Mutagenesis, Vol. 14, No. 1, 83-86,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Chromosome breakage at sites of oncogenes in a population accidentally exposed to radioactive chemical pollution
Siberian Medical University, 634050 Tomsk'a/ya 808, Russia
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Abstract |
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The purpose of the present study was to investigate the level of aberrations at fragile sites of chromosomes in peripheral blood lymphocytes of the population of an area polluted with radionuclides, following an accident at the Siberian Chemical Plant. We carried out the micronucleus test to screen people with radiation-related cytogenetic effects. Of the 1246 inhabitants of the settlement of Samus examined, 148 showed a significantly increased frequency of micronucleated erythrocytes and were selected for chromosome analysis as a radiation-exposed group. Additional analysis was carried out on 40 patients with gastric cancer and atrophic gastritis with stage II–III epithelial dysplasia. Eighty six individuals from a non-polluted area were used as a control group. Chromosomal breaks and exchanges occurred preferentially in chromosomes 3 and 6 among radiation-exposed persons and patients. The regions 3p14–25 and 6p23 were damaged most often. There was a tendency to preferential involvement of q21–25 of chromosome 6 in patients with gastric cancer and atrophic gastritis. Specific damage at certain chromosome sites was observed in the radiation-exposed population as well as in patients with gastric cancer. Most often this damage was located near oncogene loci, which could imply that chromosome damage induced by radiation is likely to be a predisposing factor to the expression of oncogenes and malignant transformation of cells in exposed individuals.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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On 6 April 1993 there was an accident at the Siberian Chemical Plant (SCP) near the town of Tomsk which resulted in extensive contamination over 250 km2 with radionuclides such as 90Sr, 137Ce and 239Pu (see Figure 1
). The highest radiation level outside the plant territory was as much as 400 µR/h.
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Our previous investigations demonstrated very low levels of DNA repair activity and increased frequencies of micronucleated erythrocytes (Almassy et al., 1978
; Ashby and Mohammed, 1986) and chromosomal aberrations in the peripheral blood T lymphocytes (Ilyinskikh et al., 1995, 1996). It is known that the fragile sites of chromosomes respond first to cytogenetic instability and contribute to the increased risk of developing neoplasms (Yunis, 1981, 1983; Yunis and Soreng, 1984). Certain sites of chromosomes have been found to be fractured in tumour cells (Shabtai et al., 1985). Furthermore, proto-oncogene expression as a result of chromosomal aberrations has been observed by many investigators (Syakste and Epenpreis, 1987). A correlation between the coincidence of oncogene localization and chromosome breakpoint has been detected for a number of oncogenes. According to most authors the fact that chromosomal aberrations are not causal for tumour cells and may be changed to oncogene expression surely indicates that chromosomal aberrations play an important role in the mechanism of oncogene activation. The aim of the present study was to investigate the level of chromosome aberrations at fragile sites in the peripheral blood lymphocytes of the population of an area polluted with radionuclides following a radiation accident at the SCP.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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During the period April–July 1993 as many as 1246 inhabitants of the settlement of Samus exposed to radiation were examined using cytogenetic methods. In no case had individuals been exposed to radiation professionally or medically. The investigation was begun within 1 week after the accident. We carried out the micronucleus test on standard haematological smears to screen people with radiation-related cytogenetic effects according to a method which has been previously described (Ilyinskikh et al., 1986, 1992
). Of the 1246 inhabitants of the settlement of Samus examined, 148 [78 male and 70 female; 38.8 ± 11.4 and 33.4 ± 13.3 years of age (mean ± SD), respectively] showed a significantly increased frequency of micronucleated erythrocytes and were selected for chromosome analysis as a radiation-exposed group (persons with radiation-related chromosome damage). Furthermore, of the 1246 persons examined, we selected 20 patients (12 male and eight female) with gastric cancer (papillary or acinar adenocarcinoma) and 20 patients (13 male and seven female) with atrophic gastritis at the II–III stage of epithelium dysplasia for a comparative analysis. The patients were clinically examined at the Clinic for Oncology, the Siberian Medical University, Tomsk, and their diagnoses were based on the results of endoscopic biopsies using gastroduodenoscopy. In no case had patients received chemotherapy or radiotherapy before cytogenetical examination. There were workers at a shipbuilding yard, fishermen, farmers, school-teachers and secondary and professional school students among the patients and radiation-exposed persons. For chromosome analysis, venous blood samples (10 ml) were obtained with informed consent from the patients and radiation-exposed group. Furthermore, peripheral blood from 86 individuals (46 male and 40 female individuals; 36 ± 5.6 and 34.2 ± 9.5 years of age, respectively) who lived in the settlement of Loskutovo, which was not contaminated with radionuclides, was used as a control group (Figure 1
). Lymphocyte cultures were prepared from the whole blood according to Yunis (1981). Briefly, the lymphocyte cultures were incubated in RPMI-1640 medium (Sigma, St Louis, MO) with phytohaemagglutinin (PHA-M; Difco, Detroit, MI) as a stimulator and 20% fetal calf serum. Cell division was inhibited by colcemid at the stage of prometaphase. After cultivation, standard chromosome preparations were made and stained using a chromosome banding method. Chromosomal aberrations were evaluated in 1000 cells/sample by three independent cytogeneticists using coded slides. The analysis was conducted in cells with a maximum possible number of chromosome bands according to recommendations given by Yunis (1981, 1983) and Yunis and Soreng (1984). In the present study the actual frequency of constitutional chromosomal damage was compared with the expected frequencies based on chromosome lengths of the human karyotype (Vogel and Motulsky, 1996). These calculations were made assuming that the probability of occurrence of structural aberrations was equal at any site. In each case, an individual examined had to fill in a questionnaire by means of which we grouped people according the their place of birth, sex, smoking habit, year of birth, anamnesis of diseases, the presence of children with abnormalities in the family, still-born children, cancer, X-ray examinations and length of residence in the locality. Each of the persons also described their diet in the questionnaire. In this study, the qualitative and quantitative evaluation of each diet was based on the method of Giovannucci et al. (1995). For each of 90 food and beverage items listed, a commonly used unit or portion size was specified and participants were asked how often, on average, they had consumed that amount of each food over the past year. Participants chose from among nine possible responses, which ranged from `never' to `six or more times per day'. We also asked about the brand, duration and frequency of multivitamin and individual vitamin supplements used and the types of fat commonly used. The questionnaire also contained an open-ended section for foods that were not listed.
Since vitamin A is antimutagenic and can decrease radiation-related chromosome damage, we have calculated vitamin A intake among the individuals examined. We calculated retinol (preformed vitamin A) intake as two thirds of the vitamin A activity (in international units) of dairy foods plus the total vitamin A activity in other animal products. Also, we added the content of vitamin E and folic acid to our nutrient database.
Statistical analysis of the data was by Student's t-test, ANOVA (analysis of variance) and correlation analysis. In the statistical analysis of the results we used procedures available in the SAS statistical package (SAS Institute Inc., 1989).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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We carried out the micronucleus test on standard haematological smears to screen people with exposure-related cytogenetic effects according to a method which has been previously described (Ilyinskikh et al., 1986, 1992
). Of the 1246 inhabitants of the settlement of Samus examined, 148 [78 male and 70 female; 38.8 ± 11.4 and 33.4 ± 13.3 years of age (mean ± SD), respectively] showed a significantly increased frequency of micronucleated erythrocytes and were selected for chromosome analysis as a radiation-exposed group.
The data of the present study indicate that both the patients and the radiation-exposed group have increased frequencies of chromosome aberrations as compared with the controls (Table I). Furthermore, the overwhelming majority of aberrations were dicentric and ring chromosomes, which are caused by exposure to radiation (Vogel and Motulsky, 1996).
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We observed no correlation between an increased level of chromosome aberrations and sex or nationality among the Samus inhabitants. Also, we observed no correlation between increased level of chromosome aberrations and smoking habit (r = –0.17, P > 0.05). It is known that deficiency of folic acid, vitamin E or carotenes can lead to cytogenetic instability in man (Ilyinskikh et al., 1990
). Both the controls and the radiation-exposed persons had approximately equal consumption of these vitamins. In this study, both groups showed no correlation between level of carotenes, vitamin E or folic acid consumption and frequency of chromosome aberrations.
A total of 453 chromosome and chromatid breaks and exchanges were detected. There were 147 in individuals with cytogenetic damage (radiation-exposed group), 122 in patients with gastric cancer, 88 in patients with atrophic gastritis and 86 in the contol group. The data obtained show that the distribution of chromosomal structural damage did not always correspond to the expected level (Table II). The closest values with regard to the expected level were observed in the population from the settlement of Loskutovo (contol group). A lack of breaks in small chromosomes was noted in nearly all cases, including the control group, which was in accordance with data obtained in a study of karyotype in healthy donors (Ilyinskikh et al., 1984Ilyinskikh et al., 1986).
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The expected frequencies of chromosome aberrations were calculated on the basis of absolute chromosome lengths (Vogel and Motulsky, 1996
). In the chromosomes of the control group and patients with gastritis the number of localized aberrations was close to the expected level, i.e. the number of aberrations was in proportion to chromosome length (the longer a chromosome the more often it was damaged). In individuals with high levels of radiation-related chromosome damage as well as in patients with gastric cancer a marked rise in fragility of chromosomes 3 and 6 was observed.
A more thorough analysis has shown that in chromosome 3 the damage is localized at sites from p14 to p25. The oncogene raf-1 is known to be localized at site p25. It has been reported that site [t(3,8)] leads to mixed thyroid cancer and deletion [del(35)] leads to small cell lung cancer (Yunis, 1981; Sandberg, 1984; Shabtai et al., 1985). The site 3p14 is known as a fragile chromosome site. It has been shown that the fragile site p14 on the short arm of chromosome 3 takes part in translocation (3;8)(p14,q24), which is typical of hereditaty renal cell carcinoma (Drabkin et al., 1985). Furthermore, deletion of the short arm 3p (p14–q23) is often observed in small cell lung cancer (Yunis, 1981; Sandberg, 1984). Damage at q21–q25 of chromosome 6 (where the oncogene myb and, close to it, the oncogene yes are located) was observed in patients with gastric cancer. Similar findings were noted in patients suffering from atrophic gastritis with epithelium dysplasia. In individuals with radiation-related chromosome damage the greater part of the observed damage was also localized on chromosome 6 at p23, where there is a fragile chromosome site. Site p23 of chromosome 6 is both a fragile site and a place of chromosome breakage in translocation (6;9)(p23;q24), which is followed by an increase in the number of bone marrow basophils in acute non-lymphocytic leukemia (Yunis, 1981). It should be noted that the fragile site 6p23 is located next to the oncogene Ki-ras-1 (6p23–q12), which may be of great importance for activation of the latter during malignant transformation. According to the literature data (Dilernia et al., 1987) an increased number of peripheral blood lymphocytes with chromosomal aberrations was noted in patients with atrophic gastritis and gastric cancer, however, multiple chromosome aberrations were found not for chromosomes 3 and 6 but for chromosome 1 and 9, i.e. at sites where there are no fragile chromosomes. We believe that the difference between our findings and those obtained by Dilernia et al. (1987) is that those authors evaluated one family in which recurrent gastric cancer was observed over three generations. Familial cancers were not registered in our samples. According to the data in Table I, the inhabitants of Samus, particularly gastritis and cancer patients and the radiation-exposed persons, who regularly consumed local fish showed increased chromosome aberrations as compared with the controls. Between 1961 and 1994 the SCP discharged radioactive liquid waste into the Tom River, which resulted in considerable contamination of local fish with long-lived radionuclides such as 90Sr, 137Ce and 239Pu (Rikhvanov, 1994). Therefore, local fish consumption may be one of the principal causes of chromosome damage and cancer development among the inhabitants. It should be noted that the radiation-related structural chromosome damage is distributed proportionally with regard to chromosome length in the human karyotype. Therefore, the specificity of damage observed in people in the area exposed to radiation cannot be explained only by the direct influence of the radiation factor. Previous data demonstrated that there was a significant reduction in immunity and enhanced circulation of some potentially oncogenic viruses among radiation-exposed people in this area (Ilyinskikh et al., 1995) The ability of viruses to induce specific damage at certain sites on chromosomes is well known (Ilyinskikh et al., 1984; Nichols, 1963).
Thus, specific damage at certain chromosome sites of T lymphocytes was observed in a population exposed to radiation after the accident at the SCP as well as in patients with gastric cancer. In most cases, the sites of chromosomal damage were located near the locus of protooncogenes, which, according to the literature, can lead to the development of neoplasms. The chromosome damage induced by radiation is likely to be a predisposing factor to the activation of oncogenes and malignant transformation of cells in exposed individuals.
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Notes |
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1 To whom correspondence should be addressed. Tel: +7 3822 413679; Fax: +7 3822 233309; Email: root{at}ecogen.tomsk.su
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Received on April 14, 1998; accepted on September 14, 1998.
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