Thiago de Salazar e Fernandes

Evidences of the static magnetic field influence on cellular systems.

Efforts to elucidate the doubtful character of the static magnetic field (SMF) influence on living cells have been made, although the topic still faces controversies because confusing reports in the scientific literature. This study intended to collect the most relevant issues separated by different topics (relating the SMF to its action on cellular systems) and analyze how the many field intensities, cell types and exposure time would affect the cell or intracellular structures. The analysis was based in the search in online databases aiming to give a general view of how the data can show conformity. It is proposed that scientists have been searching for linearity in what is actually a well characterized nonlinear system and two outputs are considered: the high sensitivity of parameters in which specific cell responses are generated and also the complexity and particularity of each cellular system. It is possible to trigger effects from a SMF, however in a stochastic way and depending on the cell system.

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.

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.

Long-term correlation of the electrocorticogram as a bioindicator of brain exposure to ionizing radiation

Understanding the effects of radiation and its possible influence on the nervous system are of great clinical interest. However, there have been few electrophysiological studies on brain activity after exposure to ionizing radiation (IR). A new methodological approach regarding the assessment of the possible effects of IR on brain activity is the use of linear and nonlinear mathematical methods in the analysis of complex time series, such as brain oscillations measured using the electrocorticogram (ECoG). The objective of this study was to use linear and nonlinear mathematical methods as biomarkers of gamma radiation regarding cortical electrical activity. Adult Wistar rats were divided into 3 groups: 1 control and 2 irradiated groups, evaluated at 24 h (IR24) and 90 days (IR90) after exposure to 18 Gy of gamma radiation from a cobalt-60 radiotherapy source. The ECoG was analyzed using power spectrum methods for the calculation of the power of delta, theta, alpha and beta rhythms and by means of the α-exponent of the detrended fluctuation analysis (DFA). Using both mathematical methods it was possible to identify changes in the ECoG, and to identify significant changes in the pattern of the recording at 24 h after irradiation. Some of these changes were persistent at 90 days after exposure to IR. In particular, the theta wave using the two methods showed higher sensitivity than other waves, suggesting that it is a possible biomarker of exposure to IR.Understanding the effects of radiation and its possible influence on the nervous system are of great clinical interest. However, there have been few electrophysiological studies on brain activity after exposure to ionizing radiation (IR). A new methodological approach regarding the assessment of the possible effects of IR on brain activity is the use of linear and nonlinear mathematical methods in the analysis of complex time series, such as brain oscillations measured using the electrocorticogram (ECoG). The objective of this study was to use linear and nonlinear mathematical methods as biomarkers of gamma radiation regarding cortical electrical activity. Adult Wistar rats were divided into 3 groups: 1 control and 2 irradiated groups, evaluated at 24 h (IR24) and 90 days (IR90) after exposure to 18 Gy of gamma radiation from a cobalt-60 radiotherapy source. The ECoG was analyzed using power spectrum methods for the calculation of the power of delta, theta, alpha and beta rhythms and by means of the α-exponent of the detrended fluctuation analysis (DFA). Using both mathematical methods it was possible to identify changes in the ECoG, and to identify significant changes in the pattern of the recording at 24 h after irradiation. Some of these changes were persistent at 90 days after exposure to IR. In particular, the theta wave using the two methods showed higher sensitivity than other waves, suggesting that it is a possible biomarker of exposure to IR.

Static magnetic field effects on proteases with fibrinolytic activity produced by Mucor subtilissimus.

The influence of a static magnetic field (SMF) on crude enzyme extracts with proteolytic activity is described and discussed. Proteolytic enzymes, which hydrolyze peptide bonds, and fibrinolytic enzymes, which dissolve fibrin clots, have industrial relevance, and applicability dependent on improvements of productivity and activity. We investigated whether a moderate SMF affects proteolysis in different in vitro tests: general proteolysis of azocasein substrate, and static and dynamic fibrinolytic processes (to compare fibrin gel configuration under exposure). Crude enzyme extracts, obtained from solid state fermentation of Mucor subtilissimus UCP (Universidade Católica de Pernambuco, Recife, Brazil) 1262, were used to carry out assays under slightly heterogeneous fields: a varied vertical SMF (for tests in Eppendorf tubes, from 0.100 to 0.170 T) and a varied horizontal SMF (for tests in Petri dishes, from 0.01 to 0.122 T), generated by two permanent magnets (NdFeB alloy). Results showed significant differences (P < 0.05) in static fibrinolysis assays after 24 h of exposure. The mean diameter of halos of fibrin degradation in the treated group increased by 21% compared to the control group; and the pixel number count of fibrin consumption (in a computational analysis of the area of each halo) enhanced by 30% with exposure. However, in dynamic fibrinolysis assays, no effects of SMF were observed. These results suggest a response of fibrin monomers to the SMF as a possible cause of the observed effects. Bioelectromagnetics. 38:109-120, 2017.

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.