Chromosomal aberrations and micronuclei in lymphocytes of workers at a phosphate fertilizer factory

Ziqiang Meng, Bo Zhang

Division of Environmental Biological Toxicology, Department of Life Sciences, Shanxi University, Taiyuan 030006, China

Abstract

The frequencies of chromosomal aberrations (CA) and micronuclei (MN) in peripheral blood lymphocytes of 40 workers at a phosphate fertilizer factory in North China, were studied. HF and SiF4 are the main air pollutants and small amounts of dust containing fluoride, NH3 and S02 were also present in the factory. It was shown that the chemicals caused an increase in both CA and MN. The mean frequencies per 100 metaphase of major CA type (chromosome rings, translocations, and dicentrics) of the workers and the non-exposed controls were 0.91 and 0.24 (p < 0.01), respectively. The average percentages of lymphocytes with MN of the workers and the controls were 1.55 ± 0.71 and 0.62 + 0.54 (p < 0.01), respectively. Both CA frequency and MN frequency of the workers increased with length of the chemical exposure period up to 10 years.

Keywords: Fluoride; Chromosomal aberration; Micronuclei; Human lymphocyte

1. Introduction

Fluoride is a ubiquitous substance found naturally in food and water, and it is also a common air pollutant in some industrial productions. A number of studies of the genotoxic effects of fluoride have been conducted in a variety of systems, including plants, bacteria, fruit flies, animals, and also human cells. However, the results on this topic have been conflicting. Some studies have reported that fluoride is a mutagenic agent and causes chromosomal damage, but other investigations have showed fluoride does not produce genotoxic effects [1- 14].

To examine possible damage to the genetic apparatus at chromosomal level caused by exposure of workers to air pollution in a phosphate fertilizer factory, in which HF and SiF4 are the main air pollutants, we analyzed the peripheral blood lymphocytes of the workers for chromosomal aberrations (CA) and micronuclei (MN). The present studies provide evidence that chronic exposure of workers to the air pollutants in the factory is associated with increased CA and MN in their blood lymphocytes.

2. Materials and methods

2. 1. Chemical pollutants

In the air of the workplace of the phosphate fertilizer factory, fluorine (F), sulfur dioxide (S02), ammonia (NH3), and floating dust (particle diameter: < 10 um) were analyzed with the selecting electrode method of fluorine ions, pararosaniline hydrochloride spectrophotometry, sodium hypochlorate-salicylate spectrophotometry, and the weight method, respectively [15]. The concentrations of these chemicals varied irregularly over one year in this workplace, the range for F concentrations was from 0.50 to 0.80 mg/m3 air at the time of investigation, for S02 was from 0.02 to 0.07 mg/m3, for NH3 was from 0.01 to 0.05 mg/m3, for the floating dust was from 0.05 to 0.20 mg/m3. These data indicated that the air pollutant in the workplace was mainly fluorine (HF and SiF4).

2.2. Subjects

The subjects were 40 workers exposed mainly to fluoride (HF, and SiF4) within the same workplace of the phosphate fertilizer factory, and 40 controls, working and studying in Shanxi University, situated in the same city as the factory, matched according to sex, age and smoking habits. It was difficult to find enough people in the factory who were not exposed to fluoride or other chemicals as a control population, so university staff in the same city were chosen as controls. The income levels of the workers and the university staff controls were generally similar at the time of investigation, but data on the socio-economical levels and nutritional status of the subjects were not collected and analyzed. All subjects were interviewed about recent viral infections, vaccinations, previous occupational exposure to chemicals, drug intake and alcohol consumption. No difference in these respects could be found between the worker and the control group. Occupational exposure to the chemicals had not caused clinical symptoms in workers and all controls were also healthy.

2.3. Cell culture

Venous blood was drawn into heparinized tubes and the samples coded and cultures established the same day according to the technique of Hungerford [16] with minor modifications. To culture lymphocytes whole blood (0.2 ml) was added to 4.8 ml of RPMI1640 medium containing 20% new calf serum, 100 units/ml penicillin, 100 ug/ml streptomycin and 2% phytohemagglutinin M (PHA). The blood was cultured at 37'C in 5% C02. For CA analysis, the cells were cultured for 48 h. For the MN assay, the cells were cultured for 72 h, and the cytochalasin B (Sigma Chemical Co., final concentration 3 ug/ml) was added to the cultures 24 h prior to harvesting, according to the method described by Fenech and Morley [17].

2.4. Analysis for CA and MN

For measuring MN frequency, cultures were centrifuged at 80-100 X g for 6-8 min after an incubation period of 72 h. The pelleted cells were treated to preserve the cytoplasm by gently resuspending in 5 ml hypotonic solution of 0.075 M KCI/saline (2:8 v/v) for 3-5 min and then resuspending twice in 5 ml cold Carnoy's fixative (methanol/glacial acetic acid, 3:1 v/v) for 20 min at room temperature. After the final centrifugation (at 100 X g for 8 min) cells were thoroughly mixed using the tip of a Pasteur pipette and were dropped from a height of 3-4 cm onto wet, clean slides. For each culture at least two slides were prepared. The slides were air-dried and stained with 10% Giemsa for approximately 10 min.

For CA analysis, the cultures were processed in the same way except that 0.1 ml of colcemid (final concentration: 2.7 x 10-5 M) was added to each culture for mitotic arrest 4 h before harvest and the hypotonic treatment was performed with prewarmed 0.075 M KCI for 15 min at 37'C. The air-dried slides were stained with 10% Giemsa.

2.5. Slide scoring

For cytogenetic analysis, preparations were coded and scored blind. Scoring of MN was limited to binucleate lymphocytes only with preserved cytoplasm [17], according to the criteria proposed by Countryman and Heddle [18]. Identification of binucleate cells in cell groups required careful visual examination of the individual cell boundaries. The results are expressed as the average percentage of micronucleated cells per 2000 binucleate cells on the two different slides from the same culture.

CA were analyzed in 200 metaphase cells for each person. Only cells with 46 chromosomes were included in the analysis. Cells analyzed for chromatid and isochromatid breaks and other types of aberrations [19].

2.6. Statistical analysis

The x2-test was used for the CA and the MN frequencies.

3. Results

The average percentages of chromosome-type, chromatid-type aberrations and their total were all significantly higher (p < 0.01) in workers than in controls (Table 1). Dicentrics, centric rings, acentric fragments and chromatid breaks were the types of aberrations that showed a significant increase (Table 2).

Table 2 also indicates that the frequencies of major chromosomal aberrations (including rings, translocations, and dicentrics) in workers and in controls were 0.91% and 0.24%, respectively. The difference between them was statistically significant (P < 0.01).

Fig. 1 shows the frequency distribution of lymphocytes with MN among the workers; 31.0% of the controls were found not to have lymphocytes with MN. Frequencies of cells with MN in 52.4% controls were in the range 0.10 to 1.00%, only 16.6% of the controls were in the range 1.00 to 2.00%, and none was over 2.00%. However, all subjects in the worker group had cells carrying MN; 75% of workers were over 1.00% and 17.50% were higher than 2.00%, these differences between the workers and the controls were statistically significant (p < 0.01).

Analysis of the correlation between the individual results for the micronucleus index and cells with aberrations indicated that the correlation factor, R, was 0.426 (p < 0.01). It implies that there is a positive correlation between MN and CA in the worker group.

There was correlation between the mean frequency of lymphocytes with CA or the mean frequency of lymphocytes with MN and length of service for which workers were exposed to the air pollutants at the factory (Table 4). Both the frequency of the cells with CA and the frequency of the cells with MN increased with length of pollutant exposure period up to 10 years. However, both frequencies begin to decrease after about 10 years of pollutant exposure period.

4. Discussion

Fluorine is a necessary biological trace element for human health [12]. However, some animal experiments have indicated that fluoride is tumorigenic, that it increases tumor growth rate and interferes with DNA repair in vitro and in vivo [14,20]. Yiamouyiannis and Burk [20] reported an increase in human cancer death rate in fluoridated areas. Studies of the relationship of non-waterborne fluorides and cancer have reported positive correlations between food fluoride levels and stomach cancer [21] and a possible correlation between airborne fluorides and lung cancer [22,23]. Some studies indicate that NaF induces chromosome aberrations in cultured human blood lymphocytes in vitro, and in bone marrow cells of mice [4,24]. Our study here provides evidence that the air pollutants at the phosphate fertilizer factory, in which HF and SiF4 are the main chemicals, could induce both CA and MN in human blood lymphocytes in vivo. Our earlier observation on sister-chromatid exchanges (SCE) of peripheral blood lymphocytes from this same population showed that the mean SCEs/cell of the workers was significantly higher than that of the controls (p < 0.01) [13]. The results of our studies imply that even if the concentration of the chemical pollutants in the air is low (e.g. F 0.50-0.80 mg/m 3), it may cause damage to genetic material at the chromosomal level, although the general health of workers in the phosphate fertilizer factory was found to be satisfactory. There is, therefore, a need to improve safety, sanitary conditions, and health surveillance for the high risk group, which had an increased frequency of major CA, SCE, and MN induced by the chemical pollutants. However, in this study data on the socio-economic and nutritional status were not collected. Although the income levels of the factory workers and the university staff controls were generally similar at the time of investigation, the potential impact of these factors were not studied, but one cannot exclude that these factors may have had some impact on the results obtained.

It has been found that fluoride affects enzymatic activities, and this effect could delay mitotic and meiotic cycles causing chromosomal breakages [25]. Such chromosomal aberrations may eventually lead to the formation of structural changes and fragmentation as observed in the present studies. Since it has been shown that fluoride can inhibit nucleic acid synthesis [26], it is suggested that chromosomal abnormalities induced by fluoride could be the results from interaction with the enzymes responsible for DNA synthesis or repair, rather than directly with DNA.

The evidence presented here showed the chromosome damage rate declines in those who have worked for more than 10 years in the factory. This might be due to an adaptation mechanism being produced to the chemical pollutants, or due to an equilibrium being reached between chromosome damages, lymphocyte death, and lymphocyte renewal, and/or DNA damage and repair in the cells.

In the phosphate fertilizer factory, HF and SiF4 are the main air pollutants. However, dust containing fluoride, ammonia (NH3), and sulfur dioxide (S02) were also released in small amounts into the air in the fertilizer production, as mentioned in our earlier paper [13]. These pollutants may also contribute to the cytogenetic damage observed [27]. Hence, health studies of workers exposed chronically to HF and SiF4 are needed to understand the meaning of the observed cytogenetic damage in their lymphocytes.

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