Belo Horizonte,MG, Brazil, October 24-28, 2011 A BRASILEIRA DE ENERGIA ... · 1000 per slide). The...

2011 International Nuclear Atlantic Conference - INAC 2011

Belo Horizonte,MG, Brazil, October 24-28, 2011

ASSOCIAÇÃO BRASILEIRA DE ENERGIA NUCLEAR - ABEN

ISBN: 978-85-99141-04-5

INAC 2011, Belo Horizonte, MG, Brazil.

MICRONUCLEI AS BIOMARKERS OF GENOTOXICITY OF GAMMA RADIATION IN AQUATIC ENVIRONMENTS

Luanna R. S. Silva1, Edvane B. Silva2, Ronaldo C. da Silva3, Francisco F.

Amâncio4, Ana M. M. A. Melo5

1Grupo de Estudos em Radioproteção e Radioecologia (GERAR),

Departamento de Energia Nuclear, Universidade Federal de Pernambuco,

Av. Prof. Luiz Freire, 1000, Recife, PE, Brazil

Email: [email protected]

2Grupo de Estudos em Radioproteção e Radioecologia (GERAR), Departamento de Energia Nuclear,

Centro Acadêmico de Vitória – UFPE Rua Alto do Reservatório, S/N – Bela Vista

CEP: 55608-680 Vitória de Santo Antão , PE, Brazil


3 Departamento de Genética, Universidade Federal de Pernambuco,

Av. Prof. Luiz Freire, 1000, Recife, PE, Brazil

e-mail: [email protected]

4 Laboratório de Radiobiologia Departamento de Biofísica e Radiobiologia,

Universidade Federal de Pernambuco, Av. Prof. Moraes Rego, s/n,

Recife, PE, Brazil Email: [email protected]

5Grupo de Estudos em Radioproteção e Radioecologia (GERAR),

Departamento de Biofísica e Radiobiologia, Universidade Federal de Pernambuco,

Av. Prof. Moraes Rego, s/n, Recife, PE, Brazil


ABSTRACT

Ionizing radiation is a genotoxic agent, inducing gene mutations and cellular death. Several efforts have been defendants in the development of techniques for measurement of radiation damage in biological systems. Among these techniques, micronuclei test has been showing as a great biomarker of DNA damage, being used in environmental monitoring to detect genotoxic agents in the environment. Additionally, organisms as Biomphalaria glabrata, freshwater molluscs, presents itself as an excellent model to assess damage caused by physical and chemical agents, due their biological and environmental characteristics. The snails were divided into groups of 5 individuals exposed to doses of 0 (control), 25, 35, 45 and 55 Gy of 60Co. After 48 hours of irradiation, the hemolymph was collected and prepared the slides, which were stained with Giemsa and analyzed the cellular changes in haemocytes Statistical analysis was accomplished through chi-square test, ANOVA and Tukey test (p< 0,05). The results indicated that B. glabrata showed to be sensitive to gamma radiation. The snails irradiated with 35 Gy showed a decrease of haemocytes, while that of 55 Gy increased. Cellular and morphological changes were observed at doses of 35, 45 and 55 Gy and the dose of 55 Gy, the most radiotoxic.




ISBN: 978-85-99141-04-5


As the frequency of micronuclei, were statistically significant differences between control and irradiated. The results suggest that morphological analysis and the frequency of micronuclei in haemocytes of B. glabrata can be considered reliable parameters in assessing the genotoxic action in aquatic environments.

1. INTRODUCTION

Ionizing radiation is considered a powerful genotoxic agent that can induce gene mutations, chromosomal aberrations and cell death [1][2]. Irradiated cells show a tendency to increase or decrease the number of chromosomes and many of the cellular effects are due to changes during replication or repair of DNA damage. These changes can cause chromosomal instability transmitted, resulting in various effects, even after several mitotic cycles [3][4]. Recently there has been great interest in developing techniques that assist in understanding of possible biological damage that occur after exposure of organisms to certain doses of ionizing radiation. We can cite: the dominant lethal test [5][6][7], Comet assay[8][9]; micronucleus assay [10][11][12] and chromosomal aberrations[13][14][15][16]. The micronucleus (MN) are structurally smaller nuclei representing the genetic material that was lost from the main core, as a consequence of genetic damage that can be caused by physical, chemical or biological agents, capable of interfering in the process of binding of the chromosome fibers zone or which may lead to loss of genetic material, such as fragments of chromosomes or whole chromosomes [17][9]. The micronucleus test has shown to be a great biomarker of action of genotoxic or mutagenic agents. For this reason it is used in the study of environmental genotoxicity for the purpose of biomonitoring [9]. The study of monitoring environments is important due to various physical and chemical agents that are released into the environment from various human activities [18]. Biomonitors organisms such as snails Biomphalaria glabrata, which are in aquatic environments can suffer damage to their biomolecules. For this reason, the development of sensitive techniques and low cost detection of these effects are of great importance for biomonitoring. One technique that has been used with other biomarkers, is using the micronucleus test in biomonitoring environmental, such as the golden mussel (Limnoperna fortunei), which was used as a biomonitor organism detection of pollutants, the parameter used was the observed micronuclei in haemocytes of the mollusk [9]. Another study accomplished with the micronucleus test in hemolymph of molluscs to biomonitoring was developed by Pavlica [10], with the mussel Dreissena polymorpha and Gastropoda Planorbarius corneus. The mollusk, Biomphalaria glabrata due to their biological characteristics and environmental, has been presented as great experimental model allowing the evaluation of effects produced by physical and chemical agents[19][20][21][7]. For its wide geographical distribution and interference on human health, as a vector of schistosomiasis mansoni intermediate, earn interest not only as a model of physiological changes induced by




ISBN: 978-85-99141-04-5


external stimuli, but also by enabling the standardization of methods of population control, or as bioindicators and biomarkers of interfering environmental [19]. The objective of this study is to evaluate cellular changes in haemocytes of the snail Biomphalaria glabrata submitted to different doses of 60Co gamma radiation and analyze the frequency of micronuclei in haemocytes. Provide support for the standardization of a new tool in biomonitoring test the action of physical and chemical agents released in aquatic environments using the micronucleus test in haemocytes of Biomphalaria glabrata.

2. METHODOLOGY 2.1. Animals For the experiments, were used hemolymph of snails Biomphalaria glabrata, obtained in the creation of mollusks Laboratory of Radiobiology, Department of Biophysics and Radiobiology, Federal University of Pernambuco (UFPE/Brasil). Were selected molluscs B. glabrata pigmented presenting the following characteristics: young adults with shell diameter 10 mm, 2 months of age and sexually mature, evidenced by the production of eggs. 2.2. Irradiation of molluscs The snails were irradiated with a 60Co source at the Department of Nuclear Energy -DEN / UFPE. The doses used were 25, 35, 45 and 55 Gy and 0 (control), with five replicates for each dose. The doses were established taking into account previous work in the Laboratory of Radiobiology Department Biophysics and Radiobiology, UFPE-Brazil and the Study Group on Radiation Protection and Radioecology –GERAR of DEN-UFPE-Brazil [20]. In which, observed that certain doses acted differently in biological systems. 2.3. Preparation of slides The hemolymph of B. glabrata was collected with the support of a micropipette. The procedure is to stimulate the release of hemolymph through successive touches in the region Foot of the animal. The region to be manipulated, suffers a retraction in the shell then releasing the hemolymph, which is collected with a micropipette. [10]. After obtaining the hemolymph, a rate of 0.1 ml was mixed with 0.1 ml of Ringer and 10 mM EDTA. The whole procedure took place on the microscope slide and then placed in a moist chamber for 30 minutes. After having spent the waiting time, the cells were fixed with 1% glutaraldehyde in Ringer's solution for 5 minutes, then rinsed with Ringerand stained. The staining method used was conventional (Giemsa), this staining was done using a Giemsa solution (Merck), Azur-eosin-methylene blue diluted in phosphate buffer, pH 6.8, obtaining a final solution to 5%. The slides were submerged in this solution for 5 minutes, then washed with distilled water and observed under an optical microscope (MEDILUX) with increased 1000x. 2.4. Cell counting




ISBN: 978-85-99141-04-5


For each stained cells 1000 were counted in with distinction intact nuclear and cellular analyzing the presence of micronuclei and cellular abnormalities, the following criteria of the [10]. The micronucleus number were scored by visual inspection (Medilux) and were identified according to the following criteria:

1. Diameter smaller than 1 / 3 the size of the nucleus;

2. Color and texture similar to the nucleus;

3. Do not be in direct contact with the nucleus;

2.5. Statistical analysis Statistical analysis of MN frequency between groups was performed using the chi-square test (χ2), significance level of 5% e p<0,05 (BioEstat program- version 5.0). The analysis between the mean numbers of haemocytes per dose were performed using ANOVA and Tukey test with p<0,05 were considered to be statistically significant

3. RESULTS 3.1. Analysis of cellular radiosensitivity of haemocytes of B. glabrata It can be observed in Table 1, the total number of haemocytes originating of B.glabrata control group (no exposed) and exposed to 60Co gamma radiation doses and of 25, 35, 45 and 55 Gy.

Table 1. Haemocytes originating in the irradiated and non irradiated snails.

As shown in Table 1, the number of micronuclei after irradiation was lower as compared to control group. The dose of 35 Gy that had the lowest number of cells per slide analyzed (≥ 1000 per slide). The hemolymph of the animals exposed to dose of 55 Gy showed an increase

Dose (Gy) Total number of haemocytes 0 5042

25 4442

35 3334

45 4799

55 5678




ISBN: 978-85-99141-04-5


in the number of haemocytes in relation to experimental other and control groups. Using ANOVA statistical test we find that the mean number of cells to doses of 35 Gy and 55 are significantly different at 5%. During the analysis of different results were observed morphological changes in haemocytes of molluscs subjected to doses of 35, 45 and 55 Gy of the gamma radiation. These modifications consist in the formation of bilobulated nucleus and nucleoplasmic bridges (Figure 1).

Figure 1. Changes found in the hemolymph of B. glabrata irradiated at doses of 35, 45 and 55 Gy. (A) bilobulated nucleus, (B) nucleoplasmic bridges (shown by red arrow). (1000x)

Another cellular alteration was exclusively observed during microscopic analysis of hemocytes originating from snails exposed to a dose of 55 Gy. The hemocytes presented misshapen nuclei and numerous vacuoles in the cytoplasm as well as marginalization of chromatin forming dense aggregates, which indicates cellular apoptosis (Figure 2).

A B




ISBN: 978-85-99141-04-5


Figure 2. vacuolization of cellular cytoplasm and marginalization of chromatin in haemocytes of Biomphalaria glabrata submitted to 55 Gy of 60Co gamma radiation (1000x).

3.2. Frequency of micronucleus

Mollusks exposed to gamma radiation we noticed an increased frequency of micronucleus in compared to control group (Figure 3). The chi-square test (χ2) showed statistically significant differences (p <0.05) between the frequency of micronucleus of control group and irradiated. The dose of 45 Gy showed a higher frequency of micronuclei when compared to other doses of radiation, showing greater significance compared to control (p = 0.0002). Although it is shown that after irradiation there was an increased frequency of MN in the control group, this increase did not occur in a dose-dependent (Figure 3) (Figure 4).




ISBN: 978-85-99141-04-5


Figure 3. Frequency of Micronuclei B. glabrata subjected to 60Co gamma radiation. * Significant (p< 0,05), ** Very significant (p<0,001) *** extremely significant (p<0,0001) compared to control.

Figure 4. Micronucleus in haemocyte of B. glabrata.

4. DISCUSSION

Noting differences in the amounts of haemocytes at the doses used, suggest the existence of a defensive behavior between different groups of mollusks irradiated with high doses of 60Co




ISBN: 978-85-99141-04-5


gamma radiation. [20], analyzing the effects of high doses of gamma radiation on fertility B. glabrata noted that the number of embryos produced by irradiated molluscs vary independently of the dose. Another aspect noted by Cantinha [20] was that the exposure dose of 40 Gy had the lowest number of embryos compared to control, while the exposure dose of 60 Gy did the reverse, this is, the dose had the highest cells in the experimental group compared to control. These data are similar to those found in this study that showed an increase in the number of haemocytes in a dose of 55 Gy and a decrease in dose of 35 Gy. It may be noted that despite the increased number of haemocytes after exposure of molluscs dose of 55 Gy, the viability of these cells can be questioned because they were found several morphological changes that made it unfeasible. [20], in their experiments, we observed that the dose of 60 Gy, close to the one used in this study had a higher number of embryos and increased cell inviability, which is similar to the results here in case of inviability haemocytes and cell observed. According [1], the different tissue types have different responses to ionizing radiation. The comparison of the radiosensitivity of different cell lines indicates that the cells that divide rapidly are more radiosensíveis the slow reproduction (law Tribondeau and Bergonié), although this rule has exceptions (lymphocytes and some melanomas). When we analyze the radiosensitivity of haemocytes of B. glabrata irradiated can see that all experimental groups showed changes in the number and structural of cells of the cells. These changes follow the principle of the law mentioned above. Were found nuclear abnormalities and increased frequency of micronuclei in oral mucosa cells of smokers and nonsmokers and found that 96% of the smokers had nuclear deformities, such as binucleated cells, nuclear buds and nucleoplasmic bridges [22], these results also found in this work, the mollusk B. glabrata exposed to gamma radiation As described by [23], the formation of bridges nucleoplasmic in lymphocytes, probably originated when the centromeres of dicentric chromosomes are pulled to opposite poles during anaphase and the resulting DNA bridge is covered by the nuclear envelope. This cellular changes described by the author may have been the same factor that led to the formation of bridges nucleoplasmic haemocytes of B. glabrata. It is suggested that these nuclear changes are caused by the action of physical and chemical agents on cells, causing mutagenicity. The characteristics found in cells irradiated at a dose of 55 Gy, as vacuolization of the cell, are suggestive of induction of apoptosis. Studies by Pavlica [10] with mussel (D. polymorha) and gastropods (P. corneus) exposed to pentachlorophenol (PCP) at concentrations of 450 and 600 mg / l, also found cells with similar characteristics, suggesting also that it was apoptosis cellular. Pavlica (2000) suggested that apoptosis of haemocytes was due to the inhibiting action of PCP Ca2+ pump. It is known that a cause of apoptosis is linked to calcium levels inside the cell [24][25]. The stimulus for apoptosis radiation-induced appears to be related to the number of breaks produced in DNA, the rate at which they occur and the speed and efficiency of repair [26]. Assuming that DNA is the initial target for apoptosis radiation-induced, the cellular response to damage in the form of DNA repair can lead to cell death [27][28]. Thus, it is suggested that




ISBN: 978-85-99141-04-5


the probable apoptosis emerged in haemocytes was irradiated by the action of radiation on the cell's DNA. The various nuclear changes cited were not the only experimental findings in this paper. It was also observed that ionizing radiation induced other changes in DNA, as the presence of micronucleated cells. Studies by [29], radiating culture of human lymphocytes with doses of 0, 0.1, 0.25, 0.5, 1 Gy of 60Co source with dose rate 0.25 Gy / min, observed that there was increased frequency of micronuclei in a dose-dependent, the different behavior found in this study for the case of B. glabrata. This difference may be due to use of sources with different dose rates. The present study used a source of 60Co with a dose rate of 6.912 kGy / h, while in experiment [29] the dose rate was 0.25 Gy / min (0.015 kGy / h). There are principles governing radiobiology which clarify the events that took place, ie, there is a dose-response relationship, where the emergence of a radiolesion depends on the radiosensitivity of biological structure and exposure to radiation, ie, the shorter the exposure time , the smaller the effects caused by radiation. Lymphocyte culture showed a higher sensitivity to radiation when compared to haemocytes, but the haemocytes were collected from subjects exposed and not cell culture. We can conclude that haemocytes showed radiosensitivity to ionizing radiation, but the radiosensitivity was not dependent dose. It is known that the haemopoietic system is one of the most radiosensitive, so the exposure of individuals to whole-body irradiation may have different biological effects. Biological dosimetry studies using lymphocytes to check the biological effects suffered by people accidentally or occupationally exposed to radiation [16]. Were observed in this study that snails exhibited similar behavior, because the defense cells [30], the haemocytes, also undergo morphological changes and the presence of MNs when exposed to ionizing radiation, suggesting the feasibility of their use in environmental biomonitoring. No data on frequency of MN in B. glabrata irradiated in the literature. But studies of other species of snails exposed to chemicals different evaluated the frequency of MN can be used in monitoring aquatic environments [31][32]. Other studies were performed using the B. glabrata as a bioindicator of ionizing radiation in aquatic environments, yet these studies assessed both macroscopically as changes in fecundity, fertility and mortality of molluscs [33], but cellular changes were not perceived by these tests. Therefore, the results in our experiments using haemocytes of B. glabrata suggests a analysis tool new that biological effects suffered by the DNA molecule after exposure to radiation and possibly to other genotoxic agents present in aquatic environments.

5. CONCLUSIONS

So we can conclude that the adult snails of B. glabrata were sensible to the effects of 60Co gamma radiation and their haemocytes can be used as an indicator of the presence of high doses of ionizing radiation in aquatic environments and the use of MN proved to be a sensitive tool for the monitoring areas affected by radiation.




ISBN: 978-85-99141-04-5


ACKNOWLEDGEMENTS

This research was financially supported by the CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). We are grateful to the Laboratory Gamalab of Department of Nuclear Energy of the Federal University of Pernambuco for access to 60Co source

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30. S. S. SOUZA, A origem das células de defesa (hemócitos) em Biomphalaria glabrata (Say, 1818). Dissertação de mestrado- Universidade Federal da Bahia. ICS, Instituto de ciências da saúde, 2006.

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