ÓÄÊ 636.2:631.523:575:576.08
GENOTYPIC AND PARATYPIC FACTORS INFLUENCING ON THE MICRONUCLEI TEST RESULTS
T.T. Glazko1, Yu.A. Stolpovskii2, V.I. Glazko1
Comparative analysis of the erythrocyte frequencies with micronuclei in peripheral blood of the Mongolian indigenous cattle breed and the yaks breeding in different ecologic-geographical areas of Mongolia, and also interspecies hybrids between them was carried out. It was shown that on the frequencies of erythrocytes with micronuclei essential influences was rendered by age of animals, environment condition, and species accessory. It was revealed that in interspecies hybrids the frequency of erythrocytes with micronuclei in 2-3 times above, than in animals of parental species in the same age groups and environment conditions. The necessity to include in consideration of the micronuclei test results the influences of the external and internal factors was concluded at the using the erythrocyte frequencies with micronuclei for bioindication of ecotoxic pollution of the inhabitancy of agricultural animal species and sources of the foodstuffs of an animal origin.
Key words: cytogenetic animalies, micronuclei test, interspecies hybrids, ecology-geographical factors.
Raising genotoxic contamination of environment necessitates the methods of their control, because food obtained in such conditions can be unsafe for humans. Traditional methods of pollutant detection are designed only for a limited number of toxicants without implied possibility of cumulative effects in biological objects. These facts suggest the development of detection methods indicating general ecotoxic effects in bioindicator mammalian species, which is especially important for testing safety conditions of food production. Farm animals can be used as such bioindicators (1, 2) as they contact with the same environmental factors as humans simultaneously being a source of food.
To assess ecotoxic effects in biological material, the most available parameter of analysis is the frequency of different cytogenetic abnormalities in blood cells - metaphase plates (polyploid and aneuploid cells, metaphases with different types of chromosomal aberrations). However, this method includes cultivation in a nutrient medium with stimulants of cell division and antibiotics, since spontaneous division of peripheral blood cells is quite rare. Such pretreatment can distort the actual mutation spectra by additional mutations, while consuming lots of time and resources, in contrast to micronucleus test having more prospects in bioindication.
Currently, there are three main types of micronucleus tests widely used in genotocicity analyses: testing nuclear-free erythrocytes (3), mononuclear lymphocytes and buccal epithelium, as well as binucleated lymphocytes with blocked cytokinesis (4-6). These tests are based on data about the frequency of spontaneous formation of micronuclei in peripheral blood cells, as well as other factors affecting results of research. Exogenous genotoxins determine the analyzed parameter along with genotypic characteristics of tested animals, which should be considered when using farm animal species as bioindicators. It has been shown the species’ differences in frequency of micronuclei formation caused by ionizing radiation (7). Humans, cattle and goats showed the closest values characterizing increase in number of cells with micronuclei in response to irradiation; birds and fish were the most resistant, rabbits and pigs demonstrated medium sensitivity. In this context, it has been suggested using cattle and goats as effective bioindicators of genotoxic pollution. At the same time, it has been reported about the pronounced genetically determined intraspecific differences in frequencies of cells with micronuclei (8). In earlier studies, the authors have revealed differences between cattle breeds of various productivity directions (9). It has been also shown the relatively high frequency of cells with micronuclei in interspecific hybrids (2).
The purpose of this study was to compare the effects of age, species, interspecific hybridization and environmental conditions on results of micronucleus test in cattle, yaks and their interspecific hybrids.
Technique. In 2009-2010, the authors studied the smears of peripheral blood obtained from local Mongolian cattle (13 goal) and yaks (14 goal) aged 3-6 years and kept in different eco-geographical regions of Mongolia (North-West Mongolia, the Biosphere Reserve of Hovsgol region; Southern Mongolia, zone of risky farming adjacent to the Gobi Desert), 2-month-old calves of local Mongolian cattle (4 goal) and interspecific hybrids (dzo, or khainag – the progeny from mating cows and yaks, 7 goal) bred by farmers of Hovsgol (the samples were collected in 2008 during an expedition).
To prepare the smears, a drop of peripheral blood was mixed on a slide with a drop of saline, evenly distributed on surface, dried, fixed with methanol for 30 min and stained with Giemsa dye (“Merk”, Germany). The number of erythrocytes with micronuclei was determined in 3000 cells and calculated to 1000 cells (‰). The smears were examined under the binocular microscope (“Motik”, China) with integrated digital camera (zoom x 1000).
Statistical reliability of intergroup differences was assessed by Student's t-test (tS).
Results. The minimum frequency of erythrocytes with micronuclei (Table) was detected in Mongolian yaks and local Mongolian cattle from the area adjacent to the Gobi Desert (respectively, 0,3 ± 0,2 and 1,8 ± 0,6 ‰). The cattle showed somewhat smaller rates of erythrocytes with micronuclei compared with yaks, and these frequencies were considerably lower than those of previously studied cattle breeds of different productivity directions (9, 10).
The frequency of erythrocytes with micronuclei was reliably higher (P <0,05) in animals of both species ranched in more favorable conditions of Hovsgol (yaks - 3,2 ± 0,6 ‰, cattle - 4,6 ± 0, 7 ‰). Local Mongolian cattle of Hovsgol demonstrated the values close to typical levels of beef cattle earlier detected by the authors (9, 10). Interspecies differences in this region were less pronounced.
The frequency of erythrocytes with micronuclei in the peripheral blood of cattle from different eco-geographical regions of Mongolia (2008 |
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Species, |
Parameter, ‰ |
Mean |
Hovsgol region |
||
Dzo (khainags) |
8,0 (104) |
10,0±1,1 |
Cattle: |
|
|
mature animals 4,5 (77) |
4,6±0,7 |
|
|
1,0 (78) |
|
|
5,0 (79) |
|
|
3,5 (80) |
|
|
6,5 (81) |
|
|
6,0 (82) |
|
|
6,0 (83) |
|
calves |
12,0 (68) |
9,5±1,1 |
|
10,5 (69) |
|
|
8,0 (70) |
|
|
7,5 (71) |
|
Yaks |
1,3 (55) |
3,2±0,6 |
the Gobi |
||
Cattle |
2,0 (111) |
1,8±0,6 |
|
0,3 (112) |
|
|
1,0 (113) |
|
|
3,0 (114) |
|
|
1,0 (117) |
|
|
0,3 (118) |
|
|
4,6 (122) |
|
Yaks |
0,6 (161) |
0,3±0,2 |
|
0 (162) |
|
|
0 (163) |
|
|
1,3 (164) |
|
|
0 (165) |
|
|
0 (166) |
|
|
0 (167) |
|
|
||
These findings reveal the trend towards a relatively lower stability of the chromosome apparatus in local Mongolian cattle than in yaks.
Both species from different regions had statistically reliable differences in frequency of erythrocytes with micronuclei, which indicates that yaks and cattle from zone of risky farming (under intense natural selection) has more stable chromosomal apparatus compared with the same species grown in more favorable conditions. It can be expected that such differences are the result of natural selection for high resistance to unfavorable environmental factors.
The analysis of data in age groups of local Mongolian cattle from Hovsgol indicated that the frequency of erythrocytes with micronuclei was significantly higher (P <0,01) in 2-month-old calves than in mature animals from parent population (Table). Apparently, the young animals showed relative immaturity of cellular components of the immune system responsible for elimination of erythrocytes with micronuclei. The literature data describe age-related increase in frequency of erythrocytes with micronuclei found in Black-and-White cattle (11), which was assumed to be connected with accumulation of spontaneous mutations in somatic cells. At the same time, this mismatch of obtained data and results of earlier studies can be explained by the fact that the authors investigated blood samples taken from 2-month-old animals, while O. Semenova (11) observed the older group with better elimination of abnormal erythrocytes.
Earlier, it has been shown the marked differences in frequencies of cytogenetic abnormalities in peripheral blood of cattle breeds of different productivity directions (10), thereby indicating the presence of fairly pronounced genetic component affecting stability of the chromosomal apparatus.
There is a lot of literature data about individual diversity of cattle within breeds for frequencies of cytogenetic abnormalities in blood cells (12, 13), about correlations between aneuploidy of lymphocytes and reduced resistance of erythrocyte membranes to acid treatment, as well as between the frequency of cytogenetic abnormalities and suppression of animals’ reproductive abilities (13, 14). In humans, it has been proved the connection between the frequency of cytogenetic abnormalities in peripheral blood cells and reproductive functions (15).
Reduced stability of the chromosomal apparatus is especially important criteria in both bioindication of genotoxic effects and in prediction of animals’ reproductive function. These facts suggest clarification of possible genetic impact on frequency of cytogenetic anomalies in somatic cells revealed by results of micronucleus test. To assess this impact, the authors compared the frequency of erythrocytes with micronuclei in mature (3-6 years) animals of local Mongolian cattle, yaks and their interspecific hybrids (khainag) farmed under identical conditions of the Biosphere Reserve Hovsgol. These hybrids are sterile, but they have no disorders of phenotypic development.
As it was found (Table), khainags demonstrated reliably (P <0,01) higher values of analyzed parameters than local cattle (10,0 ± 1,1 vs. 4,6 ± 0,7 ‰) and yaks (10,0 ± 1,1 vs. 3,2 ± 0,6 ‰). Therefore, in similar age groups of animals bred under identical environmental conditions, interspecies hybrids had 2-3 times higher frequency of erythrocytes with micronuclei than in their parents. According to the obtained data, genetic component for the frequency of cytogenetic anomalies in somatic cells can significantly affect the assessment of instability of chromosomal apparatus, which should be known when using these characteristics in bioindication of exogenous genotoxic effect.
Thus, the instability of chromosomal apparatus was evaluated upon the data on frequency of erythrocytes with micronuclei in peripheral blood, which depends on several factors including age, species and intensity of natural selection aimed at improved resistance to adverse environmental factors. Genetic component causes significant impact in variability of results at micronucleus test (2-3 times higher values in interspecific hybrids than in parental species in similar age groups and environment). The influence of these factors should be considered using the frequency of cytogenetic anomalies in somatic cells of animals in bioindication of ecotoxic contamination of environment and food of animal origin.
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1
Russian State Agrarian University - Moscow Agricultural Academy named after K.A. Timiryazev, Moscow 127550, Russia
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Received June 2, 2010
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