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.

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.

Monitoring human exposure to urban air pollutants.

A multidisciplinary study on a general population exposed to vehicle exhaust was undertaken in Pisa in 1991. Environmental factors such as air pollution and those associated with lifestyle were studied. Meanwhile, biological and medical indicators of health condition were investigated. Chromosomal aberrations, sister chromatid exchanges (SCEs), and micronuclei in lymphocytes were included for the assessment of the genotoxic risk. Because of the large number (3800) of subjects being investigated, standardization of protocols was compulsory. The results on data reproducibility are reported. To assess the reliability of the protocol on a large scale, the population of Porto Tolle, a village located in northeast Italy, was studied and compared to a subset of the Pisa population. Preliminary results showed that probable differences between the two populations and individuals were present in terms of SCE frequencies. The study was potentially able to detect the effects of several factors such as age, smoking, genetics, and environment. The in vitro treatment of lymphocytes with diepoxybutane confirmed the presence of more responsive individuals and permitted us to investigate the genetic predisposition to genetic damage. The possible influence of environmental factors was studied by correlation analyses with external exposure to air pollutants as well as with several lifestyle factors.

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.

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.

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.

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.

Bone marrow cell chromosomal aberrations and styrene biotransformation in mice given styrene on a repeated oral schedule.

Styrene's capacity to induce chromosomal aberrations was studied in bone marrow cells of CD1 male mice. No mutagenic effect could be detected after either a 4-day treatment course with daily oral doses of 500 mg/kg or a 70-day course with daily oral doses of 200 mg/kg. Urinary elimination of styrene metabolites related to styrene-7,8-oxide formation (i.e. phenylethylene glycol, mandelic acid, benzoic acid, phenylglyoxylic acid and total mercapturic acids) was quantitatively evaluated in the group of mice given the 200 mg/kg dose. In parallel, kinetic studies were made on styrene and styrene-7,8-oxide blood concentrations in the same group of animals. These determinations were carried out on days 1 and 70 of treatment by spectrophotometric, gas chromatographic and mass fragmentographic procedures. Not even nanograms of styrene-7,8-oxide were found in the blood of styrene-treated mice. This suggests that the metabolite does not migrate from the cellular compartment where it is formed being immediately metabolized or irreversibly bound to cellular structures. This observation could well explain the lack of mutagenic effects observed.

Chromosomal aberration analysis of workers in tannery industries.

A chromosomal aberration analysis was carried out on tannery workers and on matched controls. Workers were engaged in 2 different processing areas, the drum and the finishing workshop. A weak but significant increase in chromosomal aberration frequencies was shown in drum workers in comparison to controls, no difference was shown in finishers.

Kinetics of chromosomal aberrations and first mitosis division in human lymphocytes exposed to mitomycin C.

In order to understand the relationship between the chromosomal damage detectable at the first mitosis after mutagen treatment and the induced mitotic delay we studied the time pattern of both mitotic indices and chromosomal aberration frequencies in human lymphocytes treated in G1 with mitomycin C (2.5 microM) and cultured in vitro in the presence of 5-bromo-2'-deoxyuridine. Mitotic delay was observed in treated cells cultured for 81 h. At this point an increase in the frequency of chromosomal aberrations is evident and a higher proportion of abnormal cells enters mitosis, the long delay being due to the extensiveness of DNA damage. The importance of cell cycle progression for the detection of the maximal amount of induced chromosomal damage is discussed.

Induction of chromosomal aberrations and SCE by chloramphenicol.

The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with chloramphenicol (CAP), an antimicrobial agent acting by inhibiting protein synthesis. Moreover chromosomal aberrations and sister-chromatid exchanges were studied in bone marrow cells of treated mice and in Chinese hamster cell cultures (V79) respectively. While no aberrations were induced by short treatments in human lymphocytes exposed in G1 and G2 phases, high frequencies of aberrations, exclusively of the chromatid type, were induced when the drug was administered during a whole cell cycle. Aberrant metaphases were detected only at the end and a few hours after the end of treatment; at later times aberrant cells reached control values. Doses producing aberrations only slightly increased SCE both in human lymphocytes and in V79 cells. In mouse bone marrow cells CAP induced a high mitotic delay and few structural aberrations; intrachromosomal vacuoles were observed.

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.

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.

Human lymphocytes assay: cyclophosphamide metabolic activation by S9 system with low cytotoxicity.

The general suitability of exposing human lymphocytes directly to prolonged contact with an Ames-type microsomal (S9) activation system has been examined, for testing the effect of the indirect chemical mutagen, cyclophosphamide (CPA), on induction of chromosomal aberrations. Direct exposure of lymphocytes to only S9 mix produced a decrease in the mitotic index within 30-60 min, whereafter it stabilized at acceptable values. Further toxic effects following treatment with different doses of CPA and S9 mix, for the longest times of exposure were due to production of clastogenic metabolites. On the basis of these results, the low cytotoxicity of S9 mix in our conditions allows extension of the application of the test to the study of metabolites which require prolonged contact with the target cells.

C-mitosis and numerical chromosome aberration analyses in human lymphocytes: 10 known or suspected spindle poisons.

As a part of a coordinated EEC project to validate suitable assays for chemically induced genomic mutations, numerical chromosomal aberrations and spindle effects were studied in human lymphocyte cultures exposed to cadmium chloride, chloral hydrate, colchicine, diazepam, econazole, hydroquinone, pyrimethamine, thiabendazole, thimerosal and vinblastine. Chromosome number analysis was carried out after treatment for 48 and 72 h; spindle effects, i.e., increases in the mitotic indices and c-mitoses, were analyzed in cultures treated 5 h before fixation. Dose-related numerical chromosomal aberrations are induced by colchicine and vinblastine, the only chemicals that also induce c-mitotic effects in a wide range of doses. Hyperdiploidy is induced by chloral hydrate, cadmium chloride and thimerosal without dose-effect relationship; chloral hydrate and thimerosal affect spindle functions while only a weak spindle effect is produced by cadmium chloride. Tetraploid and/or endoreduplicated cells are induced without dose-effect relationship by hydroquinone, thiabendazole and thimerosal, all of them able to produce c-mitotic effects. Diazepam and econazole induce only hypodiploidy; pyrimethamine does not induce numerical chromosomal aberrations.

Micronucleated lymphocytes in people occupationally exposed to potential environmental contaminants: the age effect.

This work is part of a research project on 2 groups of tannery workers (i.e., workers employed in the tanning process and those employed in the finishing department), and 2 control groups consisting of individuals paired with each exposed person according to sex, age and smoking habit. The whole study included the evaluation of micronuclei as well as of chromosomal aberrations and sister-chromatid exchanges in peripheral blood lymphocytes. Data on micronucleus analysis in both controls and exposed persons are shown in this paper. There was no statistically significant difference between MN frequencies in the 2 groups of exposed and controls, nor any positive correlation with smoking habit. The effect of age on basal frequency of micronucleated cells clearly emerges in the present study: both controls and exposed show an increase in MN frequency due to age. This could be correlated with a higher sensitivity to breaks, rearrangements or aneuploidogenic events of circulating lymphocytes in aged people.

Chromosomal monitoring of chromium-exposed workers.

A cytogenetic analysis was carried out on peripheral blood lymphocytes of workers exposed to chromite in a ferrochromium plant, to evaluate the possible existence of genetic damage. A quantitatively limited increase in aberrant cell frequencies was detected in subjects working in the furnace sector. The data were analyzed by several statistical approaches.

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 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.

Genotoxicity, metabolism and blood kinetics of epichlorohydrin in mice.

Epichlorohydrin (ECH), a direct mutagen in vitro, did not induce chromosomal aberrations in bone-marrow cells of CD1 mice given single oral doses of 50 and 200 mg/kg in water. The ECH diol derivative (3-chloro-1,2-propanediol) was tested in vitro by a forward-mutation assay on the yeast Schizosaccharomyces pombe and showed a weak but significant mutagenic effect. The failure of ECH to induce mutagenic effects appears to be due to the rapid metabolic clearance of the compound in vivo. ECH blood kinetics at both doses, and at the same time the concentration of the diol, were determined. ECH rapidly disappeared from mouse blood, being no longer detectable 20 min after treatment. In contrast, 3-chloro-1,2-propanediol was measurable up to 5 h after dosage. No difference was observed in the kinetic and metabolic behavior of ECH after single and repeated doses (50 and 200 mg/kg/day for 7 days). When 3-chloro-1,2-propanediol was tested, neither glutathione depletion nor epoxide hydrolase inhibition (evaluated with both styrene-7,8-oxide and ECH as substrates) could be detected in mouse liver. Finally, no difference in ECH blood kinetics or metabolism were observed in experiments in which the compound was administered (200 mg/kg) intraperitoneally in water or orally as a solution in dimethyl sulfoxide.