Combined Analysis of Gamma-H2AX/53BP1 Foci and Caspase Activation in Lymphocyte Subsets Detects Recent and More Remote Radiation Exposures

Analysis of gamma-H2AX foci in blood lymphocytes is a promising approach for rapid dose estimation to support patient triage after a radiation accident but has one major drawback: the rapid decline of foci levels post-exposure cause major uncertainties in situations where the exact timing between exposure and blood sampling is unknown. To address this issue, radiation-induced apoptosis (RIA) in lymphocytes was investigated using fluorogenic inhibitors of caspases (FLICA) as an independent biomarker for radiation exposure, which may complement the gamma-H2AX assay. Ex vivo X-irradiated peripheral blood lymphocytes from 17 volunteers showed dose-and time-dependent increases in radiation-induced apoptosis over the first 3 days after exposure, albeit with considerable interindividual variation. Comparison with gamma-H2AX and 53BP1 foci counts suggested an inverse correlation between numbers of residual foci and radiation-induced apoptosis in lymphocytes at 24 h postirradiation (P = 0.007). In T-helper (CD4), T-cytotoxic (CD8) and B-cells (CD19), some significant differences in radiation induced DSBs or apoptosis were observed, however no correlation between foci and apoptosis in lymphocyte subsets was observed at 24 h postirradiation. While gamma-H2AX and 53BP1 foci were rapidly induced and then repaired after exposure, radiation-induced apoptosis did not become apparent until 24 h after exposure. Data from six volunteers with different ex vivo doses and post-exposure times were used to test the capability of the combined assay. Results show that simultaneous analysis of gamma-H2AX and radiation-induced apoptosis may provide a rapid and more accurate triage tool in situations where the delay between exposure and blood sampling is unknown compared to gamma-H2AX alone. This combined approach may improve the accuracy of dose estimations in cases where blood sampling is performed days after the radiation exposure. 

Wilms' tumor gene 1 (WT1) expression in childhood acute lymphoblastic leukemia:a wide range of WT1 expression levels, its impact on prognosis and minimal residual disease monitoring

Wilms' tumor gene 1 (WT1) is overexpressed in the majority (70-90%) of acute leukemias and has been identified as an independent adverse prognostic factor, a convenient minimal residual disease (MRD) marker and potential therapeutic target in acute leukemia. We examined WT1 expression patterns in childhood acute lymphoblastic leukemia (ALL), where its clinical implication remains unclear. Using a real-time quantitative PCR designed according to Europe Against Cancer Program recommendations, we evaluated WT1 expression in 125 consecutively enrolled patients with childhood ALL (106 BCP-ALL, 19 T-ALL) and compared it with physiologic WT1 expression in normal and regenerating bone marrow (BM). In childhood B-cell precursor (BCP)-ALL, we detected a wide range of WT1 levels (5 logs) with a median WT1 expression close to that of normal BM. WT1 expression in childhood T-ALL was significantly higher than in BCP-ALL (P<0.001). Patients with MLL-AF4 translocation showed high WT1 overexpression (P<0.01) compared to patients with other or no chromosomal aberrations. Older children (> or =10 years) expressed higher WT1 levels than children under 10 years of age (P<0.001), while there was no difference in WT1 expression in patients with peripheral blood leukocyte count (WBC) > or =50 x 10(9)/l and lower. Analysis of relapsed cases (14/125) indicated that an abnormal increase or decrease in WT1 expression was associated with a significantly increased risk of relapse (P=0.0006), and this prognostic impact of WT1 was independent of other main risk factors (P=0.0012). In summary, our study suggests that WT1 expression in childhood ALL is very variable and much lower than in AML or adult ALL. WT1, thus, will not be a useful marker for MRD detection in childhood ALL, however, it does represent a potential independent risk factor in childhood ALL. Interestingly, a proportion of childhood ALL patients express WT1 at levels below the normal physiological BM WT1 expression, and this reduced WT1 expression appears to be associated with a higher risk of relapse.

Donor leukemia following allogeneic bone marrow transplantation

Although allogeneic bone marrow transplantation has been shown to be a highly effective treatment for acute and chronic leukemia, leukemic relapse remains a significant problem. Leukemic relapse occurs in recipient cells in the majority of cases, but the paucity of donor cell leukemias may reflect the sensitivity of the investigative technique. We have developed a highly sensitive technique to identify the origin of all hematopoietic cells in the post transplant state which is based on PCR amplification of microsatellites, polymorphic tandem repetitive elements. We have identified donor leukemia (AML M5) following a sex matched BMT for severe aplastic anemia, verified a previously reported case of donor leukemia following BMT for chronic granulocytic leukemia and recently identified an acquired cytogenetic abnormality(del 11q23) in donor cells four years following an apparently successful BMT for AML. In all cases the donors have remained healthy. Postulated mechanisms include transfer to the transplanted marrow of a dormant oncogene residing in the DNA of either a virus, the chromosomes of degenerating irradiation damaged host leukemic cells or in the marrow stroma which is radioresistant and host in origin following BMT. Using sensitive techniques donor leukemia has been shown to be a more common event than was previously thought and an understanding of its pathogenesis may allow us to elucidate leukemogenic mechanisms in man.

Further evidence consistent with Yqh as an indicator of risk of gonadal blastoma in Y-bearing mosaic Turner syndrome

An 8-year-old girl with some features of Turner syndrome and karyotype 45X/46XY had developed a bilateral gonadoblastoma in her rudimentary ovaries. Her normal Y chromosome showed the characteristic distal fluorescence, as seen in her father's. Another mosaic, this time 45X/46XidicY, and also with some Turner features had rudimentary ovaries, but no gonadoblastoma had developed at age 14. The nature of her idicY, which showed no fluorescent distal Yq and had one of the centromeres inactivated, was confirmed by in situ hybridisation with a Yp-specific probe. Using primers from a human Yp-specific sequence, we amplified DNA extracted from paraffin-embedded ovarian tissue from both cases, and from a normal testicle and a normal ovary as controls. The finding of the expected Y-derived PCR product in the rudimentary gonads from these mosaic patients indicates the presence of their Y chromosome in both. We discuss the validity of the findings, and the possible role of sequences in or near the fluorescent part of Yq in the origin of gonadoblastoma in Y-bearing mosaic Turner syndrome.

Recurrent deletions and reciprocal duplications of 10q11.21q11.23 including CHAT and SLC18A3 are likely mediated by complex low-copy repeats.

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.

Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αβ) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell-associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

Évaluation de la fréquence des micronoyaux et du potentiel clastogène et/ou aneugène du benzo-a-pyrène suite à une exposition in vitro des lymphocytes humains

Le benzo-a-pyrène (BaP) est un cancérogène reconnu pour l'homme, contaminant présent dans notre environnement. Il cause des dommages à l'ADN que nous avons mesurés dans les lymphocytes exposés à de faibles concentrations de BaP, provenant de 20 jeunes volontaires non fumeurs et en santé. Suite à l’exposition, la fréquence des micronoyaux (MN) augmente significativement et décrit une courbe dose-réponse non linéaire, suggérant le déclenchement du processus de détoxification et la réparation de l’ADN. Des différences entre les individus et entre les sexes sont présentes dans la réponse génotoxique produite par le BaP. Le test des aberrations chromosomiques montre que le pourcentage de chromosomes cassés augmente significativement dans les cellules exposées au BaP. Combinés avec l'augmentation de la fréquence des MN, nos résultats confirment l'effet clastogène du BaP déjà rapporté dans la littérature. L’hybridation in situ en fluorescence (FISH) des MN avec une sonde pancentromérique est aussi utilisée pour établir leur mécanisme de formation. La FISH révèle que la majorité des MN formés après une exposition au BaP contient un centromère et plus, ce qui est significativement différent de la condition non exposée. Plus précisément, dans nos conditions expérimentales, les MN induits par le BaP contiennent surtout trois centromères et plus, indiquant également la présence d'un effet aneugène. L'effet clastogène du BaP est relié à son rôle d'initiateur dans la cancérogenèse, alors que l'effet aneugène le relierait à l'étape de progression. Ces résultats sont importants puisque l'exposition aux composés de la classe du BaP est de longue durée (cigarette, air pollué).

Alteraciones en el material genético de pintores de carros expuestos a solventes orgánicos en una localidad de Bogotá

Los solventes orgánicos son sustancias químicas que por sus propiedades físico-químicas son fácilmente inhalados o absorbidos por la piel, pueden causar daños de diversa índole en la salud. En Colombia existen normas que contemplan las medidas de protección, sin embargo persiste la informalidad en el sector de pintores de autos, por lo cual los trabajadores expuestos, a largo plazo pueden ver afectada su salud. En este estudio se analizó la relación entre individuos expuestos laboralmente a los solventes orgánicos versus no expuestos con respecto a la longitud de sus telómeros y formación de fragilidades. Se emplearon muestras de sangre extraídas por venopunción, recolectada en dos tubos: uno con Heparina, destinado al cultivo de linfocitos, para obtener cromosomas metafásicos y evaluar en ellos la presencia de fragilidades; el otro tubo con EDTA, fue empleado para la extracción de ADN y se utilizó para obtener los valores de longitud telomérica mediante la técnica de PCR cuantitativa. Los análisis estadísticos se realizaron aplicando la prueba de rangos de Wilcoxon, en el caso de la presencia de fragilidades se analizó la razón No.Fragilidades/No.Metafases, aplicando el método de Wilcoxon se encontró que existe diferencia estadísticamente significativa entre expuestos y no expuestos (p = 0,036), en donde los expuestos presentan mayor frecuencia de fragilidades. Por otra parte el valor relativo de longitud telomérica del grupo de expuestos fue mayor que el observado en el grupo de no expuestos, esta diferencia fue estadísticamente significativa (Wilcoxon, p = 0.002).

Avaliação da genotoxicidade das águas do lençol freático sob a Universidade Federal do Rio Grande do Norte

The groundwater represents the most important freshwater supply of planet. Dailly, in all world a great amount of toxic and genotoxic material reaches the aquatic systems, mainly the aquifers. The Barreiras aquifer through of five water wells is responsible for the supplying of Universidade Federal do Rio Grande do Norte (UFRN). All water wells are polluted with nitrate and some heavy metals, two of them were disabled. The genotoxicity of groundwater samples from Barreiras Aquifer in UFRN was assessed using the Allium cepa test, the Ames test and the Salmonella typhymurium microsuspension test (Kado test). For the Allium cepa test the influence of the groundwater samples collected on macroscopic (root length, colour and form) and microscopic (root tip mitotic index, chromosome aberrations and micronucleus) parameters was examined. All water samples caused a significant increase of the chromosome and mitotic aberration frequency and reduction on the rooth growth compared to negative control. Bridges and chromosome stickness were the most frequent kind of aberration in dividing cells. Furthermore, breaks were also observed. No significant increase in the number of micronuclei was found in relation to the negative controls. For Ames test were used the Salmonella typhymurium strains TA98 and TA100 without metabolic activation, applying the direct method. Prior to the Kado test, organic fractions from the water samples were obtained through XAD resin concentration. The mutagenicity organic extracts were evaluated by Kado test using TA98 and TA100 strains, in the absence and presence of S9 mix (metabolic activation). The concentrations of seven heavy metal ions were measured in water samples, but only Ni, Cu and Cr levels exceeded the permissible maximum concentration for the natural reservoirs. The results obtained for mutagenic activity using the Ames test were negative in all raw water samples analyzed. Positive results in XAD4 extracts of water samples were obtained for TA98 in the presence of S9 mix for two stations. Concentrations of heavy metals and nitrate can be correlated with the toxicity and genotoxicity of water analyzed. The mutagenic effect detected with TA98 strain suggested that organic compounds (after metabolization) are involved with the mutagenicity detected in the samples analyzed. The data set obtained in this work indicated the presence of at least two classes of mutagens: organic and inorganic compounds

Lapachol Induces Clastogenic Effects in Rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of the genotoxic potential of Austroplenckia populnea (Reiss) Lundell chloroform fraction from barkwood extract in rodent cells in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The mutagenic potential of Clusia alata (Clusiaceae) extract based on two short-term in vivo assays

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Genotoxicity and mutagenicity of Rosmarinus officinalis (Labiatae) essential oil in mammalian cells in vivo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

DNA damage in lymphocytes and buccal mucosa cells of children with malignant tumours undergoing chemotherapy

The aim of the present study was to evaluate DNA damage (micronucleus) in cytokinesis-blocked lymphocytes and exfoliated buccal mucosa cells from children with malignant tumours and under chemotherapy. Micronucleated cells (MNCs) were assessed from children before and during chemotherapy. A total of 21 healthy children (controls), matched for gender and age, were used as control. The results pointed out higher frequencies of micronucleated lymphocytes in children with malignant tumour before any therapy when compared to healthy probands. Furthermore an increase of micronucleated lymphocytes during chemotherapy was detected when compared to the data obtained before chemotherapy. No statistically significant increases of MNCs were noticed in buccal mucosa cells at any of the timepoints evaluated. Taken together, these data indicate that the presence of malignant tumours may increase the frequency of DNA damage in circulating lymphocytes, these cells being more sensitive for detecting chromosome aberrations caused by anti-cancer drugs.

Classic and molecular cytogenetic analyses reveal chromosomal gains and losses correlated with survival in head and neck cancer patients

Purpose: Genetic biomarkers of head and neck tumors could be useful for distinguishing among patients with similar clinical and histopathologic characteristics but having differential probabilities of survival. The purpose of this study was to investigate chromosomal alterations in head and neck carcinomas and to correlate the results with clinical and epidentiologic variables.Experimental Design: Cytogenetic analysis of short-term cultures from 64 primary untreated head and neck squamous cell carcinomas was used to determine the overall pattern of chromosome aberrations. A representative subset of tumors was analyzed in detail by spectral karyotyping and/or confirmatory fluorescence in situ hybridization analysis.Results: Recurrent losses of chromosomes Y (26 cases) and 19 (14 cases), and gains of chromosomes 22 (23 cases), 8 and 20 (11 cases each) were observed. The most frequent structural aberration was del(22)(q13.1) followed by rearrangements involving 6q and 12p. The presence of specific cytogenetic aberrations was found to correlate significantly with an unfavorable outcome. There was a significant association between survival and gains in chromosomes 10 (P = 0.008) and 20 (P = 0.002) and losses of chromosomes 15 (P = 0.005) and 22 (P = 0.021). Univariate analysis indicated that acquisition of monosomy 17 was a significant (P = 0.0012) factor for patients with a previous family history of cancer.Conclusions: the significant associations found in this study emphasize that alterations of distinct regions of the genome may be genetic biomarkers for a poor prognosis. Losses of chromosomes 17 and 22 can be associated with a family history of cancer.