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Bioindicators in radiation protection

Abstracts

Biodosimetry is the evaluation of absorbed dose using bioindicators. Among chromosomal aberrations, scoring of dicentrics from peripheral human blood has been used as gold standard for biodosimetry, although in case of large scale incidents its use presents some drawbacks. Advances in technology have led to new investigations allowing or permitting the use of new methods which not only improve this "classical" biodosimetry but permits the design of other bioindicators making possible faster analyses, particularly in events where many persons may have been exposed. This report presents an overview of some recent studies developed by the "Grupo de Estudos em Radioproteção e Radioecologia - GERAR", Nuclear Energy Department of UFPE - Brazil, involving biodosimetry.

Biodosimetry; Physical Dosimetry; Radioprotection; Bioindicators; Protein expression


Biodosimetria pode ser definida como a avaliação da dose absorvida individualmente usando bioindicadores. Entre as aberrações cromossômicas, a quantificação de discêntricos em sangue periférico humano tem sido usada como padrão ouro in biodosimetria, embora essa técnica possua várias limitações em casos de incidentes envolvendo um grande número de indivíduos. Os avanços tecnológicos têm proporcionado novas ferramentas de investigações, resultando no desenvolvimento de novos métodos com intuito de otimizar essa dosimetria biológica "clássica", bem como na descoberta de novos bioindicadores, com o objetivo de possibilitar avaliação de exposição individual de forma mais rápida, em particular em situações envolvendo grande número de indivíduos expostos. Este texto apresenta um breve relato de alguns dos estudos desenvolvidos pelo Grupo de Estudos em Radioproteção e Radioecologia - GERAR, do Departamento de Energia Nuclear da UFPE - Brasil, associados ao emprego dos "clássicos" e novos bioindicadores em biodosimetria.


RADIOBIOLOGY, RADIOPROTECTION AND DOSIMETRY

Bioindicators in radiation protection

Ademir Amaral* * Author for correspondence ; Thiago Salazar Fernandes; Mariana Brayner Cavalcanti

Grupo de Estudos em Radioproteção e Radioecologia; Departamento de Energia Nuclear; Universidade Federal de Pernambuco; Av. Professor Luiz Freire, 1000; 50740540; amaral@.ufpe.br; Recife - PE - Brasil

ABSTRACT

Biodosimetry is the evaluation of absorbed dose using bioindicators. Among chromosomal aberrations, scoring of dicentrics from peripheral human blood has been used as gold standard for biodosimetry, although in case of large scale incidents its use presents some drawbacks. Advances in technology have led to new investigations allowing or permitting the use of new methods which not only improve this "classical" biodosimetry but permits the design of other bioindicators making possible faster analyses, particularly in events where many persons may have been exposed. This report presents an overview of some recent studies developed by the "Grupo de Estudos em Radioproteção e Radioecologia - GERAR", Nuclear Energy Department of UFPE - Brazil, involving biodosimetry.

Key words: Biodosimetry, Physical Dosimetry, Radioprotection, Bioindicators, Protein expression

RESUMO

Biodosimetria pode ser definida como a avaliação da dose absorvida individualmente usando bioindicadores. Entre as aberrações cromossômicas, a quantificação de discêntricos em sangue periférico humano tem sido usada como padrão ouro in biodosimetria, embora essa técnica possua várias limitações em casos de incidentes envolvendo um grande número de indivíduos. Os avanços tecnológicos têm proporcionado novas ferramentas de investigações, resultando no desenvolvimento de novos métodos com intuito de otimizar essa dosimetria biológica "clássica", bem como na descoberta de novos bioindicadores, com o objetivo de possibilitar avaliação de exposição individual de forma mais rápida, em particular em situações envolvendo grande número de indivíduos expostos. Este texto apresenta um breve relato de alguns dos estudos desenvolvidos pelo Grupo de Estudos em Radioproteção e Radioecologia - GERAR, do Departamento de Energia Nuclear da UFPE - Brasil, associados ao emprego dos "clássicos" e novos bioindicadores em biodosimetria.

INTRODUCTION

The discovery of ionizing radiations (IR) may be considered as a watershed in human history for improving the quality of life, especially as an important tool in medicine and as source of energy. However, the initial lack of knowledge about the physical, chemical and biological phenomena involved in the interactions of IR with living tissues was responsible for deaths among the pioneering radiation workers and exposed patients (Kathren, 1962; Caufield, 1990). Since then, radiation protection (radioprotection) was introduced as the science in charge of protecting people (workers or not) and the environment from the harmful effects of radiation. Radioprotection has been continuously reviewed, and well documented in detail over the years. The importance of radiation protection programs has grown due to the increase in the application of ionizing radiation as much as because of the public interest on potential risks associated with radiation. Up today, radioprotection management is based on the evaluation of specific dosimetric quantities. Absorbed dose, the amount of energy delivered to matter by IR per unit of mass, is the fundamental physical quantity to evaluate potential biological response resulting from exposure to radiation. Although for the International System of Units, the unit of absorbed dose is the gray (1 Gy = 1 J.kg-1) (ICRP, 1991), the rad unit (1 rad = 0,01 Gy = 1 cGy) is commonly used principally when medical radiology is performed.

In general, absorbed dose can be directly determined by physical dosimeters (such as film or TLD badges, semiconductors, ionization chambers) or, indirectly, by numerical models. However, in most cases of real or suspected accidental exposures to IR, physical dosimetry cannot be straightforwardly performed for retrospective estimates, mainly due to the lack of information about the irradiation conditions.

In such situations, biological dosimetry (biodosimetry) has been proposed as an alternative method, which is based on the investigation of cellular and molecular changes (bioindicators) induced by IR, in order to correlate them with the radiation dose.

In terms of radioprotection, bioindicators can be defined as all individual biological endpoints (macro-or microscopic changes) used to indicate an exposure to IR, principally, representing an early event that occurs as a result of IR interaction with living tissues (Bonassi and Au, 2002).

First Bioindicators: Physical Symptoms

The first observed bioindicators were the early physical symptoms resulting from individual exposure to ionizing radiation, corresponding to the prodromal stage following an acute irradiation, such as nausea, vomiting, and diarrhea. The severity and duration of those symptoms are related to the absorbed dose and physical characteristics of the radiation, since equal absorbed doses from different forms of radiations (such as X and g rays; electrons, protons neutrons and α particles) do not imply the same level of biological response. For example, for 1 to 2 Gy of gamma irradiation, nausea and vomiting appear within 6 h after exposure (Roman et al., 1997; Lushbaugh et al., 1982). On the other hand, an absorbed dose of 2 Gy, as a result of external skin contamination, without absorption, will certainly not lead to such physical symptoms.

Another important clinical bioindicator is erythema, which also depends of the type of radiation and the skin condition, having a median dose estimate of 6 Gy for its appearance. All these symptoms are important "macroscopic" bioindicators of acute irradiation.

Although those "visual" symptoms should be considered as part of the radiation history, they are still found as a result of overexposure to ionizing radiation. Fig. 1 shows the result of chronic overexposure of the left hand of a surgeon-traumatologist studied in 2004 in Recife (Brazil) (Fernandes, 2006).

Fig. 1

Chromosomal Aberrations and Micronulcei

Chromosome aberrations (CA) in circulating lymphocytes of human blood is the most extensively studied system (Bender, 1964; 1969; Lloyd et al., 1986; Lloyd et al., 2000; Amaral, 2005).

Some unstable chromosome-type aberrations (such as dicentrics and rings) are generally considered to be specific to radiation exposure, although in certain circumstances a few chemical agents can also induce them. Several studies have shown no significantly differences between in vivo and in vitro CA in irradiated blood lymphocytes -(Dossou et al., 2000; IAEA, 2001). Thus, the dose-effect relationship obtained after in vitro irradiation of blood is generally employed as a calibration curve to estimate effects from in vivo irradiations (Ramalho et al., 1995).

Nowadays, two cytogenetic methods have become commonly used: scoring unstable CA (principally dicentrics) and the FISH (fluorescence in situ hybridization), the latter, based on the use of fluorescence probes to visualize dicentrics and translocations without the prerequisite of the time-consuming karyotyping (Fernandes et al., 2008).

Fig. 2 presents a dicentric evidenced by FISH, making clear the distinction of two centromeres highlighted in red color.


Works developed by Fernandes et al. (2008) evaluated the relative accuracy of this analysis for unstable chromosomal aberrations in lymphocyte metaphases using four cell staining options, namely: Giemsa; 4 6-Diamidine-2'-phenylindole techniques highlight centromeres. This study suggests that standard block staining method is the choice for routine sampling because it reduces cost and time for slide preparation. On the other hand, centromere highlighting methods, such FISH, allow a more precise detection of dicentrics and provide confirmation, resolving unequivocal identification of suspected dicentrics.

Dose estimates based on the scoring of chromosome aberrations take into account the uniform whole body irradiation. This is a simple assumption since the majority of accidental or overexposures involve partial-body irradiation, and interpreting the yield in such cases become an outstanding difficulty. In situations such the case of surgeon doctor (Fig. 1), where the overexposure was focused on a very small percentage of the body volume (left hand), cytogenetic studies presented no deviation from background (Fernandes et al., 2006). On the other hand, if a large proportion of the body is overexposed, it is possible to detect dicentrics present in the fraction of lymphocytes from the irradiated part of the body. Fig. 2 presents a dicentric evidenced by FISH, making clear the distinction of two centromeres highlighted in red color (Fernandes et al., 2008).

Micronulei

Micronuclei (MN) are cytoplasm chromatin, masses that arise from centric or acentric products of damaged chromosomes (Uma Devi et al., 1998; IAEA, 2001). In other words, MN are a kind of unstable CA byproduct. They have the appearance of small nuclei, in addition to the cell's nucleus, as shown in binucleated cells of the Fig. 3, and they are identified during mitogen-activated human lymphocytes division, blocking at cytokinesis stage. As the scoring of MN is more sensitive and faster than the scoring of CA, improvements in MN methodology for biodosimetry have been tested (IAEA, 2001). As for unstable chromosomal aberration, in order to interpret the scoring of MN in terms of radiation dose, a calibration curve (frequency MN versus dose) is also necessary.


Measuring Protein Expression

IR can cause different injuries in DNA, which induce the expression of several proteins in order to repair such damages. Among the proteins expressed during the DNA repair process, p53 has an important role concerning the genome integrity conservation. This protein is found in the cytoplasm in small concentration and has a short average life. However, a variety of physicochemical agents, after damaging the DNA molecule, trigger the expression of p53 increasing its concentration and its average half-life,making possible its detection (Levine, 1997; Rössner Jr. et al., 2004). Thus, the correlation between the increasing p53 expression and the irradiation may constitute a fast and reliable method of individual monitoring in cases of accidental or suspected exposures to IR.

The advent of fluorescent techniques, particularly flow cytometry, opened new possibilities in terms of detection of intracellular bioindicators. This technique can allow the measurement of multiparameters of cells, such as: size, granularity and complexity (Becton Dickinson and Company, 2000). Hence, Cavalcanti et al. (2008) were the first to propose the evaluation p53 protein expression levels as bioindicator of individual exposure to ionizing radiation by flow cytometry. The authors observed that the p53 expression increased with the absorbed doses.

Using the same methodology described by the authors (Cavalcanti et al., 2008), peripheral blood sample from a healthy donor were exposed to a 60Co source (dose-rate: 196.67 cGy/min). Fig. 4 presents the results obtained for two blood samples without irradiation (A), and irradiated with 4 Gy (B), following 72 h of incubation in a 5% CO2 at 37 ºC with phytohemagglutinin (PHA). For the non-irradiated sample the p53 expression level (region UL) was 2.76%, while for the irradiated one the percentage of this protein expression was 23.62%. It is possible to verify that the p53 expression levels in samples have increased for all absorbed dose.


The high expression level of p53 in the dot-plot B emphasized the potential of this methodology as screen method to investigate accidental exposures. Obviously, for this, one should keep in mind that individual radiosensitivity plays an important role in this kind of studies.

CONCLUSION

In order to evaluate individual irradiation, rapid and reliable dose estimates are crucial for risk assessments. In individual evaluation, biodosimetry may represent more than a complementary methodology to physical dosimetry, especially when this latter is not feasible. Advances in molecular biology together with the appearance of new techniques, such flow cytometry, opened the possibility for correlating intracellular changes with individual absorbed dose.

ACKNOWLEDGMENTS

The authors are grateful to the Centro de Radioterapia de Pernambuco (CERAPE-Brazil), for providing laboratory facilities, to the Fundação Cooperação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil) for financial support.

Received: August 26, 2008;

Revised: September 11, 2008;

Accepted: September 13, 2008

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  • *
    Author for correspondence
  • Publication Dates

    • Publication in this collection
      20 Feb 2009
    • Date of issue
      Dec 2008

    History

    • Received
      26 Aug 2008
    • Reviewed
      11 Sept 2008
    • Accepted
      13 Sept 2008
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