Mutagenesis, Vol. 17, No. 2, 157-162,
March 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
REVIEW |
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
Istituto Superiore Sanità, Laboratory of Comparative Toxicology and Ecotoxicology, Viale Regina Elena 299, 00161 Rome, Italy
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Abstract |
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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
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. |
Introduction |
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The toxic and leukaemogenic properties of benzene, a widely used solvent and gasoline component, have been known for decades (IARC, 1982
). Despite its harmful properties, benzene is still one of the largest volume industrial chemicals (Fishbein, 1988) and a major environmental pollutant in urban areas (WHO, 1993) due to gasoline evaporation and combustion (Merian and Znader, 1982). The widespread human exposure to benzene raises considerable concern and several estimates have been made to evaluate the risk of long-term effects under various exposure scenarios (US EPA, 1998; WHO, 1999). However, despite half a century of investigations, the mechanism of leukaemogenesis by benzene has not been fully elucidated (Snyder et al., 1993; Smith, 1996) and low dose effects cannot presently be estimated with confidence. To this end, valuable information can be provided by investigations on biological effects in human populations exposed to low benzene concentrations. Cytogenetic analyses in particular are an effective and informative methodology, because of the distinct clastogenicity of benzene and the likely involvement of chromosomal alterations in the mechanism of benzene-induced leukaemia (Irons and Stillman, 1996; Smith, 1996).
Conventional metaphase analysis has been successfully used to demonstrate the clastogenic effect of benzene in peripheral lymphocytes at concentrations just above current occupational standards (Picciano, 1979; Sarto et al., 1984; Yardley-Jones et al., 1990). However, more sensitive approaches are required to explore the potential clastogenicity of low benzene concentrations. Recent studies on benzene genotoxicity in humans have exploited progress in molecular cytogenetics to focus the analysis on benzene-specific alterations. Fluorescence in situ hybridization (FISH) has been used for the painting of whole chromosomes involved in benzene-induced myelodisplasies (Smith et al., 1998) or for the hybridization of chromosome arms involved in aberrations commonly observed among leukaemia patients (Zhang et al., 1998a,b) Actually, leukaemia-related chromosome changes should be regarded as a prepathological alteration, mechanistically associated with the final adverse outcome of exposures, and their utility in the biomonitoring of subjects exposed to low benzene levels seems questionable. Less specific but more sensitive end-points are more likely to play a role in the assessment of the biological effects of low benzene exposure. The tandem labelling method, based on FISH of adjacent centromeric and pericentromeric regions (e.g. 1cen and 1q12 on chromosome 1), has been proposed as a sensitive and reliable technique to this end (Au et al., 1999; Marcon et al., 1999). The hybridized target was in fact shown to be particularly sensitive to several chemical mutagens, including benzene metabolites (Eastmond et al., 1994; Rupa et al., 1997; Murg et al., 1999a). Moreover, this approach allows the simultaneous detection of chromosome breakage and hyperploidy of the hybridized chromosome in both metaphase and interphase nuclei, enabling assessment of the occurrence of chromosomal alterations in virtually any cell type, with no need for in vitro cultivation. Previous studies have demonstrated the suitability of the tandem labelling methodology for the biomonitoring of populations occupationally exposed to pesticides (Rupa et al., 1995; Au et al., 1999), tobacco smoke (Rupa and Eastmond, 1997) and petrochemical products (Marcon et al., 1999).
In this study, the tandem labelling methodology was used to assess the aneugenic and clastogenic effects of occupational exposure to petroleum fuels and low benzene concentrations in a group of gasoline station attendants. Peripheral lymphocytes were hybridized with and classical satellite probes in different phases of the cell cycle, i.e. unstimulated lymphocytes, 48 h stimulated cells, metaphase lymphocytes and binucleated lymphocytes, to gain some insights into the persistence and biological meaning of the chromosomal alterations identified, and the results are compared with fuel exposure markers.
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Materials and methods |
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Study subjects
The study was performed on 12 gasoline station attendants and 12 unexposed controls matched by age. All subjects were male and non-smokers. Average benzene exposure (7 h TWA) of station attendants was 0.1 ± 0.01 p.p.m. Other characteristics of the study subjects have been reported elsewhere (Carere et al., 1998
). Detailed information on medical history and other potential confounding factors, such as smoking habit, alcohol or drug consumption and diagnostic exposure to X-rays, was collected by questionnaire. Informed consent was obtained from all participants in the study.
Cell sample preparation
A sample (7 ml) of venous blood was collected in heparinized tubes from each subject by venipuncture. After collection, blood was aliquoted and immediatly processed following standardized procedures. For the analysis of unstimulated lymphocytes, one aliquot of blood (2 ml) was incubated in 0.075 M KCl at room temperature for 30 min and then fixed three times in cold methanol/acetic acid (3:1). The remaining amount was used to establish replicated whole blood cell cultures in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 20% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 2% phytohaemagglutinin (PHA HA-15; Abbott), penicillin (5000 IU/ml) and streptomycin (5000 mg/ml) (both from Flow Laboratories, Irvine, UK). All cultures were incubated at 37°C in a humidified 5% CO2 atmosphere. For the analysis of stimulated lymphocytes in interphase and metaphase, cells were harvested 48 h after stimulation. Colchicine (10-5 M final concentration) was added 3 h prior to harvest. Cells were fixed following a standard protocol (10 min at 37°C in 0.075 M KCl, three times in 3:1 methanol/acetic acid). Parallel cell cultures, supplemented with 6 µg/ml cytochalasin B (Sigma) 44 h after stimulation, were used to obtain binucleated lymphocytes. Cell cultures were harvested 22 h after cytochalasin B supplementation, incubated for 2 min in hypotonic solution at room temperature and fixed three times in 5:1 methanol/acetic acid. Fixed cell samples were stored at –20°C until slide preparation and hybridization.
Fluorescence in situ hybridization (FISH)
FISH was performed using an satellite biotinylated probe (D1Z5; ONCOR, Gaithersburg, MD) to label the centromeric region (1cen) and a classical satellite digoxigenated probe (Boehringer-Mannheim) to detect the pericentromeric region (1q12) of chromosome 1. Slide hybridization was performed according to standard procedures (Rupa et al., 1997). Detection of the biotinylated probe was performed with FITC–avidin (Vector Laboratories, Burlingame, CA), while amplification of the signal was obtained using biotinylated anti-avidin D (Vector Laboratories) and FITC–avidin. The digoxigenin probe was detected with rhodamine-conjugated anti-digoxigenin antibody (Boehringer-Mannheim) and Texas Red-conjugated secondary antibodies (Vector Laboratories). Cells were counterstained with 1 µg/ml DAPI Vectashield (Vector Laboratories). A fluorescence microscope equipped with a triple bandpass filter (Chroma) was used to analyse the hybridized slides. Digital images for the three flurochromes were sequentially taken with a Spot camera (Diagnostic Instruments) and merged by dedicated software (FISH 2000; Delta Sistemi, Italy).
Slide scoring
For each individual, 2000 unstimulated cells, 2000 stimulated lymphocytes and, wherever possible, 500 metaphases and 2000 nuclei from binucleated cells were scored on randomized, coded slides by three scorers. The analysis of interphase cells was performed according to previously published criteria (Eastmond et al., 1994). The presence of a green spot adjacent to a red signal was interpreted as an intact chromosome 1 (Figure 1A). Cells showing two adjacent red and green signals and one isolated red spot were classified as breaks within the pericentromeric region (Figure 1B), while cells showing a clear separation between the red and green fluorochromes were classified as breaks between the centromeric and the pericentromeric regions of chromosome 1 (Figure 1C); the two types of separations between the fluorescent signals described above were recorded as breaks when the distance between the spots was greater than the width of the probes. Occasionally, nuclei with separated fluorescent signals connected by a thin thread were also observed (Figure 1E and F): these figures were interpreted as chromatin decondensation and included with normal cells containing intact chromosomes 1. Finally, cells with three or more red spots adjacent to an equal number of green spots were classified as hyperploids. Identical criteria were applied in the scoring of binucleated cells, considering the single nucleus as the unit of analysis. The occasional appearance of a red signal in a micronucleus, indicating loss of a fragment of chromosome 1, was included as a break. Similarly, in the analysis of metaphases the displacement of red and green signals was considered to indicate a break between the centromeric and pericentromeric regions, whereas the appearance of an extra red spot on a fragment or translocated to another chromosome (Figure 1D) was taken as evidence of breakage within the pericentromeric region. In both interphase and metaphase cells all breaks identified under triple bandpass filter observation were also inspected for confimation with specific filters for fluorescein, Texas Red and DAPI. In order to control for scorer differences and verify their concordance of judgement, a sample of slides were randomly selected and analysed by all scorers for cross-comparison. Moreover, most potentially abnormal figures were checked by all scorers.
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Statistical analysis
The two-tailed Mann–Whitney U-test was used to compare exposed and control groups. Correlations between variables were investigated by two-tailed Pearson correlation analysis of natural logarithmic transformed data. All statistical analyses were performed using the SPSS statistical package (v.9.0).
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Results |
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Dual colour FISH was used to detect chromosome breakages targeting the 1cen–1q12 region in peripheral lymphocytes of gasoline station attendants and matched controls. The results obtained are summarized in Table I
, where individual data on the incidence of breaks in different cell types are shown.
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The separation of fluorescent signals suggested the occurrence of breaks within 1cen–1q12 in interphase cells of all study subjects. The distribution of breaks among the subjects was slightly skewed on the right side, with some cases significantly deviating from normality. Therefore, a non-parametric test was used for the statistical analysis of results. Breaks were relatively more frequent among benzene-exposed workers than in unexposed controls: the difference between groups did not attain statistical significance for unstimulated cells (P = 0.1, Mann–Whitney test), but it was highly significant for mitogen-stimulated interphase cells (P < 0.001, Mann–Whitney test).
Despite the above evidence of an exposure-related increase in chromosome damage, no parallel increase in micronuclei in cytokinesis-blocked cells was observed in a previous study on the same subjects (Carere et al., 1998). This discrepancy prompted further work to assess the incidence of breaks in 1cen–1q12 in metaphase cells. To this end, at least 5000 metaphases were scored in both groups from the same slides used for the analysis of stimulated interphase cells. Only two breaks in 5157 scored metaphases and four breaks in 5000 metaphases were observed in the exposed and control groups, respectively. Even though these data do not allow a reliable estimation of individual frequencies of breaks, they indicate that the overall incidence of breaks in metaphase was at least one order of magnitude lower than observed in interphase (Table I
). A plausible explanation for this result is selective elimination of damaged cells during the cell cycle and/or prevention of their entering mitosis. To check this possibility, tandem hybridization of 1cen–1q12 was performed on a further set of slides, prepared for the cytogenetic analysis of binucleated cells. In this way the incidence of breaks in 1cen–1q12 could be evaluated in a cell population which had passed through mitosis, thus enabling us to confirm or disprove the hypothesis of selective elimination of damaged cells at mitotic checkpoints. Despite some technical problems related to probe penetration, at least 1000 nuclei could be scored in binucleated cells from 19 subjects. The results obtained show an overall incidence of breaks which is basically comparable with the incidence observed in G0 and in stimulated interphase cells, i.e. at least one order of magnitude higher than observed in metaphase cells (Table I). Interestingly, in binucleated cells the figures classified as 1cen–1q12 breaks were also relatively more frequent in benzene-exposed workers than in controls, with a difference close to statistical significance (P = 0.058, Mann–Whitney test).
In order to shed some light on the intriguing results obtained, namely the evidence for chromosomal alterations related to chemical exposure expressed only in interphase cells, the data were re-analysed considering breaks between the and classical satellites (–c breaks) and breaks within the classical satellite (c–c breaks) separately. The data in Table II show that –c breaks alone were responsible for the difference between the exposed and control groups, showing a statistically significant prevalence in both unstimulated cells (P = 0.045) and stimulated lymphocytes (P = 0.001) of benzene-exposed workers and also a bordeline excess in binucleated cells (P = 0.054). Interestingly, –c breaks in unstimulated lymphocytes were significantly correlated with –c breaks observed in stimulated lymphocytes and in binucleated lymphocytes (P = 0.018 and P = 0.015, respectively, two-tailed Pearson correlation analysis on natural logarithm transformed values) (Figures 2 and 3, respectively). On the other hand, only c–c breaks were observed in metaphase cells of both study groups, with an overall incidence 3- to 9-fold lower than observed in interphase cells.
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No significant difference in trisomy incidence was observed between the study groups. The overall frequencies of chromosome 1 hyperploidy in stimulated interphase cells (0.7
) and in metaphases (1.0) were in the range of values measured, in the same subjects, for hyperploidy of chromosomes 7, 11, 18 and X (Carere et al., 1999).
A correlation analysis was carried out to probe the relationships between incidence of –c or c–c breaks (natural logarithm transformed) in different cell types and smoking habit, fuel exposure markers (urinary benzene and trans-muconic acid, lead in blood and external benzene measured in the breathing zone) and other cytogenetic end-points (micronuclei and chromosome missegregation) measured in previous investigations on the same subjects (Carere et al., 1998, 1999). To avoid casual correlations, only highly significant (P < 0.01) or recurrent correlations were considered. Two-tailed Pearson correlation coefficients highlighted a significant correlation between frequency of –c breaks in stimulated lymphocytes and urinary benzene level (P = 0.001, Figure 4) and between –c breaks in unstimulated lymphocytes and blood lead concentration (P = 0.007, Figure 5). No significant correlation was observed between 1cen–1q12 breaks and smoking habit or other cytogenetic end-points.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study the tandem labelling approach, a methodology based on dual colour FISH of adjacent centromeric and pericentromeric regions (Eastmond et al., 1994
; Rupa et al., 1995), was applied to detect chromosomal alterations in peripheral lymphocytes of gasoline station attendants. The analysis was carried out on lymphocytes harvested in different phases of the cell cycle (i.e. before and after mitogen stimulation, at first metaphase and on post-mitotic binucleated cells) in order to obtain some insights into the nature and stability of the lesions. The experimental results obtained indicate a higher frequency of lesions affecting 1cen–1q12 in lymphocytes of gasoline station attendants compared with age-matched controls. A prevalence of signal separation was observed in both unstimulated (G0) cells and, to a greater extent, in interphase cells harvested after 48 h mitogen stimulation. In principle, the possibility that easier detection of fluorescent signals in larger, stimulated cells could contribute to the latter result cannot be ruled out. However, the fact that successful probe hybridization, with bright signals and >90% efficiency, was achieved in both stimulated and unstimulated cells suggests a limited role of technical factors. On the other hand, the prevalence of chromosomal alterations in stimulated cells of exposed subjects may indicate the involvement of lesions convertible into visible alterations during S phase, reinforcing the biological plausibility of an effect of chemical exposure. In this respect it has to be recalled that similar results were obtained in a previous study on another population exposed to low benzene levels (Marcon et al., 1999), where increased incidences of breaks in 1cen–1q12 as well as in the pericentromeric region of chromosome 9 were detected in lymphocytes of exposed subjects.
The results obtained in this work basically support the use of the tandem approach in biomonitoring of low level chemical exposures. However, this study also raised some questions as to the nature of the lesions which lead to signal displacement in interphase nuclei. Even though it is generally assumed that signal separation reflects structural chromosome damage, such as chromatid and chromosome breaks and chromosome exchanges (Rupa et al., 1995; Murg et al., 1999b), the possible involvement of chromatin unwinding and unfolding (Salama et al., 2001) has to be taken into account. Actually, the parallel analysis of metaphase spreads and interphase nuclei carried out in this study strongly supports the latter mechanism.
The lower incidence of breaks in metaphase compared with interphase observed in this study could be due either to selective arrest and removal of damaged cells at a G2/M checkpoint or to chromatin decondensation at 1cen–1q12 in interphase. The occurrence of relatively high frequencies of nuclei with signal separation in binucleated cells, which have just terminated mitosis, strongly argues against the hypothesis of a block at G2/M. On the other hand, chromatin decondensation was observed in several interphase nuclei showing split signals connected by a thread of decondensed chromatin (Figure 1E and F). It is conceivable that more extensive chromatin decondensation might lead to faded signals, below the detection power of standard FISH techniques, thus mimicking a breakage in the interphase chromosome. Such decondensation mainly affects the boundary between the two repetitive DNA regions hybridized, as indicated by the prevalence of displacement of the centromeric and pericentromeric fluorescent signals (–c breaks) in interphase. On the other hand, the analysis of metaphase spreads indicates that this region is not a preferential breakage point, given that no true –c breaks could be detected in >10 000 scored metaphases.
In conclusion, it is proposed that chemical exposure leads to modification(s) of chromatin organization in 1cen–1q12 which results in a mild decondensation of the region. The observation that the alterations detected in interphase, operationally defined breaks, are highly intercorrelated among different cell types (i.e. unstimulated and stimulated lymphocytes and post-mitotic binucleated cells) suggests some stability of this modification. The available information does not allow us to disclose the nature and mechanism(s) underlying such putative epigenetic modification, nor the causative agent(s). However, considering the exposure profile of the study subjects, the involvement of benzene should not be dismissed, in view of the interference exerted by benzene metabolites on the activity of topoisomerase II (Chen and Eastmond, 1995; Frantz et al., 1996; Hutt and Kalf, 1996) and their interactions with other chromatin proteins (Smith, 1996).
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Acknowledgments |
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This study was partially funded by the Italian Ministry of the Environment (Project PR 22-IS).
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Notes |
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1 To whom correspondence should be addressed. Tel: +39 0649902680; Fax: +39 0649387139; Email: marcon{at}iss.it
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References |
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Received on June 28, 2001; accepted on October 31, 2001.
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