Protective effects of bark ethanolic extract from Spondias dulcis Forst F. against DNA damage induced by benzo[a]pyrene and cyclophosphamide.

This study evaluated the genotoxicity, mutagenicity, antigenotoxicity, and antimutagenicity effects on biochemical parameters of oxidative stress of the Spondias dulcis bark ethanolic extract on mice. The extract was evaluated in the doses of 500, 1000, and 1500 mg/kg bw via gavage. To evaluate the protective effects of the extract, benzo[a]pyrene (B[a]P) and cyclophosphamide (CP) were chosen as DNA damage inducers. Genotoxicity and antigenotoxicity were evaluated by the comet assay. Cytotoxicity, mutagenicity, and antimutagenicity were evaluated by the micronucleus test in bone marrow and peripheral blood. The biochemical parameters of oxidative stress were evaluated by the quantification of catalase activity (CAT) and reduced glutathione (GSH) in total blood, liver and kidney, and malondialdehyde (MDA), in liver and kidney. No genotoxic, cytotoxic, or mutagenic effect was found on mice exposed to the extract. The extract depleted the number of damaged nucleoids in total blood and the number of micronucleus (MN) in both cell types. The extract was able to increase CAT activity and GSH levels and decrease MDA levels after treatment with B[a]P and CP. The results indicate that the S. dulcis extract has potential to be used as preventive compound against DNA damage caused by CP and B[a]P.

Cytogenetic and molecular analysis of MLL rearrangements in acute lymphoblastic leukaemia survivors.

The successful treatment of paediatric malignancies by multimodal therapy has improved outcomes for children with cancer, especially those with acute lymphoblastic leukaemia (ALL). Second malignant neoplasms, however, represent a serious complication after treatment. Depending on dosage, 2-12% of patients treated with topoisomerase II inhibitors and/or alkylating agents develop treatment-related acute myeloid leukaemia characterized by translocations at 11q23. Our goal was to study MLL rearrangements in peripheral lymphocytes using cytogenetic and molecular methods in order to evaluate the late effects of cancer therapy in patients previously treated for childhood ALL. Chromosomal rearrangements at 11q23 were analysed in cytogenetic preparations from 49 long-term ALL survivors and 49 control individuals. Patients were subdivided depending on the inclusion or omission of topoisomerase II inhibitors (VP-16 and/or VM-26) in their treatment protocol. The statistical analysis showed significant (P = 0.007) differences between the frequency of translocations observed for the groups of patients and controls. These differences were also significant (P = 0.006) when the groups of patients (independent of the inclusion of topoisomerase II inhibitors) and controls were compared (P = 0.006). The frequencies of extra signals, however, did not differ between groups of patients and controls. Several MLL translocations were detected and identified by inverse polymerase chain reaction, followed by cloning and sequencing. Thirty-five patients (81%) presented putative translocations; among those, 91% corresponded with t(4;11) (q21;q23), while the other 9% corresponded with t(11;X), t(8;11)(q23;q23) and t(11;16). Our results indicate an increase in MLL aberrations in childhood ALL survivors years after completion of therapy. The higher frequency in this cohort might be associated with therapy using anti-tumoural drugs, independent of the inclusion of topoisomerase II inhibitors. Even though the biological significance of these rearrangements needs further investigation, they demonstrate a degree of genome instability, indicating the relevance of cytogenetic and molecular studies during the follow-up of patients in complete clinical remission.

Fluorescent in situ hybridization in liver cell touch preparations from autopsy.

The objective of the present study was to develop a simplified and low-cost protocol for the investigation of congenital anomalies of chromosomal etiology by fluorescent in situ hybridization (FISH) using probes for chromosomes X, 18, 13/21 in liver cell touch preparations obtained from autopsies performed at the University Hospital of the Faculty of Medicine of Ribeirão Preto. Liver touch preparations were obtained from 11 autopsy cases and fixed in 95% ethanol or methanol:acetic acid (3:1). The FISH technique was carried out according to the protocol of Pinkel with modifications, using probes for chromosomes X, 18, 13/21. There was no significant difference in labeling intensity, quantity of nuclei, or number of signals present per nucleus between the materials fixed with the two fixatives. Similar results were obtained with different times of storage up to 14 months at -20 degrees C. We concluded that the use of touch preparations pretreated with acetic acid and fixed in 95% ethanol represents an efficient, practical, and low-cost method of cell preparation for FISH analysis.

Translocation analysis by the FISH-painting method for retrospective dose reconstruction in individuals exposed to ionizing radiation 10 years after exposure.

Fluorescence in situ hybridization (FISH) is a powerful method largely used for detecting chromosomal rearrangements, translocations in particular, which are important biomarkers for dose assessment in case of human exposure to ionizing radiation. To test the possibility of using the translocation analysis by FISH-painting method in retrospective dose assessment, we carried out in vitro experiments in irradiated human lymphocytes, in parallel with the analysis of translocations in lymphocytes from 10 individuals, who were exposed to 137cesium in the Goiânia (Brazil) accident (samples collected 10 years after exposure). The in vitro dose-response curve for the genomic translocation frequencies (FGs) fits a linear quadratic model, according to the equation: Y=0.0243X(2)+0.0556X. The FG values were also calculated for the individuals exposed to 137cesium, ranging from 0.58 to 5.91 per 100 cells, and the doses were estimated and compared with the results obtained by dicentric analysis soon after the accident, taking the opportunity to test the validity of translocation analysis in retrospective biodosimetry. A tentative of retrospective dosimetry was performed, indicating that the method is feasible only for low level exposure (below 0.5Gy), while for higher doses there is a need to apply appropriate correction factors, which take into consideration mainly the persistence of chromosomal translocations along with time, and the influence of endogenous and exogenous factors determining the inter-individual variability in the cellular responses to radiation.

Primary monophasic synovial sarcoma of the kidney: a case report and review of literature.

Primary synovial sarcoma (SS) of the kidney is a rare neoplasm and its presenting features are similar to other common renal tumors, making early diagnosis difficult. To date, few cases have been reported in the literature. Primary renal SSs can exist in either a monophasic or a biphasic pattern, the former being more common and tending to have a better prognosis than the biphasic variant. Herein we describe a case of primary renal SS that was diagnosed based on histopathology and immunohistochemistry after radical nephrectomy. Fusion gene product analysis was also done by FISH and RT-PCR. Patient follow-up and literature review are presented, focused on systemic therapy. We highlight that these tumors should be correctly diagnosed as clinical results and specific treatment are distinct from primary epithelial renal cell carcinoma. Adjuvant chemotherapy should be tailored for each patient in the management of disease, although its role still remains unclear.

Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism.