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PURPOSE: To analyze the effects of extending lymphocyte cultivation time on the Mitotic Index, frequency of first-division cells, and dose estimation after irradiating blood samples with different doses of radiation. MATERIALS AND METHODS: Blood samples from two healthy male volunteers were separately irradiated with three doses (3, 5, and 6 Gy) using a 60Co gamma source (average dose rate: 1.48 kGy.h-1) and cultivated in vitro for conventional (48 h) and extended (56, 68, and 72 h) amounts of time. Colcemid (0.01 µg.mL-1) was added at the beginning of the culture period. Cells were fixed, stained with fluorescence plus Giemsa (FPG), and analyzed under a light microscope. The effects of prolonged culture duration on the Mitotic Index (MI), frequency of first-division cells (M1 cells), and the First-Division Mitotic Index (FDMI) were investigated. The estimation of delivered doses was conducted using a conventional 48h-culture calibration curve. RESULTS: Overall, cells presented higher MI (up to 12-fold) with the extension of culture, while higher radiation doses led to lower MI values (up to 80% reduction at 48 h). Cells irradiated with higher doses (5 and 6 Gy) had the most significant increase (5- to 12-fold) of MI as the cultivation was prolonged. The frequency of M1 cells decreased with the prolongation of culture for all doses (up to 75% reduction), while irradiated cells presented higher frequencies of M1 cells than non-irradiated ones. FDMI increased for all irradiated cultures but most markedly in those irradiated with higher doses (up to 10-fold). The conventional 48h-culture calibration curve proved adequate for assessing the delivered dose based on dicentric frequency following a 72-hour culture. CONCLUSION: Compared to the conventional 48-hour protocol, extending the culture length to 72 hours significantly increased the Mitotic Index and the number of first-division metaphases of irradiated lymphocytes, providing slides with a better scorable metaphase density. Extending the culture time to 72 hours, combined with FPG staining to score exclusively first-division metaphases, improved the counting of dicentric chromosomes. The methodology presented and discussed in this study can be a powerful tool for dicentric-based biodosimetry, especially when exposure to high radiation doses is involved.

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Radiotherapy (RT) is an important treatment for cervical cancer. The quality of life of patients undergoing RT may be compromised during and following treatment by nausea, diarrhea, vomiting, burns, erythema and fistula. Cytokinesis-block micronucleus (CBMN) assays may be useful for predicting adverse effects of RT for cancer. The CBMN test is easy to perform and is reproducible for screening subjects exposed to ionizing radiation. We investigated the use of the frequency of micronuclei (MN) from peripheral blood samples, irradiated in vitro, as a possible biomarker to predict the side effects of RT in patients with cervical cancer. We used 10 patients with cervical cancer receiving RT and chemotherapy. We found a strong relation between the frequency of MN and the appearance of acute side effects of RT for cervical cancer. We suggest that the methodology presented here may be useful for predicting side effects of RT for patients affected by cervical cancer and who have undergone chemotherapy.

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This research was designed to evaluate the influence of the irradiation process of the leaf extracts of Libidibia ferrea (Leguminosae) on the production of secondary chemical compounds, including their biological activity. Leaves were collected and prepared to obtain the crude extract, which was then aliquoted and separately exposed to a Co-60 source with different doses, namely: 5, 7, 10, 12, 15, 20, 25, and 30 kGy. From irradiated and control samples, tests of toxicity were carried out with the microcrustacea Artemia salina Leach at three moments: 24 h, 60 and 180 days after the irradiation of the samples. Bioassays showed an increase in the toxicity of the irradiated extracts, correlated with the dose. The toxicity level did not change with the storage time, indicating the excellent stability of the samples. To assess the phytochemical profile of the crude and irradiated extracts, three techniques were employed: thin-layer chromatography (TLC), liquid chromatography coupled to mass spectrometry (LC-MS), and gas chromatography coupled to mass spectrometry (GC-MS). The phytochemical results emphasized the presence of phenols, tannins, and triterpenes. The analytical tests confirmed the role of ionizing radiation in breaking down macromolecules into simpler chemical species responsible for increasing chemical activity of the extract. This report presents and discusses ionizing radiation as an outstanding tool for enhancing active chemical compounds in leaf extracts of Libidibia ferrea, which reflects on their biochemical properties.

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This study evaluated the antibacterial activity of crude and fractionated leaf extracts of Anacardium occidentale, after receiving 10 kGy from 60Co, against multiresistant strains of Staphylococcus aureus in vitro. Minimum Inhibitory Concentrations-MIC and Minimum Bacteriostatic Concentrations-MBC were respectively assessed by serial microdilution technique in multiwall plates and Petri dishes, against standard strains and clinical isolates of multiresistant S. aureus. The results pointed out a significantly increase of the antibacterial activity of the such extracts after irradiation, emphasizing the role of gamma radiation on leaf extracts of A. occidentale to improve bioactive substances, offering new raw material for antibacterial drugs.

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Several molecules and events involved in cell response to radiation-induced damage have been investigated towards a personalized radiotherapy. Considering the importance of active caspase-3 in the proteolytic cascade that ensures radiation-induced apoptosis execution, this research was designed to evaluate the expression levels of this protein as a bioindicator of individual radiosensitivity. Peripheral blood samples of 10 healthy individuals were gamma-irradiated (cobalt-60 source) with 1, 2 and 4 Gy (control: non-irradiated samples), and active caspase-3 expression levels were measured in lymphocytes, by flow cytometry, ex vivo and after different times of in vitro incubation (24, 48 and 72 hours). Short-term incubation of 24 h was the most adequate condition to evidence correlations between dose radiation and active caspase-3 expression. For each radiation dose, it was observed a significant inter-individual variation in active caspase-3 expression intensity, suggesting that this parameter may be suitable for evidence individual radiosensitivity. The methodology presented and discussed in this work may help to predict healthy tissues response to radiation exposure toward the better patient outcome.

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ABSTRACT Several molecules and events involved in cell response to radiation-induced damage have been investigated towards a personalized radiotherapy. Considering the importance of active caspase-3 in the proteolytic cascade that ensures radiation-induced apoptosis execution, this research was designed to evaluate the expression levels of this protein as a bioindicator of individual radiosensitivity. Peripheral blood samples of 10 healthy individuals were gamma-irradiated (cobalt-60 source) with 1, 2 and 4 Gy (control: non-irradiated samples), and active caspase-3 expression levels were measured in lymphocytes, by flow cytometry, ex vivo and after different times of in vitro incubation (24, 48 and 72 hours). Short-term incubation of 24 h was the most adequate condition to evidence correlations between dose radiation and active caspase-3 expression. For each radiation dose, it was observed a significant inter-individual variation in active caspase-3 expression intensity, suggesting that this parameter may be suitable for evidence individual radiosensitivity. The methodology presented and discussed in this work may help to predict healthy tissues response to radiation exposure toward the better patient outcome.

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This work evaluated the effects of ionizing radiation on the physico-chemical and sensory characteristics of the potato cultivar Ágata (Solanum tuberosum L.), including budding and deterioration, with the end goal of increasing shelf life. For this, four groups of samples were harvested at the maturation stage. Three of them were separately exposed to a Co-60 source, receiving respective doses of 0.10, 0.15 and 2.00 kGy, while the non-irradiated group was kept as a control. All samples were stored for 35 days at 24 °C (± 2) and at 39% relative humidity. The following aspects were evaluated: budding, rot, loss of weight, texture, flesh color, moisture, external and internal appearance, aroma, soluble solids, titratable acidity, vitamin C, protein, starch and glucose. The results indicated that 0.15 kGy was the most effective dose to reduce sprouting and post-harvest losses, under the conditions studied.

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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.

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This paper focuses on the application of dicentric chromosome assay biodosimetry in cases of low-dose overexposures to professionals working in nuclear medicine and discusses how to present the results and associated uncertainties, to make possible a better understanding of biodosimetric reports. Five examples are presented of low or possibly zero exposure dose that are illustrative of typical problems that arise in occupational settings, in this instance in nuclear medicine departments. This is a scenario of minor concern in terms of health consequences but it is relevant in legal terms. They pose dilemmas for investigators but biological dosimetry can make a valuable contribution to resolving the cases.

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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.

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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.

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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.

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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.

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.

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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.

Este trabalho avaliou a estimativa da dose de radiação simulando uma exposição parcial do corpo através da irradiação in vitro de amostras de sangue misturadas com amostras não irradiadas. Foi observado que o prolongamento do tempo de cultura permite que a real fração de linfócitos em M1 contendo aberrações cromossômicas seja detectada, propiciando melhores estimativas de dose, sem a necessidade de correções matemáticas.

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The aim of this work was to establish a protocol to evaluate ionizing radiation effects on P-glycoprotein (P-gp) activity. For this, human peripheral blood samples were irradiated in vitro with different doses and P-gp activity was analyzed for CD4 and CD8 T lymphocytes through rhodamine123-efflux assay by flow cytometry. By simultaneous employment of percentage and mean fluorescence index parameters, subject-by-subject analysis pointed out changes in P-gp activity for some individuals and irradiated samples. Based on this work, the proposed protocol was considered adequate for evaluating P-gp activity on cells after radioactive stress. Besides, this research suggests that P-gp activity could be an important factor to define patient-specific protocols in combined chemo-and radiotherapy, particularly when radiation exposure precedes chemical treatment.

O objetivo deste trabalho foi estabelecer um protocolo para avaliar o efeito da radiação ionizante na atividade da glicoproteína-P (gp-P). Para isto, amostras de sangue periférico humano foram irradiadas in vitro com diferentes doses, e a atividade da gp-P foi analisada para os linfócitos T CD4 e CD8 através do ensaio do efluxo da rodamina123 por citometria de fluxo. Por emprego simultâneo dos parâmetros de porcentagem e índice médio de fluorescência, a análise indivíduo por indivíduo apontou mudanças na atividade da gp-P para alguns indivíduos e amostras irradiadas. Com base neste trabalho, o protocolo proposto foi considerado adequado para avaliar a atividade da gp-P em células após estresse radioativo. Além disso, esta pesquisa sugere que a atividade da gp-P poderia ser um importante fator para definir protocolos paciente-específicos envolvendo quimio e radioterapia, particularmente quando a exposição à radiação precede o tratamento químico.

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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 (chi(2) 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.

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Ionizing radiation (IR) can cause various lesions in DNA, which induce the increase of p53 expression levels in order to repair radiation induced damage. Thus, the correlation between the increase of p53 expression and an irradiation may constitute a fast and powerful method of individual monitoring in cases of accidental or suspected exposures to IR. In this context, the aim of this research was to evaluate changes in lymphocyte p53 expression levels, based on flow cytometry, after in vitro irradiation of peripheral blood samples. For the measurement of such expression levels of p53 protein, an investigation was carried out in order to establish a methodology of analysis based on flow cytometry. Hence, relationships among levels of expression of p53 protein with the absorbed dose have been verified. The results presented in this report emphasized flow cytometry as an important tool for the fast evaluation of p53 protein expression levels as bioindicator of individual exposure to acute ionizing radiation.

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The objective of this work was to evaluate radiation doses delivered to technologists engaged in different tasks involving positron emission tomography (PET) studies with FDG (fluorodeoxyglucose). This investigation was performed in two French nuclear medicine departments, which presented significant differences in their arrangements and radiation safety conditions. Both centers administered about 300 MBq per PET/CT study, although only one of them is a dedicated clinical PET center. Dose equivalent Hp(10) and skin dose Hp(0.07) were measured using Siemens electronic personnel dosimeters. For assessment dose absorbed by hands during drawing up of tracer and injection into the patient, a Polimaster wristwatch gamma dosimeter was employed. Absorbed dose and the time spent during each investigated task were recorded for a total of 180 whole-body PET studies. In this report, the methodology employed, the results and their radioprotection issues are presented as well as discussed.

O objetivo deste trabalho foi o de avaliar doses absorvidas por profissionais de saúde em diferentes tarefas relacionadas à tomografia por emissão de pósitrons com [18F]-FDG (fluordesoxiglicose). Esta pesquisa foi realizada em dois centros de medicina nuclear na França, os quais apresentavam diferenças significativas em sua organização e radioproteção. Esses centros aplicavam aproximadamente 300 MBq por exame PET/CT, embora apenas um deles correspondesse a um serviço de medicina nuclear dedicado a exames por PET. A dose equivalente (Hp(10)) e a dose na pele Hp(0,07) foram medidas usando dosímetros eletrônicos (Siemens). Para avaliação da dose nas mãos do tecnologista durante a preparação do radiofármaco e durante injeção no paciente, um dosímetro tipo relógio de pulso (Polimaster) foi empregado. A dose absorvida e o tempo empregado durante cada tarefa foram registrados para um total de 180 exames de corpo inteiro através da PET. Neste trabalho, a metodologia empregada, os resultados e suas conseqüências na dose absorvida para o profissional de saúde são apresentados e discutidos.

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The increase in ionizing radiation (IR) applications, especially nuclear, has been followed by the growth of public concern for the potential associated risks. The publicÆs perception of such risks is often based on the philosophy that IR is harmful at any exposure level. On the other hand, although radiation workers have knowledge about the nature of IR and its potential health effects, the relationship between absorbed dose and risk is not well understood, principally for low doses. This report presents an overview of physical and biological dosimetry as complementary methodologies, as well as their possible contribution for improving risk perception in radioprotection.

O crescente aumento das aplicações das radiações ionizantes, em particular as radiações de origem nuclear, tem sido acompanhado pelo aumento do interesse público em relação aos riscos associados a essas aplicações. A percepção de tais riscos por parte da população é freqüentemente baseada na filosofia que a radiação ionizante é perigosa independentemente dos níveis de exposição. Por outro lado, apesar dos trabalhadores ocupacionalmente expostos terem conhecimento da natureza da IR e seus possíveis efeitos á saúde, a relação entre dose absorvida e risco não é bem entendida por estes, em particular para baixos valores de dose. Este artigo resume aspectos da dosimetria física e biológica como metodologias complementares na melhoria da percepção dos riscos em radioproteção.

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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.

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