specific common fragile sites: a biomarker of exposure to pesticides.

In this work, we analyzed the aphidicolin-sensitive common fragile sites in seven females and four males occupationally exposed to pesticides and in ten controls. The same males had been monitored one year earlier in a previous study by the same authors. Results showed enhanced expression in exposed subjects at eight bands, namely, 6q25, 7p22, 7q22, 7q32, 13q14, 14q24, 16q22, and 16q23. Most of these bonds were fragile sites and breakpoints involved in chromosome rearrangements found in hematopoietic tumors. Moreover, six of these bands were already detected, with enhanced expression, in the first monitoring carried out on male subjects. These results indicated that fragile sites analysis is a reproducible cell response to human exposure to pesticides.

Modulating factors of individual sensitivity to diepoxybutane: chromosome aberrations induced in vitro in human lymphocytes.

Peripheral blood lymphocytes from a sample of 62 randomly selected donors were analysed for spontaneous and diepoxybutane (DEB)-induced chromosomal aberrations (CA). These individuals were part of a larger sample of 122 subjects whose DEB responsiveness was evaluated by means of sister chromatid exchange (SCE) analysis. Confounding factors (such as smoking, wine and coffee consumption, occupation and haematological factors) were analysed for their effect on individual DEB-responsiveness, but no statistically significant associations were observed. Interestingly, a bimodal distribution of aberrant cell frequencies was clearly detectable, showing the existence of DEB-sensitive subjects belonging to the second mode (CA frequencies > 19%). When responsiveness evaluated by means of CA induction was compared with SCE responsiveness, it was noted that all SCE-inducible subjects (> 110.9 SCEs/cell) belonged to the second mode of CA frequency distribution. On the other hand, highly CA inducible individuals did not necessarily show a higher SCE-response, although their DEB-induced SCE frequencies were above average (92 SCEs/cell). DEB-induced CA frequency correlated with baseline levels, indicating that DEB-sensitive individuals also showed higher spontaneous chromosome damage (3.6 versus DEB-resistant 2%, P < 0.05). Finally, when simple and multiple regression analyses were carried out, DEB-sensitivity appeared negatively related to haematic concentrations of proteins and uric acid (intercept 0.131 +/- 0.011, slope -0.029 +/- 0.0116, r = -0.39; P < 0.01), probably due to its antioxidant activity. This finding confirmed previous observations on the scavenger activity of plasma factors on DEB mutagenicity.

Modulating factors of individual sensitivity to diepoxybutane: sister chromatid exchanges induced in vitro in human lymphocytes.

Spontaneous and diepoxybutane (DEB)-induced sister chromatid exchanges (SCEs) were examined in cultured peripheral lymphocytes (PBL) from 122 healthy donors. SCE-inducing activity under defined experimental conditions and individual sensitivity to genotoxic stress were assessed. SCE means distribution appeared asymmetrical, identifying about 22% of subjects characterized by a 'high-respondent' phenotype with more than 111 SCEs/cell. Confounding factors, such as smoking habit, wine and coffee consumption, work activity and hematological factors, showed a limited capacity to affect individual SCE responsiveness, however hemoglobin and uric acid seemed to antagonize DEB genotoxicity.

Chromosome aberrations in humans in relation to site of residence.

Baseline frequencies of chromosome aberrations (CAs) were assessed in three samples of healthy individuals, 60 living in a rural area (Po Delta), 134 in Pisa downtown and 116 in Cascina, a small town near Pisa, Italy. The three groups were similar for average age, sex ratio, smoking, drinking habit, and occupation. Multifactor ANOVA showed that CA frequencies increased significantly with age (p < 0.0001 excluding and including gaps), and with smoking habit (p = 0.0045 including gaps; p = 0.04 excluding gaps). Gender, drinking habit and occupation exerted no statistically significant effects. Multifactor ANOVA showed also a significant effect of the site of residence on the frequency of the CA, including gaps (p = 0.0003) and excluding gaps (p = 0.03). The CA frequency of the Pisa samples was statistically significantly higher than that of the Po Delta samples. Air pollution was considered to be a possible factor in determining the observed differences among the sites of residence, as levels of air pollutants (SO2 and TSP, total suspended articles) were more elevated in Pisa and Cascina than in the Po Delta. In addition, respiratory symptoms used as indirect indicators of air pollution at individual level were significantly more frequent in the Pisa population than in Cascina or in the Po Delta. These findings might support the hypothesis that air-pollution levels, even within E.E.C. (European Economic Community) air-quality standards, may influence baseline CA frequencies.

Spontaneous and aphidicolin-sensitive fragile site 3cen co-localizes with the (TTAGGG)n telomeric sequence in Chinese hamster cells.

Aphidicolin-sensitive fragile sites were analyzed in immortalized Chinese hamster embryonal fibroblast cells (CHEF18) at three different passages along their spontaneous progression toward tumorigenicity. Five fragile sites (viz., 12q22, 3cen, 3p21, 3q31, and Xq21) were detected. Three of these sites carry spontaneous aberrations and are thus regions of chromosomal instability; however, they were not involved in the formation of the clonal rearrangements that are characteristic of CHEF 18 cells. The presence of the (TTAGGG)n telomeric sequence in chromosome bands associated with fragile sites was investigated using fluorescence in situ hybridization and primed in situ labeling. A common location of fragile sites and telomeric sequence was found at the centromere of chromosome 3.

Induction of chromosomal aberrations and spindle disturbances in Chinese hamster epithelial liver cells in culture by pyrene and benzo[a]pyrene quinones.

Regioisomers of pyrene and benzo[a]pyrene quinones were tested for their ability to induce structural and numerical aberrations and spindle disturbance in Chinese hamster epithelial liver (CHEL) cells in culture. All quinones tested were clastogenic. Pyrene-1,8-quinone (P-1,8-Q) and benzo[a]pyrene-3,6-quinone (BP-3,6-Q) induced strikingly high levels of triradials. In addition, dicentrics and ring chromosomes were very common in BP-3,6-Q-treated cultures. Isomers of these compounds, pyrene-1,6-quinone (P-1,6-Q) and benzo[a]pyrene-1,6-quinone (BP-1,6-Q), induced unobtrusive patterns of chromosomal aberrations. We suspect that the P-1,8-Q and BP-3,6-Q moieties bound to the DNA were still reactive, and formed crosslinks and/or underwent redox cycling leading to high local concentrations of reactive oxygen species. In addition, P-1,8-Q and BP-3,6-Q induced c-mitoses, hyperdiploidies and polyploidies, in particular endoreduplications. These effects were not seen with the other two test compounds, or they were only detected at the highest concentrations used, which were strongly cytotoxic (c-mitoses with P-1,6-Q, polyploidies with BP-1,6-Q).

Enhanced expression of common fragile site with occupational exposure to pesticides.

The expression of common fragile sites (FS), induced by aphidicolin, in subjects with occupational history of exposure to pesticides has been studied. Results showed a higher frequency of FS in exposed subjects; in particular, there was an elevated expression of FS at the cancer breakpoints 3p14, 5q31, 7q22, 7q32, 14q24, and 16q22, involved in leukemias and non-Hodgkin's lymphoma. Moreover, the frequency of breaks in chromosomal bands carrying oncogenes or tumor suppressor genes involved in aberrations was significantly higher in exposed subjects at sites 1q25, 3p25, 7p22, 8q24.1, and 13q14.

Enhancement of SCE frequency by alpha-naphthoflavone in cultured lymphocytes in relation to environmental factors.

One hundred and nine healthy subjects living in an urban area of Tuscany were monitored using sister chromatid exchange (SCE) analysis on lymphocytes cultured in standard or alpha-naphthoflavone (ANF)-supplemented medium in order to collect the most complete data possible for those constitutional and environmental factors with which genotoxic risk can be associated. ANF genotoxicity depends on its metabolic activation by cellular P-450 monooxygenase systems whose activity can be modulated by exposure to carcinogenic but nongenotoxic xenobiotics. Lymphocytes grown in standard conditions showed a significant increase of SCE frequency associated with smoking habits and age. Although the addition of ANF caused an upward shift of SCE frequency in all subjects, smokers, coffee drinkers, and blue-collar workers showed a significantly higher SCE level; this suggests that potential risk factors rising from a modified cell metabolism are present in these categories. These results indicate that in vitro ANF treatment of lymphocytes could be a useful tool in the detection of environmental exposure to those classes of chemicals involved in metabolic activation of promutagens.