Analía Seoane

Genotoxic ability of cadmium, chromium and nickel salts studied by kinetochore staining in the cytokinesis-blocked micronucleus assay.

The aneugenic and clastogenic ability of cadmium chloride(II), cadmium sulfate(II), nickel chloride(II), nickel sulfate(II), chromium chloride(III) and potassium dichromate(IV) have been evaluated through kinetochore-stained micronucleus test. Traditional genotoxicity assays evaluate DNA damage, gene mutations and chromosome breakage. However, these tests are not adequate to detect aneugenic agents that do not act directly on DNA. Staining kinetochores in the cytokinesis-blocked micronucleus assay is a useful way to discriminate between clastogens and aneuploidogens and may allow a rapid identification of aneuploidy-inducing environmental compounds. Human diploid fibroblasts (MRC-5) were employed. All compounds increased micronuclei frequency in a statistically significant way. However, increases in kinetochore-positive micronuclei frequencies were higher than in kinetochore-negative ones. The present work demonstrates the genotoxic ability of the cadmium and chromium salts studied. Aneugenic as well as clastogenic ability could be observed with this assay. Nickel salts, as it was expected because of their known weak mutagenicity, showed lower genotoxic effects than the other metal salts studied. As the test employed only allows the detection of malsegregation, it is proposed that this mechanism is at least one of those by which the tested metal salts induced aneuploidy. On the other hand, visualization of kinetochores in all experiments suggests that the compounds studied did not act by damaging these structures.

Chromosomal aberrations in peripheral blood lymphocytes from native Andean women and children from northwestern Argentina exposed to arsenic in drinking water.

For conducting an adequate human cancer risk assessment of inorganic arsenic (As) in the low-dose region, it is important to establish its mode of action. In this context, the nature of genotoxic effects induced by this agent is of considerable interest. However, the results from such investigations in human have been conflicting. In an attempt to resolve this issue, the clastogenic and aneugenic potential of As was investigated in women and children from native population exposed to high levels (around 0.2 mg/l) of natural As via drinking water in San Antonio de los Corbes in the Andean region of Salta, Northwestern Argentina. The water did not contain elevated levels of heavy metals, such as lead or cadmium, nor was the investigated population exposed to significant industrial pollution or to pesticides. An ethnically similar control group from Rosario de Lerma, Salta, where only extremely low concentration of arsenic in drinking water could be detected, was used as a control. To evaluate the genotoxic effects in peripheral blood lymphocytes, micronuclei (MN) in binucleated cells, sister-chromatid exchanges (SCEs) and the fluorescence in situ hybridization technique (FISH) in combination with chromosome specific DNA libraries were employed. The data obtained clearly indicate a highly significant increase in the frequency of MN and of trisomy in lymphocytes from exposed children and women in comparison with controls, but no notable effects were found on the frequencies of SCEs, specific translocations, or on cell cycle progression. As supported by FISH analysis, at least a proportion of MN appears to originate from whole chromosome loss. An additional finding was the unusually low background levels of MN in unexposed individuals from this ethnic group as compared to other populations, e.g., Caucasians.

Aneugenic effects of some metal compounds assessed by chromosome counting in MRC-5 human cells.

Development of a comprehensive test battery is necesary for the evaluation and detection of aneugenic chemicals. The chromosome couting method was used in the present study. The aneugenic ability of cadmium choride (1.0, 2.0 and 4.0x10(-3) mM), cadmium sulfate (3. 3, 6.7x10(-5) and 1.3x10(-4) mM), potassium dichromate (2.5, 5. 0x10(-4) and 1.0x10(-3) mM) and cacodilic acid (1.25, 2.5 and 5. 0x10(-2) mM) were analysed using MRC-5 cells which have a modal diploid number of 2 n=46 with a spontaneous aneuploid or polyploid cells not higher than 13% and 8%, respectively.All compounds induced significant increments of aneuploid cells in relation to negative controls. The frequency of aneuploid cells increased in all treatments with cadmium chloride. Cadmium sulfate induced significant increments of aneuploid cells with the two higher doses. All the doses of potassium dichromate increased the frequency of aneuploid cells although to a lesser degree than the other compounds. In these cases, differences were in the borderline of statistical significance (p<0.05). Moreover, a low number of cells could be analysed in treatments with the highest dose due to the decrease in the mitotic index. Results obtained are coincident with previous reports using the same methodology in the sense that induced aneuploidy was mainly evidenced by the increase of hypodiploid but not hyperdiploid cells. In addition, anaphase-telophase analysis of the effects of the same doses of these metal compounds in CHO cells showed significant increments of lagging chromosomes and increased frequencies of kinetochore positive micronuclei in MRC-5 cells. These findings could be considered as an indication that the main cause of unequal chromosome separation is the failure of kinetochores to attach the spindle apparatus either by alteration of its protein components or by the altered chromatid separation in anaphase.

Biological Dosimetry Intercomparison Exercise: An Evaluation of Triage and Routine Mode Results by Robust Methods

Abstract Well-defined protocols and quality management standards are indispensable for biological dosimetry laboratories. Participation in periodic proficiency testing by interlaboratory comparisons is also required. This harmonization is essential if a cooperative network is used to respond to a mass casualty event. Here we present an international intercomparison based on dicentric chromosome analysis for dose assessment performed in the framework of the IAEA Regional Latin American RLA/9/054 Project. The exercise involved 14 laboratories, 8 from Latin America and 6 from Europe. The performance of each laboratory and the reproducibility of the exercise were evaluated using robust methods described in ISO standards. The study was based on the analysis of slides from samples irradiated with 0.75 (DI) and 2.5 Gy (DII). Laboratories were required to score the frequency of dicentrics and convert them to estimated doses, using their own dose–effect curves, after the analysis of 50 or 100 cells (triage mode) and after conventional scoring of 500 cells or 100 dicentrics. In the conntional scoring, at both doses, all reported frequencies were considered as satisfactory, and two reported doses were considered as questionable. The analysis of the data dispersion among the dicentric frequencies and among doses indicated a better reproducibility for estimated doses (15.6% for DI and 8.8% for DII) than for frequencies (24.4% for DI and 11.4% for DII), expressed by the coefficient of variation. In the two triage modes, although robust analysis classified some reported frequencies or doses as unsatisfactory or questionable, all estimated doses were in agreement with the accepted error of ±0.5 Gy. However, at the DI dose and for 50 scored cells, 5 out of the 14 reported confidence intervals that included zero dose and could be interpreted as false negatives. This improved with 100 cells, where only one confidence interval included zero dose. At the DII dose, all estimations fell within ±0.5 Gy of the reference dose interval. The results obtained in this triage exercise indicated that it is better to report doses than frequencies. Overall, in both triage and conventional scoring modes, the laboratory performances were satisfactory for mutual cooperation purposes. These data reinforce the view that collaborative networking in the case of a mass casualty event can be successful.

Genetic instability induced by low doses of x-rays in hamster cells.

Purpose: Genomic instability involves time delayed events and can be manifested as elevated rates of heritable changes in the progeny of irradiated cells. To study the induction of chromosomal instability by very low doses of radiation Chinese Hamster Ovary (CHO) cells were exposed to 10 – 50 milisieverts (mSv) (≈10 – 50 miligrays (mGy)) of x-rays. Materials and methods: Control and irradiated cell populations were assayed for chromosomal aberrations and assessed using a micronucleus test and anaphase-telophase analysis at the first cell division post-irradiation and at every four population doublings thereafter up to 16 population doublings post-irradiation. Results: Frequencies of micronuclei, anaphase-telophase alterations and chromosomal aberrations were increased when the cells were analysed immediately after x-ray exposure. Micronuclei and anaphase-telophase alterations showed significantly increased frequencies when they were analysed at 12 and 16 population doublings after exposure to 50 mSv. Chromosomal aberrations increased significantly at 12 and 16 population doublings after exposure to 10 mSv and 50 mSv. Conclusions: Our results are consistent with the presence of a phenomenon by which the initial DNA damage in the surviving cells is memorized. Micronuclei and achromatic lessions were the main cytogenetic damage observed in cells exposed to very low doses of x-rays, indicating that these low doses are able to induce genetic instability.

Protein-energy malnutrition contributes to increased structural chromosomal alteration frequencies in Argentinean children

The relationship between protein-energy malnutrition and genetic damage has been studied in human beings and laboratory animals, but results are still conflicting. The aim of the present study was to assess the induction of structural chromosomal aberrations in peripheral blood lymphocytes of children with primary protein-energy malnutrition. A case-control study was performed. Samples were obtained from 25 primary malnourished infants (mean age, 22 months; range, 1-66 months). The control group consisted of 25 eutrophic children from the same population who were matched 1:1 by age and sex. Anthropometric and clinic evaluations were performed to assess nutritional condition. Before blood collection, we interviewed each individuals parent to complete a semi-structural survey specifying age, dietary habits, viral or bacterial diseases; previous exposure to diagnostic x-rays; and use of therapeutic drugs. After 48 hours, 100 cultured lymphocytes were analyzed per patient. Statistical analysis was performed using the Epi Dat 3.0 program (P < or = .05). The chromosomal aberration frequency was nearly 7 times higher in malnourished infants than in controls (14.61% vs 2.2%, respectively). This difference was statistically significant (P < .001) and may be explained by the occurrence of achromatic lesions, breaks, and telomeric associations. DNA damage could be attributed to several factors: severe deficiency of essential nutrients (ie zinc, iron, and vitamin A) required in the synthesis of DNA maintenance factors; deterioration of repair mechanisms allowing the persistence of an unusually high number of structural chromosomal aberrations; and/or the absence of specific factors needed to protect the cell against oxidative DNA damage.

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90–95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.

Effects of copper sulphate concentrations during in vitro maturation of bovine oocytes

THE OBJECTIVES WERE TO EVALUATE: 1) copper (Cu) concentrations in plasma and follicular fluid (FF) from cattle ovaries; 2) the effects of supplemental Cu during in vitro maturation (IVM) on DNA damage of cumulus cells and glutathione (GSH) content in oocytes and cumulus cells; and 3) supplementary Cu during IVM on subsequent embryo development. Copper concentrations in heifer plasma (116 ± 27.1 μg/dL Cu) were similar (P > 0.05) to concentrations in FF from large (90 ± 20.4 μg/dL Cu) and small (82 ± 22.1 μg/dL Cu) ovarian follicles in these heifers. The DNA damage in cumulus cells decreased with supplemental Cu concentrations of 4 and 6 μg/mL (P < 0.01) in the IVM medium (mean ± SEM index of DNA damage was: 200.0 ± 27.6, 127.6 ± 6.0, 46.4 ± 4.8, and 51.1 ± 6.0 for supplementation with 0, 2, 4, and 6 μg/mL Cu respectively). Total GSH concentrations increased following supplementation with 4 μg/mL Cu (4.7 ± 0.4 pmol in oocytes and 0.4 ± 0.04 nmol/10(6) cumulus cells) and 6 μg/mL Cu (5.0 ± 0.5 pmol in oocytes and 0.5 ± 0.05 nmol/10(6) cumulus cells, P < 0.01) compared with the other classes. Cleavage rates were similar (P ≥ 0.05) when Cu was added to the IVM medium at any concentration (65.1 ± 2.0, 66.6 ± 1.6, 72.0 ± 2.1, and 70.7 ± 2.1 for Cu concentrations of 0, 2, 4, and 6 μg/mL). Percentages of matured oocytes that developed to the blastocyst stage were 18.7 ± 0.6, 26.4 ± 0.03, and 29.0 ± 1.7% for 0, 2, and 4 μg/mL Cu, and was highest (33.2 ± 1.6 %) in oocytes matured with 6 μg/mL Cu (P > 0.01). There was an increase (P > 0.05) in mean cell number per blastocyst obtained from oocytes matured with 4 and 6 μg/mL Cu relative to 0 Cu (IVM alone) and 2 μg/mL Cu. In conclusion, Cu concentrations in the FF and plasma of heifers were similar. Adding copper during oocyte maturation significantly increased both intracellular GSH content and DNA integrity of cumulus cells. Since embryo development was responsive to copper supplementation, we inferred that optimal embryo development to the blastocyst stage was partially dependent on the presence of adequate Cu concentrations during IVM.

Differential response of two cell lines sequentially irradiated with low X-ray doses

An experiment was designed to compare the effect of repeated low doses of X-rays in two different cell lines: one transformed, epithelial like and aneuploid Chinese hamster ovary K-1 (CHO-K1); the other originated from a human primary culture, fibroblast, diploid and non-transformed, MRC-5. CHO and MRC-5 cells were cultured for 14 or eight passages, respectively. Irradiation was performed once per passage when cells were in the quiescent state (90 – 95% in G1/G0). Cells were exposed to 10.0 mSv X-ray doses. Ionizing radiation did not induce apoptosis or necrosis in the exposed CHO cell population. Significant increases of low-level damaged cells (degrees 1 and 2) were found for the 14 cycles of radiation when compared with controls, except for the first irradiation cycle. No significant increases in the frequency of cells with severe damage were observed. The frequency of MRC-5 cells with low-level damage increased significantly when compared with controls for radiation cycles seven and eight. Significant increases of apoptosis, necrosis and severe damage were found only for the highest dose. Transformed and non-transformed cell types responded differently to direct and indirect damage using low-dose repeat exposures to ionizing radiation. Though more investigation is needed to understand the mechanisms of radiation effects in chronic low-dose-exposed cell populations, cellular type should be taken into account in the design of in vitro experiments for understanding low-dose-irradiation effects.

Cytogenetic analysis in Chinese hamster cells chronically exposed to low doses of X‐rays

Purpose: It is important to develop simple experimental models to assess the induction of DNA damage and study the different factors involved under controlled conditions. This paper describes the cytogenetic analysis carried out in Chinese hamster cells (CHO) sequentially exposed to very low doses of X‐rays. Materials and methods: CHO cells were cultured for 14 passages. Irradiation treatment was performed once per passage, and three irradiation doses were employed: 2.5, 5.0 and 10.0 mSv. Results: Sequential irradiation of CHO cells did not increase the yield of chomatid‐ or chromosome‐type aberrations. However, a significant increase of achromatic lesions (gaps) was found after the first or second X‐ray dose, with all three irradiation doses employed. Conclusions: The variation in the frequency of gaps as well as that in the mitotic index during the 14 cycles of radiation could be an indication of the induction of genomic instability. According to this, continuous rises and falls in the frequency of gaps as well as in the mitotic index reflects the simultaneous induction of endogenous DNA damage, cell death and cell survival.