Mutagenesis, Vol. 14, No. 4, 391-396,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
CREST staining of micronuclei from free-living rodents to detect environmental contamination in situ
1 Center for Evolutionary Genetics (CNR), Via degli Apuli 4, 00185 Rome, and 2 Nucleic Acid Center (CNR), Department of Genetics and Molecular Biology and 3 Department of Animal and Human Biology, `La Sapienza' University, 4 Department of Biology, `Roma Tre' University, Rome, Italy and 5 Institute of Vertebrate Biology, Academy of Sciences of the Czech Republic, Brno, Czech Republic
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Abstract |
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In this work immunofluorescent antikinetochore (CREST) staining was used to analyse bone marrow micronuclei (MN) from free-living animals belonging to four different rodent species. Yellow-necked mice (Apodemus flavicollis) and bank voles (Clethrionomys glareolus) were trapped in the Czech Republic, Algerian mice (Mus spretus) in Spain and house mice (Mus musculus domesticus) in Italy. Animals were collected in areas displaying low or high environmental pollution in order to investigate the sensitivity of CREST analysis on bone marrow MN as a biomarker of environmental stress in situ. Differences in total MN frequencies between animals collected in control or contaminated areas were statistically significant for two species, whereas the differences in CREST+ MN were statistically significant for three species. Interestingly, the percentages of CREST+ MN in animals collected in the control areas were very low (3.2–8.7%), suggesting that activities inducing alterations in the distribution of chromosomes are very rare in natural conditions. The increased frequencies of CREST+ MN observed in areas with high environmental impact indicate that activities producing loss of chromosomes at mitosis may be characteristic of anthropogenic environments such as industrial settlements around petrochemical factories. Our data suggest that the analysis of CREST+ MN may represent a sensitive end-point for the detection of environmental contamination by genotoxic xenobiotics, offering the advantage of providing information on the mechanism of action of environmental contaminants.
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Introduction |
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Human activities induce changes in ecosystems by releasing pollutants into the environment. The need to detect and estimate the impact of pollution on natural environments has led to the search for sentinel species, so-called `biomonitors' and sensitive end-points.
Free-living small mammals are suitable organisms to monitor environmental pollution on terrestrial ecosystems in situ, because they are known to concentrate the pollutants present in the ecosystem, such as pesticides, radionuclides, heavy metals and other contaminants (Talmage and Walton, 1991
). The micronucleus (MN) test has been utilized on wild rodents since 1978 to investigate the genetic damage induced by environmental pollution (Materiy and Maslova, 1978; Cristaldi et al., 1985; Tice et al., 1987; Eckl and Riegler, 1997). Significant correlations between pesticides (McBee and Bickham, 1988), radioactivity (Cristaldi et al., 1991), heavy metal contamination (Ieradi et al., 1992, 1996; Tull-Singleton et al., 1994) and MN or chromosome aberration frequencies have been detected in wild rodents living in contaminated areas.
MN are formed when an entire chromosome or a chromosome fragment fails to migrate in one of the two daughter nuclei during mitosis. Conventional microscopic analysis, however, does not discriminate MN with respect to their content. It has been shown that the presence or absence of kinetochore proteins in a MN could be used to identify MN deriving from chromosome loss (kinetochore-positive) or deriving from chromosome breakage (kinetochore-negative). A large number of studies have demonstrated that the MN assay could distinguish clastogens from aneugenic agents using immunofluorescent antikinetochore (CREST) staining of MN following treatment with agents with different mechanisms of action. The methodology has been successfully applied both in cultured mammalian cells (Degrassi and Tanzarella, 1988; Eastmond and Tucker, 1989) and in bone marrow cells from laboratory mice (Gudi et al., 1990; Miller and Adler, 1990; Miller et al., 1991). At present, little data are available concerning CREST staining in free-living rodents (Tanzarella et al., 1997).
Previous studies using four different species of wild living rodents, house mouse (Mus musculus domesticus), Algerian mouse (Mus spretus), yellow-necked mouse (Apodemus flavicollis) and bank vole (Clethrionomys glareolus), showed that MN frequencies in peripheral blood and bone marrow erythrocytes from Giemsa stained slides were increased in animals collected in contaminated areas from three European countries, i.e. Italy, Spain and the Czech Republic (Ieradi et al., 1996, 1998, 1999, Zima et al., 1999).
The present study applied immunofluorescent antikinetochore staining using the CREST antibody to bone marrow MN from animals collected in the areas that had been previously studied in the above-mentioned European countries, in order to determine the sensitivity of the analysis of CREST-positive MN in wild rodents as a biomarker of environmental stress. Furthermore, the comparison of results obtained in rodent species collected in different European countries provided important information for the identification of model species for ecotoxicology studies within Europe.
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Materials and methods |
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Trapping sites
A total of 128 animals were collected from October 1994 to October 1997 in several locations. Sixteen (nine male and seven female) yellow-necked mice (A.flavicollis) and 16 (seven male and nine female) bank voles (C.glareolus) were collected in Litvinov (Northern Bohemia, Czech Republic); 16 (nine male and seven female) yellow-necked mice and 16 (eight male and eight female) bank voles were collected in Ceska Kamenice (Decin district, Northern Bohemia, Czech Republic). The first site is located in the Most Basin, a coal mining area with a high concentration of petrochemical factories and thermal power plants in one of the most polluted regions in central Europe. According to data from 1996 (Czech Ecological Institute, 1997
), yearly average concentrations of 68 mg/m3 SO2, 69 mg/m3 NOx and 70 mg/m3 dusty aerosols were recorded in the area. The animals were trapped at the edges of an old beech forest situated ~30 m from a road and urban settlements. The second site was the village of Filipov, near the town of Ceska Kamenice, situated within the Labe River Sandstone Protected Landscape Area. This area also suffers from global air pollution caused by industrial emissions in this part of central Europe. However, the yearly average concentrations of pollutants are considerably lower than in the Most Basin and there are no important local sources of industrial pollution. No frequented road communications were situated in the vicinity of the trapping site.
Sixteen (five male and eleven female) Algerian mice (M.spretus) were collected in a marshland site in the Doñana Biological Reserve and 16 (ten male and six female) Algerian mice were collected in a marshland site close to the town of Huelva in Andalusia (Spain). One site was within the Doñana National Park, which is located at the mouth of the Guadalquivir river; the second site was located in an industrial settlement consisting of a large petrochemical complex situated in the estuary formed by the mouths of the Odiel and Tinto rivers near Huelva. This site includes several phosphoric acid plants specializing in the production of fertilizers from phosphorites and some chalcopyrite transformation plants (Bolivar et al., 1995; Martin et al., 1995).
Sixteen (nine male and seven female) house mice (M.m.domesticus) were collected in Rome (Italy) along a road exposed to high traffic flows (Muro Torto Avenue; on average 3000 vehicles/h during the trapping period; Traffic Office of Roman Municipality) and 16 (eight male and eight female) house mice were trapped in one area practically closed to traffic (the city Zoo), which served as controls. The trappings were performed at the same sites where a previous survey showed a significant increase in MN frequency in mice exposed to high motor vehicle flow (Ieradi et al., 1996).
Among the 128 collected animals, only five were sexually immature.
Characteristics of selected species
Apodemus flavicollis inhabits mainly the undergrowth of both coniferous and deciduous woodlands, where it feeds predominantly on seeds, but also on animal food. In the temperate forest it is often syntopic with the bank vole (C.glareolus), a typical inhabitant underground and in the undergrowth of thick woods, whose diet mainly consists of green parts of plants, seeds, roots and berries during spring and of insects in the winter.
The standard chromosome complement of A.flavicollis species comprises 48 acrocentric chromosomes and a variable number of B chromosomes have been reported in various European populations. At the two sites studied, a total of 156 individuals were karyotyped from 1979 to 1996 and relatively high frequencies of the supernumerary B chromosomes were recorded (Zima and Macholan, 1995; Zima et al., 1999). Karyotyping of the yellow-necked mice trapped during this survey showed the presence of individuals with relatively high frequencies of B chromosomes in both localities (1–7 and 0–3 supernumerary B chromosomes per individual in Ceska Kamenice and Litvinov, respectively). All the C.glareolus individuals from the two collecting sites that were examined had the standard karyotype of the species (2N = 56).
Mus spretus, the western Mediterranean mouse, is found outdoors in sympatry with the house mouse (M.m.domesticus). This species eats mainly seeds and is found at its highest population density in marshlands (Cagnin et al., 1998). Mus m.domesticus is the most common synanthropic mammal from prehistoric times and has been transported by humans to almost all areas of the world. This mouse is omnivorous and usually lives inside buildings, but may live outdoors when the wild sibling species are absent. Both species have a standard complement of 2N = 40 chromosomes. Robertsonian translocations were not found in the population of M.m.domesticus from Rome (Capanna et al., 1977).
Slide preparation and CREST staining
Animals were captured in live traps and transported to the laboratory where they were killed by cervical dislocation and prepared immediately for analyses. For the MN test both femurs were excised and bone marrow was flushed into a test tube with fetal calf serum and 25 mM EDTA. The cell suspension was centrifuged at 800 r.p.m. for 5 min, the pellet was suspended in a small amount of the same solution and cells were smeared on clean slides. Bone marrow smears were fixed in absolute methanol and maintained at –20°C until staining.
Air-dried slides were used for May–Grunwald Giemsa staining as already described (Ieradi et al., 1996). 2000 erythrocytes (both normochromatic and polychromatic) were analysed for each individual on Giemsa stained slides.
The procedure of Chen et al. (1994) was followed for antikinetochore staining, using FITC-conjugated rabbit anti-human antibody as secondary antibody and FITC-conjugated anti-rabbit antibody as tertiary antibody. Slides were counterstained by immersion in 0.2 µg/ml DAPI for 10 min and mounted in 2 µg/ml propidium iodide in antifade solution (Vector Laboratories). MN were located using a Zeiss Axiophot microscope with UV and blue-violet excitation and successively classified for the kinetochore reaction under a blue-violet filter. In most cases, 2000 erythrocytes (both polychromatic and normochromatic) were analysed from each animal (mean value 1820 scored cells/animal).
Microscopic examination of CREST labelling in bone marrow nucleated cells showed no detectable differences in antigen reaction between the four species examined, in accordance with the positive staining of kinetochores observed on cells from different mammalian species such us Indian muntjac cells (Brinkley et al., 1985), Chinese hamster cells (Degrassi and Tanzarella, 1988), laboratory mouse (Miller and Adler, 1990) and laboratory rat (de Stoppelaar et al., 1997).
Statistical analyses
As the absolute values of MN were low and not normally distributed, the Cox transformation (xtr = ÷x + 0.5) was applied to stabilize the variance. The analysis of variance (ANOVA) was applied to transformed data and the t-test was used to calculate the levels of significance for differences in the frequencies of total MN and CREST+ MN between mouse populations. The level of significance was established at P 
0.05. All analyses were carried out using the Statistica package.
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Results |
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Total (CREST+ and CREST–) MN and CREST+ MN were observed in bone marrow erythrocytes of eight groups consisting of 16 wild rodents belonging to four different species collected in areas with low or high environmental contamination. A graphic representation of the distribution within each group of the individual frequencies of MN/1000 cells is reported in Figure 1
to describe the observed interindividual variability. Approximately the same number of males and females (see Materials and methods) were included in each group and data are presented pooling together the sexes, since the t-test analysis did not show significant differences in MN frequencies between female and male animals within the same group.
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Mean frequencies of MN for each group are presented in Table 1
and graphically illustrated in Figure 2. Table 1 shows that the mean frequencies of MN and CREST+ MN/1000 erythrocytes varied considerably among species. The variations between species were statistically significant using the ANOVA test (total MN, F = 21.89, P < 0.000001; CREST+ MN, F = 13.48, P < 0.00001). The level of MN or CREST+ MN was generally lower in the species with the higher chromosome number, i.e. C.glareolus, suggesting that the probability of losing a chromosome is not related to the number of chromosomes of the species. Moreover, in A.flavicollis no significant correlation was observed between CREST+ MN frequencies and number of supernumerary B chromosomes (r = –0.350, P = 0.058). Previous work at the same localities in the Czech Republic also failed to show a relationship between incidence of B chromosomes in A.flavicollis individuals and MN frequencies after Giemsa staining (Zima et al., 1999).
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In the Czech Republic, animals from two different species (A.flavicollis and C.glareolus) living in the same territory were collected. This provided us with the opportunity to evaluate the relative sensitivity of the two rodent species to the same environmental contamination. A statistically significant higher frequency of total MN was observed in yellow-necked mice (A.flavicollis) in comparison with bank voles (C.glareolus) in both the control (t = 4.57, P = 0.000078) and the contaminated area (t = 5.18, P = 0.000014; Table 1
and Figure 2). When comparing the frequencies of MN observed in the two areas displaying different degrees of contamination, the data showed that yellow-necked mice collected in the industrial area (Litvinov) had statistically significant higher frequencies of MN as compared with animals from the same species trapped in the protected area (Ceska Kamenice) (t = 2.33, P = 0.02). The difference in MN frequencies between bank voles collected in the two areas was not statistically significant (t = 0.80, P = 0.42).
A significantly higher frequency (t = 3.76, P = 0.0007) of MN was observed in erythrocytes from M.spretus samples collected near fertilizer-producing plants in the Huelva industrial area as compared with specimens captured in the Doñana Biological Reserve in Spain (Table 1 and Figure 2). A higher frequency of MN was also observed in M.m.domesticus collected in Rome along a road with high traffic flow (Muro Torto Avenue) as compared with animals trapped within the Rome Zoo, although the difference was not statistically significant (Table 1 and Figure 2; t = 1.31, P = 0.19).
The frequencies of CREST+ MN were then compared between low and high environmental impact areas to investigate whether industrial pollution and motor vehicle traffic were associated with chemical activities producing chromosome loss or breakage (Table 1 and Figure 3). The frequency of CREST+ MN was found to be significantly higher in animals from Litvinov both for A.flavicollis (t = 3.83, P = 0.0006) and C.glareolus (t = 2.15, P = 0.03) in comparison with animals from Ceska Kamenice. When comparing the two species collected in the Litvinov area, yellow-necked mice showed a statistically significant higher frequency of CREST+ MN (P = 0.0004) in comparison with bank voles. Mus spretus collected in the Huelva industrial area also showed higher frequencies of CREST+ MN (t = 3.90, P = 0.0005) when compared with animals from Doñana Park. The CREST+ MN frequency did not show significant differences between M.m.domesticus collected in the traffic-exposed area (Muro Torto Avenue) or in the Zoo area closed to traffic.
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We also compared the relative sensitivity of the fluorescent staining method, which uses both DAPI and propidium iodide, with conventional Giemsa staining in detecting MN in wild living rodents. Out of the total 128 animals collected, Giemsa stained slides were analysed for 118 in parallel to obtain two independent estimates for the frequencies of MN/animal. Both normochromatic and polychromatic erythrocytes were scored on Giemsa slides, because the two types of cells are indistinguishable after DAPI and propidium iodide staining. Comparison of the individual data obtained using DAPI and propidium iodide with those obtained with Giemsa staining showed a statistically significant difference between the two staining methods, when using the t-test for dependent samples (t = 5.33, P = 0.000001). The mean frequency of total MN in the 118 analysed animals was found to be 2.69 (SD = 2.53) MN/1000 erythrocytes in DAPI stained slides and 1.51 (SD = 1.30) MN/1000 erythrocytes in Giemsa samples. The difference was statistically significant (t = 4.42, P = 0.000015).
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Discussion |
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The present results show the feasibility of CREST staining of bone marrow MN from free-living rodents to identify MN containing entire chromosomes and indicate that CREST+ MN analysis in wild rodents may help to identify the mechanism of action of environmentally present chemical or physical agents. The data also show that the analysis of CREST+ MN is a useful method to investigate the genetic damage induced in wild rodents living in areas neighbouring industrial settlements and, thereby, monitor environmental contamination in situ.
Total MN frequencies, obtained by scoring slides stained using both DAPI and propidium iodide, and CREST+ MN frequencies were compared in rodent species living in areas contaminated with multiple chemicals and in areas with low environmental contamination. Differences in total MN frequencies between control and contaminated areas were statistically significant in two species (M.spretus in Spain and A.flavicollis in the Czech Republic), whereas CREST+ MN frequencies were significantly different in three species (M.spretus in Spain, A.flavicollis and C.glareolus in the Czech Republic).
Therefore, the analysis of CREST+ MN in erythrocytes allowed us to identify aneuploidy-inducing activities in areas contaminated by industrial settlements in the Czech Republic and Spain. Furthermore, the increased frequencies of CREST+ MN observed in areas with high environmental impact suggest that activities producing loss of chromosomes at mitosis may be characteristic of anthropogenic environments such as industrial settlements. Higher frequencies of total MN or CREST+ MN were also observed in animals collected in an area subjected to high traffic flow in Rome (Muro Torto Avenue) in comparison with the control area (the Zoo), although the differences were not statistically significant. This result is divergent from that obtained in a previous survey on the same areas (Ieradi et al., 1996). The difference may be explained by the recent decrease in motor vehicle flow on Muro Torto Avenue (Traffic Office of Roman Municipality) and the widespread use of unleaded fuel in Rome.
Our data show a very low proportion of MN containing entire chromosomes in animals collected in control areas, ranging from 3.2 to 8.7% of total MN among the four species examined. This result suggests that internal conditions and/or environmental activities disturbing the fidelity of chromosome distribution in natural habitats are present at much lower levels as compared with conditions or activities that produce chromosome structural breakage and CREST– MN. Interestingly, the percentages of CREST+ MN in mouse species collected in control areas (3.2% in M.spretus and 4.1% in M.m.domesticus) were much lower than those observed in untreated laboratory mice, which ranged between 30 and 49% in (102/E1xC3H/E1) F1 mice (Miller and Adler, 1990; Miller et al., 1991; Schriever-Schwemmer and Adler, 1994). This result also indicates that laboratory mouse stocks suffer an increased incidence of chromosome aneuploidy as compared with free-living rodents subjected to natural selection.
The conventional analysis of MN frequencies in free-living rodents has already been shown to be a reliable indicator of environmental stress in several studies (Materiy and Maslova, 1978; Cristaldi et al., 1985, 1990, 1991; Ieradi et al., 1996, 1998; Eckl and Riegler, 1997). However, the higher frequencies of total MN observed in slides stained with the fluorescent dyes DAPI and propidium iodide suggest that the use of both fluorochromes as counterstaining in CREST analysis may be useful in identifying MN of small size or faintly stained after Giemsa.
The comparative analysis of results obtained in A.flavicollis and C.glareolus collected in the same areas suggests a higher suitability of the first species in terrestrial ecogenotoxicological studies. In accordance with our results, laboratory studies investigating the response of four species of wild rodents to experimental irradiation by cytofluorimetric methods indicated that Apodemus may be a suitable genus for biomonitoring studies using MN frequencies (Abramsson-Zetterberg et al., 1997). However, it would be interesting to investigate the reasons underlying the resistance of Clethrionomys sp. to the induction of MN in terms of genetic make up, ecological characteristics and feeding habits of the species. Furthermore, in countries of Mediterranean climate, such us Spain and Italy, Mus sp. may be more readily available and suitable for these studies due to its restricted home range and relative abundance (Cristaldi et al., 1990; Ieradi et al., 1996).
In conclusion, our data suggest that the analysis of CREST+ MN may represent a sensitive end-point for the detection of environmental contamination by genotoxic xenobiotics, in addition to providing information on the mechanism of action of environmental pollutants. Finally, our study suggests that the application of molecular cytogenetic techniques such as CREST staining and fluorescent in situ hybridization using centromeric probes may be very useful not only in mechanistic studies but also in applied investigations on environment quality.
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Acknowledgments |
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We would like to thank S. Moreno for logistic support in Andalusia and the Director of the `Schola Humanitas', Mr M. Stovicek, for laboratory use and accommodations in Litvinov (Czech Republic). S. Sabelli, E. Liudi and M. Coriandri are acknowledged for assistance with CREST staining. We are also grateful to Dr C. Fuselli and Dr C. Maltese for Rome pollution data. This work was partially supported by the Human Capital and Mobility Programme of the European Community through the Doñana Biological Station Facility and by The Italian National Research Council (no. 96.00084.CT04, CNR, Italy). Studies performed in the Czech Republic were supported by the Grant Agency and Ministry of Education of the Czech Republic (no. 206/97/0850 and VS97102, granted to J.Z.)
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Notes |
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6 To whom correspondence should be addressed. Tel: +39 06 4 457 527; Fax: +39 06 4 457 529; Email: degrassi{at}axcasp.caspur.it
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References |
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Abramsson-Zetteberg, L., Grawé, J. and Zetteberg, G. (1997) Spontaneous and radiation-induced micronuclei in erythrocytes from four species of wild rodents: a comparison with CBA mice. Mutat. Res., 393, 55–71.[Web of Science][Medline]
Bolívar, J.P., García-Tenorio, R. and García-León, M. (1995) Enhancement of natural radioactivity in soils and salt marshes surrounding a non-nuclear industrial complex. Sci. Total Environ., 173/174, 125–136.
Brinkley, B.R., Tousson, A. and Valdivia, M.M. (1985) The kinetochore of mammalian chromosomes: structure and function in normal mitosis and aneuploidy. In Dellarco, V.L., Voytek, P.E. and Hollaender, A. (eds), Aneuploidy, Etiology and Mechanisms. Plenum Press, New York, NY, pp. 279–290.
Cagnin, M., Moreno, S., Aloise, G., Garofalo, G., Villafuerte, R., Gaona, P. and Cristaldi, M. (1998) A comparative study of Spanish and Italian terrestrial small mammal coenoses of different biotopes in Mediterranean Peninsular tip regions. J. Biogeogr., 25, 1105–1113.
Capanna, E., Civitelli, M.V. and Cristaldi, M. (1977) Chromosomal rearrangement, reproductive isolation and speciation in mammals. The case of Mus musculus. Boll. Zool., 44, 213–246.
Chen, H., Rupa, D.S., Tomar, R. and Eastmond, D. (1994) Chromosomal loss and breakage in mouse bone marrow and spleen cells exposed to benzene in vivo. Cancer Res., 54, 3533–3539.
Cristaldi, M., Ieradi, L.A., Licastro, E., Lombardi Boccia, G. and Simeone, G. (1985) Environmental impact of nuclear power plants on wild rodents. Acta Zool. Fenn., 173, 205–207.
Cristaldi, M., D'Arcangelo, E., Ieradi, L.A., Mascanzoni, D., Mattei, T. and Van Axel Castelli, I. (1990) 137-Cs determination and mutagenicity tests in wild Mus musculus domesticus before and after the Chernobyl accident. Environ. Pollut., 64, 1–9.[Medline]
Cristaldi, M., Ieradi, L.A., Mascanzoni, D. and Mattei, T. (1991) Environmental impact of the Chernobyl accident: mutagenesis in bank voles from Sweden. Int. J. Radiat. Biol., 59, 31–40.[Web of Science][Medline]
Czech Ecological Institute (1997) Statistical Environmental Yearbook of the Czech Republic 1997. The Ministry of the Environment of the Czech Republic and Czech Statistical Office, Prague, Czech Republic.
Degrassi, F. and Tanzarella, C. (1988) Immunofluorescent staining of kinetochores in micronuclei: a new assay for the detection of aneuploidy. Mutat. Res., 203, 339–345.[Web of Science][Medline]
de Stoppelaar, J.M., de Roos, B., Mohn, G.R. and Hoebee, B. (1997) Analysis of DES-induced micronuclei in binucleated rat fibroblasts: comparison between FISH with a rat satellite I probe and immunocytochemical staining with CREST serum. Mutat. Res., 392, 139–149.[Web of Science][Medline]
Eastmond, D.A. and Tucker, J.D. (1989) Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody. Environ. Mol. Mutagen., 13, 34–43.[Web of Science][Medline]
Eckl, P.M. and Riegler, D. (1997) Levels of chromosomal damage in hepatocytes of wild rats living within the area of a waste disposal plant. Sci. Total Environ., 196, 141–149.[Medline]
Gudi, R., Shandu, S.S. and Atwhal, S. (1990) Kinetochore identification in micronuclei in mouse bone marrow erythrocytes: an assay for the detection of aneuploidy-inducing agents. Mutat. Res., 234, 263–268.[Web of Science][Medline]
Ieradi, L.A., Giordano, D., Amarena, D., Cardarelli, E., Campanella, L. and Grossi, R. (1992) Heavy metal contamination in small rodents from urban areas. In Bohac, J. (ed.), Proceedings of the VI International Conference Bioindicatores Deteriorisationis Regionis, Ceské Budejovice, Czechoslovakia, 15–21 September 1991, pp. 219–227.
Ieradi, L.A., Cristaldi, M., Mascanzoni, D., Cardarelli, E., Grossi, R. and Campanella, L. (1996) Genetic damage in urban mice exposed to traffic pollution. Environ. Pollut., 92, 323–328.[Medline]
Ieradi, L.A., Moreno, S., Bolivar, J.P., Cappai, A., Di Benedetto, A. and Cristaldi, M. (1998) Free living rodents as bioindicators of genetic risk in natural protected areas. Environ. Pollut., 102, 265–268.
Ieradi, L.A., Zima, J., Sartoretti, A., Allegra, F., Wlosokova, E. and Cristaldi, M. (1999) Integrated system of evaluation of genotoxic damage on wild rodents from polluted areas (Czech Republic). In Proceedings of the 9th International Symposium on Bioindicatores, University Putra Malaysia, 24–27 November 1997, in press.
Martin, J.E., Bolivar, J.P., Respaldiz, M.A., Garcia-Tenorio, R. and da Silva, M.F. (1995) Environmental impact of fertilizer industries evaluated by PIXE. Nucl. Instrum. Methods Phys. Res., 103, 477–481.
Materiy, L.D. and Maslova, K.I. (1978) Micronuclei in peripheral blood cells of Microtus oeconomus Pall. living in areas of enhanced natural radioactivity. Radiobiologya, 18, 919–922.
McBee, K. and Bickham, J.W. (1988) Petro-chemical related DNA damage in wild rodents detected by flow cytometry. Bull. Environ. Contam. Toxicol., 40, 343–349.[Web of Science][Medline]
Miller, B.M. and Adler, I.D. (1990) Application of antikinetochore antibody staining (CREST staining) to micronuclei in erythrocytes induced in vivo. Mutagenesis, 5, 411–415.
Miller, B.M., Zitzelsberger, H., Weier, H.U. and Adler, I.D. (1991) Classification of micronuclei in murine erythrocytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated satellite DNA. Mutagenesis, 6, 297–302.
Schriever-Schwemmer, G. and Adler, I.D. (1994) Differentiation of micronuclei in bone marrow cells: a comparison between CREST staining and fluorescent in situ hybridization with centromeric and telomeric DNA probes. Mutagenesis, 9, 333–340.
Talmage, S.S. and Walton, B.T. (1991) Small mammals as monitors of environmental contaminants. Rev. Environ. Contam. Toxicol., 119, 47–108.[Web of Science][Medline]
Tanzarella, C., Degrassi, F., Zima, J., Ieradi, L.A. and Cristaldi, M. (1997) CREST-staining of micronuclei in free-living rodents. Mutat. Res., 379 (suppl. 1), S120.
Tice, R.R., Ormiston, B.G., Boucher, R., Luke, C.A. and Paquette, D.E. (1987) Environmental biomonitoring with feral rodent species. Environ. Sci. Res., 36, 175–179.
Tull-Singleton, S., Kimball, S. and McBee, K. (1994) Correlative analysis of heavy metal bioconcentration and genetic damage in white-footed mice (Peromyscus leucopus) from hazardous waste site. Bull. Environ. Contam. Toxicol., 52, 667–672.[Web of Science][Medline]
Zima, J. and Macholan (1995) B chromosomes in the wood mice (genus Apodemus). Acta Theriol., 3 (suppl.), 75–86.
Zima, J., Ieradi, L.A., Allegra, F., Sartoretti, A., Wlosokova, E and Cristaldi, M. (1999) The quantitative incidence of B chromosomes is not directly related to mutagenic environmental effects. Folia Zool., in press.
Received on November 24, 1998; accepted on March 18, 1999.
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