Mutagenesis, Vol. 15, No. 6, 517-523,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Increased cytogenetic damage detected by FISH analysis on micronuclei in peripheral lymphocytes from alcoholics
Department of Pharmacology, University of Bologna, Via Irnerio 48, 40126 Bologna, 2 Centro per lo Studio ed il Trattamento Multidisciplinare dell'Uso Inadeguato dell'Alcol `G.Fontana' Sant'Orsola Hospital, Via Massarenti 9, 40138 Bologna and 3 Medical Clinic Unit, `Ospedale degli Infermi', Faenza, Italy
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Abstract |
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Alcohol abuse greatly increases the risk of various malignancies, including cancer of the liver and digestive tract. Although it is thought that this may be due, at least partially, to the mutagenic properties of ethanol, little is known about the genotoxic effects of ethanol in humans. We investigated the chromosomal damage in lymphocytes from 20 alcoholics and 20 controls using the micronucleus (MN) assay combined with fluorescence in situ hybridization (FISH) with a pancentromeric DNA probe capable of differentiating centromere positive (C+) from centromere negative (C–) MN. The frequency of MN in binucleate lymphocytes was significantly higher in alcoholics than in controls (12.0 ± 5.4 and 7.6 ± 1.6, respectively; P < 0.05). FISH revealed significantly higher frequencies of C+ MN in alcoholics than in controls (8.2 ± 4.8 and 3.4 ± 1.4, respectively; P < 0.05). In the alcoholics, no association was found between years of alcohol abuse and frequency of MN or C+ MN. However, age influenced MN and C+ MN frequency both in alcoholics and controls. These results indicate that alcohol abuse may well induce chromosome loss in humans, suggesting a possible aneugenic mechanism of alcohol. This effect could contribute to the health hazards related to alcoholism such as cancer risk.
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Introduction |
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Alcohol abuse is a significant public health problem. Chronic excessive ingestion of ethanol is associated with serious social, medical and economic problems, including the development of neurological and mental disorders and potentially life-threatening damage to most of the major organ systems.
In 1988, the International Agency for Research on Cancer concluded that, while experimental studies did not show a carcinogenic effect for ethanol as such, epidemiological data were conclusive enough to classify alcoholic drinks as group 1 carcinogens in humans for cancer of the upper airways, digestive tract and liver (IARC, 1988
). Alcohol probably increases the risk of colorectal cancer and breast cancer (Garro et al., 1992; Seitz et al., 1992; Longnecker, 1995). Moreover, alcohol consumption by pregnant women can result in fetal alcohol effects and fetal alcohol syndrome (Lieber, 1992). Even though the toxic effects of alcohol are well documented, the mechanisms by which alcohol and alcoholic drinks affect cancer risk are poorly understood. The toxic effects of alcohol are mediated at least partially by damage to DNA: chronic consumption and metabolism of alcohol result in the generation of several classes of DNA-damaging molecules, including reactive oxygen species, lipid peroxidation products and acetaldehyde (Brooks, 1997). Alcohol and acetaldehyde (the initial metabolite of ethanol) have been examined in experimental test systems (for a review, see Obe and Anderson, 1987). In human and mammalian cells in vitro, ethanol is generally non-genotoxic, but it induces sister chromatin exchanges (SCEs) in human lymphocytes in vitro in the presence of alcohol dehydrogenase. In vivo results with ethanol have been variable. Small increases in SCE frequency have been recorded, but Obe and Anderson (1987) found that micronuclei (MN) were not induced. Various in vitro and in vivo assays have shown that alcohol can induce chromosome mis-segregation (Kaufman, 1983, 1985, 1997; Kafer, 1984; Kaufman and Bain, 1984; Crebelli et al., 1989; Rey et al., 1992).
Acetaldehyde has also been tested in a variety of in vitro systems. The evidence of mutagenic activity in bacteria is controversial (Obe and Anderson, 1987). However, It has been demonstrated that acetaldehyde does induce gene mutations in cultured human lymphocytes (He and Lambert, 1990) and in mouse lymphoma cells (Wangenheim and Bolcsfoldi, 1988). Furthermore, chromosomal aberrations and MN arise in human lymphocytes after acetaldehyde treatment (Böhlke et al., 1983; Migliore et al., 1996).
In humans, there is evidence of increased chromosome damage and changes in the number of chromosomes in peripheral blood lymphocytes of heavy drinkers (Obe et al., 1980; Obe and Anderson, 1987; Hütter et al., 1999). Increased frequencies of micronucleated buccal mucosa cells have been detected in smoking heavy drinkers (Stick and Rosin, 1983). Increased frequencies of SCE have been observed in lymphocytes from chronic alcoholics (Butler et al., 1981; Hender et al., 1984). Moreover, habitual drinkers with the deficient aldehyde dehydrogenase 2 isozyme (ALDH2) enzyme have significantly higher frequencies of SCE than those from ALDH2-proficient individuals (Morimoto and Takeshita, 1996). DNA adducts of acetaldehyde have been detected in lymphocytes of alcohol abusers (Fang and Vaca, 1997), suggesting that the genotoxic action of acetaldehyde is an important mechanism underlying the carcinogenicity associated with chronic alcohol ingestion.
We recently confirmed these effects at the chromosomal level on a small group of 11 alcoholics and found significantly more MN in the peripheral lymphocytes of alcoholics than in age- and gender-matched controls (Castelli et al., 1999).
MN originate from acentric chromosome fragments or whole chromosomes that are not included in the main daughter nuclei during nuclear division. Consequently, the frequency of MN provides a measure of both chromosome breakage and chromosome loss, and can be taken as an indicator of genotoxic response to carcinogenic agents (Fenech, 1993). However, the MN assay lacks specificity due to the heterogeneous cytogenetic origin of the endpoint analysed. The fluorescent in situ hybridization (FISH) technique involves probes that can label the centromeric region of all chromosomes, and so provides a more powerful tool for determining the content of MN (Becker et al., 1990; Norppa et al., 1993a). This makes FISH suitable for evaluating the genotoxic effects induced in vitro by chemical and physical agents (Migliore et al., 1995; Kirsch-Volders et al., 1996; Parry et al., 1996) and for investigating cytogenetic damage in humans (Ramìrez et al., 1997; Surralès et al., 1997; Chang et al., 1999; Migliore et al., 1999).
For the present study, we selected a new group of 20 alcoholics and 20 controls. Alongside the MN assay, we also applied FISH analysis with a pancetromeric probe to assess the frequency of chromosomal damage in their peripheral lymphocytes and to study the mechanism underlying MN formation. Correlations between donor age, length of alcohol abuse, smoking history and the observed MN frequency were also investigated.
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Materials and methods |
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Study population
The study involved 20 alcoholics (seven females and 13 males) and 20 controls (seven females and 13 males). All the subjects were in regular employment or were pensioners. After providing informed consent, each participant was extensively interviewed by a specialized physician, with a detailed questionnaire being filled in to provide important information for the study. Gender, date of birth, smoking habit, work-related exposure to hazard agents, dietary habits, use of therapeutic drugs, and alcohol consumption were all reported. All subjects in both groups were smokers, the number of cigarettes smoked daily varying from 20 to 30. None of the subjects had genetic disorders or a history of severe medical illness or occupational chemical exposure. The alcoholics included in the study suffered from alcohol dependence syndrome and had received periodic psychotherapy sessions and clinical examinations for the treatment of the alcohol-related diseases. Each alcoholic had a daily intake of pure alcohol of >120 g. The beverages commonly consumed by the subjects included wine, beer and spirits. None of the alcoholics had a deficient diet or had any major change in dietary habits during their alcohol dependence. They were all in a fair state of general nutrition, as assessed by triceps skin-fold measurement (>10 mm), urinary creatinine:height ratio and haemoglobin values (>12). Liver function tests did not indicate any progressive liver disease. The alcohol consumption of controls ranged from 8 to 13 g/day, and was lower than the mean Italian value of 17.3 g/day reported by Bollettino Epidemiologico della Società Italiana di Alcologia (1996).
Lymphocyte cultures and slide preparation
The MN test was performed using the cytochalasin B technique (Fenech and Morley, 1985). Peripheral lymphocytes were isolated from heparinized blood by Histopaque gradient centrifugation (Sigma, St Louis, MO, USA). Two replicate cultures were set up using 2x106 lymphocytes for each sample in 5 ml RPMI 1640 (Sigma) medium, containing 15% fetal calf serum (Sigma), 1% phytohaemagglutinin (Sigma), L-glutamine 1 mM (Sigma), 100 IU of penicillin and 100 µg/ml streptomycin (Sigma). They were incubated at 37°C in 5% CO2 for 72 h. Cytochalasin B (Sigma) was added (final concentration 6 mg/ml) for the last 28 h.
Cells were collected and treated with a mild hypotonic treatment (one part RPMI1640 medium and one part distilled water, at 37°C) for 2 min and were fixed in 3:1 methanol:glacial acetic acid. Cells from duplicate cultures of each subject were pooled and used for setting up the slides. The slides were prepared by cytocentrifugation (ALC 4236, Bologna, Italy) and air-dried. For each subject, one or two slides were stained with conventional May–Grünwald–Giemsa staining (Sigma), while the others were stored at –20°C in N2 until FISH analysis (Miller and Nüsse, 1993).
FISH analysis
The origin of MN was assessed by FISH using a synthetic centromeric digoxigenin-labelled probe specific for all human centromeres (Oncor, Gaithersburg, MD, USA), a 171 bp tandem random repeat considered to be a selection of alphoid sequences which hybridized to the centromere of all human chromosomes (Titenko-Holland et al., 1994). Molecular hybridization and immunofluorescence detection of the probe were carried out according to the protocol provided with the Oncor chromosome in situ kit. The slide was pretreated with pepsin (2 mg/ml; Sigma), for 5 min. The dried slides were denatured with 70% formamide in 2x SSC (saline–sodium citrate buffer) at 70°C for 2 min. The digoxigenin-labelled human 
-satellite probe (Oncor) was denatured in a 70°C water bath for 5 min. Hybridization was carried out overnight at 37°C in a humidified chamber. After hybridization, slides were washed in 2x SSC for 5 min at 72°C. Centromeric signals were detected using a fluorescein isothyocianate (FITC)-labelled anti-digoxigenin antibody (Oncor). The cells were counterstained with propidium iodide (0.3 g/ml antifade).
Slide scoring
In accordance with standard criteria (Fenech, 1993), MN analysis was performed on coded slides by scoring 2000 binucleate (BN) lymphocytes for each subject. Cell cycle parameters were evaluated by classifying 1000 cells according to the number of nuclei. The nuclear division index (NDI) was calculated from the formula: NDI = (M1 + 2M2 + 3M3 + 4M4)/N, where M1 to M4 are the number of cells with one to four nuclei, respectively, and N indicates the total number of cells scored (Eastmond and Tucker, 1989). For the FISH analysis, slides were scored using a 100x objective of a fluorescence microscope (Zeiss) with filters for FITC (green). Only BN lymphocytes showing >20 very bright centromeric signals within both red main nuclei were scored (Chang et al., 1999). The MN found were classified as centromere-positive (C+) if they showed a fluorescent signal and as centromere-negative (C–) if there was no such signal; and the frequencies of C+ MN/1000 BN and C– MN/1000 BN were calculated for each individual.
Statistical analysis
Student's t-test was used to compare the frequencies of MN in alcoholics and controls. Linear regression analysis was used to evaluate the influence of age and smoking habits on MN frequencies of both groups.
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Results |
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Table I
summarizes the demographic and lifestyle characteristics of the study population. The two populations (the alcoholics and the controls) were fairly similar as regards gender, age and smoking history. The alcoholics had a mean age of 49.1 ± 9.9 years (range: 36–62 years) and an average length of alcohol abuse of 19.6 ± 8.8 years (range: 4–40 years). The mean age of controls (47.7 ± 10.2 years; range: 33–62 years) was similar to that of the alcoholics. All subjects in both groups had a history of uninterrupted smoking for 
15 years before the sampling time. No difference was observed between the two groups in terms of years of smoking exposure (28.0 ± 9.1 years in alcoholics as compared with 25.5 ± 7.2 years in controls).
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Individual results of the MN assay are shown in Table II
. This table indicates the frequencies of BN cells with MN (BNMN) and the total number of MN per 1000 lymphocytes. If we consider the cell as the unit of mutation, the number of BNMN is a better parameter for measuring genotoxicity than the number of MN (Surralès et al., 1994). BNMN frequencies were significantly greater in alcoholics than in controls (12.0 ± 5.4 and 7.6 ± 1.6, respectively; P = 0.01). There was no statistically significant difference in the mean frequency of BNMN in female alcoholics and male alcoholics (12.3 ± 7.0 and 11.9 ± 4.7, respectively). The mean NDI for alcoholics was not significantly different from that for the controls (1.38 ± 0.16 and 1.37 ± 0.05, respectively).
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No significant correlation was found between the BNMN frequencies and the length of alcohol abuse (r = 0.035, P = 0.885). A significant correlation was found between the age of subjects of both groups and the BNMN frequencies (alcoholics: r = 0.470, P = 0.037; controls: r = 0.646, P = 0.002). The duration of smoking, expressed as years of exposure, had no effect on the BNMN frequencies in the alcoholics (r = 0.157, P = 0.510) or the controls (r = 0.196, P = 0.409).
The centromeric content of MN as measured by FISH are shown in Table III. The frequencies of C+ and C– MN were determined after classifying 50 MN. The frequencies of C+ MN in alcoholics were significantly higher than those in controls (8.2 ± 4.8 and 3.4 ± 1.4 per 1000 BN, respectively; P = 0.001). The mean C– MN for alcoholics was not significantly different from that for controls (4.6 ± 2.0 and 4.4 ± 0.7 per 1000 BN, respectively). The frequencies of C+ MN/1000 BN and C– MN/1000 BN were not associated with the duration of alcohol exposure in years (r = 0.041, P = 0.864 and r = –0.021, P = 0.930, respectively). Regression analysis indicated a significant correlation between the age and the frequencies of C+ MN both in alcoholics (r = 0.491, P = 0.028) and controls (r = 0.740, P = 0.001), whereas C– MN frequencies in both groups were unrelated to age (alcoholics: r = 0.113, P = 0.635; controls: r = 0.337, P = 0.146). No consistent association between the smoking habits and the C– or C+ MN frequencies emerged in alcoholics (C+ MN: r = 0.162, P = 0.495; C– MN: r = 0.094, P = 0.694) or controls (C+ MN: r = 0.302, P = 0.195; C– MN: r = 0.076, P = 0.751).
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Discussion |
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In this study, we assessed the levels of cytogenetic damage, apparent as MN frequencies, of 20 alcoholics and 20 controls. FISH with a pancentromeric DNA probe was then employed to investigate the mechanisms underlying MN formation. To our knowledge, this is the first time that FISH has been used for this purpose in alcoholics.
There was a significantly greater frequency of MN in lymphocytes of alcoholics than in those of controls, while there was no difference in cell proliferation parameters, as measured by NDI, between the two groups. These results confirm our previous findings on a smaller group of subjects (Castelli et al., 1999), indicating that alcohol abuse in humans is associated with genetic damage, which can be detected as MN. FISH analysis showed higher frequencies of C+ MN in the BN of alcoholics than in those of controls. This suggests that high alcohol consumption could be associated with chromosome loss in human cells. A possible aneugenic effect of ethanol has been demonstrated in different assays. Ethanol induces mis-segregation and nondisjunction in Aspergillus nidulans (Kafer, 1984; Crebelli et al., 1989), nondisjunction in Drosophila melanogaster (Rey et al., 1992) and mis-segregation of chromosomes during oogenesis and spermatogenesis in rodents (Kaufman, 1983, 1985, 1997; Kaufman and Bain, 1984). In the light of this information, it has been suggested that ethanol acts on the cytoskeletal elements of the spindle apparatus or on its precursor elements (Kaufman, 1985, 1997). Moreover, a possible aneugenic effect of acetaldehyde cannot be excluded, since this molecule induces high percentages of MN-containing whole chromosomes, as demonstrated by the centromere signal inside the MN in human lymphocytes treated in vitro (Migliore et al., 1996). Numerical chromosomal abnormalities have been reported in mesothelial cells and leucocytes taken from the ascitic fluid of patients with alcoholic cirrhosis (To et al., 1981) and in peripheral lymphocytes from heavy drinkers (Obe et al., 1980; Obe and Anderson, 1987). Moreover, an increase in aneuploidy has been found in the sperm cells of alcoholics (Robbins et al., 1997).
The increased chromosome damage recorded in the alcoholics studied by us did not appear to be associated with the duration of alcohol abuse. There are several possible reasons for this. Alcoholic beverages are highly complex solutions, which contain different organic and inorganic compounds, including known and suspected carcinogens such as nitrosamines, polycyclic aromatic hydrocarbons and mycotoxins, as well as a variety of ethers, phenolic and other compounds derived from the interaction between the original plant material and the production process (IARC, 1988). Although many compounds have been identified as being common to all alcoholic beverages, they are present in different quantities in different beverages (IARC, 1988). Even though the alcoholics all had a daily intake of pure alcohol of >120 g, they had consumed various types of beverage in different quantities over the years of their dependence. These factors could make it difficult to assess with any degree of reliability the existence of any relationship between the duration of alcohol abuse and MN frequency.
An association was found between age and MN frequency in both alcoholics and controls. Evidence of an age-related increase in MN frequency has come from biomonitoring studies (Fenech, 1998; Bolognesi, et al., 1999). It has been postulated that this could reflect an increase in spontaneous chromosome instability with age, associated with an accumulation of DNA damage due to a progressive impairment of overall DNA repair capacity (Barnett and King, 1995). A similar pattern of chromosomal instability occurs in various premature ageing syndromes characterized by defective DNA-damage defence mechanisms (Weirich-Schwaiger et al., 1994).
FISH revealed that the C+ MN frequency significantly correlated with age in all subjects. The age-dependent increase of MN in human lymphocytes is known to be due to MN harbouring whole chromosomes rather than acentric fragments. Although this phenomenon seems to be mainly related to the loss of the X chromosome in females and of the Y chromosome in males (Guttenbach et al., 1995; Stone and Sandberg, 1995; Catalàn et al., 1998), some studies have also reported the involvement of autosomes (Richard et al., 1994; Catalàn et al., 1995). Since our study employed an -DNA probe specific for the centromere of all human chromosomes for FISH analysis, it was not possible for us to identify involvement of specific autosomes or sex chromosomes in MN.
In our study, the smoking habit, considered in terms of years of exposure, was not associated with cytogenetic damage in alcoholics or controls. These results are in keeping with various other studies (Sorsa et al., 1988; Migliore et al., 1991; Bolognesi et al., 1993; Norppa et al., 1993b; Stierum et al., 1993; Van Hummelen et al., 1993; Pitarque et al., 1996; Thierens et al., 1996; Bolognesi et al., 1997; Barale et al., 1998) but contrast with the results of several other studies that have found that cigarette smoking affects MN frequency (Hogstedt et al., 1983; Tomanin et al., 1991; da Cruz et al., 1994). These conflicting data give the overall impression that the MN assay is not specific in detecting smoking effects (Barale et al., 1998).
In conclusion, our results indicate that there may well be a risk of a change in the number of chromosomes arising from the micronucleation of whole chromosomes in alcoholics. The data from this first study employing centromere-specific FISH in MN analysis of alcoholics provide plausible biological evidence that alcohol abuse could cause chromosome loss in humans. The potential aneugenic effect of ethanol may be an important cause of the toxicity associated with alcohol abuse, such as fetal alcohol syndrome and cancer risk. The detrimental influence of aneuploidy on human health is widely accepted. Its effects include birth defects, spontaneous abortion and infertility (Abruzzo and Hassold, 1995). Tumour cells frequently have alterations in chromosome number (Rew, 1994) and several specific aneuploidies have been associated with tumor development (Mitelmam, 1994; Tucker and Preston, 1996). Recently Li et al. (2000) proposed an interesting two-stage model to explain how carcinogens may cause cancer via aneuploidy. In the first stage, carcinogens generate an aneuploid cell by chemically or physically altering proteins of the spindle apparatus or chromosomes, as has been reported previously (Oshimura and Barrett, 1986; Jensen et al., 1993; Li et al., 1997; Matsuoka et al., 1997). In the second stage, aneuploidy can lead to destabilization of the karyotype and thus initiate autocatalytic evolution of the karyotype. This process would generate new lethal, preneoplastic and eventually neoplastic karyotypes (Li et al., 1997; Duesberg, 1999; Rasnick and Duesberg, 1999). Autocatalytic karyotype variations would also explain the genetic instability and phenotypic heterogeneity of cancer cells (Duesberg et al., 1998). In this light, the results of the present study deepen our understanding of the possible relationship between chromosomal damage and chronic alcohol exposure and reinforce the concept that genotoxic mechanisms are important in alcohol-related carcinogenesis.
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Acknowledgments |
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We are grateful to Robin M.T.Cooke for editing. This work was supported jointly by a MURST (Ministry of the University and of the Scientific Technology Research) grant (ex. 60%) and a University of Bologna, Special Grant to the project `Sviluppo di sonde fluorescenti'.
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Notes |
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1 To whom correspondence should be addressed. E-mail: fmaffei{at}biocfarm.unibo.it
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References |
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Received on May 23, 2000; accepted on August 3, 2000.
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