Effects of gamma radiation on the vector competence of Aedes aegypti (diptera: Culicidae) to transmit Zika virus.

One of the limitations of the Sterile Insect Technique (SIT), conventionally performed by ionizing radiation, regards separating males from females, which is not 100% effective. Some irradiated females may be released together with males in the field at SIT. The present study aimed to evaluate the influence of ionizing radiation on the ability of Aedes aegypti mosquitoes to transmit the Zika virus after exposing female pupae to a 40 Gy of gamma radiation. The results suggest that the genetic damage induced by exposure of females to this dose level promotes their total sterility, but it does not influence their vector competence. However, our data point out that ionizing radiation may decrease the proportion of infective mosquitoes.

Effects of gamma radiation on the reproductive viability of Aedes aegypti and its descendants (Diptera: Culicidae).

This work evaluated the genetic damage in descendants of male pupae of Aedes (Stegomyia) aegypti (Diptera: Culicidae) separately exposed to 20, 30, and 40 Gy of gamma radiation in the context of Sterile Insect Technique (SIT). Despite the transmission of the dominant lethal mutation, the employed dose levels did not promote a marked reduction in adult mosquito emergence and fertility. This study emphasized that semi-sterilizing doses < 50 Gy for SIT of Aedes aegypti are not recommended.

Micronucleus assay for predicting side effects of radiotherapy for cervical cancer.

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.

Gamma irradiation for enhancing active chemical compounds in leaf extracts of Libidibia ferrea (Leguminosae).

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.

Antibacterial activity of irradiated extracts of Anacardium occidentale L. on multiresistant strains of Staphylococcus aureus.

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.

Evaluation of Pb-210 in urine and frequency of micronuclei in exfoliated cells as indicators of exposure to cigarettes.

This study aimed at analyzing the frequency of micronuclei (MN) in exfoliated cells as well as the levels of Pb-210 in urine samples to evaluate the association between the smoking habit and toxic stress of transitional epithelial cells. The frequency of MN was scored from Giemsa-stained slides while exchange resin and beta counting techniques were employed to measure the concentrations of this radioisotope. Urine samples of smokers had levels of Pb-210 up to 158.65 mBq L-1. For nonsmokers, the median was below the detection limit (45 mBq L-1). The analyses of mononucleated cells showed a significant increase of the frequency of MN in smokers when compared to nonsmokers. Statistical tests showed a tight relation between the cigarette consumption and the increase of the frequency of MN, rather than with the levels of Pb-210 present in smoke particles. The results indicate the usefulness of the methodology for the evaluation of human health risks related to chronic contamination with Pb-210.

Active caspase-3 expression levels as bioindicator of individual radiosensitivity.

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.

Active caspase-3 expression levels as bioindicator of individual radiosensitivity

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.

Physico-chemical and sensory evaluation of potato (Solanum tuberosum L.) after irradiation.

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.

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.

Image processing in biodosimetry: A proposal of a generic free software platform.

The scoring of chromosome aberrations is the most reliable biological method for evaluating individual exposure to ionizing radiation. However, microscopic analyses of chromosome human metaphases, generally employed to identify aberrations mainly dicentrics (chromosome with two centromeres), is a laborious task. This method is time consuming and its application in biological dosimetry would be almost impossible in case of a large scale radiation incidents. In this project, a generic software was enhanced for automatic chromosome image processing from a framework originally developed for the Framework V project Simbio, of the European Union for applications in the area of source localization from electroencephalographic signals. The platforms capability is demonstrated by a study comparing automatic segmentation strategies of chromosomes from microscopic images.

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.

Biological dose assessment after low-dose overexposures in nuclear medicine.

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.

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.

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.

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.

Bioindicators in radiation protection

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.

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.

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.

Flow cytometry protocol to evaluate ionizing radiation effects on P-glycoprotein activity

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.

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 (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.