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Cytogenetic biomonitoring of workers from laboratories of clinical analyses occupationally exposed to chemicals
Mutation Research 520 (2002) 73–82
Cytogenetic biomonitoring of workers from laboratories of clinical analyses occupationally exposed to chemicals A. Testa a,∗ , R. Ranaldi a , L. Carpineto a , F. Pacchierotti a , D. Tirindelli a , L. Fabiani b , A.R. Giuliani b , M. Urso c , A. Rossini c , F. Materazzo c , M. Petyx c , V. Leoni c b
a Section of Toxicology and Biomedical Science, E.N.E.A, CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy Chair of Hygiene, Department of Internal Medicine and Public Health, Aquila University, Via S.Siro 67100, L’Aquila, Italy c Chair of Environmental Hygiene, Department of Public Health Sciences, University of Rome “La Sapienza”, Piazza A. Moro 5, 00185 Rome, Italy
Received 26 March 2002; received in revised form 20 June 2002; accepted 28 June 2002
Abstract A cytogenetic monitoring study was carried out on a group of workers in clinical analysis laboratories to investigate the risk of occupational exposure to chronic low levels of chemicals. Thirty-four clinical laboratories have been involved in the study. In these laboratories, toxicants and analytical procedures utilized have been characterized. The individual occupational exposure of workers was assessed by use of a questionnaire concerning the chemical substances utilized. About 300 different chemicals have been identified. Cytogenetic analyses (chromosomal aberration and micronucleus tests) were carried out on a strictly selected group of 50 workers enrolled from these laboratories and compared to 53 controls (healthy blood donors) matched for gender and age. The exposed group shows a significantly higher frequency of genetic damage than the control group. Both chromatid and chromosome aberration frequencies in workers appear significantly higher than in controls. Similarly, comparison between micronucleated cells rates of exposed and unexposed groups show significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls. © 2002 Published by Elsevier Science B.V. Keywords: Cytogenetic biomonitoring; Workers; Clinical analysis laboratory; Chromosomal aberration; Micronuclei
1. Introduction Occupational exposure of workers in clinical analysis laboratories is very complex because they are potentially exposed to a high number of chemical substances used or stored at work. In addition, there is a great variability between laboratories in terms of instruments and analytical methods used for the same ∗ Corresponding author. Tel.: +39-6304-86654; fax: +39-6304-83644. E-mail address: [email protected] (A. Testa).
type of analysis. Furthermore, products and techniques rapidly change, which makes the assessment of occupational risks very difficult. Although few data have been published for chemical exposure in laboratory workers, they indicate organic solvents, aldehydes and metals as the most significant inhalation hazards. A number of studies have shown an increased risk of cancer in chemists working in clinical analysis laboratories. Some of these studies report an increase in mortality due to all types of cancer for these workers in comparison to the general population. An increase in mortality due to lymphoematopoietic and cerebral
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tumors was found in workers involved in analyses related to organic chemistry [1–4]. On the other hand, some data have indicated a reduction of mortality and morbidity due to cancer in chemists compared to other kinds of workers and to the general population [5]. Hence, these observations did not provide definite data and the risk of cancer in workers in the field of chemistry is still matter of debate. It must be noted that the methodologies used in these studies often did not exclude the risk of bias nor errors in evaluating the type of exposure. Indeed, the kind of toxic agent was often deduced just by evaluating possible agents that this category of worker comes into contact with rather than directly evaluating each case or getting the workers to fill in a questionnaire. As far as genotoxic effects from this kind of occupational exposure are concerned, only very few data are available. A significant increase of sister chromatid exchanges (SCE) was found in a group of non-smoking laboratory workers compared with non-smoking control subjects. However, no such difference was found between smoking controls and laboratory workers [6]. In a cytogenetic study on laboratory personnel of chemical laboratories, significantly increased frequencies of chromatid and isochromatid breaks were found in workers in comparison with the controls [7]. No increase of genetic damage in terms of sister chromatid exchange and micronucleus induction was found in peripheral lymphocytes of a group of laboratory employees [8]. The aim of this study was to evaluate the genotoxic effects of chronic low-dose exposure to chemicals in a selected group of subjects working in clinical analysis laboratories by cytogenetic monitoring (using chromosome aberration and micronucleus assays) on peripheral blood lymphocytes (PBLs). Since cytogenetic monitoring studies are very time-consuming, it is necessary, as suggested by Tucker and Preston [9], before “embarking” on such a study, to “design the experiments in such a way that data obtained will be of maximum possible benefit for characterizing and quantifying adverse human health effects, particularly cancer”. Particular consideration should be given to the various factors affecting the DNA damage level. Hence, in order to exclude bias due to the “confounding factors” of lifestyle we have enrolled only carefully pre-selected subjects in our study.
2. Materials and methods 2.1. Selection of subjects The study was carried out in 34 laboratories of clinical analyses in two Italian hospitals. All individuals provided written informed consent according to the law in force. All subjects involved received a letter concerning the aims of the research study, in which they were informed that the results would be published and that they would not be receiving any personal results. They were personally interviewed by filling in two different questionnaires: (a) the “personal health questionnaire” by Carrano and Natarajan [10] for the evaluation of “lifestyle confounding factors”, and (b) a specific questionnaire on the individual professional exposure (kind of laboratory, methodologies, chemical substances utilized, number of determinations per week, waste disposal methods) [11]. We excluded the following from the investigation: smokers, alcohol drinkers, subjects who had been exposed to radiation within the last 6 months or with recent viral or bacterial infections, subjects recently taking drugs or having vaccinations, individuals with known genetic defects in the family or major illnesses, and subjects aged over 50 years. Only 50 exposed subjects (15 males, 35 females; mean age 37 years) out of the 300 clinical laboratory workers interviewed for the study met all the above criteria and were included in this study. The same selection criteria were used for the control group. A total of 53 controls (23 males, 30 females; mean age 33 years) were selected from usual blood donors. Both exposed and control subjects live in urban areas in the middle and the south of Italy. So they are probably exposed to the same amount and type of pollution. 2.2. Exposure assessment Individual exposure was assessed according to the analytical methods used in the laboratory and to the quantity and type of chemical substance. Table 1 shows the distribution of laboratory workers among the different laboratories, the number of annual determinations and the length of service. Overall mean length of service was 10.62 ± 7.15 years and the standard working time was 36 h per week.
A. Testa et al. / Mutation Research 520 (2002) 73–82 Table 1 Distribution of exposed subjects among different laboratories Laboratory
Number of subjects
Number of annual determinations
Mean years/ work (±S.D.)
Microbiology
11
48,700
7.9 (±8.4)
Clinical chemistry Immunology Microbiology
9
1,076
14.7 (±6.1)
Hystology
3
17,500
8.0 (±5.3)
Clinical chemistry Immunology
12
367,566
9.5 (±6.0)
Immunology Hystology Molecular biology
4
4,850
9.5 (±5.4)
Clinical chemistry Hystology
3
262,350
Various
8
–
9.7 (±11.5) 12.3 (±8.6)
Briefly, about 300 different chemical substances were identified. Of these, according to the European Community Classification of Chemicals, 40% are the corrosive, 28% toxic if inhaled or ingested, 12% flammable and 4% carcinogens. The overall quantity of each reagent used in the different laboratories was determined. However, a quantitative definition of individual exposure was hindered by varying factors including the equipment available (manual or automatic), the working environment, and waste storage methods. 2.3. Lymphocyte cultures Venous blood was drawn into heparinized tubes. Lymphocyte cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI 1640 medium (Flow) supplemented with 10% heat-inactivated fetal calf serum (Gibco, Gaithersburg, MD, USA), 2% phytohaemagglutinin (Murex), 1.5% penicillin–streptomycin (5000 IU/ml–5000 g/ml, respectively (Sigma) and 1% l-glutamine (Sigma). Cultures were grown at 37 ◦ C. Slides were analyzed blind by two different researchers who scored the same number of cells for each subject. For the chromosome aberrations (CA) assay, cultures were fixed at 48 h according to the standard
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methods following a 90 min treatment with 0.2 g/ml colcemid (Sigma). Air-dried metaphase spreads were stained by the conventional unbanded Giemsa method. A total of 100 well-spread metaphases containing 46 ± 1 centromeres for workers and 200 for controls were examined on coded slides. Chromosome- and chromatid-type aberrations were recorded. For the micronucleus (MN) study, cultures were incubated at 37 ◦ C for 72 h and, 44 h from the beginning, cytochalasin-B (Cyt-B, Sigma) at a final concentration of 6 g/ml was added to arrest cytokinesis. Air-dried preparations were stained by the conventional Giemsa method. The presence of MN was evaluated by scoring a total of 1000 binucleated cells (BN) with well-preserved cytoplasm for each donor. In addition, 1000 lymphocytes were scored to evaluate the percentage of cells with 1–4 or more nuclei. The nuclear division index (NDI) and the cytokinesis block proliferation index (CBPI) were used for measuring cell proliferation kinetics. 2.4. Statistical analysis The distribution of all parameters evaluated by the Kolmogoroff–Smirnoff test showed significant departures from the normal distribution. Therefore, both CA and MN data were analyzed by the Mann–Whitney U-test (significance taken as P < 0.05). To estimate the existence of any relationship between CA and MN frequencies and the variables age and sex, the Spearman rank correlation test was used. The same test was also utilized to evaluate the correlation between the two different cytogenetic assays. The data were analyzed using the STATSOFT package.
3. Results Tables 2 and 3 summarize chromosome aberrations and micronucleus frequencies in female and male workers, respectively. Tables 4 and 5 show the data obtained from the control subjects. The NDI and the CBPI individual values are also reported in the tables. The exposed group shows a significantly higher frequency of genetic damage than the control group. As shown in Table 6, chromatid and chromosome aberration frequencies in workers
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Table 2 Chromosome aberration and micronucleus frequencies in female clinical laboratory workers Subjects
Age
Chromosome aberrations Del
Dic + CR
Exchange
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 E27 E28 E29 E30 E31 E32 E33 E34 E35
30 31 35 35 33 38 43 34 40 42 39 31 34 40 40 25 27 37 41 29 36 31 32 31 26 27 34 48 48 44 43 40 37 46 34
0 2 1 1 0 0 1 0 1 1 0 1 2 0 1 0 2 0 1 0 2 2 0 2 1 0 0 0 0 1 2 3 1 1 2
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
35
36
31
3
0
Mean ±S.D.
CsA (%) 0 2 1 1 0 0 1 0 1 1 1 1 2 0 1 0 2 0 1 0 2 2 0 2 2 0 0 0 0 1 3 3 1 1 2
Chromatid aberrations Del
Exchange
2 3 1 1 1 0 4 1 1 2 1 1 2 0 1 0 1 1 0 2 2 3 0 1 0 3 2 3 2 2 2 1 2 1 2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
51
0
0.97 0.92
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
2 3 1 1 1 0 4 1 1 2 1 1 2 0 1 0 1 1 0 2 2 3 0 1 0 3 2 3 2 2 2 1 2 1 2
2 5 2 2 1 0 5 1 2 3 2 2 4 0 2 0 3 1 1 2 4 5 0 3 2 3 2 3 2 3 5 4 3 2 4
11 10 10 24 17 15 17 13 16 13 9 4 4 10 7 4 4 4 6 5 3 12 6 3 4 6 5 8 8 5 7 9 4 10 8
11 10 10 27 17 17 17 15 16 13 9 4 4 10 7 4 4 4 6 5 3 15 7 3 4 6 5 8 8 5 8 10 4 10 8
1.6 1.8 1.7 1.6 1.7 1.8 1.8 1.8 1.8 1.8 1.8 1.9 1.6 1.6 2.0 1.9 1.8 1.9 2.3 1.6 1.8 1.6 1.6 1.8 1.7 1.8 1.8 1.7 1.8 1.7 1.2 1.2 1.9 1.9 1.7
1.6 1.7 1.6 1.6 1.6 1.7 1.7 1.7 1.7 1.7 1.7 1.8 1.6 1.6 1.9 1.7 1.7 1.7 2.2 1.5 1.7 1.5 1.6 1.7 1.6 1.7 1.7 1.6 1.7 1.7 1.2 1.2 1.8 1.7 1.7
1.46 1.01
2.43 1.46
1.7 0.2
1.7 0.1
8.6 4.89
8.9 5.38
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation.
appear significantly higher than in controls. Similarly, comparison between micronucleated cell rates of the exposed and unexposed group shows significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls.
As far as the correlation between sex and chromosome aberration frequency is concerned, no statistically significant differences were found between males and females in both the groups. A slightly significant increase (P = 0.012) in micronucleus rates was found in the female control group.
A. Testa et al. / Mutation Research 520 (2002) 73–82
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Table 3 Chromosome aberration and micronucleus frequencies in male clinical laboratory workers Subjects
Age
Chromosome aberrations Dic + CR
Exchange
2 1 2 0 1 1 3 4 0 3 0 0 0 1 1
0 0 2 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19
3
0
Del E36 E37 E38 E39 E40 E41 E42 E43 E44 E45 E46 E47 E48 E49 E50
49 42 33 49 38 43 27 49 38 47 36 35 39 40 42
15 Mean ±S.D.
40.5
CsA (%) 2 1 4 0 1 1 3 5 0 3 0 0 0 1 1
Chromatid aberrations Del
Exchange
3 1 1 1 1 5 3 3 0 3 0 0 2 2 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
25
0
1.47 1.59
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
3 1 1 1 1 5 3 3 0 3 0 0 2 2 0
5 2 5 1 2 6 6 8 0 6 0 0 2 3 1
10 16 9 8 4 4 3 6 4 8 4 4 4 4 14
11 16 9 8 4 4 3 6 4 8 4 4 4 4 16
1.8 1.9 1.8 1.8 1.5 1.8 1.7 1.4 1.7 1.6 1.9 2.1 1.8 1.7 1.3
1.7 1.8 1.8 1.7 1.6 1.7 1.6 1.4 1.6 1.5 1.8 2.0 1.7 1.7 1.3
1.67 1.49
3.13 2.64
1.7 0.2
1.7 0.2
6.8 4.00
7.0 4.34
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation.
Our results also indicate an effect of age on micronucleus frequency. As can be observed in Fig. 1, there is a significant positive correlation (P < 0.05) between micronucleus frequency and age in both the groups with similar slopes but the occurrence of BNMN is higher in the exposed group than in controls. No statistical differences in NDI and CBPI values were found between groups. As far as the correlation between different cytogenetic parameters is concerned, a positive correlation was found between chromosome and chromatid aberration frequencies both in the exposed group (P = 0.006, r = 0.4) and in the control group (P = 0.009, r = 0.4). No correlation was found between the micronucleus test and the chromosome aberration assay.
4. Discussion Workers in biomedical laboratories are potentially exposed to numerous occupational hazards: chemicals, radioactive materials and biological agents. The exposure levels are generally low and the different
sensitivity of analytical methods, for the different sources of exposure, precludes a precise dose assessment. Moreover, because of the great variability between laboratories regarding analytical techniques and the quantity of chemicals used for the same kind of analysis, biomedical laboratories cannot be considered as a single unit. As far as epidemiological data are concerned, no large studies have been undertaken for people handling chemicals in clinical and research biomedical laboratories. Some of these data are indicative, although not yet conclusive, for increased mortality risk due to malignant lymphoma, leukemia and cancer of the gastrointestinal tract among laboratory workers [12–14]. A cluster of cancer cases, especially sarcomas and lymphomas, was detected among subjects engaged in molecular biology, genetics, and mutagenicity testing in the Pasteur Institute in Paris [15]. Another investigation on laboratory workers carried out between 1971 and 1980 in England and Wales reported significantly increased risk for cancer of the brain, stomach and nervous system and a decreased risk for cancer of the cervix [12].
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Table 4 Chromosome aberrations and micronucleus frequencies in female control subjects Subjects
Age
Chromosome aberrations Del
Dic + CR
Exchange
6 8 10 11 17 18 19 20 21 22 24 27 28 30 34 36 37 39 40 43 44 45 46 47 48 49 50 51 52 53
25 28 30 32 24 24 29 31 37 40 26 33 29 26 31 34 41 45 46 25 31 43 43 25 25 30 26 29 25 30
1 2 1 2 0 1 0 1 0 0 0 0 0 0 0 0 3 0 1 2 0 1 2 1 1 0 1 1 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
30
31
22
1
2
Mean ±S.D.
CsA (%) 0.5 1 0.5 1 0 0.5 0 0.5 0 0 0.5 0 0 0 0 0 2 0 0.5 1 0 0.5 1.5 0.5 0.5 0 0.5 0.5 0 0.5
Chromatid aberrations Del
Exchange
2 1 1 2 1 3 0 2 0 0 0 2 2 1 2 1 1 0 1 3 0 1 3 1 0 2 1 2 2 4
0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
41
1
0.42 0.41
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
1 0.5 0.5 1 0.5 1.5 0 1 0 0 0 1 1 0.5 1 0.5 0.5 0 0.5 1.5 0 0.5 1.5 0.5 0 1 0.5 1 1 2
1.5 1.5 1 2 0.5 2 0 1.5 0 0 0.5 1 1 0.5 1 0.5 2.5 0 1 2.5 0 1 3 1 0.5 1 1 1.5 1 2.5
6 3 3 6 3 2 2 4 4 5 4 4 3 4 1 4 9 17 3 6 6 7 7 2 3 4 4 5 4 5
6 3 3 6 3 2 2 4 4 5 4 4 3 4 1 4 12 20 4 7 7 7 7 3 3 4 4 5 4 5
1.7 1.7 1.8 1.8 1.8 1.8 1.8 1.8 1.8 2.2 1.9 1.7 2.2 1.6 1.9 1.7 1.6 1.7 2.0 1.6 1.8 1.3 1.7 2.1 1.4 2.0 2.0 2.1 1.9 1.7
1.6 1.7 1.7 1.75 1.7 1.7 1.7 1.7 1.7 1.9 1.8 1.6 1.9 1.5 1.7 1.6 1.6 1.6 1.9 1.5 1.7 1.3 1.6 1.9 1.2 1.9 1.8 1.1 1.8 1.6
0.68 0.53
1.1 0.82
1.8 0.2
1.7 0.2
4.7 3.0
5 3.5
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation.
Subsequently, a mortality study on workers employed by the research staff of the Italian National Institute of Health revealed excess of brain, pancreatic, lymphohematopoietic and breast cancers [16]. Finally, a recent review on cancer risk assessment among employees in biological, medical and agronomic research laboratories suggests a low overall risk of cancer albeit that a higher risk may be suggested for cancers of pancreas, brain and for non-Hodgkin’s lymphoma [17].
In this study, a group of strictly selected clinical laboratory workers and a comparable control group were monitored for chromosome aberrations and micronuclei. Our results show that the group of workers under study exposed to chronic low levels of chemical toxicants has a highly significant increase of genetic damage in terms of chromatid and chromosome aberration and micronucleus frequencies compared with the control group. Besides the elevated frequency of chromatid aberrations usually related to exposure to
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Table 5 Chromosome aberration and micronucleus frequencies in male control subjects Subjects
Age
Chromosome aberrations Del
Dic + CR
Exchange
1 2 3 4 5 7 9 12 13 14 15 16 23 25 26 29 31 32 33 35 38 41 42
49 41 43 25 36 38 36 50 32 32 28 31 34 35 32 30 29 31 25 24 31 39 26
2 0 0 0 0 0 2 1 1 3 0 1 0 0 0 0 1 0 1 0 0 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
23
34
14
1
1
CsA (%) 1 0 0 0 0.5 0 1 0.5 0.5 1.5 0 0.5 0 0 0 0.5 1 0 0.5 0 0 0.5 0.5
Mean ±S.D.
Chromatid aberrations Del
Exchange
3 1 0 2 2 0 6 1 1 4 1 0 0 1 1 0 3 2 0 0 0 1 4
0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
33
2
0.37 0.43
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
1.5 0.5 0 1 1.5 0 3 0.5 0.5 2 0.5 0 0 1 0.5 0 1.5 1 0 0 0 0.5 2
2.5 0.5 0 1 2 0 4 1 1 3.5 0.5 0.5 0 1 0.5 0.5 2.5 1 0.5 0 0 1 2.5
1 3 4 2 2 4 4 5 3 3 2 2 4 3 3 3 6 4 2 1 3 2 3
1 3 4 2 2 4 4 5 3 3 3 2 4 4 3 3 6 4 2 1 3 2 3
1.9 1.8 1.7 1.9 1.9 1.8 1.8 1.7 1.7 1.8 1.7 1.8 1.8 1.9 1.8 1.7 1.5 1.7 1.6 1.6 2.0 1.7 1.6
1.8 1.7 1.6 1.8 1.8 1.7 1.7 1.6 1.7 1.7 1.7 1.7 1.9 18 17 16 1.4 1.6 1.5 1.6 1.8 1.6 1.5
0.76 0.82
1.13 1.15
3 1.2
3.1 1.2
1.7 0.1
1.7 0.1
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation. Table 6 Comparison of chromosome aberration and micronucleus frequencies between exposed and control subjects Groups
Number of CsA (%) subjects
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
0.75 (±0,46) 0.68 (±0.53) 0.76 (±0.82)
1.14 (±0.95) 1.1 0 (±0.82) 1.13 (±1.15)
3.94 (±2.45) 4.7 (±3.0) 3.0 (±1.2)
4.0 (±2.9) 5.0 (±3.5) 3.1 (±1.2)
1.77 (±0.17) 1.70 (±0.14) 1.79 (±0.2) 1.68 (±0.2) 1.70 (±0.1) 1.68 (±0.1)
Controls All 53 Females 30 Males 23
0.41 (±0.46) 0.42 (±0.41) 0.37 (±0.43)
Exposed All 50 Females 35 Males 15
1.12∗∗ (±1.17) 1.52∗∗ (±1.16) 2.68∗∗∗ (±1.89) 8.06∗∗∗ (±4.68) 8.38∗∗∗ (±5.1) 1.74 (±0.2) 0.97∗ (±0.92) 1.46∗∗ (±1.01) 2.43∗∗ (±1.46) 8.6∗∗∗ (±4.89) 8.9∗∗ (±5.38) 1.7 (±0.2) 3.13∗ (±2.64) 6.8∗∗∗ (±4.0) 7.0∗∗ (±4.3) 1.72 (±0.2) 1.47∗ (±1.59) 1.67 (±1.49)
CBPI
1.67 (±0.2) 1.7 (±0.1) 1.7 (±0.2)
CsA = chromosome aberration; CtA = chromatid aberration; CA tot = total of chromosome aberrations; BNMN = binucleated cell with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis-block proliferation index; ±S.D. = standard deviation. ∗ P< 0.05. ∗∗ P < 0.001. ∗∗∗ P < 0.0001.
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Fig. 1. Relationship between BNMN frequency and age in exposed and control subjects.
chemical agents, the results show also severe chromosome damages. Particular attention should be paid to this finding because it has been demonstrated that chromosome aberration frequency is itself an indicator of cancer risk rather than just a reflection of exposure, and that the study of aberration levels may directly predict an increased cancer risk [18–20]. In these studies, the authors trichotomized the levels of CA found in large populations. They found a doubling of mortality for all cancers in subjects with a frequency of chromosome aberrations corresponding to the upper thirtile. Our results on chromosome aberration frequencies found in laboratory workers suggest that they could have an increased risk for cancer. As far as the micronucleus data are concerned, workers show very highly statistically increased values compared with the controls. A positive correlation between micronucleus frequency and age in both sexes with a steeper linear regression line for females (data not shown) was also found, confirming other literature data [21]. Even if the relationship of cancer risk with micronuclei is not
as well substantiated as that with chromosome aberration, this method has proven useful as a “biological marker of early effects” in biomonitoring studies on human population exposed to genotoxic agents. The lack of correlation between the two tests is possibly due to the fact that both chromosome loss and chromosome breakage contribute to micronucleus formation suggesting that aneuploidy may be induced by this kind of exposure. The initial hypothesis of a very low-dose exposure for subjects employed in clinical laboratory analysis suggested us to perform a very strict selection of biological, environmental or lifestyle factors affecting DNA damage levels (confounding factors) for subjects involved in this study. In this way, we increased the resolution of the methods used and obtained a more accurate risk assessment. The background of chromosomal aberration and micronucleus frequencies found in our control subjects is very low in respect of the corresponding values reported by other authors [22–25]. This finding allow us to think that normal
A. Testa et al. / Mutation Research 520 (2002) 73–82
healthy individuals are exposed to a variety of such agents as part of their daily life and that the exclusion of these do result in a decrease in genetic damage. In conclusion, the increased chromosome aberrations and micronucleus frequencies found in workers indicate potential genetic hazards. The causative agents could be specific chemicals or a complex mixture of chemicals in the laboratory environment. Unfortunately, very little information is available on adverse human health effects of chemical mixtures [26]. This observation shows the need to increase occupational preventive programs and to improve supervision of the working environment in clinical laboratories.
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Acknowledgements [13]
We would like to thank Prof. G. Girelli of the Department of Experimental Medicine and Pathology of the University “La Sapienza” of Rome and her expert staff for technical assistance in the blood collection. We are grateful to Prof. Renata Cozzi for comments, helpful discussions and reading the manuscript. This study has been partially supported by Grant of the Ministry of Labour (Research Contract 737/97). References [1] J.M. Harrington, H.S. Shannon, Mortality study of pathologist and medical laboratory technicians, Br. Med. J. 4 (1975) 329–332. [2] R. Olin, Leukaemia and Hodgkin’s disease among Swedish chemistry graduates, Lancet 23 (1966) 916. [3] R. Olin, The hazards of chemical laboratory environment—a study of the mortality in two cohorts of Swedish chemists, Am. Ind. Hyg. Assoc. J. 39 (1978) 557–562. [4] R. Olin, A. Ahlbom, Cancer mortality among three Swedish male academic cohorts: chemists, architects and mining engineers/metallurgists, Ann. New York Acad. Sci. (1982) 197–201. [5] S.K. Hoar, S. Pell, A retrospective cohort study of mortality and cancer incidence among chemists, J. Occ. Med. 23 (7) (1981) 485–494. [6] B. Lambert, A. Lindblad, Sister chromatid exchange and chromosome aberrations in lymphocytes of laboratory personnel, J. Toxicol. Environ. Health 6 (1980) 1237–1243. [7] F. Funes-Cravioto, C. Zapata-Gayon, B. Kolmodin-Hedman, B. Lambert, J. Lindsten, E. Norberg, M. Nordenskjold, R. Olin, A. Swensson, Chromosome aberrations and sister chromatid exchange in workers in chemical laboratories and
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Cytogenetic biomonitoring of workers from laboratories of clinical analyses occupationally exposed to chemicals A. Testa a,∗ , R. Ranaldi a , L. Carpineto a , F. Pacchierotti a , D. Tirindelli a , L. Fabiani b , A.R. Giuliani b , M. Urso c , A. Rossini c , F. Materazzo c , M. Petyx c , V. Leoni c b
a Section of Toxicology and Biomedical Science, E.N.E.A, CR Casaccia, Via Anguillarese 301, 00060 Rome, Italy Chair of Hygiene, Department of Internal Medicine and Public Health, Aquila University, Via S.Siro 67100, L’Aquila, Italy c Chair of Environmental Hygiene, Department of Public Health Sciences, University of Rome “La Sapienza”, Piazza A. Moro 5, 00185 Rome, Italy
Received 26 March 2002; received in revised form 20 June 2002; accepted 28 June 2002
Abstract A cytogenetic monitoring study was carried out on a group of workers in clinical analysis laboratories to investigate the risk of occupational exposure to chronic low levels of chemicals. Thirty-four clinical laboratories have been involved in the study. In these laboratories, toxicants and analytical procedures utilized have been characterized. The individual occupational exposure of workers was assessed by use of a questionnaire concerning the chemical substances utilized. About 300 different chemicals have been identified. Cytogenetic analyses (chromosomal aberration and micronucleus tests) were carried out on a strictly selected group of 50 workers enrolled from these laboratories and compared to 53 controls (healthy blood donors) matched for gender and age. The exposed group shows a significantly higher frequency of genetic damage than the control group. Both chromatid and chromosome aberration frequencies in workers appear significantly higher than in controls. Similarly, comparison between micronucleated cells rates of exposed and unexposed groups show significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls. © 2002 Published by Elsevier Science B.V. Keywords: Cytogenetic biomonitoring; Workers; Clinical analysis laboratory; Chromosomal aberration; Micronuclei
1. Introduction Occupational exposure of workers in clinical analysis laboratories is very complex because they are potentially exposed to a high number of chemical substances used or stored at work. In addition, there is a great variability between laboratories in terms of instruments and analytical methods used for the same ∗ Corresponding author. Tel.: +39-6304-86654; fax: +39-6304-83644. E-mail address: [email protected] (A. Testa).
type of analysis. Furthermore, products and techniques rapidly change, which makes the assessment of occupational risks very difficult. Although few data have been published for chemical exposure in laboratory workers, they indicate organic solvents, aldehydes and metals as the most significant inhalation hazards. A number of studies have shown an increased risk of cancer in chemists working in clinical analysis laboratories. Some of these studies report an increase in mortality due to all types of cancer for these workers in comparison to the general population. An increase in mortality due to lymphoematopoietic and cerebral
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tumors was found in workers involved in analyses related to organic chemistry [1–4]. On the other hand, some data have indicated a reduction of mortality and morbidity due to cancer in chemists compared to other kinds of workers and to the general population [5]. Hence, these observations did not provide definite data and the risk of cancer in workers in the field of chemistry is still matter of debate. It must be noted that the methodologies used in these studies often did not exclude the risk of bias nor errors in evaluating the type of exposure. Indeed, the kind of toxic agent was often deduced just by evaluating possible agents that this category of worker comes into contact with rather than directly evaluating each case or getting the workers to fill in a questionnaire. As far as genotoxic effects from this kind of occupational exposure are concerned, only very few data are available. A significant increase of sister chromatid exchanges (SCE) was found in a group of non-smoking laboratory workers compared with non-smoking control subjects. However, no such difference was found between smoking controls and laboratory workers [6]. In a cytogenetic study on laboratory personnel of chemical laboratories, significantly increased frequencies of chromatid and isochromatid breaks were found in workers in comparison with the controls [7]. No increase of genetic damage in terms of sister chromatid exchange and micronucleus induction was found in peripheral lymphocytes of a group of laboratory employees [8]. The aim of this study was to evaluate the genotoxic effects of chronic low-dose exposure to chemicals in a selected group of subjects working in clinical analysis laboratories by cytogenetic monitoring (using chromosome aberration and micronucleus assays) on peripheral blood lymphocytes (PBLs). Since cytogenetic monitoring studies are very time-consuming, it is necessary, as suggested by Tucker and Preston [9], before “embarking” on such a study, to “design the experiments in such a way that data obtained will be of maximum possible benefit for characterizing and quantifying adverse human health effects, particularly cancer”. Particular consideration should be given to the various factors affecting the DNA damage level. Hence, in order to exclude bias due to the “confounding factors” of lifestyle we have enrolled only carefully pre-selected subjects in our study.
2. Materials and methods 2.1. Selection of subjects The study was carried out in 34 laboratories of clinical analyses in two Italian hospitals. All individuals provided written informed consent according to the law in force. All subjects involved received a letter concerning the aims of the research study, in which they were informed that the results would be published and that they would not be receiving any personal results. They were personally interviewed by filling in two different questionnaires: (a) the “personal health questionnaire” by Carrano and Natarajan [10] for the evaluation of “lifestyle confounding factors”, and (b) a specific questionnaire on the individual professional exposure (kind of laboratory, methodologies, chemical substances utilized, number of determinations per week, waste disposal methods) [11]. We excluded the following from the investigation: smokers, alcohol drinkers, subjects who had been exposed to radiation within the last 6 months or with recent viral or bacterial infections, subjects recently taking drugs or having vaccinations, individuals with known genetic defects in the family or major illnesses, and subjects aged over 50 years. Only 50 exposed subjects (15 males, 35 females; mean age 37 years) out of the 300 clinical laboratory workers interviewed for the study met all the above criteria and were included in this study. The same selection criteria were used for the control group. A total of 53 controls (23 males, 30 females; mean age 33 years) were selected from usual blood donors. Both exposed and control subjects live in urban areas in the middle and the south of Italy. So they are probably exposed to the same amount and type of pollution. 2.2. Exposure assessment Individual exposure was assessed according to the analytical methods used in the laboratory and to the quantity and type of chemical substance. Table 1 shows the distribution of laboratory workers among the different laboratories, the number of annual determinations and the length of service. Overall mean length of service was 10.62 ± 7.15 years and the standard working time was 36 h per week.
A. Testa et al. / Mutation Research 520 (2002) 73–82 Table 1 Distribution of exposed subjects among different laboratories Laboratory
Number of subjects
Number of annual determinations
Mean years/ work (±S.D.)
Microbiology
11
48,700
7.9 (±8.4)
Clinical chemistry Immunology Microbiology
9
1,076
14.7 (±6.1)
Hystology
3
17,500
8.0 (±5.3)
Clinical chemistry Immunology
12
367,566
9.5 (±6.0)
Immunology Hystology Molecular biology
4
4,850
9.5 (±5.4)
Clinical chemistry Hystology
3
262,350
Various
8
–
9.7 (±11.5) 12.3 (±8.6)
Briefly, about 300 different chemical substances were identified. Of these, according to the European Community Classification of Chemicals, 40% are the corrosive, 28% toxic if inhaled or ingested, 12% flammable and 4% carcinogens. The overall quantity of each reagent used in the different laboratories was determined. However, a quantitative definition of individual exposure was hindered by varying factors including the equipment available (manual or automatic), the working environment, and waste storage methods. 2.3. Lymphocyte cultures Venous blood was drawn into heparinized tubes. Lymphocyte cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI 1640 medium (Flow) supplemented with 10% heat-inactivated fetal calf serum (Gibco, Gaithersburg, MD, USA), 2% phytohaemagglutinin (Murex), 1.5% penicillin–streptomycin (5000 IU/ml–5000 g/ml, respectively (Sigma) and 1% l-glutamine (Sigma). Cultures were grown at 37 ◦ C. Slides were analyzed blind by two different researchers who scored the same number of cells for each subject. For the chromosome aberrations (CA) assay, cultures were fixed at 48 h according to the standard
75
methods following a 90 min treatment with 0.2 g/ml colcemid (Sigma). Air-dried metaphase spreads were stained by the conventional unbanded Giemsa method. A total of 100 well-spread metaphases containing 46 ± 1 centromeres for workers and 200 for controls were examined on coded slides. Chromosome- and chromatid-type aberrations were recorded. For the micronucleus (MN) study, cultures were incubated at 37 ◦ C for 72 h and, 44 h from the beginning, cytochalasin-B (Cyt-B, Sigma) at a final concentration of 6 g/ml was added to arrest cytokinesis. Air-dried preparations were stained by the conventional Giemsa method. The presence of MN was evaluated by scoring a total of 1000 binucleated cells (BN) with well-preserved cytoplasm for each donor. In addition, 1000 lymphocytes were scored to evaluate the percentage of cells with 1–4 or more nuclei. The nuclear division index (NDI) and the cytokinesis block proliferation index (CBPI) were used for measuring cell proliferation kinetics. 2.4. Statistical analysis The distribution of all parameters evaluated by the Kolmogoroff–Smirnoff test showed significant departures from the normal distribution. Therefore, both CA and MN data were analyzed by the Mann–Whitney U-test (significance taken as P < 0.05). To estimate the existence of any relationship between CA and MN frequencies and the variables age and sex, the Spearman rank correlation test was used. The same test was also utilized to evaluate the correlation between the two different cytogenetic assays. The data were analyzed using the STATSOFT package.
3. Results Tables 2 and 3 summarize chromosome aberrations and micronucleus frequencies in female and male workers, respectively. Tables 4 and 5 show the data obtained from the control subjects. The NDI and the CBPI individual values are also reported in the tables. The exposed group shows a significantly higher frequency of genetic damage than the control group. As shown in Table 6, chromatid and chromosome aberration frequencies in workers
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Table 2 Chromosome aberration and micronucleus frequencies in female clinical laboratory workers Subjects
Age
Chromosome aberrations Del
Dic + CR
Exchange
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 E27 E28 E29 E30 E31 E32 E33 E34 E35
30 31 35 35 33 38 43 34 40 42 39 31 34 40 40 25 27 37 41 29 36 31 32 31 26 27 34 48 48 44 43 40 37 46 34
0 2 1 1 0 0 1 0 1 1 0 1 2 0 1 0 2 0 1 0 2 2 0 2 1 0 0 0 0 1 2 3 1 1 2
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
35
36
31
3
0
Mean ±S.D.
CsA (%) 0 2 1 1 0 0 1 0 1 1 1 1 2 0 1 0 2 0 1 0 2 2 0 2 2 0 0 0 0 1 3 3 1 1 2
Chromatid aberrations Del
Exchange
2 3 1 1 1 0 4 1 1 2 1 1 2 0 1 0 1 1 0 2 2 3 0 1 0 3 2 3 2 2 2 1 2 1 2
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
51
0
0.97 0.92
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
2 3 1 1 1 0 4 1 1 2 1 1 2 0 1 0 1 1 0 2 2 3 0 1 0 3 2 3 2 2 2 1 2 1 2
2 5 2 2 1 0 5 1 2 3 2 2 4 0 2 0 3 1 1 2 4 5 0 3 2 3 2 3 2 3 5 4 3 2 4
11 10 10 24 17 15 17 13 16 13 9 4 4 10 7 4 4 4 6 5 3 12 6 3 4 6 5 8 8 5 7 9 4 10 8
11 10 10 27 17 17 17 15 16 13 9 4 4 10 7 4 4 4 6 5 3 15 7 3 4 6 5 8 8 5 8 10 4 10 8
1.6 1.8 1.7 1.6 1.7 1.8 1.8 1.8 1.8 1.8 1.8 1.9 1.6 1.6 2.0 1.9 1.8 1.9 2.3 1.6 1.8 1.6 1.6 1.8 1.7 1.8 1.8 1.7 1.8 1.7 1.2 1.2 1.9 1.9 1.7
1.6 1.7 1.6 1.6 1.6 1.7 1.7 1.7 1.7 1.7 1.7 1.8 1.6 1.6 1.9 1.7 1.7 1.7 2.2 1.5 1.7 1.5 1.6 1.7 1.6 1.7 1.7 1.6 1.7 1.7 1.2 1.2 1.8 1.7 1.7
1.46 1.01
2.43 1.46
1.7 0.2
1.7 0.1
8.6 4.89
8.9 5.38
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation.
appear significantly higher than in controls. Similarly, comparison between micronucleated cell rates of the exposed and unexposed group shows significantly higher frequencies of binucleated cells with micronucleus (BNMN) and of total micronuclei (MN tot) in workers than in controls.
As far as the correlation between sex and chromosome aberration frequency is concerned, no statistically significant differences were found between males and females in both the groups. A slightly significant increase (P = 0.012) in micronucleus rates was found in the female control group.
A. Testa et al. / Mutation Research 520 (2002) 73–82
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Table 3 Chromosome aberration and micronucleus frequencies in male clinical laboratory workers Subjects
Age
Chromosome aberrations Dic + CR
Exchange
2 1 2 0 1 1 3 4 0 3 0 0 0 1 1
0 0 2 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
19
3
0
Del E36 E37 E38 E39 E40 E41 E42 E43 E44 E45 E46 E47 E48 E49 E50
49 42 33 49 38 43 27 49 38 47 36 35 39 40 42
15 Mean ±S.D.
40.5
CsA (%) 2 1 4 0 1 1 3 5 0 3 0 0 0 1 1
Chromatid aberrations Del
Exchange
3 1 1 1 1 5 3 3 0 3 0 0 2 2 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
25
0
1.47 1.59
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
3 1 1 1 1 5 3 3 0 3 0 0 2 2 0
5 2 5 1 2 6 6 8 0 6 0 0 2 3 1
10 16 9 8 4 4 3 6 4 8 4 4 4 4 14
11 16 9 8 4 4 3 6 4 8 4 4 4 4 16
1.8 1.9 1.8 1.8 1.5 1.8 1.7 1.4 1.7 1.6 1.9 2.1 1.8 1.7 1.3
1.7 1.8 1.8 1.7 1.6 1.7 1.6 1.4 1.6 1.5 1.8 2.0 1.7 1.7 1.3
1.67 1.49
3.13 2.64
1.7 0.2
1.7 0.2
6.8 4.00
7.0 4.34
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation.
Our results also indicate an effect of age on micronucleus frequency. As can be observed in Fig. 1, there is a significant positive correlation (P < 0.05) between micronucleus frequency and age in both the groups with similar slopes but the occurrence of BNMN is higher in the exposed group than in controls. No statistical differences in NDI and CBPI values were found between groups. As far as the correlation between different cytogenetic parameters is concerned, a positive correlation was found between chromosome and chromatid aberration frequencies both in the exposed group (P = 0.006, r = 0.4) and in the control group (P = 0.009, r = 0.4). No correlation was found between the micronucleus test and the chromosome aberration assay.
4. Discussion Workers in biomedical laboratories are potentially exposed to numerous occupational hazards: chemicals, radioactive materials and biological agents. The exposure levels are generally low and the different
sensitivity of analytical methods, for the different sources of exposure, precludes a precise dose assessment. Moreover, because of the great variability between laboratories regarding analytical techniques and the quantity of chemicals used for the same kind of analysis, biomedical laboratories cannot be considered as a single unit. As far as epidemiological data are concerned, no large studies have been undertaken for people handling chemicals in clinical and research biomedical laboratories. Some of these data are indicative, although not yet conclusive, for increased mortality risk due to malignant lymphoma, leukemia and cancer of the gastrointestinal tract among laboratory workers [12–14]. A cluster of cancer cases, especially sarcomas and lymphomas, was detected among subjects engaged in molecular biology, genetics, and mutagenicity testing in the Pasteur Institute in Paris [15]. Another investigation on laboratory workers carried out between 1971 and 1980 in England and Wales reported significantly increased risk for cancer of the brain, stomach and nervous system and a decreased risk for cancer of the cervix [12].
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Table 4 Chromosome aberrations and micronucleus frequencies in female control subjects Subjects
Age
Chromosome aberrations Del
Dic + CR
Exchange
6 8 10 11 17 18 19 20 21 22 24 27 28 30 34 36 37 39 40 43 44 45 46 47 48 49 50 51 52 53
25 28 30 32 24 24 29 31 37 40 26 33 29 26 31 34 41 45 46 25 31 43 43 25 25 30 26 29 25 30
1 2 1 2 0 1 0 1 0 0 0 0 0 0 0 0 3 0 1 2 0 1 2 1 1 0 1 1 0 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0
30
31
22
1
2
Mean ±S.D.
CsA (%) 0.5 1 0.5 1 0 0.5 0 0.5 0 0 0.5 0 0 0 0 0 2 0 0.5 1 0 0.5 1.5 0.5 0.5 0 0.5 0.5 0 0.5
Chromatid aberrations Del
Exchange
2 1 1 2 1 3 0 2 0 0 0 2 2 1 2 1 1 0 1 3 0 1 3 1 0 2 1 2 2 4
0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
41
1
0.42 0.41
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
1 0.5 0.5 1 0.5 1.5 0 1 0 0 0 1 1 0.5 1 0.5 0.5 0 0.5 1.5 0 0.5 1.5 0.5 0 1 0.5 1 1 2
1.5 1.5 1 2 0.5 2 0 1.5 0 0 0.5 1 1 0.5 1 0.5 2.5 0 1 2.5 0 1 3 1 0.5 1 1 1.5 1 2.5
6 3 3 6 3 2 2 4 4 5 4 4 3 4 1 4 9 17 3 6 6 7 7 2 3 4 4 5 4 5
6 3 3 6 3 2 2 4 4 5 4 4 3 4 1 4 12 20 4 7 7 7 7 3 3 4 4 5 4 5
1.7 1.7 1.8 1.8 1.8 1.8 1.8 1.8 1.8 2.2 1.9 1.7 2.2 1.6 1.9 1.7 1.6 1.7 2.0 1.6 1.8 1.3 1.7 2.1 1.4 2.0 2.0 2.1 1.9 1.7
1.6 1.7 1.7 1.75 1.7 1.7 1.7 1.7 1.7 1.9 1.8 1.6 1.9 1.5 1.7 1.6 1.6 1.6 1.9 1.5 1.7 1.3 1.6 1.9 1.2 1.9 1.8 1.1 1.8 1.6
0.68 0.53
1.1 0.82
1.8 0.2
1.7 0.2
4.7 3.0
5 3.5
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation.
Subsequently, a mortality study on workers employed by the research staff of the Italian National Institute of Health revealed excess of brain, pancreatic, lymphohematopoietic and breast cancers [16]. Finally, a recent review on cancer risk assessment among employees in biological, medical and agronomic research laboratories suggests a low overall risk of cancer albeit that a higher risk may be suggested for cancers of pancreas, brain and for non-Hodgkin’s lymphoma [17].
In this study, a group of strictly selected clinical laboratory workers and a comparable control group were monitored for chromosome aberrations and micronuclei. Our results show that the group of workers under study exposed to chronic low levels of chemical toxicants has a highly significant increase of genetic damage in terms of chromatid and chromosome aberration and micronucleus frequencies compared with the control group. Besides the elevated frequency of chromatid aberrations usually related to exposure to
A. Testa et al. / Mutation Research 520 (2002) 73–82
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Table 5 Chromosome aberration and micronucleus frequencies in male control subjects Subjects
Age
Chromosome aberrations Del
Dic + CR
Exchange
1 2 3 4 5 7 9 12 13 14 15 16 23 25 26 29 31 32 33 35 38 41 42
49 41 43 25 36 38 36 50 32 32 28 31 34 35 32 30 29 31 25 24 31 39 26
2 0 0 0 0 0 2 1 1 3 0 1 0 0 0 0 1 0 1 0 0 1 1
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0
23
34
14
1
1
CsA (%) 1 0 0 0 0.5 0 1 0.5 0.5 1.5 0 0.5 0 0 0 0.5 1 0 0.5 0 0 0.5 0.5
Mean ±S.D.
Chromatid aberrations Del
Exchange
3 1 0 2 2 0 6 1 1 4 1 0 0 1 1 0 3 2 0 0 0 1 4
0 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0
33
2
0.37 0.43
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
CBPI
1.5 0.5 0 1 1.5 0 3 0.5 0.5 2 0.5 0 0 1 0.5 0 1.5 1 0 0 0 0.5 2
2.5 0.5 0 1 2 0 4 1 1 3.5 0.5 0.5 0 1 0.5 0.5 2.5 1 0.5 0 0 1 2.5
1 3 4 2 2 4 4 5 3 3 2 2 4 3 3 3 6 4 2 1 3 2 3
1 3 4 2 2 4 4 5 3 3 3 2 4 4 3 3 6 4 2 1 3 2 3
1.9 1.8 1.7 1.9 1.9 1.8 1.8 1.7 1.7 1.8 1.7 1.8 1.8 1.9 1.8 1.7 1.5 1.7 1.6 1.6 2.0 1.7 1.6
1.8 1.7 1.6 1.8 1.8 1.7 1.7 1.6 1.7 1.7 1.7 1.7 1.9 18 17 16 1.4 1.6 1.5 1.6 1.8 1.6 1.5
0.76 0.82
1.13 1.15
3 1.2
3.1 1.2
1.7 0.1
1.7 0.1
Del = deletions (breaks); Dic = dicentrics; CR = centric ring; CsA = chromosome aberrations; CtA = chromatid aberrations; CA tot = total of chromosome aberrations; BNMN = binucleated cells with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis block proliferation index; ±S.D. = standard deviation. Table 6 Comparison of chromosome aberration and micronucleus frequencies between exposed and control subjects Groups
Number of CsA (%) subjects
CtA (%)
CA tot (%)
BNMN (‰)
MN tot (‰)
NDI
0.75 (±0,46) 0.68 (±0.53) 0.76 (±0.82)
1.14 (±0.95) 1.1 0 (±0.82) 1.13 (±1.15)
3.94 (±2.45) 4.7 (±3.0) 3.0 (±1.2)
4.0 (±2.9) 5.0 (±3.5) 3.1 (±1.2)
1.77 (±0.17) 1.70 (±0.14) 1.79 (±0.2) 1.68 (±0.2) 1.70 (±0.1) 1.68 (±0.1)
Controls All 53 Females 30 Males 23
0.41 (±0.46) 0.42 (±0.41) 0.37 (±0.43)
Exposed All 50 Females 35 Males 15
1.12∗∗ (±1.17) 1.52∗∗ (±1.16) 2.68∗∗∗ (±1.89) 8.06∗∗∗ (±4.68) 8.38∗∗∗ (±5.1) 1.74 (±0.2) 0.97∗ (±0.92) 1.46∗∗ (±1.01) 2.43∗∗ (±1.46) 8.6∗∗∗ (±4.89) 8.9∗∗ (±5.38) 1.7 (±0.2) 3.13∗ (±2.64) 6.8∗∗∗ (±4.0) 7.0∗∗ (±4.3) 1.72 (±0.2) 1.47∗ (±1.59) 1.67 (±1.49)
CBPI
1.67 (±0.2) 1.7 (±0.1) 1.7 (±0.2)
CsA = chromosome aberration; CtA = chromatid aberration; CA tot = total of chromosome aberrations; BNMN = binucleated cell with micronucleus; MN tot = total of micronuclei; NDI = nuclear division index; CBPI = cytokinesis-block proliferation index; ±S.D. = standard deviation. ∗ P< 0.05. ∗∗ P < 0.001. ∗∗∗ P < 0.0001.
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Fig. 1. Relationship between BNMN frequency and age in exposed and control subjects.
chemical agents, the results show also severe chromosome damages. Particular attention should be paid to this finding because it has been demonstrated that chromosome aberration frequency is itself an indicator of cancer risk rather than just a reflection of exposure, and that the study of aberration levels may directly predict an increased cancer risk [18–20]. In these studies, the authors trichotomized the levels of CA found in large populations. They found a doubling of mortality for all cancers in subjects with a frequency of chromosome aberrations corresponding to the upper thirtile. Our results on chromosome aberration frequencies found in laboratory workers suggest that they could have an increased risk for cancer. As far as the micronucleus data are concerned, workers show very highly statistically increased values compared with the controls. A positive correlation between micronucleus frequency and age in both sexes with a steeper linear regression line for females (data not shown) was also found, confirming other literature data [21]. Even if the relationship of cancer risk with micronuclei is not
as well substantiated as that with chromosome aberration, this method has proven useful as a “biological marker of early effects” in biomonitoring studies on human population exposed to genotoxic agents. The lack of correlation between the two tests is possibly due to the fact that both chromosome loss and chromosome breakage contribute to micronucleus formation suggesting that aneuploidy may be induced by this kind of exposure. The initial hypothesis of a very low-dose exposure for subjects employed in clinical laboratory analysis suggested us to perform a very strict selection of biological, environmental or lifestyle factors affecting DNA damage levels (confounding factors) for subjects involved in this study. In this way, we increased the resolution of the methods used and obtained a more accurate risk assessment. The background of chromosomal aberration and micronucleus frequencies found in our control subjects is very low in respect of the corresponding values reported by other authors [22–25]. This finding allow us to think that normal
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healthy individuals are exposed to a variety of such agents as part of their daily life and that the exclusion of these do result in a decrease in genetic damage. In conclusion, the increased chromosome aberrations and micronucleus frequencies found in workers indicate potential genetic hazards. The causative agents could be specific chemicals or a complex mixture of chemicals in the laboratory environment. Unfortunately, very little information is available on adverse human health effects of chemical mixtures [26]. This observation shows the need to increase occupational preventive programs and to improve supervision of the working environment in clinical laboratories.
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