High frequency of constitutive alkali-labile sites in mouse major satellite DNA, detected by DNA breakage detection-fluorescence in situ hybridization.

DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) is a new procedure for detecting and quantifying DNA breaks and alkali-labile sites in single cells. Cells trapped within an agarose matrix are deproteinized and treated with an alkaline unwinding solution that transforms DNA breaks and alkali-labile sites into single-strand DNA (ssDNA) motifs starting from the end of the break. These ssDNA motifs are susceptible to being hybridized with whole genome or specific DNA probes, and detected using current FISH procedures. As DNA breaks increase in a target region, more ssDNA is produced and more DNA probe hybridizes, thus increasing the FISH signal, which may be captured and analyzed using a digital image analysis system. This increase can be reflected in the surface area, mean and whole fluorescence intensity of the signal. When intact mouse splenocytes were processed with this technique using a whole genome probe, a very strong background signal was evident when compared with human blood leukocytes. In fact, when using 0.03M NaOH as the alkaline unwinding solution at 22 degrees C for 2.5min, the whole fluorescence intensity from mice cells was 50 times higher than that from human cells, thus suggesting the existence of a high frequency of constitutive alkali-labile sites in the DNA from mouse cells. Furthermore, when alkaline unwound mouse cells were simultaneously hybridized with the whole genome probe (FITC-revealed, green) and a major satellite DNA probe (Cy-3-labeled, red) both signals appeared co-localized. This result demonstrates that the high frequency of constitutive alkali-labile sites detected in the mouse genome is mainly located in the major satellite DNA sequences, resembling the findings from human 5bp classical satellite DNA sequences.

DBD-fish on neutral comets: simultaneous analysis of DNA single- and double-strand breaks in individual cells.

Humanblood leukocytes exposed to X-rays were immersed in an agarose microgel on a slide, extensively deproteinized, and electrophoresed under neutral conditions. Following this single-cell gel electrophoresis assay, characteristics of DNA migration (i.e., area of the comet) are related to the DNA double-strand breaks (dsbs) yield. After electrophoresis, comets were briefly incubated in an alkaline unwinding solution, transforming DNA breaks and alkali-labile sites into restricted single-stranded DNA (ssDNA) motifs. These motifs behave as target sites for hybridization with a whole genome probe, following the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure. As DNA breakage increases with dose, more ssDNA is produced in the comet by the alkali and more DNA probe hybridizes, resulting in an increase in the mean fluorescence intensity. Since radiation-induced DNA single-strand breaks (ssbs) are far more frequent than dsbs, the mean fluorescence intensity of the DBD-FISH signal from the comet is related to the ssb level, whereas the surface area of the same comet signal is indicative of the dsb yield. Thus, both DNA break types may be simultaneously analyzed in the same cell. This was confirmed in a repair assay performing the DBD-FISH on neutral comets from a human cell line defective in the repair of dsbs. Otherwise, treatment with hydrogen peroxide, a main inducer of ssbs, increased the mean fluorescence intensity, but not the surface, of X-ray-exposed human leukocytes.

Evidence of abundant constitutive alkali-labile sites in human 5 bp classical satellite DNA loci by DBD-FISH.

Human blood leukocytes within an agarose matrix were deproteinized and exposed to an alkaline denaturation that generates single-stranded DNA (ssDNA) starting from the ends of spontaneous basal DNA breaks and alkali-labile sites. Since the amount of ssDNA produced within a specific sequence area may be detected by hybridization with a specific probe, we quantified this in situ in different satellite DNA loci (DBD-FISH: DNA Breakage Detection FISH). The DBD-FISH signal, corrected for the respective FISH signals in metaphase, was remarkably strong in the 5bp classical satellite DNA domains analyzed (D1Z1, D9Z3, DYZ1), intermediate in the classical satellite 1 DNA sequences, and low in the alphoid satellite regions (D1Z5, DXZ1, all centromeres). This result is evidence of a high density of constitutive alkali-labile sites, probably abasic sites, within the 5bp satellite DNA sequences in human blood leukocytes. The presence and relative abundance of alkali-labile sites could explain the high frequency of spontaneous breakage and rearrangements in pericentromeric heterochromatin of chromosomes 1, 9, and 16, but not in Yqh, when this chromatin is undercondensed through spontaneous or induced demethylation, i.e. ICF syndrome or 5-azacytidine treatment.

[Risk of occupational exposure to hepatitis B virus in Venezuelan health workers. Multicenter study]. / Riesgo de exposición ocupacional al virus de la hepatitis B en personal de salud Venezolano. Estudio multicéntrico.

In a multicenter study, 970 serum samples were collected from venezuelan health workers. Presence of HBV serological markers (HBsAg, antiHBs and total anticore) were screened by microELISA. Two-hundred and forty-seven samples (27%) showed at least one positive marker, being the most frequently found the antisurface antibody which was present in different working areas, including those considered at the low exposure level (medicine 24.7%). The proportion of antisurface positive samples also was significantly higher in the group with more than 10 years of service compared to the prevalence showed by the group with less than 5 years (19.1 vs 12.8%). Groups considered to be at low risk, contrary to the expected results, showed a similar or higher prevalence compared to the groups classified as continues or frequent exposure (17.7 vs 8.6%). Our findings suggest a permanent HBV load circulation at the venezuelan hospitals environment.