On April 6, 1993, near the town of Tomsk (Russia) there was an accident at the Siberian Chemical Plant (SCP

) which resulted in extensive contamination of an area of 250 km2 to the north of SCP with long-lived radionuclides such as 239Pu, 137Cs and 90Sr. Cytogenetic methods and electron spin resonance (ESR) spectrometry of tooth enamel were used to estimate the radiation doses received by the population. The ESR signal intensity and the chromosomal aberration frequency in lymphocytes of the tooth donors showed a good correlation. The data showed that 15% of the inhabitants of the Samus settlement received a radiation dose >90 cGy. The exceptions were results of an examination of fishermen, where ESR gave high values (80–210 cGy) but both the chromosome assay and the cytokinesis block micronucleus method gave lower ones (8–52 cGy). A large increase in chromosome damage was observed in people born between 1961 and 1969. It was found that during these years several serious accidents at the Siberian Chemical Plant had occurred causing radiation pollution of the area. The number of cells with chromosome aberrations was significantly less among the people arriving in Samus after 1980. We found good correlations between the level of carotene consumption and a decrease in frequency of both micronuclei in binucleated lymphocytes (r = 0.68, P < 0.01) and chromatid aberrations (r = 0.61, P < 0.01) among the inhabitants. We also examined the inhabitants of Samus for opisthorchis infection, which was present in 30% of the population. The Samus inhabitants affected by Opisthorchis felineus showed significantly increased levels of micronuclei in binucleated lymphocytes and chromatid aberrations as compared with the controls 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. The PathfinderTM Cellscan system allows the detection of nuclei and micronuclei within cells of a test sample for evaluation of compound toxicity. The presence of micronuclei containing very small quantities of DNA, allows the quantification of induced DNA damages. Rapid and high quality image capture assures accurate and reliable quantitation

on In Vivo and In Vitro Micronuclei Assay Technical Performance: Total time for automated capture and analysis: Between 6 and 15 min per 1000 cells at 20x. cell number) Automated and fast focusing Automated nuclei and micronuclei detection and separation Data analysis and automated display of results as graphics: histograms. In vivo assay The percentage of micronuclei is given within subpopulation of erythorcytes (normochromatophils et polychromatophils).using FDA 21 CFR Part 11 compliant technology with user-friendly and powerful analysis software. The application includes: • • • • • • Streamlined fully automated and time-saving image acquisition Pre-setting for capture on each slide. walk-away capture and analysis On-line determination of stopping criteria (ex. Chromosomal aberrations and micronuclei in lymphocytes of workers at a phosphate fertilizer . Acridine Orange…) or Brightfield Microscopy (Giemsa). scatterplots for all populations Assay types: Fluorescence (DAPI. tri-nucleated or whole population). In vitro assay The percentage of micronuclei is given by the number of micronuclei versus the number of the studied population of nuclei (bi-nucleated.

54 (p < 0.91 and 0.55 ± 0. fruit flies.62 + 0. were studied. Chemical pollutants In the air of the workplace of the phosphate fertilizer factory. Materials and methods 2.14]. Shanxi University. Chromosomal aberration. It was shown that the chemicals caused an increase in both CA and MN. However. translocations. Taiyuan 030006. fluorine (F). 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. 1. Volume 393. The mean frequencies per 100 metaphase of major CA type (chromosome rings. Chromosomal aberrations and micronuclei in lymphocytes of workers at a phosphate fertilizer factory Ziqiang Meng. including plants. respectively. HF and SiF4 are the main air pollutants and small amounts of dust containing fluoride.71 and 0. Both CA frequency and MN frequency of the workers increased with length of the chemical exposure period up to 10 years. Human lymphocyte 1. bacteria. respectively. in which HF and SiF4 are the main air pollutants. Some studies have reported that fluoride is a mutagenic agent and causes chromosomal damage. Keywords: Fluoride. Pages 283-288. and floating dust (particle diameter: < 10 um) were analyzed with the selecting . animals. 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. Introduction Fluoride is a ubiquitous substance found naturally in food and water.01). the results on this topic have been conflicting. Bo Zhang Division of Environmental Biological Toxicology. we analyzed the peripheral blood lymphocytes of the workers for chromosomal aberrations (CA) and micronuclei (MN). and also human cells. 2. A number of studies of the genotoxic effects of fluoride have been conducted in a variety of systems.24 (p < 0. but other investigations have showed fluoride does not produce genotoxic effects [1. ammonia (NH3). and it is also a common air pollutant in some industrial productions. Micronuclei. The average percentages of lymphocytes with MN of the workers and the controls were 1. and dicentrics) of the workers and the non-exposed controls were 0.01). Department of Life Sciences. sulfur dioxide (S02). To examine possible damage to the genetic apparatus at chromosomal level caused by exposure of workers to air pollution in a phosphate fertilizer factory.factory Mutation Research Year 1997. NH3 and S02 were also present in the factory.

electrode method of fluorine ions.05 mg/m3. Slide scoring For cytogenetic analysis.8 ml of RPMI1640 medium containing 20% new calf serum. To culture lymphocytes whole blood (0.50 to 0. age and smoking habits. previous occupational exposure to chemicals.4. so university staff in the same city were chosen as controls. preparations were coded and scored blind. For each culture at least two slides were prepared.. The pelleted cells were treated to preserve the cytoplasm by gently resuspending in 5 ml hypotonic solution of 0. For the MN assay. final concentration 3 ug/ml) was added to the cultures 24 h prior to harvesting. 2. All subjects were interviewed about recent viral infections. clean slides. sodium hypochlorate-salicylate spectrophotometry. 100 units/ml penicillin. for the floating dust was from 0. For CA analysis.20 mg/m3. working and studying in Shanxi University. It was difficult to find enough people in the factory who were not exposed to fluoride or other chemicals as a control population. 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.07 mg/m3. The blood was cultured at 37'C in 5% C02. and the weight method.02 to 0.01 to 0. The concentrations of these chemicals varied irregularly over one year in this workplace.05 to 0.2. For CA analysis.80 mg/m3 air at the time of investigation. pararosaniline hydrochloride spectrophotometry. 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. 100 ug/ml streptomycin and 2% phytohemagglutinin M (PHA). and 40 controls. 3:1 v/v) for 20 min at room temperature.5.1 ml of colcemid (final concentration: 2. 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. Scoring of MN was limited to . drug intake and alcohol consumption. 2. the cells were cultured for 72 h. for NH3 was from 0. vaccinations.075 M KCI for 15 min at 37'C. the cells were cultured for 48 h. The air-dried slides were stained with 10% Giemsa. Occupational exposure to the chemicals had not caused clinical symptoms in workers and all controls were also healthy. the cultures were processed in the same way except that 0. cultures were centrifuged at 80-100 X g for 6-8 min after an incubation period of 72 h. and the cytochalasin B (Sigma Chemical Co. according to the method described by Fenech and Morley [17]. Subjects The subjects were 40 workers exposed mainly to fluoride (HF. situated in the same city as the factory. matched according to sex.3. the range for F concentrations was from 0. 2. These data indicated that the air pollutant in the workplace was mainly fluorine (HF and SiF4).2 ml) was added to 4. for S02 was from 0. No difference in these respects could be found between the worker and the control group. The slides were air-dried and stained with 10% Giemsa for approximately 10 min. 2. respectively [15].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.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. and SiF4) within the same workplace of the phosphate fertilizer factory. Analysis for CA and MN For measuring MN frequency.

and dicentrics) in workers and in controls were 0.65 + 181 (2.32 + 104 (1. chromatid-type aberrations and their total were all significantly higher (p < 0. translocations. respectively.binucleate lymphocytes only with preserved cytoplasm [17].09) 0. acentric fragments and chromatid breaks were the types of aberrations that showed a significant increase (Table 2).01). CA were analyzed in 200 metaphase cells for each person.30 + aberrations (frequency. centric rings. Table 2 also indicates that the frequencies of major chromosomal aberrations (including rings. 2. The results are expressed as the average percentage of micronucleated cells per 2000 binucleate cells on the two different slides from the same culture. Types of chromosomal aberrations of lymphocytes Group Control Worker Number of subjects 42 40 Number of cells 8400 8000 Chromosome-type aberrations Dicentrics Acentric rings Centric rings 3 (0.01) in workers than in controls (Table 1).08) a 0. Identification of binucleate cells in cell groups required careful visual examination of the individual cell boundaries.08) b 55 (0.18) b Aberrant cells with chromatid-type 28 (0.27) b (a) Mean frequency per 100 metaphase.26 + Total number of aberrant cells 0.6500) c . 3. The difference between them was statistically significant (P < 0.6.0250) 52 (0.33 + 77 (0.96 + aberrations (frequency. Statistical analysis The x2-test was used for the CA and the MN frequencies.24%. %) 0. Cells analyzed for chromatid and isochromatid breaks and other types of aberrations [19]. Frequencies of lymphocytes with chromosomal aberrations Group Control Worker Number of persons 42 40 Number of metaphases 8400 8000 Aberrant cells with chromosome-type 27 (0.01 TABLE 2. % (b) Significantly different from control by x2-test at p < 0. according to the criteria proposed by Countryman and Heddle [18].0119) 13 (0.91% and 0. Only cells with 46 chromosomes were included in the analysis. Dicentrics.1548) 11 (0. %) 0. TABLE 1.1375) b 2 (0. Results The average percentages of chromosome-type.07) 0.0357) a 1 (0.

01).2750) c (a) Mean frequency per 100 metaphase. %. TABLE 3.9625) c Total chromosomal aberrations 55 (0.62 + 1.6% of the controls were in the range 1.3214) 28 (0. Frequencies of lymphocytes with micronuclei (MN) Group Control Worker Number of persons 42 40 Number of binucleate cells observed 84 000 80 000 Number of binucleate cells with MN 520 1240 Frequencies of binucleate cells with MN (X + 0.00%. It implies that there is a positive correlation between MN and CA in the worker group. both frequencies begin to decrease after about 10 years of pollutant exposure period.0625) c 105 (1. 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).05 (c) Significantly different from control group by x2-test at p < 0. these differences between the workers and the controls were statistically significant (p < 0.3000) c 8 (0.00% and 17. Analysis of the correlation between the individual results for the micronucleus index and cells with aberrations indicated that the correlation factor.50% were higher than 2.10 to 1. only 16. R.Acentric fragments Translocations Minutes Gaps Total Chromatid-type aberrations Breaks 2 (0.55 + 0.0595) 6 (0.71 SE a. (b) Significantly different from control group by x2-test at p < 0.01). %) 0.54 b (a) Standard error of the means from all persons of each group.6707) 182 (2. However. .3333) 24 (0.0000) 4 (0. Fig. was 0.1000) 85 (1. 75% of workers were over 1.3125) c 73 (0. However.0500) Interchanges Total 28 (0. Frequencies of cells with MN in 52.426 (p < 0.4% controls were in the range 0.3333) 77 (0.01 TABLE 4. 31.01). 1 shows the frequency distribution of lymphocytes with MN among the workers. all subjects in the worker group had cells carrying MN. (b) Significantly different from control by x2-test at p < 0.01 Table 3 shows the frequencies of cells with MN in peripheral blood lymphocytes of workers were statistically significantly higher than controls (p < 0.0238) 3 (0.00%.00 to 2.00%. 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.9125) c 0 (0.0% of the controls were found not to have lymphocytes with MN. and none was over 2.1000) 8 (0.00%.0714) 27 (0.0357) 5 (0.

35 + 5-10 10 2000 72 0. which had an increased frequency of major CA. Discussion Fluorine is a necessary biological trace element for human health [12]. It has been found that fluoride affects enzymatic activities.42 + > 10 6 1200 22 12 000 170 0.81 + 1. could induce both CA and MN in human blood lymphocytes in vivo.60 + 2.01 versus < 5 group by x2-test 4.25 + <5 24 4800 87 48 000 600 0.Chromosomal aberrations (CA) or micronuclei (MN) and length of service in workers at the phosphate fertilizer factory Chromosomal aberrations Micronuclei No.12 a a 1.41 20 000 470 0. of No. a need to improve safety. SCE.g. Some studies indicate that NaF induces chromosome aberrations in cultured human blood lymphocytes in vitro. 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. although the general health of workers in the phosphate fertilizer factory was found to be satisfactory. it may cause damage to genetic material at the chromosomal level. of cells cells % (X No. and health surveillance for the high risk group. rather than directly with DNA. and this effect could delay mitotic and meiotic cycles causing chromosomal breakages [25]. However. in this study data on the socio-economic and nutritional status were not collected. in which HF and SiF4 are the main chemicals. lymphocyte death.83 + 1. and in bone marrow cells of mice [4. The evidence presented here showed the chromosome damage rate declines in those who have worked for more than 10 years in the factory. of Group No. it is suggested that chromosomal abnormalities induced by fluoride could be the results from interaction with the enzymes responsible for DNA synthesis or repair.26 0.53 (a) p < 0.23].80 mg/m 3).01) [13]. However. F 0. This might be due to an adaptation mechanism being produced to the chemical pollutants.51 0. Our study here provides evidence that the air pollutants at the phosphate fertilizer factory. or due to an equilibrium being reached between chromosome damages. and/or DNA damage and .50-0.20]. Although the income levels of the factory workers and the university staff controls were generally similar at the time of investigation. There is.61 3. and lymphocyte renewal. therefore. and MN induced by the chemical pollutants. The results of our studies imply that even if the concentration of the chemical pollutants in the air is low (e. Studies of the relationship of nonwaterborne 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. Such chromosomal aberrations may eventually lead to the formation of structural changes and fragmentation as observed in the present studies.24]. that it increases tumor growth rate and interferes with DNA repair in vitro and in vivo [14. the potential impact of these factors were not studied. of No. Since it has been shown that fluoride can inhibit nucleic acid synthesis [26]. sanitary conditions. but one cannot exclude that these factors may have had some impact on the results obtained. of cells cells % (X (years) persons observed with + SE) observed with + SE) CA CA 1. Yiamouyiannis and Burk [20] reported an increase in human cancer death rate in fluoridated areas. some animal experiments have indicated that fluoride is tumorigenic.

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