Radiation Protection Dosimetry (2005), Vol. 115, No. 1–4, pp. 455–460 doi:10.1093/rpd/nci072
USE OF CYTOGENETIC INDICATORS IN RADIOBIOLOGY A. S. Rodrigues1,2, N. G. Oliveira1,3, O. Monteiro Gil1,4, A. Le´onard5 and J. Rueff1, 1 Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa, R. da Junqueira 96, P 1349-008 Lisbon, Portugal 2 Universidade Lus ofona, Lisbon, Portugal 3 Faculty of Pharmacy, Universidade de Lisboa, Lisbon, Portugal 4 Nuclear and Technological Institute, Sacave´m, Lisbon, Portugal 5 Catholic University of Louvain, Brussels, Belgium The study of ionising radiation has systematically relied on cytogenetic indicators to evaluate the biological effects and has led to theoretical approaches to explain observations associated with radiation exposure. In many of the early studies on radiobiology, the induction of chromosomal aberrations was the method of choice to evaluate dose–response relationships. But progressively, this and other cytogenetic biomarkers were used to obtain mechanistic insight on the biological effects induced by radiation. This paper attempts to give a view on the use of cytogenetic indicators in the study of various radiationrelated phenomena, including radiation dosimetry, mechanisms involved in the various cellular responses to radiation, such as bystander effects, chromosomal instability and adaptive response, as well as DNA repair pathways. One future direction may involve the use of cytogenetic indicators to evaluate various molecular determinants in individuals’ susceptibility to radiation, using other techniques such as fluorescence in situ hybridisation (FISH) and linking them to specific gene functions and single nucleotide polymorphisms.
INTRODUCTION Biomarkers are used in preventing chemical and radiation-induced health effects, by providing an indication of risk associated with exposure. The complexity of bio-monitoring is underlined by the various methods of assessment and their association with exposure as depicted in Figure 1(1), in which external dose (assessed by environmental monitoring) is linked stepwise to late developing disease, in essence an irreversible effect (assessed by health surveillance). Cytogenetic biomarkers can be classified in this scheme as early biological effects, which also include gene mutations and unscheduled DNA synthesis(2). It has been shown in prospective follow-up studies(3,4) that subjects with elevated levels of structural chromosomal aberrations (CAs) may be at an elevated risk for cancer, whereas no such association was observed with other biomarkers. This places the CAs assay as a reliable indicator of risk. On the other hand, cytogenetic biomarkers as a whole can be helpful in evaluating the in vitro and in vivo mechanisms underlying DNA damage from ionising radiation, namely the involvement of DNA repair. This paper gives a broad view of the use of cytogenetic markers in the study of diverse radiationrelated effects (Table 1).
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DNA DAMAGE AND BIOLOGICAL RADIATION DOSIMETRY Ionising radiation can induce a wide range of DNA lesions, including damage to bases, DNA–DNA and DNA–protein cross-links, DNA single (SSB) and double strand breaks (DSBs). It is generally agreed, however, that the formation of DSBs is the critical radiation-induced damage that leads to chromosomal aberrations such as dicentrics, reciprocal translocations and rings, which involve interaction of DSBs with each other. One of the critical factors related to the study of biological effects of ionising radiation is the estimation of dose. The majority of such studies use human lymphocytes, which besides their availability are known to be very sensitive to ionising radiation. Biological monitoring of humans exposed to ionising radiation has relied heavily on cytogenetic indicators such as unstable chromosomal aberrations, especially dicentric chromosomes (DIC), which are suitable indicators of a recent exposure to ionising radiation(5). Stable chromosomal aberrations, namely reciprocal translocations (fluorescence in situ hybridisation (FISH)/chromosome painting), also are an area of growing interest in radiation dosimetry because they can adequately be used to detect a retrospective exposure to ionising radiation. Other cytogenetic biomarkers such as micronuclei (MN), and to a lesser extent, sister-chromatid exchanges (SCEs) in peripheral lymphocytes, have also been used in dosimetry studies as well as in predicting cancer risk. The use of cytogenetic biomarkers, mentioned above, has been helpful in confirming the exposure
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A. S. RODRIGUES ET AL.
External dose
Genetic factors
Environmental monitoring
Susceptibility monitoring
Internal dose Biological monitoring Dose at critical site Biological effect monitoring Early adverse effects Environmental monitoring Health surveillance
Late developing disease (Irreversible) Health surveillance
Exposure
Effect
Figure 1. Scheme of the levels and methodologies used in the biological monitoring of genotoxicity associated with exposure to ionising radiation and chemicals.
Table 1. Usefulness of cytogenetic biomarkers in radiobiology. Application
Biomarkers
Purpose
Radiation dosimetry Recent exposure Retrospective exposure
DIC; MN Translocations
Evaluation of an exposure to ionising radiation and determination of the absorbed dose
High vs. low-LET radiation exposure
CAs—DIC and ‘Crea’; multi-aberrant Evaluation of the type of exposure by the cells, Translocations (increased genotoxicity pattern of the biomarker complexity), MN and multimicronucleated cells, SCEs
Mechanistic studies Bystander effect Chromosomal instability CAs; Translocations; MN; SCEs Adaptive response DNA repair studies NHEJ and HR repair PARPs Excision repair Susceptibility studies
Study of key radiation-induced phenomena by using cytogenetic biomarkers under different experimental protocols Study of key DNA repair pathways by using cytogenetic biomarkers in DNA repair defective cells or in pharmacologically inhibited cells (e.g. DNA repair inhibitors)
CAs; Translocations; MN
MN; CAs; Translocations
Functional characterisation of genetic polymorphisms of genes involved in the individual susceptibility to ionising radiation (e.g. cell cycle control, DNA repair, and apoptosis)
CAs, chromosomal aberrations; Crea, complex rearrangements; DIC, dicentric chromosomes; HR, homologous recombination; LET, linear energy transfer; MN, micronuclei; NHEJ, non-homologous end-joining; PARPs, Poly(ADPribose) polymerases; and SCEs: sister-chromatid exchanges.
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USE OF CYTOGENETIC INDICATORS IN RADIOBIOLOGY Table 2. Examples of the use of cytogenetic biomarkers in different radiation exposure scenarios. Exposure Atomic bomb Long-term atomic bomb survivors from Hiroshima and Nagasaki Environmental Domestic radon Nuclear power plant vicinity Fall-out from nuclear tests Occupational Nuclear power plant workers Uranium miners Medical workers High altitude aircraft crew Astronauts
The formation of DSBs can result in acentric DNA fragments that eventually are lost by the cell during division. These acentric fragments can be incorporated in micronuclei in cytokinesisblocked binucleated cells. Dose–effect relationships of micronuclei are similar, albeit lower than those for CAs, since not all acentric fragments give rise to micronuclei. In human lymphocytes, the persistence of cytogenetic damage over time depends on various factors, including the type of biomarker and the severity of the outcome to the cell, which can induce mitosis-linked cell death and/or apoptosis, or renewal of the lymphocyte population. A more complete review on the usefulness and limits of cytogenetic markers in biological dosimetry is given in the accompanying article by Le´onard et al. HIGH-LET vs. LOW-LET RADIATION
Accidental Chernobyl Goiania Medical External radiotherapy 60Co 131 I (e.g. thyroid carcinoma; hyperthyroidism) Diagnostic X rays Long-term thorotrast survivors
to ionising radiation and/or in evaluating the extent of DNA damage in different scenarios, namely after an environmental, occupational, accidental or medical exposure (Table 2). Of particular interest is the follow-up study of cancer patients treated with ionising radiation. Recently, we studied for a period of 24 months, a group of 19 thyroid cancer patients (papillary and follicular carcinoma) after 131I therapy(6). Lymphocytes from these patients revealed an increase of the frequencies of the cytogenetic parameters studied (CAs, DIC, MN) demonstrating the existence of a mild but persistent DNA damage pattern. In such biological dosimetry studies, the dose of ionising radiation received can be derived by extrapolation from human data on the cytogenetic biomarker studied, compared with dose–response relationships obtained in vitro. Dicentric chromosomes and centric rings (centric r) have been extensively validated as highly sensitive biomarkers for recent radiation exposure. The shape of the dose–effect relationship for the induction of such aberrations is linear– quadratic in general, but linear for low doses of ionising radiation. For dicentrics, linearity can be demonstrated down to 20 mGy, but for lower doses, statistical variations mask the effect of radiation(7). Repair processes can reduce the quadratic component of the curve as the dose rate decreases.
Mechanistic knowledge of DNA and cell damage by high-linear-energy-transfer (LET) radiation, such as heavy ions or alpha-particles, is less extensive compared to that of low-LET radiation. Although alpha-particles are only capable of traversing a fraction of a cell volume, they are very effective in producing a high density of localised lesions(8). DNA lesions produced by alpha-particles are characterised by clustering, inducing DSBs, which are difficult to repair. Complex chromosome rearrangements, sometimes referred to as ‘Crea’(9), are thus induced, leading to enhanced cell death. This leads to a greater biological effectiveness per unit dose for high-LET radiation. Comparison of the relative biological effectiveness (RBE) for different types of radiation can provide information on the underlying mechanisms of damage induced by radiation, on assessment of risk associated with accidental, occupational and environmental exposure (e.g. to radon), and can also allow different therapy applications for human disease. One instance of high-LET radiation application is the use of the boron neutron capture (BNC) reaction in the treatment of malignant glioma and melanoma patients, the boron neutron capture therapy (BNCT). This reaction depends on the capture of thermal neutrons by the minor stable isotope of boron, 10B in boronated compounds, e.g. p-borono-L-phenylalanine (BPA), with the release of alpha and lithium particles. The propagation of the alpha and lithium particles in biological tissues is characterised by a short-range and a high-LET with a remarkable destructive power(10). The exposure of human melanoma cells to BNC reaction revealed a characteristic pattern with the presence of multi-micronucleated cells (MNs)(11). In fact, for increasing doses of alpha particles the frequency of micronucleated cells containing one MN per cell decreased, whereas the frequencies of micronucleated melanoma cells with two, and especially
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three or more MNs per cell notoriously increased. These results are consistent with the formation of multiple damaged sites on the DNA molecule. Similar observations occur with CAs, with an increase of complex rearrangements involving two or more chromosomes as well as in multi-aberrant cells. Long-term biological consequences of exposure to high-LET radiation may be linked to misrepaired lesions or complex insertions after repair, which may occur even at low doses. The pattern of genotoxicity may be used to evaluate the quality of radiation involved, e.g. high- vs. low-LET radiation.
MECHANISTIC STUDIES: BYSTANDER EFFECT, CHROMOSOMAL INSTABILITY AND ADAPTIVE RESPONSE Cytogenetic biomarkers may also be useful for the knowledge of the mechanisms underlying some intriguing phenomena induced by ionising radiation: the bystander effect, the chromosomal instability and the adaptive response. One interesting issue related to radiobiology is the observation that some effects arising from radiation exposure may be non-targeted, that is, effects arising from nonnuclear or even non-cellular exposure. Nagasawa and Little(12) showed that in CHO cells irradiated with very low doses of alpha particles, >30% of cells had induced SCEs, even though
