Assessment of the neutrophil/lymphocyte ratio in patients with supraventricular tachycardia.

OBJECTIVE: The neutrophil/lymphocyte ratio (NLR) has been evaluated as a new predictor of cardiovascular risk. Inflammation has been shown to be associated with various arrhythmias including supraventricular tachycardias (SVTs). In this study, we aimed to investigate the relation between NLR and SVT in patients with a documented atrial tachyarrhythmia. METHODS: The study used a retrospective cross-sectional design. Patients who had SVT but were otherwise healthy were included. The exclusion criteria included drug use (except antiarrhythmic agents), morbid obesity, acute or chronic infection, inflammatory diseases, systemic diseases, and cancer. Total and differential leukocyte counts and routine biochemical tests were performed before the ablation procedure. RESULTS: The study included 150 patients with SVT and 98 healthy controls. The biochemical and hematological parameters were comparable between the groups, except neutrophil and lymphocyte counts. The neutrophil count was significantly higher (4.7±1.5x103/µL versus 4.1±1.0x103/µL; p<0.001) and lymphocyte count was significantly lower (2.2±0.6x103/µL versus 2.5±0.6x103/µL; p=0.001) in the SVT group than in the control group. As a result, the SVT group had significantly higher NLR values than the control group (2.2±0.9 versus 1.7±0.5; p<0.001). In addition, NLR values were higher in patients in whom tachycardia was induced during an electrophysiological study (EPS) (2.3±0.9 versus 2.0±0.8; p=0.02). The association between NLR and SVT remained significant after multivariate analysis (odds ratio: 1.5, 95% confidence interval: 1.001-2.263, p=0.049). CONCLUSION: Our study indicated that NLR values were significantly higher in patients with documented SVT than in control subjects. Inducibility of SVT during EPS was associated with higher NLR values.

Assessment and Implications of Scattered Microbeam and Broadbeam Synchrotron Radiation for Bystander Effect Studies.

Synchrotron radiation is an excellent tool for investigating bystander effects in cell and animal models because of the well-defined and controllable configuration of the beam. Although synchrotron radiation has many advantages for such studies compared to conventional radiation, the contribution of dose exposure from scattered radiation nevertheless remains a source of concern. Therefore, the influence of scattered radiation on the detection of bystander effects induced by synchrotron radiation in biological in vitro models was evaluated. Radiochromic XRQA2 film-based dosimetry was employed to measure the absorbed dose of scattered radiation in cultured cells at various distances from a field exposed to microbeam radiotherapy and broadbeam X-ray radiation. The level of scattered radiation was dependent on the distance, dose in the target zone and beam mode. The number of γ-H2AX foci in cells positioned at the same target distances was measured and used as a biodosimeter to evaluate the absorbed dose. A correlation of absorbed dose values measured by the physical and biological methods was identified. The γ-H2AX assay successfully quantitated the scattered radiation in the range starting from 10 mGy and its contribution to the observed radiation-induced bystander effect.

EPI-CT: in vitro assessment of the applicability of the γ-H2AX-foci assay as cellular biomarker for exposure in a multicentre study of children in diagnostic radiology.

PURPOSE: To conduct a feasibility study on the application of the γ-H2AX foci assay as an exposure biomarker in a prospective multicentre paediatric radiology setting. MATERIALS AND METHODS: A set of in vitro experiments was performed to evaluate technical hurdles related to biological sample collection in a paediatric radiology setting (small blood sample volume), processing and storing of blood samples (effect of storing blood at 4°C), the reliability of foci scoring for low-doses (merge γ-H2AX/53BP1 scoring), as well as the impact of contrast agent administration as potential confounding factor. Given the exploratory nature of this study and the ethical constraints related to paediatric blood sampling, blood samples from adult volunteers were used for these experiments. In order to test the feasibility of pooling the γ-H2AX data when different centres are involved in an international multicentre study, two intercomparison studies in the low-dose range (10-500 mGy) were performed. RESULTS: Determination of the number of X-ray induced γ-H2AX foci is feasible with one 2 ml blood sample pre- and post-computed tomography (CT) scan. Lymphocyte isolation and fixation on slides is necessary within 5 h of blood sampling to guarantee reliable results. The possible enhancement effect of contrast medium on the induction of DNA DSB in a patient study can be ruled out if radiation doses and the contrast agent concentration are within diagnostic ranges. The intercomparison studies using in vitro irradiated blood samples showed that the participating laboratories, executing successfully the γ-H2AX foci assay in lymphocytes, were able to rank blind samples in order of lowest to highest radiation dose based on mean foci/cell counts. The dose response of all intercomparison data shows that a dose point of 10 mGy could be distinguished from the sham-irradiated control (p = 0.006). CONCLUSIONS: The results demonstrate that it is feasible to apply the γ-H2AX foci assay as a cellular biomarker of exposure in a multicentre prospective study in paediatric CT imaging after validating it in an in vivo international pilot study on paediatric patients.

Assessment of simulated high-dose partial-body irradiation by PCC-R assay.

The estimation of the dose and the irradiated fraction of the body is important information in the primary medical response in case of a radiological accident. The PCC-R assay has been developed for high-dose estimations, but little attention has been given to its applicability for partial-body irradiations. In the present work we estimated the doses and the percentage of the irradiated fraction in simulated partial-body radiation exposures at high doses using the PCC-R assay. Peripheral whole blood of three healthy donors was exposed to doses from 0-20 Gy, with 6°Co gamma radiation. To simulate partial body irradiations, irradiated and non-irradiated blood was mixed to obtain proportions of irradiated blood from 10-90%. Lymphocyte cultures were treated with Colcemid and Calyculin-A before harvest. Conventional and triage scores were performed for each dose, proportion of irradiated blood and donor. The Papworth's u test was used to evaluate the PCC-R distribution per cell. A dose-response relationship was fitted according to the maximum likelihood method using the frequencies of PCC-R obtained from 100% irradiated blood. The dose to the partially irradiated blood was estimated using the Contaminated Poisson method. A new D0 value of 10.9 Gy was calculated and used to estimate the initial fraction of irradiated cells. The results presented here indicate that by PCC-R it is possible to distinguish between simulated partial- and whole-body irradiations by the u-test, and to accurately estimate the dose from 10-20 Gy, and the initial fraction of irradiated cells in the interval from 10-90%.

Safety assessment of nanoparticles cytotoxicity and genotoxicity of metal nanoparticles in vitro.

There is an urgent need to investigate the mechanisms of action of NMs and to develop testing strategies to assess potential environmental and human hazard. Our study has shown that metal nanoparticles such as iron oxide may induce genotoxic effect. It was inferred that coating and surface properties influence NPs cytotoxicity and genotoxicity.

Assessment of cytogenetic damage and oxidative stress in personnel occupationally exposed to the pulsed microwave radiation of marine radar equipment.

Due to increased usage of microwave radiation, there are concerns of its adverse effect in today's society. Keeping this in view, study was aimed at workers occupationally exposed to pulsed microwave radiation, originating from marine radars. Electromagnetic field strength was measured at assigned marine radar frequencies (3 GHz, 5.5 GHz and 9.4 GHz) and corresponding specific absorption rate values were determined. Parameters of the comet assay and micronucleus test were studied both in the exposed workers and in corresponding unexposed subjects. Differences between mean tail intensity (0.67 vs. 1.22) and moment (0.08 vs. 0.16) as comet assay parameters and micronucleus test parameters (micronuclei, nucleoplasmic bridges and nuclear buds) were statistically significant between the two examined groups, suggesting that cytogenetic alterations occurred after microwave exposure. Concentrations of glutathione and malondialdehyde were measured spectrophotometrically and using high performance liquid chromatography. The glutathione concentration in exposed group was significantly lower than in controls (1.24 vs. 0.53) whereas the concentration of malondialdehyde was significantly higher (1.74 vs. 3.17), indicating oxidative stress. Results suggests that pulsed microwaves from working environment can be the cause of genetic and cell alterations and that oxidative stress can be one of the possible mechanisms of DNA and cell damage.

Genotoxic assessment in peripheral blood lymphocytes of post-polio individuals using sister chromatid exchange analysis and micronucleus assay.

Environmental pollution is a complex issue because of the diversity of anthropogenic agents, both chemical and physical, that have been detected and catalogued. The consequences to biota from exposure to genotoxic agents present an additional problem because of the potential for these agents to produce adverse change at the cellular and organism levels. Past studies in virus have focused on structural damage to the DNA of environmental species that may occur after exposure to genotoxic agents and the use of this information to document exposure and to monitor remediation. In an effort to predict effects at the population, community and ecosystem levels, in the present study, we attempt to characterize damage occurring through genotoxic agents like 5-bromo-2-deoxyuridine, BrdU, using sister chromatid exchange technique and the formation of micronuclei (MN) in the peripheral lymphocytes of the post-polio syndrome sequelae affected by poliovirus. Analysis of structural chromosomal aberrations (CAs) and involvement of the specific chromosome break were pursued in this study. They revealed a significantly higher incidence of CAs (chromatid and chromosome breaks) in patients compared with controls, where the specific chromosome break has emerged as specific. Also, the maximum numbers of breaks were found to be in chromosome 1 at the position 1p36.1. The results also suggest a correlation between CAs and content of MN.

Regulação cruzada entre peroxidases e indolamina 2.3 dioxigenase no controle da metabolização do triptofano / Peroxidases and indoleamine 2.3 dioxygenase crosstalk modulating tryptophan metabolization

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no...

Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by...

Transcriptome-wide assessment of human brain and lymphocyte senescence.

BACKGROUND: Identifying biological pathways that vary across the age spectrum can provide insight into fundamental mechanisms that impact disease and frailty in the elderly. Few methodological approaches offer the means to explore this question on as broad a scale as gene expression profiling. Here, we have evaluated mRNA expression profiles as a function of age in two populations; one consisting of 191 individuals with ages-at-death ranging from 65-100 years and with post-mortem brain mRNA measurements of 13,216 genes and a second with 1240 individuals ages 15-94 and lymphocyte mRNA estimates for 18,519 genes. PRINCIPAL FINDINGS: Among negatively correlated transcripts, an enrichment of mitochondrial genes was evident in both populations, providing a replication of previous studies indicating this as a common signature of aging. Sample differences were prominent, the most significant being a decrease in expression of genes involved in translation in lymphocytes and an increase in genes involved in transcription in brain, suggesting that apart from energy metabolism other basic cell processes are affected by age but in a tissue-specific manner. In assessing genomic architecture, intron/exon sequence length ratios were larger among negatively regulated genes in both samples, suggesting that a decrease in the expression of non-compact genes may also be a general effect of aging. Variance in gene expression itself has been theorized to change with age due to accumulation of somatic mutations and/or increasingly heterogeneous environmental exposures, but we found no evidence for such a trend here. SIGNIFICANCE: Results affirm that deteriorating mitochondrial gene expression is a common theme in senescence, but also highlight novel pathways and features of gene architecture that may be important for understanding the molecular consequences of aging.

Bone marrow-derived progenitor cells and renal fibrosis.

Bone marrow-derived cells modulate solid organ diseases. For example, the different immune cell populations mediate tissue inflammation and damage, whereas progenitor cell populations are thought to enhance tissue regeneration. However, in the process of tissue fibrosis the contribution of either cell type is less clear. Here we discuss current concepts on how different bone marrow-derived progenitor cell populations may contribute to renal fibrosis.

Flexibility of single microvilli on live neutrophils and lymphocytes.

We measured the flexural stiffness of single microvilli on live human neutrophils and lymphocytes using 40-nm fluorescent beads. The beads were bound to the tips of the microvilli by anti-L-selectin antibodies. Digital bead images were acquired with an exposure time of 3 s at high magnification. Using a Gaussian point spread function, we obtained an analytical expression that relates the image profile to the flexural stiffness. We found that the flexural stiffnesses were 7 and 4 pN/microm for single microvilli on human neutrophils and lymphocytes, respectively. We also verified with live cells that 75% of neutrophil L-selectin and 72% of lymphocyte L-selectin were on the microvillus tips. Our results indicate that the leukocyte microvilli in contact with the endothelium or other surfaces will bend easily under physiological shear stresses.

An assessment of P-glycoprotein expression and activity in peripheral blood lymphocytes of transplant candidates.

BACKGROUND: P-glycoprotein (P-gp) is involved in the transport of the xenobiotic immunosuppressive agents and many cytokines, such as IL-2 and IFN-gamma. Hence, P-gp activity on peripheral blood lymphocytes (PBLs) could affect the pharmacologic response to xenobiotic immunosuppressants and immune responsiveness. The objectives of this study were to (1) determine the level of P-gp expression and activity on PBLs of kidney transplant candidates; and (2) determine whether P-gp expression correlates with P-gp activity. METHODS: We measured P-gp expression and activity on CD3(+)/CD8(+), CD3(+)/CD4(+), B lymphocytes, and NK cells of 36 kidney transplant candidates using a flow cytometric assay. P-gp activity was determined for each subpopulation of cells by the ratio of the mean Rhodamine 123 fluorescence (MFI Rh123) in the presence of verapamil divided by the MFI Rh123 in the absence of verapamil. P-gp expression was noted as the percentage of P-gp(+) cells. RESULTS: NK cells exhibited the greatest amount of P-gp activity (MFI Rh123 = 20.2 +/- 16.4) compared with other cell populations (P < .05). P-gp efflux activity was also significantly elevated in CD3(+)/CD8(+) cells (13.9 +/- 10.5) compared with B lymphocytes (4.9 +/- 2.7; P < .05) and CD4/CD3(+) cells (2.4 +/- 1.0; P < .05). P-gp expression was significantly higher in B lymphocytes (11.7 +/- 9.5) and NK cells (10.2 +/- 7.3) when compared with CD3(+)/CD8(+) cells (7.3 +/- 6.9) and CD3(+)/CD4(+) cells (6.4 +/- 3.8). P-gp expression was highly variable and did not correlate with P-gp activity (P > .05). CONCLUSIONS: CD3(+)/CD8(+) cells and NK cells, exhibited significantly increased P-gp activity compared with the other cell populations. P-gp expression is not a good correlate of P-gp activity. These findings may have important implications for the use of immunosuppressive drugs posttransplant and immune responsiveness after transplantation.

Assessment of DNA damage in peripheral blood of heavy smokers with the comet assay and the micronucleus test.

The comet assay (single-cell gel electrophoresis, SCG) is being increasingly used in human biomonitoring for the detection of genotoxic exposures. Cigarette smoking is a well-documented source of a variety of potentially mutagenic and carcinogenic compounds. Therefore, smoking should represent a relevant mutagenic exposure and lead to genotoxic effects in exposed cells. However, our previous investigations as well as several other published studies on human biomonitoring failed to show an effect of smoking on DNA migration in the comet assay, while some other studies did indicate such an effect. Although many factors can contribute to the generation of discrepant results in such studies, clear effects should be obtained after high exposure. We therefore performed a comparative study with healthy male heavy smokers (>20 cigarettes per day) and non-smokers (n=12 in each group). We measured the baseline comet assay effects in fresh whole blood samples and isolated lymphocytes. In addition, the amount of 'formamidopyrimidine DNA-glycosylase (FPG)-sensitive sites' was determined by a combination of the standard comet assay with the bacterial FPG protein. Furthermore, the influence of a repair inhibitor (aphidicolin, APC) on baseline DNA damage was comparatively analysed. Duplicate slides from each sample were processed and analysed separately. In all experiments, a reference standard (untreated V79 cells) was included to correct for assay variability. Finally, to compare the comet assay results with another genetic endpoint, all blood samples were investigated in parallel by the micronucleus test (MNT). Baseline and gamma radiation-induced micronucleus frequencies were determined. None of these approaches revealed a significant difference between heavy smokers and non-smokers with regard to a genotoxic effect in peripheral blood cells.

Assessment of immunosuppressive drug interactions: inhibition of lymphocyte function in peripheral human blood.

Cyclosporin (CsA) or tacrolimus (TRL) is routinely combined with either sirolimus (SRL) or mycophenolate mofetil (MMF) in immunosuppressive regimes in organ transplantation. The aim of our study was to establish a specific human blood assay of lymphocyte function in order to assess interactions of these drug combinations. Different concentrations (10(6)-10(9) nM) of CsA, TRL, SRL or mycophenolic acid (MPA, the active metabolite of MMF) was added to whole blood of five human volunteers. Drug combinations were studied by adding 250, 500 or 1000 nM of MPA to different concentrations of CsA, TRL, or SRL or by adding 1, 10 or 25 nM of SRL to different concentrations of CsA or TRL. After concanavalin-A stimulation, whole blood cultures were analyzed by flow cytometry detecting lymphocyte proliferation and activation by bivariate expression of proliferating cell nuclear antigen (PCNA)/DNA content and T cell-surface activation antigens (e.g. CD25, CD95, and CD154). We found an order of potency inhibiting lymphocyte function with SRL>TRL>CsA>MPA. In addition, we observed enhanced inhibition of PCNA, CD25, CD95 or CD154, if either CsA or TRL was combined with low concentrations of MPA, or SRL alone or if SRL was combined with low concentrations of MPA. Data analysis revealed an independent functional synergism or partial agonism in most combinations. This human blood assay is able to assess lymphocyte function and to monitor immunosuppressive therapy. The assay also permits pharmacological analysis of drug interactions, which will lead to improved safety and therapeutic efficacy in transplanted patients.

A simple cryopreservation method for dendritic cells and cells used in their derivation and functional assessment.

BACKGROUND: Cryopreservation and storage permitting multiple treatments with single donations is of practical importance to cellular therapies. HES and DMSO, used successfully in simple clinical procedures for freezing marrow and peripheral blood progenitor cells at -80 degrees C, was tested on antigen-presenting dendritic cells (DCs) and cells used in their derivation. STUDY DESIGN AND METHODS: DCs cultured in serum-free media from adherent or CD14+ apheresis MNCs (n = 36) in the presence of GM-CSF + IL4 +/- TNFalpha were frozen and stored at -80 degrees C in 6-percent HES, 5-percent DMSO, and 4-percent HSA. Apheresis MNCs, CD14+ monocytes, and lymphocytes were similarly frozen and later thawed for culture. Cells were assayed for viability, DC phenotype, mixed lymphocyte reaction, and antigen presentation before and 3, 6, 9, 12 or more months after freezing. RESULTS: DCs retained viability (82 +/- 2.3%) for at least 24 months. Mature and immature phenotype and function were preserved. Thawed MNCs and CD14+ cells differentiated to DCs and lymphocytes maintained high functional viability (92 +/- 3%) comparable to prefreeze levels. CONCLUSION: A simple -80 degrees C freezing and storage method that combines extracellular (HES) and intracellular (DMSO) agents is practical and preserves functional viability of DCs, MNCs, CD14+ monocytes, and lymphocytes.

Assessment of DNA damage in women using oral contraceptives.

The effect of the use of an oral contraceptive (OC) on the frequency of sister chromatid exchanges (SCEs) and on the response in the alkaline comet assay (single-cell gel electrophoresis (SCGE)) was investigated in 18 women taking contraceptive pills daily for 24 months. As controls, fertile women were included with regular menstrual cycles who received no OC drugs. A significant increase in the number of lymphocytes with DNA migration and an increased frequency of SCE per metaphase were observed in OC users as compared with their age-matched untreated controls (P<0.005). As higher incidences of spontaneous SCEs in peripheral blood lymphocytes have been reported to occur in females during pregnancy due to profound changes in the levels of certain sex hormones such as progesterone and estrogen, particularly during the last trimester, 17 pregnant women served as positive controls in this study in order to test the rate of genetic damage due to those changes. Higher frequencies of SCEs and comet responses were observed in pregnant women than in their matched controls. However, no statistically significant difference in DNA damage was observed between OC users and pregnant women (P>0.05). This study underscores the fact that prolonged and extensive use of these drugs in our daily life may be hazardous and also, that OC users should be aware of multifactorial risk factors (environmental, genetic and life style patterns) that may be responsible for additional DNA damage.

Nuclear architecture and radiation induced chromosome aberrations: models and simulations.

Knowledge of radiation track structure and its interaction with biological targets is a fundamental starting point in understanding the mechanisms underlying the induction of biological damage. In this context Monte Carlo codes are a powerful tool of investigation, allowing one to simulate both track structure and the features of the target(s) of interest at different scales, from nanometres (linear dimensions of DNA) to micrometres (linear dimensions of human cell nuclei and interphase chromosome territories). In the light of recent experimental findings on nuclear architecture, different approaches in modelling chromosome structure and aberration induction are discussed. In particular, a model is presented in which chromosome territories were explicitly described as subnuclear regions and aberration induction was modelled by coupling the structure of the target with that of the radiation track. Comparisons between model predictions and experimental results from the literature are also reported.

Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.

Assessment of DNA damage induced by high-LET ions in human lymphocytes using the comet assay.

The alkaline single-cell gel electrophoresis assay (comet assay) was used to analyze DNA damage induced in human lymphocytes by irradiation with high linear energy transfer (LET) ions. Our aim was to measure DNA breaks and to demonstrate the heterogeneity of the damage levels in a lymphocyte population irradiated with ions of different energies and LETs. Four experiments with heavy ions (Ar, C and U), as well as gamma-ray exposure, were conducted to enable comparisons. We demonstrated that the comet assay is able to assess the variability in DNA damage induced at the single cell level. The amount of DNA damage and its heterogeneity increased with particle fluence and LET, but saturated at high LETs. However, when expressed in terms of the mean dose, gamma-rays were more efficient than most of the ions used. The comet assay also allowed the detection of highly damaged cells (HDC), which were previously described as cells in late apoptotic stages. The rapid emergence of HDC in this study suggests that they were generated following ion irradiation-induced creation of DNA break clusters induced by ion exposure. Another clue was that the proportion of HDC increased with LET and fluence. We hypothesized that the LET threshold observed and the higher efficiency of low-LET radiation might be linked to the impossibility of measuring small DNA fragments in HDC.