Ademir Amaral

Current status of biodosimetry based on standard cytogenetic methods

Knowledge about dose levels in radiation protection is an important step for risk assessment. However, in most cases of real or suspected accidental exposures to ionizing radiation (IR), physical dosimetry cannot be performed for retrospective estimates. In such situations, biological dosimetry has been proposed as an alternative for investigation. Briefly, biodosimetry can be defined as individual dose evaluation based on biological endpoints induced by IR (so-called biomarkers). The relationship between biological endpoints and absorbed dose is not always straightforward: nausea, vomiting and diarrhoea, for example, are the most well-known biological effects of individual irradiation, but a precise correlation between those symptoms and absorbed dose is hardly achieved. The scoring of unstable chromosomal-type aberrations (such as dicentrics and rings) and micronuclei in mitogen-stimulated peripheral blood, up till today, has been the most extensively biodosimetry assay employed for such purposes. Dicentric assay is the gold standard in biodosimetry, since its presence is generally considered to be specific to radiation exposure; scoring of micronuclei (a kind of by-product of chromosomal damages) is easier and faster than that of dicentrics for dose assessment. In this context, the aim of this work is to present an overview on biodosimetry based on standard cytogenetic methods, highlighting its advantages and limitations as tool in monitoring of radiation workers’ doses or investigation into accidental exposures. Recent advances and perspectives are also briefly presented.

Dose assessment by quantification of chromosome aberrations and micronuclei in peripheral blood lymphocytes from patients exposed to gamma radiation

Scoring of unstable chromosome aberrations (dicentrics, rings and fragments) and micronuclei in circulating lymphocytes are the most extensively studied biological means for estimating individual exposure to ionizing radiation (IR), which can be used as complementary methods to physical dosimetry or when the latter cannot be performed. In this work, the quantification of the frequencies of chromosome aberrations and micronuclei were carried out based on cytogenetic analyses of peripheral blood samples from 5 patients with cervical uterine cancer following radiotherapy in order to evaluate the absorbed dose as a result of partial-body exposure to 60Co source. Blood samples were collected from each patient in three phases of the treatment: before irradiation, 24 h after receiving 0.08 Gy and 1.8 Gy, respectively. The results presented in this report emphasize biological dosimetry, employing the quantification of chromosome aberrations and micronuclei in lymphocytes from peripheral blood, as an important methodology of dose assessment for either whole or partial-body exposure to IR.

Non-specific interactions of CdTe/Cds Quantum Dots with human blood mononuclear cells.

In order to study biological events, researchers commonly use methods based on fluorescence. These techniques generally use fluorescent probes, commonly small organic molecules or fluorescent proteins. However, these probes still present some drawbacks, limiting the detection. Semiconductor nanocrystals - Quantum Dots (QDs) - have emerged as an alternative tool to conventional fluorescent dyes in biological detection due to its topping properties - wide absorption cross section, brightness and high photostability. Some questions have emerged about the use of QDs for biological applications. Here, we use optical tools to study non-specific interactions between aqueous synthesized QDs and peripheral blood mononuclear cells. By fluorescence microscopy we observed that bare QDs can label cell membrane in live cells and also label intracellular compartments in artificially permeabilized cells, indicating that non-specific labeling of sub-structures inside the cells must be considered when investigating an internal target by specific conjugation. Since fluorescence microscopy and flow cytometry are complementary techniques (fluorescence microscopy provides a morphological image of a few samples and flow cytometry is a powerful technique to quantify biological events in a large number of cells), in this work we also used flow cytometry to investigate non-specific labeling. Moreover, by using optical tweezers, we observed that, after QDs incubation, zeta potentials in live cells changed to a less negative value, which may indicate that oxidative adverse effects were caused by QDs to the cells.

A comparison of different cytological stains for biological dosimetry

Purpose: This paper examines the relative accuracy of analysis of unstable chromosomal aberrations (dicentrics, rings and fragments) in lymphocyte metaphases using four microscope slide staining options, widely used to assess radiation overdose or to survey occupationally exposed subjects. Materials and methods: Peripheral blood lymphocytes from a healthy donor were irradiated with 1.5 and 3.0 Gy of X-rays at a dose rate of 0.715 Gy/min. Dicentrics were scored by different cytological stains in order to compare block staining: Giemsa and 4′, 6-Diamidine-2′-phenylindole dihydrochloride (DAPI); with techniques that highlight centromeres: C-banding and Centromere Multiplex Fluorescence in situ Hybridization (CM-FISH). Results: At each of the two doses, the values for dicentrics per cell observed with each staining method were compared. In terms of dose estimation, no statistical difference was observed between the evaluated methods (χ2 p: 0.27 and 0.64, respectively; analysis of variance – ANOVA, p > 0.99). Therefore, the evidence of centromeres by C-banding and CM-FISH did not promote an increased discovery of dicentrics. On the other hand, when confirmation of unequivocal identification of dicentrics is needed, C-banding and CM-FISH can be a suitable method to confirm its presence. Economical and social factors must be taken into account in the decision of method as well. Conclusion: For routine use where several hundreds of cells need to be reliably processed and analyzed daily, processing slides by block staining with Giemsa and DAPI is preferable. However, to assist in resolving the minority of images that are ambiguous, C-banding and CM-FISH provide a better identification of suspected dicentrics.

Unstable chromosomal aberrations and micronuclei analyses in the biomonitoring of workers occupationally exposed to ionising radiation

The quantification of unstable chromosomal aberrations and micronuclei in peripheral blood lymphocytes is a method commonly used in biodosimetry by cytogenetic analysis, especially when physical dosimetry cannot be performed. In this context, the aim of this research was to compare these methods in the biomonitoring of health professionals occupationally exposed to ionising radiation. In parallel, the C-banding technique was applied to confirm the presence of unstable chromosomal aberrations (dicentrics and rings). For this, samples of peripheral blood from health professionals of three hospitals (Recife – Brazil) were collected and lymphocyte cultures were carried out based on classical cytogenetic techniques. The number of cells scored per subject was the same (1000) for each assay. Among the individuals, those who do not usually wear a lead apron had higher frequencies of unstable chromosomal aberrations and micronuclei than the ones who carefully observe the radioprotection rules.

Biodosimetry for dose assessment of partial-body exposure: a methodological improvement

This study has explored the possibility of combining culture times with extending the duration for which Colcemid is present in cell culture in order to obtain better dose estimations following partial-body exposures. Irradiated and unirradiated blood was mixed to simulate a partial-exposure. Dicentric frequencies and resultant dose estimations were compared from 48 and 72 h cultures with Colcemid added at the beginning, after 24 h or for the final 3 h. The frequencies of dicentrics in first division cells increased with the cell culture time, providing better dose estimations. Unwanted excessive contraction of chromosomes caused by prolonged contact with Colcemid was measured and ways to avoid this are discussed. It is suggested that the combination of a lower than usual concentration of this drug combined with its earlier addition and longer culture time may provide metaphases better suited for interpreting partial-body exposures.

SIMULTANEOUS ANALYSIS OF P53 PROTEIN EXPRESSION AND CELL PROLIFERATION IN IRRADIATED HUMAN LYMPHOCYTES BY FLOW CYTOMETRY

P53 protein has an intrinsic role in modulating the cellular response against DNA radioinduced damages and has been pointed out as an indirect indicator of individual radiosensitivity. The rate of cell proliferation is also a parameter that has been related to tissue sensitivity to radiation. However, this feature is yet understudied. In this context, the aim of this work was to employ Flow Cytometry (FC) for simultaneously assessing of p53 protein expression levels together with cellular proliferation rate of irradiated human lymphocytes. From in vitro irradiated human blood samples, mononuclear cells were isolated and labeled with Carboxylfluorescein Diacetate Succinimidyl Ester (CFSE) prior to phytohaemagglutinin (PHA) stimulation in culture for 96 hours. Cells were also labeled with anti-p53 monoclonal antibody PE-conjugated in order to analyze either proliferation rate or p53 expression levels by FC. It was verified a reduction in the proliferation rate of irradiated lymphocytes and, in parallel, a rise in the p53 expression levels, similar for quiescent and proliferating lymphocytes. The results emphasize the importance of the use of CFSE-stained lymphocytes in assays associated to proliferation rate and the use of this methodology in several studies, such as for evaluating individual radiosensitivity.

Non-linear dynamics of chromosome condensation induced by colcemid

This study investigated the dynamical process of chromosome condensation after colcemid treatment. Two pairs of human chromosomes, #2 and #3, were highlighted for the accurate identification by fluorescence in situ hybridization (FISH). A computerized image analysis system was used to measure the lengths of the two pairs of chromosomes averaged over 50 metaphases of different cultures with colcemid (0.5 µg/mL) added either at 3 or 48 h of a total 72 h culture period. For determining whether the process of chromosome condensation was chaotic or random, the algorithm of Detrended Fluctuation Analysis (DFA) was used. In order to evaluate the power of the method, the data were shuffled and DFA was performed again. It was found that colcemid prolonged treatment induced a significantly greater chromosome condensation (p<0.05), and the dynamics of this process was determined by the DFA and showed to be chaotic, with scaling exponents with range values 0.5< α<1.0. When the data were shuffled, the scaling exponent αreduced around to 0.5, which was characteristic of random events. These findings reinforced the idea that colcemid could interfere in some manner with the structure of chromosomes and the dynamics of chromosome condensation was non-linear.

A dose-response curve for biodosimetry from a 6 MV electron linear accelerator

Biological dosimetry (biodosimetry) is based on the investigation of radiation-induced biological effects (biomarkers), mainly dicentric chromosomes, in order to correlate them with radiation dose. To interpret the dicentric score in terms of absorbed dose, a calibration curve is needed. Each curve should be constructed with respect to basic physical parameters, such as the type of ionizing radiation characterized by low or high linear energy transfer (LET) and dose rate. This study was designed to obtain dose calibration curves by scoring of dicentric chromosomes in peripheral blood lymphocytes irradiated in vitro with a 6 MV electron linear accelerator (Mevatron M, Siemens, USA). Two software programs, CABAS (Chromosomal Aberration Calculation Software) and Dose Estimate, were used to generate the curve. The two software programs are discussed; the results obtained were compared with each other and with other published low LET radiation curves. Both software programs resulted in identical linear and quadratic terms for the curve presented here, which was in good agreement with published curves for similar radiation quality and dose rates.Biological dosimetry (biodosimetry) is based on the investigation of radiation-induced biological effects (biomarkers), mainly dicentric chromosomes, in order to correlate them with radiation dose. To interpret the dicentric score in terms of absorbed dose, a calibration curve is needed. Each curve should be constructed with respect to basic physical parameters, such as the type of ionizing radiation characterized by low or high linear energy transfer (LET) and dose rate. This study was designed to obtain dose calibration curves by scoring of dicentric chromosomes in peripheral blood lymphocytes irradiated in vitro with a 6 MV electron linear accelerator (Mevatron M, Siemens, USA). Two software programs, CABAS (Chromosomal Aberration Calculation Software) and Dose Estimate, were used to generate the curve. The two software programs are discussed; the results obtained were compared with each other and with other published low LET radiation curves. Both software programs resulted in identical linear and quadratic terms for the curve presented here, which was in good agreement with published curves for similar radiation quality and dose rates.

Correlation between radiation dose and p53 protein expression levels in human lymphocytes

The aim of this research was to evaluate the relationship between p53 protein levels and absorbed doses from in vitro irradiated human lymphocytes. For this, samples of blood from 23 donors were irradiated with 0.5; 1; 2; and 4 Gy from a Cobalt-60 source, and the percentages of lymphocytes expressing p53 were scored using Flow Cytometry. The subjects were divided into 3 groups, in accordance with the p53 levels expressed per radiation dose: low (Group I), high (Group II), and excessive levels (Group III). For all groups, the analyses showed that the p53 expression levels increase with the absorbed dose. Particularly for groups I and II, the correlation between this protein expression and the dose follows the linear-quadratic model, such as for radioinduced chromosomal aberrations. In conclusion, our findings indicate possible applications of this approach in evaluating individual radiosensitivity prior to radiotherapeutical procedures as well as in medical surveillance of occupationally exposed workers. Furthermore, due to the rapidity of flow-cytometric analyses, the methodology here employed would play an important role in emergency responses to a large-scale radiation incident where many people may have been exposed.