The Comet assay for the detection of DNA damage in Mus spretus from Doñana National Park

Doñana Park (Spain), a protected area in Europe, was affected by an environmental disaster in April 1998 that caused the spreading of acidic water and mud full of toxic metals from the Aznalcollar pyrite mine. In order to assess the contamination in the area and to monitor the possible biological effects of the toxic spill, a series of coordinated studies was performed utilizing several animal species living in that area. We performed genotoxicity monitoring using the Comet assay on peripheral blood leukocytes of the Algerian mouse (Mus spretus), a nonprotected rodent suitable as a bioindicator. The mice were sampled in different areas 6 months after the ecological disaster and again 1 year later. Our results showed that in 1998 all the areas examined were contaminated, as determined by an increase in the Comet assay parameters in the analyzed animals, whereas a significant decrease in the values of these parameters was observed in the 1999 samples, which were collected in a riverside area subject to tide flows. Thus, the Comet assay has proven to be an interesting and sensitive tool in studies of environmental genotoxicity. © 2002 Elsevier Science (USA). All rights reserved.Peer Reviewe

Biomonitoring of Doñana National Park using the Algerian mouse (Mus spretus) as a sentinel species

The aim of this work was to analyse the biological effects of man-made activities on Algerian mice (Mus spretus Lataste, 1883) living in Donana National Park (DNP) and its surroundings (SW Spain). Biomonitoring was performed, before and after the accident in the Aznalcollar pyrite mine (April 1998), to investigate the environmental risk due to the release of chemical waste in the Guadiamar River, which reaches the DNP marshlands. Mice (N=420) were collected from different sites, inside and outside the Park, over a period of five years (1995-1999). The experimental design consists of using different biomarkers to detect the possible biological effects induced by xenobiotics in somatic and germ cells. Micronucleus test, sperm abnormality assay and Comet assay were performed to assess genotoxic damage. In addition, concentrations of some heavy metals (Pb, Cd, Zn, Cu) and arsenic were measured in animal livers. Data obtained indicate that the biological damage observed in mice is the effect of the contaminants released by the flooding and toxic spill after the accident of the Aznalcollar mine. In particular, micronuclei frequencies were significantly higher in mice collected from the sites outside the Park, directly affected by the spread of the contaminated mud, compared to those from areas inside the Park not directly affected. Furthermore, results showed that a chronic contamination was present also before the mining accident in some areas of DNP, and this effect was still present one year after the disaster. Therefore, the study suggests that biomonitoring had to be carried out in Doñana to determine the long-term biological effects of environmental pollution and the risk for the ecosystems in this protected area. In conclusion, on the basis of the important results found in this multidisciplinary study, we should conclude that the collaboration of different competences is desirable to create a pilot center for territorial monitoring.Peer Reviewe

Biomonitoring of Doñana National Park using the Algerian mouse (Mus spretus) as a sentinel species.

The aim of this work was to analyse the biological ef-fects of man-made activities on Algerian mice (Mus spretus Lataste, 1883) living in Doñana National Park (DNP) and its surroundings (SW Spain). Biomonitoring was per-formed, before and after the accident in the Aznalcollar pyrite mine (April 1998), to investigate the environmental risk due to the release of chemical waste in the Guadiamar River, which reaches the DNP marshlands. Mice (N=420) were collected from different sites, in-side and outside the Park, over a period of five years (1995-1999). The experimental design consists of using different biomarkers to detect the possible biological effects induced by xenobiotics in somatic and germ cells. Micronucleus test, sperm abnormality assay and Comet assay were per-formed to assess genotoxic damage. In addition, concentra-tions of some heavy metals (Pb, Cd, Zn, Cu) and arsenic were measured in animal livers. Data obtained indicate that the biological damage observed in mice is the effect of the contaminants released by the flooding and toxic spill after the accident of the Aznalcollar mine. In particular, micro-nuclei frequencies were significantly higher in mice col-lected from the sites outside the Park, directly affected by the spread of the contaminated mud, compared to those from areas inside the Park not directly affected. Further-more, results showed that a chronic contamination was present also before the mining accident in some areas of DNP, and this effect was still present one year after the disaster. Therefore, the study suggests that biomonitoring had to be carried out in Doñana to determine the long-term biological effects of environmental pollution and the risk for the ecosystems in this protected area. In conclusion, on the basis of the important results found in this multidisciplinary study, we should conclude that the collaboration of different competences is desirable to create a pilot center for territorial monitoring

Genotoxic monitoring on wild rodents living in protected areas

The aim of this work was to carry out a genotoxic monitoring on small rodents living in protected areas to determine the baseline values and to establish if some of these areas may be used as reference points for successive genotoxic monitoring. To this purpose, peripheral blood micronucleus test was applied for the first time to a high number of free living rodents (N=500). Six protected areas, located in the Centre of Italy, were studied. In total 241 Apodemus sylvaticus, and 259 Apodemus flavicollis were analysed. Results obtained show that the mean frequency of micronucleated erythrocytes is lower than the threshold value (2ME/1000E) established for laboratory mice. Micronuclei frequency and the percentage of animals with ME/1000E >= 2 are significantly (p = 2 significantly higher (p < 0.005) than mice from "Gran Sasso Logo" National Park and "Lago di Penne" Regional Natural Reserve. In conclusion, data obtained indicate that "Castelporziano" Estate and "Circeo" National Park are exposed to non negligible genotoxic impact. Results may be the basis for a genotoxic monitoring which has to be performed applying, at regular intervals, peripheral blood micronucleus test also in animals living in protected areas. This study highlights the need to consider the risk of environmental contamination inside the protected areas, to understand the related problems and, consequently, to carry out an adequate management and control program of the territory

Free-living rodents as bioindicators of genetic risk in natural protected areas

A study was carried out in the south of the Iberian Peninsula in an industrial area (Huelva city) and in two natural areas ('Reserva Biologica de Doñana' in Doñana National Park and 'Isla Cristina' Marshland Natural Park), located windward and leeward of the industrial area, to estimate genetic risk induced by environmental pollution in wild mice. Mutagenetic effects in Algerian mice (Mus spretus) free living in the industrial area and the range of contamination on other populations of the same species living in the two natural protected areas, were investigated. Micronucleus test on bone marrow and peripheral blood was used to detect genetic alterations. A statistically significant increase in the frequency of micronuclei was observed in animals from the industrial area and from the Doñana Biological Reserve in comparison with those from 'Isla Cristina' marshlands. The results suggest that the mutagenicity level in natural populations living in protected areas should be controlled, and wild mice could be used as key organisms in pollution monitoring and environmental conservation. © 1998 Elsevier Science Ltd. All rights reserved.Peer Reviewe

Genotoxic damage in free-living Algerian mouse (Mus spretus) after the Coto Doñana ecological disaster

The Donana National Park (Spain), one of the most important wildlife sites in the West of Europe, was affected (25 April 1998) by the spill of acidic waste rich in toxic metals (mainly zinc, lead, copper, etc.), arsenic and aromatic amines from the Aznalcollar mine accident. Micronuclei test with May Grunwald-Giemsa and with CREST-antikinetocore staining using DAPI as counterstaining were performed on peripheral blood erythrocytes from Algerian mice to evaluate genotoxic damage. Animals were collected in four locations each differently affected by the disaster. Higher frequencies of micronuclei and CREST-positive micronuclei were observed in the sites, which were reached by toxic sludge and contaminated water in comparison with those located within the park. The results obtained applying the two methods indicate that DAPI staining is more sensitive in detecting micronuclei. Genotoxic biomonitoring should be further carried out in the area to control the mutagenetic level in natural populations. Copyright © 2001 Elsevier Science Ltd.Peer Reviewe

Genotoxic damage in free-living Algerian mouse (Mus spretus) after the Coto Doñana ecological disaster.

The Doñana National Park (Spain), one of the most important wildlife sites in the West of Europe, was affected (25 April 1998) by the spill of acidic waste rich in toxic metals (mainly zinc, lead, copper, etc.), arsenic and aromatic amines from the Aznalcollar mine accident. Micronuclei test with May Grunwald-Giemsa and with CREST-antikinetocore staining using DAPI as counter-staining were performed on peripheral blood erythrocytes from Algerian mice to evaluate genotoxic damage. Animals were collected in four locations each differently affected by the disaster. Higher frequencies of micronuclei and CREST-positive micronuclei were observed in the sites, which were reached by toxic sludge and contaminated water in comparison with those located within the park. The results obtained applying the two methods indicate that DAPI staining is more sensitive in detecting micronuclei. Genotoxic biomonitoring should be further carried out in the area to control the mutagenetic level in natural populations