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Fil: Vera Candioti, Josefina. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Catedra de Citologia; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

The in-vitro effects of flurochloridone and its formulations Twin Pack Gold® (25% a.i.) and Rainbow® (25% a.i.) were evaluated in Chinese Hamster Ovary K1 (CHO-K1) cells. The cytokinesis-block micronucleus cytome (CBMN-cyt) and single-cell gel electrophoresis (SCGE) assays were used. The activities were tested within the range of final concentrations of 0.25-15 μg flurochloridone/mL. The results demonstrated that both the flurochloridone and Rainbow® were not able to induce micronuclei (MN). On the other hand, Twin Pack Gold® only increased the frequency of MN at 5 μg/mL. Furthermore, 10 and 15 μg/mL of both formulations resulted in a cellular cytotoxicity demonstrated by alterations in the nuclear division index and cellular death. SCGE assay appeared to be a more sensitive bioassay for detecting primary DNA strand breaks at lower concentrations of flurochloridone than MN did. A marked increase in the genetic damage index was observed when 5 and 15 μg/mL of both flurochloridone and Rainbow® but only when 15 μg/mL of Twin Pack Gold® were used. This is the first report demonstrating that flurochloridone and its two commercial formulations are able to induce single-strand DNA breaks in vitro on mammalian cells.

The genotoxic effects of 2,4-D and its commercial derivative 2,4-D DMA were studied by measuring sister chromatid exchange (SCE), cell-cycle progression and mitotic index in human whole blood (WBC) and plasma leukocyte cultures (PLC). Concentrations of 10, 25, 50 and 100 microg herbicide/ml were used during 72 h. In WBC, a significant increase in SCE frequency was observed within the 10-50 microg 2,4-D/ml and 25-100 microg 2,4-D DMA/ml dose range. Contrarily, in PLC, none of the concentrations employed affected the SCEs frequency. A significant delay in cell proliferation was observed in WBC after treatments with 25 and 50 microg 2,4-D/ml and 50 and 100 microg 2,4-D DMA/ml. In PLC, only 100.0 microg 2,4-D/ml altered cell-cycle progression. For both chemicals, a progressive dose-related inhibition of mitotic activity was observed. The results demonstrated that the presence of erythrocytes in the culture system modulated the DNA and cellular damage inflicted by 2,4-D and 2,4-D DMA into human lymphocytes in vitro as well as both 2,4-D and 2,4-D DMA were more potent genotoxic agents in the presence of human red cells.

To contribute to a more accurate characterization of the mutagenic and aneugenic effects of thiabendazole (TBZ), a widely used antiparasitic and food preservative drug, the induction of sister chromatid exchanges (SCEs) and mitotic spindle anomalies as cytogenetic end-points were investigated. Studies were carried out in Chinese hamster ovary (CHO) cells and human peripheral blood lymphocytes. A significant dose-dependent increase in SCE frequency was observed in CHO cells with S9-Mix (P < 0.01) in the 50-100 microg ml(-1) dose-range, while in the absence of S9-Mix, an enhancement of the SCE frequency was exhibited at the highest dose (P < 0.01). In CHO-K1 cells a significant increase in mitotic spindle anomalies (P < 0.01) was observed with the highest concentration assayed reflecting the specific effect of TBZ formulation at the microtubule level. Cell proliferation kinetics (CPK) were not modified by the addition of this pharmaceutical product. In human lymphocyte cultures, exposure to 100 microg ml(-1) TBZ formulation resulted in a significant decrease of the mitotic index (MI) (P < 0.003) and changes in the replication index (RI) (P < 0.05).

Summary. The expression of apoptosis-related genes BCL2, BAX, BCL2L1, BCL2A1, MCL1, DAPK1 and MYC was studied by quantitative real-time polymerase chain reaction on total RNA samples from patients with acute lymphoblastic leukaemia (ALL, n = 16), acute myeloid leukaemia (AML, n = 27), chronic myeloid leukaemia (CML, n = 12), mantle cell lymphoma (MCL, n = 19) and chronic lymphoid leukaemia (CLL, n = 32). BCL2, BAX, BCL2A1, MCL1, DAPK1 and MYC were overexpressed in all patient groups. BCL2L1 was underexpressed in CLL and CML, but not in AML, ALL and MCL. MCL1 levels were significantly higher in CD13 and CD33-positive ALL, and in CD56-positive AML samples. BCL2, BCL2L1, BCL2A1 and MCL1 were overexpressed and DAPK1 was underexpressed in CLL samples with a 11q23 deletion. MYC overexpression was significantly associated with shorter overall survival in MCL (P

The effect of monocytes (MNs) on baseline SCEs and kinetics of human lymphocytes in plasma leukocyte (PLCs) and whole blood cultures (WBCs) was studied. Baseline SCEs in PLCs were nearly two-fold over WBCs. No differences in SCEs were observed between PLCs and MN-depleted PLCs, indicating that SCEs from PLCs are independent of MNs. MNs titration into PLCs decreased proportionally SCEs. Reconstitution of depleted PLCs with concentration of MNs equivalent or higher than those of PLC decreased SCEs. No variations of lymphocyte kinetics in PLCs were observed in the absence/presence of MNs. The proportion of B and T-cell subsets among interphasic lymphocytes were similar in PLC in the absence/presence of MNs, but a significant increase in the proportion of mitotic T8 lymphocytes was observed. Accordingly, MNs modulate both the in vitro basal SCEs and the mitotic activity of T8, but not their cell-cycle kinetics.

BACKGROUND. Radiotherapy is a known risk factor for sarcoma development. Postirradiation sarcomas arise within the radiation field after a latency period of several years and usually are highly malignant. Very little is yet known about their genetic changes. METHODS. Twenty-seven postirradiation sarcomas were analyzed by comparative genomic hybridization, which allows genome-wide screening of DNA sequence copy number changes. RESULTS. Copy-number aberrations were detected in 20 (74%) tumors. The mean number of aberrations per tumor was 5.3 with gains outnumbering losses. The most frequent gains affected the minimal common regions of 7q11.2-q21 and 7q22 in 30% and 7p15-pter in 26%. Gain of 8q23-qter was detected in 22%. The most frequent losses affected 11q23-qter and 13q22-q32 in 22%. In osteosarcomas, the most frequent aberration was loss of 1p21-p31, in malignant fibrous histiocytomas (MFH) gain of 7cen-q22, and in fibrosarcomas gain of 7q22. The findings in postirradiation osteosarcomas and MFHs were compared with findings in sporadic osteosarcomas and MFHs, reported previously by the authors. In sporadic osteosarcomas, gains outnumbered losses, but, in postirradiation osteosarcomas, losses were more frequent than gains. Loss at 1p was rare in sporadic osteosarcoma (3%) but frequent (57%) in postirradiation osteosarcomas. Gains at 7q were frequent both in postirradiation and sporadic MFH. CONCLUSIONS. According to previous studies on different types of sporadic sarcomas, gains at 7q or 8q are associated with poor prognosis or large tumor size. Thus, the frequent gains at 7q and 8q might have been responsible in part for the poor prognosis of postirradiation sarcomas. Also, however, some of their clinical features, i.e., high malignancy grade, late diagnosis, and central location, are associated with a poor prognosis.

The structure of the 17p amplicon from 9 human sarcoma specimens evaluated by comparative genomic hybridization (CGH) has been studied by analyzing 28 microsatellite markers by PCR. Eleven sarcoma specimens showing no DNA copy number increases at 17p by CGH were analyzed as control samples. Five specimens were analyzed by Southern blotting using probes that have previously shown amplification at the 17p12 region in astrocytoma and high-grade osteosarcoma samples. Microsatellite marker analyses revealed that all samples but 1 showing copy number increases at 17p by CGH displayed allelic imbalance that confirmed the CGH findings. Seven of these 9 cases displayed gain in copy number by microsatellite marker analysis. Four cases displaying gain in copy number were associated with loss of heterozygosity at other loci. Southern blot analysis showed amplification in 3 cases, all of them had shown copy number increases by CGH and microsatellite marker analysis, except one case, which was not included in the microsatellite marker analysis. Our results reveal the complexity of the 17p amplicon in sarcomas, suggesting that multiple target genes are involved in tumorigenesis. Int. J. Cancer 82:329–333, 1999. © 1999 Wiley-Liss, Inc.

The effect of pig monocytes (MNs) on the baseline frequency of sister chromatid exchanges (SCEs) and cell-cycle progression of pig lymphocytes was studied in plasma leukocyte (PLCs) and whole blood leukocyte cultures (WBCs). No variation in SCE frequency was observed between control WBC and PLC, nor did the addition of pig MNs to PLCs modify the baseline frequency of SCEs. Cell proliferation was slower in PLCs than in WBCs. Variations in cell-cycle progression of pig lymphocytes from PLC were observed both in the absence and presence of adherent cells in the culture. In MN-free cultures, lymphocytes proliferated faster than in parallel PLC cultures. However, when MNs were seeded into the cultures, cell-cycle progression gradually slowed as a function of the concentration of adherent cells present in the cultures. This finding shows that pig MNs modulate the in vitro cell-cycle progression of pig lymphocytes in a dose-dependent manner and that the low baseline SCE frequency of pig lymphocytes is independent of the presence or absence of MNs in the culture. Environ. Mol. Mutagen. 33:86 ‐90, 1999 © 1999 Wiley-Liss, Inc.

DNA sequence copy number changes were studied by comparative genomic hybridization (CGH) in 28 desmoid tumors. Changes were detected in 12 tumors (43%) with a mean of 1.4 changes per sample (range: 1 to 7). Out of 12 tumors associated with pregnancy or Gardner's syndrome, only two displayed changes. The minimal common regions of the most frequent gains were 1q21 (39%), chromosome 20 (32%), and 9p12 (21%). No high-level amplifications were detected. Losses of DNA sequences were two times less frequent than gains and the minimal common regions of the most frequent losses were 6q16–q21 (14%), 5q14 (11%), and 13q21–q31 (11%). Genes Chromosomes Cancer 23:183–186, 1998. © 1998 Wiley-Liss, Inc.

In this study, we investigated whether fluorescein isothiocyanate (FITC)-labeling of test DNA and Texas-red (TR) labeling of reference DNA in comparative genomic hybridization (CGH) experiments cause the results to differ from those obtained using the opposite combination (reverse labeling). Analysis was performed on a total of 20 DNA specimens consisting of 13 frozen bone marrow aspirates from patients with acute myeloid leukemia, and fresh peripheral blood samples from seven healthy donors. For CGH, one aliquot from each test DNA sample was labeled using nick-translation with FITC-dUTP and another with TR-dUTP. Afterwards, the FITC-dUTP and TR-dUTP-labeled test DNAs were hybridized to TR-dUTP- and FITC-dUTP-labeled normal reference DNAs, respectively. The results using the two combinations were compared with each other and with the results of G-banding karyotype analysis. Karyotype data was used to detect artifacts known to occur in some chromosome regions in CGH analysis. The control DNAs labeled with FITC or TR showed no DNA copy number changes. Regardless of the fluorochrome employed for labeling, no DNA copy number changes were detected using CGH in patients with normal karyotypes, nor in patients whose karyotype aberrations were present in less than 40% of cells. In the remaining patients, CGH revealed DNA copy number changes that coincided with the results of the G-banding analysis. Hybridization artifacts known to occur in CGH experiments affecting chromosome regions 1p33-pter, 16p, 17p, 19, and 22 were observed in 15-23% of the tumor samples labeled with FITC, but not in samples labeled with TR. In addition, other previously unreported overrepresentations affecting 7q21, 9q34, 16q, 17q, and chromosome 20 were observed at very low frequencies in up to 10% of the samples when FITC was used to label test DNA. However, when TR was used, overrepresentations were observed at 4q13-q21, 11q21-q23, 13q21-qter, and Xq21-q22, whereas 19p was underrepresented. The results demonstrate that TR-labeling confirms abnormalities detected using FITC-labeling and reduces hybridization artifacts in the known problematic regions of the human genome.

We studied DNA copy number changes in gastric cancer (GC) using comparative genomic hybridization (CGH) analysis on 35 resected gastric carcinomas (22 of the intestinal type and 13 of the diffuse type). Eighty-three percent of the cases showed DNA copy number changes. Gains were more common than losses (median of 3 and 1 in primary tumors of the intestinal and diffuse type, respectively). The most common gains were detected on 20q [46%; 12 intestinal type (55%) and four diffuse type (31%)], 8q [37%; 10 intestinal type (45%) and three diffuse type (23%)], and 17q12-21 [29%; all but one intestinal type (41%)]. The most frequent losses were detected on 18q [26%; all intestinal type (41%)] and on 4q [23%; all intestinal type (32%)]. High-level amplifications were observed in the intestinal type of tumors at 17q12-21 (three tumors), 20q (three tumors). 2q (one tumor), and 18q (one tumor). In the diffuse type, high-level amplification was detected once at 13q.

Lymphoid involvement in acute myeloid leukemia (AML) was studied using the morphology-antibody-chromosome (MAC) combination technique in one patient. The chromosomal aberration, trisomy 8, was demonstrated in AML blasts, glycophorin A-positive erythroblasts, and CD3- and CD20/22-positive small lymphocytes. This suggests that in this patient even lymphocytes belonged to the leukemic clone.

Fil: Molinari, Gabriela Beatriz. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

The ploidy of human bone marrow cells belonging to the megakaryocytic, granulocytic-monocytic and erythrocytic lineages was studied by in situ hybridization using the biotin-labelled Y chromosome-specific DNA probe pY431 and the chromosome 1-specific probe pUC1.77 on cells identified morphologically and immunologically by the MAC procedure. Cells of the granulocytic-monocytic and erythrocytic lineages were seen to be 2N in ploidy, whereas the ploidy of the megakaryocytic lineage ranged from 2N to 32N, with the ploidy classes 4N and 8N being predominant. The frequency of megakaryocytes with 2N chromosomes was also high., Facultad de Ciencias Naturales y Museo

Sister chromatid exchange (SCE) and chromosome aberration induction were determined for several inorganic metal salts. Arsenic, nickel, and beryllium salts at concentrations effective in causing transformation of Syrian hamster cells (HEC) induced SCE and chromosome aberrations of HEC and human lymphocytes, whereas sodium tungstate, a non-transforming chemical, neither induced SCE nor chromosome aberrations. Normal human and hamster cells exhibited equal sensitivity to SCE induction; nontoxic concentrations of sodium arsenite, beryllium sulfate, and nickel sulfate caused an increase of 8-10 SCE/cell over control values. Sodium arsenite, a trivalent arsenic, and sodium arsenate, a pentavalent arsenic, produced increases in SCE but the former was effective at lower concentrations. Both arsenic salts were less efficient in inducing SCE in human whole blood than in purified lymphocyte cultures. Sodium arsenite, sodium arsenate, nickel sulfate, and beryllium sulfate also caused damage consisting primarily of chromatid type of aberrations. In HEC, with doses most effective in SCE induction , all four metals produced aberrations in 16-21% of cells. In human lymphocytes, 34 and 30% of the cells had chromosome damage after sodium arsenite and sodium arsenate, respectively, whereas beryllium sulfate or nickel sulfate caused damage in about 10% of the cells. The induction of SCE andmore » chromosomal aberrations by metals reemphasizes the sensitivity of cytological assays and their importance for detecting genetic damage caused by carcinogens.« less

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) in conditions that produce enhancement of ultraviolet light (UV) and x-irradiation Syrian hamster embryo cell (HEC) transformation did not cause further increase in the sister chromatid exchange (SCE) frequency induced by UV and x-irradiation, two physical carcinogens that differ in their mode of DNA interaction and efficiency of SCE induction. Several factors which might influence SCE induction by TPA were studied on HEC and Chinese hamster V79-4 cells. Heat-inactivated serum was used because of the possibility that a serum component may interfere with TPA ability to cause SCE. TPA effect on SCE was determined at the first and second division post treatment on cells exposed to different 5-bromodeoxyuridine (BrdUrd) concentrations. Independent of BrdUrd concentration (1-10 microgram/ml medium) and the number of cell divisions post treatment TPA (0.01-2 microgram/ml medium) was ineffective in inducing SCE in exponentially and stationary HEC cultures cultivated in medium supplemented with heat-inactivated serum. Also, TPA did not increase the SCE frequency in V79-4 Chinese hamster cells cultured in heat-inactivated or noninactivated serum. Although SCE induction, a cellular response to carcinogen-induced DNA damage, may be important for the induction of transformation by environmental agents, the enhancement of transformation frequency caused by TPA occurs without further DNA alterations involved in SCE formation.