F.N. Dulout

A unique metabolism of inorganic arsenic in native Andean women

The metabolism of inorganic arsenic (As) in native women in four Andean villages in north-western Argentina with elevated levels of As in the drinking water (2.5, 14, 31, and 200 micrograms/1, respectively) has been investigated. Collected foods contained 9-427 micrograms As/kg wet weight, with the highest concentrations in soup. Total As concentrations in blood were markedly elevated (median 7.6 micrograms/1) only in the village with the highest concentration in the drinking water. Group median concentrations of metabolites of inorganic As (inorganic As, methylarsonic acid (MMA) and dimethylarsinic acid (DMA)) in the urine varied between 14 and 256 micrograms/1. Urinary concentrations of total As were only slightly higher (18-258 micrograms/1), indicating that inorganic As was the main form of As ingested. In contrast to all other populations studied so far, arsenic was excreted in the urine mainly as inorganic As and DMA. There was very little MMA in the urine (overall median 2.2%, range 0.0-11%), which should be compared to 10-20% of the urinary arsenic in all other populations studied. This may indicate the existence of genetic polymorphism in the control of the methyltransferase activity involved in the methylation of As. Furthermore, the percentage of DMA in the urine was significantly higher in the village with 200 micrograms As/1 in the water, indicating an induction of the formation of DMA. Such an effect has not been observed in other studies on human subjects with elevated exposure to arsenic.

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.

Genotoxic evaluation of the pyrethroid lambda-cyhalothrin using the micronucleus test in erythrocytes of the fish Cheirodon interruptus interruptus

In order to develop experimental models able to detect genotoxic effects of pollutants in aquatic organisms, the genotoxicity of the pyrethroid lambda-cyhalothrin was studied using the micronucleus test in erythrocytes of Cheirodon interruptus interruptus. The frequency of micronuclei was examined in blood smears obtained from fishes exposed in vivo to three different concentrations (0.05; 0. 01; 0.001 ug/l) of the compound and sacrificed at nine sampling times (24, 48, 72, 96 h and 8, 12, 15, 19 and 23 days). As a positive control fishes were exposed to 5 mg/l of cyclophosphamide. Results obtained demonstrated the genotoxic effects of the pyrethroid in the experimental model employed. The variation in the micronuclei frequencies in the different sampling times could be related to the blood cell kinetics and the erythrocyte replacement. The results could be considered as a validation of the MN test in fishes for the assessment of genotoxic pollutants.

DNA damage by cadmium and arsenic salts assessed by the single cell gel electrophoresis assay

Human exposure to metals is frequent due to their ubiquity, wide use in industry, and environmental persistence. Direct and indirect genotoxic effects of cadmium (Cd) and arsenic (As) were reported. However, the mechanisms of induction of genetic damage are not well known. The aim of the present work was to evaluate the degree of damage induced by Cd and As salts in a human lung fibroblasts cell line using the single cell gel electrophoresis assay (SCGE). MRC-5 cells were treated with cadmium chloride (CdCl(2)), cadmium sulfate (CdSO(4)), sodium arsenite (NaAsO(2)) and cacodylic acid (C(2)H(7)AsO(2)). A significant dose-dependent increment in the extent of DNA migration as well as in the percentage of cells with tails was observed (P<0.001) after treatment with CdSO(4) and NaAsO(2). Treatment with CdCl(2) induced a relatively low level of DNA strand breaks in comparison with that induced by CdSO(4). The increase migration observed with the three compounds could be originated either by the direct induction of DNA lesions or by the inhibition of excision repair mechanisms. On the other hand, cells treated with C(2)H(7)AsO(2) showed a decrease in the migration length with the three doses employed (P<0.001). The decrease in the rate of DNA migration could be a consequence of the induction of DNA cross-links by organic arsenicals.Cd and As salts induced DNA damage in fibroblast cells, detected as DNA migration in the single cell gel (SCG) assay. The distribution of DNA migration among cells as a function of dose revealed that the majority of exposed cells showed more DNA damage than cells obtained from control cultures. The potential for human exposure to both metals has been increased over the years due to the increment in their use. For this reason, elucidation of carcinogenic mechanisms is very important.

The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and early stage embryos.

Hyaluronic acid (HA) is a high molecular weight polysaccharide found in the extracellular matrix of most animal tissues, that exerts a profound influence on cell behavior. HA is one of the most abundant glycosaminoglycans (GAGs) in the uterine, oviductal and follicular fluids in mouse, pig, human and cattle. CD44, the principal cell membrane receptor for HA, is expressed from the 1- to 8-cell stage in human embryos, during post-implantation mouse embryogenesis and on the surface of differentiated embryonic stem cells. In the present study, we have analyzed by immunofluorescence, whether CD44 is present in bovine oocytes, fertilized oocytes and early stage embryos. Bovine cumulus-oocyte complexes (COCs) were aspirated from follicles (2-5mm) and were selected for IVM and incubated for 24h. Oocytes showing an expanded cumulus (generally 90-95%) were used for IVF. Fertilized oocytes were separated for immunofluorescence assay after 16h of sperm incubation in order to fix the eggs at the pronuclear stage. The embryos were cultured for 8 days and the different stages of development for immunofluorescence assay were separated every 24h of culture. The CD44 receptor was detected at every observation time examined. Fluorescence-tagged HA for the internalization assay was prepared by mixing fluorescein amine, Isomer I and 1mg of HA from umbilical cord. Fluorescence-tagged HA was internalized in 2-, 4-, 8- and 16-cell-stage embryos, morulae and blastocysts. CD44 is expressed on the surface and in the cytoplasm of bovine oocytes and embryos in different stages of development.

Micronuclei induction in Rana catesbeiana tadpoles by the pyrethroid insecticide lambda-cyhalothrin

Abstract Pyrethroid lambda-cyhalothrin genotoxicity was evaluated using the micronucleus test in Rana catesbeianatadpoles.Theeffectsofconcentrationandexposuretimeonthemicronucleifrequencywerestudiedinbloodsmearsobtained from tadpoles exposed to four concentrations (0.02, 0.1, 0.2 and 0.4 µg/L) of the compound for 24, 48, 72and96hand8,15,20and30days.Asapositivecontrol,tadpoleswereexposedtocyclophosphamide(5mg/L).Themicronucleated cell frequency was expressed per 1,000 cells.R. catesbeianatadpoles exposed to increasing concentrations of lambda-cyhalothrin showed an increase in themicronuclei frequency in peripheral blood. Tadpoles exposed to cyclophosphamide (CP) also showed a significantincrease in micronucleated erythrocytes which peaked after 15 days. These results suggest that R. catesbeianatadpoles may provide a useful model for monitoring water pollution. Key words: genotoxicity, micronucleus test, lambda-cyhalothrin, tadpoles . Received: June 21, 2000; accepted: December 4, 2002.

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.

Cytogenetic evaluation of butylated hydroxytoluene

Abstract The genotoxicity of butylated hydroxytoluene (BHT) was evaluated using different cytogenetic short-term tests. Chromosome aberrations and anaphase-telophase alterations were analyzed in Chinese hamster ovary (CHO) cells. The induction of sister chromatid exchanges (SCEs) was evaluated in CHO cells as well as in human peripheral blood lymphocytes treated during all the culture time or for the last 24 h of incubation. The cell cycle progression was determined in cultures with BrdU added. Doses of 0.10, 0.25 and 0.50 μg/ml of the compound diluted in DMSO were employed. Untreated and DMSO-treated cultures were used as controls. Results obtained showed that BHT induced a significant increase of chromatid and chromosome breaks with the three doses employed, but did not increase the SCE frequencies either in CHO cells or in human lymphocytes under the experimental conditions employed. In anaphase-telophase, the compound induced a significant increase of multipolar mitoses. On the other hand, cytotoxicity of BHT was evidenced by the lack of cells in second mitosis with the highest dose and the decrease of the mitotic index in CHO cells as well as by the delay of the cell cycle progression both in CHO cells and human lymphocytes. Despite this, chromosomal damage was only induced by doses raising the cytotoxic level. The results obtained can be considered as evidence of BHT genotoxicity.

Genetic variability and population structure in loci related to milk production traits in native Argentine Creole and commercial Argentine Holstein cattle

Many cattle breeds have been subjected to high selection pressure for production traits. Consequently, population genetic structure and allelic distribution could differ in breeds under high selection pressure compared to unselected breeds. Analysis of k-casein, aS1-casein and prolactin gene frequencies was made for Argentine Creole (AC) and Argentine Holstein (AH) cattle herds. The calculated FST values measured the degree of genetic differentiation of subpopulations, depending on the variances of gene frequencies.The AC breed had considerably more variation among herds at the aS1-casein and k-casein loci. Conservation strategies should consider the entire AC population in order to maintain the genetic variability found in this native breed.