Mutagenesis Advance Access originally published online on April 7, 2005
Mutagenesis 2005 20(3):187-191; doi:10.1093/mutage/gei024
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Incidence of cytogenetic damage in lead–zinc mine workers exposed to radon
Institute of Occupational Safety, SI 1000 Ljubljana, Chengdujska 25, Slovenia and 1Institute of Oncology, SI 1000 Ljubljana, Zalo
ka 7, Slovenia
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Abstract |
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The purpose of this study was to detect cytogenetic damage in mine workers working in a lead–zinc mine, which could be associated with a combined exposure to radon and heavy metals. Our study involved 70 mine workers from the lead–zinc mine. We used peripheral blood lymphocytes as the target material. The total share of structural chromosome aberration (SCA) decreased significantly over the 3 years of monitoring, from 5.08/200 analyses of metaphases in 1995 to 3.28 in 1997, owing to the decrease in exposure during the process of mine closure. The share of SCA was significantly different from the group of local people, who had never worked in the mine (1.43), as well as from the control group of Slovene residents (1.88). The share of micronuclei (MN) in mine workers also decreased in the monitored period, from 14.65/500 cytokinesis-blocked cells in 1995 to 11.77 in 1997, while the sister chromatic exchange (SCE) level did not change much (from 8.105/50 analysed cells in 1995 to 7.73 in 1997). Owing to the closure activities, the received concentrations of contaminants were falling constantly, particularly concentrations of radon. This was particularly evident in the level of SCA and the MN incidence, while the SCE values remained nearly on the same level. This indicates that the incidence of SCE is probably more strongly influenced by heavy metals than by radon.
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Introduction |
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Although radon is a physical agent present in the everyday life of living organisms, it is also very potent, causing a great deal of damage to DNA. Radon is a radioactive noble gas which decays via a number of radioactive heavy metal isotopes to the end product stable lead. The decay of radon generates high energy alpha particles, and some of its decay daughter products also generate high energy alpha particles and beta particles as well (218Po, 214Po, 214Pb and 214Bi). These decay products can become attached to airborne particles and inhaled. They undergo further radioactive decay in lungs, the high energy alpha particles penetrating the epithelium cells that line the bronchi and alveoli, which is believed to initiate the process of carcinogenesis. Recent studies have referred to the occupational, environmental and experimental exposure of humans, animals and cell cultures to radon (1
–3). In addition to its mutagenic characteristics, radon is considered by many to be a factor that significantly increases the risk of lung cancer in humans (4).
Comprehensive studies on radon as a mutagenic and carcinogenic agent have been performed in cases of increased concentration in buildings, spas, caves and underground mines (5–8).
The International Agency for Research on Cancer (IARC) has classified inorganic lead compounds as possibly carcinogenic for humans (9). This classification is based on animal studies with stimuli and the development of renal tumours in mice and rats. However, such proof is not available for humans. At the same time, the clastogenic properties of lead have been studied on lymphocytes in occupationally exposed persons. Some investigators have established a positive relationship between chromosomal aberrations and the concentration of lead in the blood, while others have not confirmed such results.
A special problem is represented by the long-term exposure to lead. Such an exposure can lead to anemia, nephritis, mental insufficiency, premature ageing, reduced libido, impotence, menstrual and ovarian cycle disturbances, etc. (10).
There are many unknown factors with respect to the individual effects of trace elements and other chemical genotoxic agents. It is impossible to separate the individual effects of different agents present in the lead–zinc mine, where radon and a cocktail of metals occur in the polluted work environment. Biological effects must therefore be interpreted en bloc.
The research, carried out in the mine and its surroundings for some decades before its closure, was intensified after 1990 by intense monitoring of radon in different parts of the mine and calculating the radiation doses received by the mine workers. Later, this study involved heavy metal biological monitoring, where special emphasis was given to the measurements of concentrations of lead in the blood and to mutagen tests.
The radon concentrations in some areas of the mine were relatively high (from 3000 to 5000 Bq/m3). Throughout the years of monitoring, the mine workers were involved in closure tasks that included a substantially reduced exposure to radon. By measuring radon levels and performing medical examinations, substantially reduced exposure doses of radiation and an important decrease of cytogenetic mutations were established.
Radon and heavy metals induce long-lasting changes in the genomes of living cells. As the most sensitive target of genotoxic agents, DNA can be affected directly or indirectly. The aim of this study was to prepare an analysis over the period of 3-year measurements based on the changes in concentrations of radon and heavy metals in the mine and to establish the effects of the changing working environment (lower levels of radiation) on the DNA of the mine workers.
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Materials and methods |
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The radon level in the mine was continuously measured by means of Sarad EQF-3010 and Sarad EQF-3020 instruments. The method involves the measurement of alpha particles emitted by radon and its decay products. Measurements were performed at different locations in the mine in 1995, 1996 and 1997. Based on the data obtained, the arithmetic mean of the effective equivalent dose for the mine was calculated and according to the hours worked in the mine (some mine workers worked longer hours than the others), the individual effective equivalent doses were estimated.
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The method used for lead measurements was ICP-MS, with measurements carried out on Perkin-Elmer ELAN 6000 apparatus. The exposures to lead were adjusted to daily average exposures. Exposure to zinc was not measured.
Peripheral blood lymphocytes were used as the target material for the cytogenetic methods and blood samples were taken simultaneously for the structural chromosome aberration (SCA), sister chromatic exchange (SCE) and micronuclei (MN) test. Standard 48-h in vitro lymphocyte cultures were used for SCA analysis. A total of 0.3 ml of heparinized whole blood was added to 5 ml of Chromosome med 1A-GIBCO culture mean (Chromosome mean 1A MSDS Number: 31670, Invitrogen Ltd, UK). The first in vitro cell division cycle was established with the addition of 5 µg/ml of BrdU (Sigma); 0.075 M potassium chloride was used for the hypotonic procedure. Fixing was performed in a mixture of glacial acetic acid and methyl alcohol at a ratio of 1:3. The cell suspension was pipetted onto cold glass slides; specimens were air-dried and stained with Giemsa (Sigma). The analyses of the SCA were carried out only with homogeneously coloured metaphases with the full number of chromosomes. The first 200 in vitro metaphases were analysed for each subject. Structural damage to chromosomes was categorized as chromatid breaks, isochromatid-chromosomal breaks, acentric fragments—individual fragments without the possibility of identifying the remainder of the affected chromosome with this method, dicentrics and ring chromosomes (11). Gaps were not included in the total number of chromosomal aberrations.
SCE tests were performed on the same culture mean as the SCA test. Lymphocytes cultured for 72 h were added to 10 µg/ml BrdU and prepared under dark conditions. The procedure used was according to Kato (12). Fifty cells per subject were analysed, SCEs were counted and presented as mean number per cell. The range of SCE frequencies was also recorded for every subject.
For the MN test, 3 µg/ml of cytochalasin B (Sigma) was added to each in vitro lymphocyte culture in the 43-h of cultivation. The Fenech–Morley method was used (13). The hypotonic procedure was omitted, and specimens were stained with May-Grünwald-Giemsa. Cells with clearly blocked cytokinesis (CB cells), i.e. binuclear cells, were analysed. Five hundred cells per person were examined. The results were presented as the number of micronuclei (MN) per 500 CB cells.
The study involved 70 mine workers from the lead–zinc mine during the process of closure (the mine was closed in 1998), 38 of them being smokers. Most of the mine workers participated in all 3 years of studies (1995, 1996 and 1997). Their average age was 38.73 years (SD = 5.71), age range 28–47 years. They had worked in the mine between 9 and 28 years. They all worked on closure tasks in the mine and were exposed to ionizing radiation. The maximum dose received in 1995 was 37.6 mSv, and the average LEED (annual effective dose for the same year) was 14.1 mSv (14). In the closure period the estimated acquired doses were lower and some workers did not work in the mine all the time.
The test results were compared with two control groups. One was the group of local residents, who were not occupationally exposed to genotoxins, but were exposed to the polluted environment outside the mine (e.g. Pb in the soil from 131 to 2250 µg/g—alimentary chain) (15). The other group represents Slovene residents living outside the wider mine area and corresponding to the group of mine workers in all the other parameters. Both control groups consisted of males in the same age group as the mine workers and all were chosen randomly from the population.
Data were analysed by using standard methods of parametric statistics. The differences between the mean values for individual groups were tested using the T-test (analysis of the difference between mean values), with Pearson correlation coefficient and the time differences featuring trends with the SPSS computer program.
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Results |
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After measuring the concentration of radon and calculating the local doses, the mean of all the calculated doses in the mine amounted to 14.103 ± 10.509 mSv in 1995. The highest measured concentration of radon was 3563 Bq/m3. In 1996 and 1997, the values were almost identical—4.494 and 5.828 mSv, approximately one third of the values in 1995. The reason for such a substantial difference between the calculated doses is the fact that workers were spending more time in areas of higher concentration in 1995 than in 1996 or 1997.
The concentrations of blood lead in the group of mine workers amounted to an average of 280 µg/l. However, in 16% of the subjects the blood lead level was more than the permissible value of 400 µg/l, according to the World Health Organization recommendation of 1980 (WHO-TRS 647). The lowest measured value for mine workers was 37 µg/l and the highest was 800 µg/l. At the same time, the mean value in the control group of local population amounted to only 72 µg/l blood lead.
In 1995, 1996 and 1997, 67, 63 and 66 examinations of SCA in mine workers carrying out closure tasks were performed. We were very interested in the impact of the decreasing dose the workers were exposed to during the monitored period (Table I).
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The mean value of chromatid breaks was 3.8 (±1.89). In comparison with the lower dose received in 1996 and 1997, this number was considerably lower than in 1995; however, the differences of mean values were not statistically significant (Table I).
The share of chromosome breaks was 
30% lower compared with the chromatid ones. The average share of chromosome breaks decreased with the reduced received dose, more precisely from 3.38 in 1995 to 1.87 in 1997 (Table I).
The average share of acentrics was also reduced along with the decreasing dose, and with it, the share of the persons examined, where acentrics were not established at all. In 1995 there were eight cases without acentrics, and in 1997 there were twenty-two. The average share of acentrics in 1995 was 2.4 ± 2.02 and in 1997, only 0.924 ± 0.79. The differences between the years of monitoring were statistically significant (Table I).
The number of people with established dicentrics decreased with time and the difference in the average number of ascertained dicentrics was not statistically significant. Rings were found only in the first year of monitoring (Table I).
The total share of SCA also decreased from 5.08 ± 2.10 in 1995 to 3.28 ± 0.82 in 1997. The average value was 4.27 ± 0.17, and the difference between the years was statistically significant.
The share of SCA in the mine workers was statistically significantly different from the local control group, as well as from the group of Slovene residents. The average SCA share of the local group was 1.43 ± 0.59 and 1.88 ± 1.06 in the group of Slovene residents. The difference of mean values was statistically significant (16,17) (Table I).
The share of MN in mine workers also decreased in the monitored period from 14.65 (min 9, max 23) in 1995 to 11.77 (min 7, max 21) in 1997; and the difference of mean values was statistically significant (Table II). However, the SCE level did not change considerably (from 8.05 in 1995 to 7.73 in 1997); the difference of mean values was statistically significant (Table II).
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A comparison of the above given values to the values measured in the local control group, exposed exclusively to heavy metals from the alimentary chain and to some higher values of radon in their residences in the vicinity of the mine (Tables III, IV and V) (the external radiation dose in the region of the mine in 1995 was 994 µSv/year, while the Slovene average is 856 µSv/year) lead us to believe that the level of exposure to radiation is higher in the vicinity of the mine than in other parts of Slovenia (18
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The difference in the SCA share between smokers (38) and non-smokers (29) in 1995 was not statistically significant, contrary to the shares of SCE and MN, where the difference is significant (Table VI).
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Discussion |
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The concentrations of lead and other heavy metals at the surface of the mine were reduced due to the redevelopment and closure of the mine. The mine workers involved in closure operations performed only the most urgent tasks (disassembly of the equipment, etc.), while most activities were carried out on higher levels of the mine (where radon concentrations were fundamentally lower) or outside the cave (where they were exposed just to the concentration of the natural background of the mine area). Owing to the reduced exposure, particularly to radon, some ‘corrections’ were established regarding the cytogenetic level. This is particularly evident in the case of the level of SCA and the MN incidence, while the SCE values remained practically on the same level (16
). This suggests that the SCE incidence may be more strongly influenced by chemical contamination by heavy metals than by radon. Three validated biological ends, i.e. SCA, SCE and MN, were used. These endpoints depend on different origins of primary DNA damage. Results of the analyses demonstrated that the values of both control groups (Table II) were almost identical and did not deviate from the data of the general population. The values of all mutagenicity tests in the mine workers were increased, and although the deviations were (on average) not excessive, they were statistically significant. Chromatid breaks prevailed among the chromosomal aberrations and should be considered as S-dependent events (DNA synthesis). The presence of isochromatid breaks in significantly higher numbers than in the control samples, and the presence of dislocated acentric fragments and unstable aberrations, indicates G1 damage, i.e. in vivo exposure.
The average 1-year irradiation dose was calculated to be 9.091 mSv. By analysing the relationship between the irradiation dose and the percentage of total chromosomal aberrations, an evident trend of dependence can be established. A weak correlation between chromosomal aberrations and years of employment in the mine was established, indicating that irradiation doses in individual parts of the mine are different and that there are differences with respect to contamination with other agents.
SCE can be considered a marker of high-LET radiation damage, but not of low-LET radiation (19,20). The incidence of SCE in this study was significantly higher in mine workers than in the control groups, although the dose in mSv and the number of SCE did not show a trend of dependence.
We believe that the comparison of the results of the mine workers and the local control group suggests an association between cytogenetic aberrations and the work environment. Increased frequency of genome damage was not found in the control group of local residents, indicating that metals did not act as clastogens at the level of current environmental pollution.
The Cox regression models, which accounted for the age at the time of first cytogenetic assay, radon exposure and smoking, showed strong and statistically significant associations between the incidence of cancer and frequency of chromatid breaks and frequency of aberrant cells, respectively. A 1% increase in the frequency of aberrant cells was paralleled by a 62% increase in the risk of cancer (P < 0.001). An increase in the frequency of chromatid breaks by 1/100 cells was followed by a 99% increase in the risk of cancer (P < 0.001). Similar results were obtained when analysing the incidence of lung cancer and the incidence of cancers other than lung cancer separately (21,22).
Smoking is obviously an additional factor that can provoke changes in the genome cells. In mine workers who work full time in an environment contaminated by heavy metals and radon, smoking is a pathway through which pollutants can enter the organism. Tobacco smoke has a synergistic effect in combination with radon exposure (23). Estimates are that the increased risk of lung cancer to smokers from radon exposure is 10–20 times higher than to people who have never smoked. However, 20% of the lifetime risk of lung cancer in non-smokers may be associated with radon exposure (11). Smoking increases the possibility of adherence of a greater concentration of radon into the lung with particles of smoke and resin, which, with the primary toxicity of the cigarette components, can cause separate cytogenetic alterations in the lymphocytes.
It is difficult to determine reliably the actual share of smoking as a genotoxic factor in this cocktail of physical and chemical agents. The result of cytogenetic damage in the case of mine workers cannot, therefore, be discussed as a causal connection with either irradiation, smoking or separately with heavy metals. We believe that in this case, the overall work environment and cytogenetic damage are in a cause–effect relationship.
The results of this research lead us to the conclusion that working under exposure to radon and heavy metals can result in dramatic consequences for the human DNA, which, if damaged, is believed to be a significant factor in the development of different forms of cancer.
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Notes |
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* To whom reprint requests should be addressed. Tel: +386 01 43 20 253; Fax: +386 01 23 12 562; Email: marjan.bilban{at}zvd.si
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Received on September 18, 2003; revised on December 13, 2004; accepted on March 10, 2005.
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