Use of the anaphase-telophase test to detect aneugenic compounds : Effects of propionaldehyde and cadmium chloride

Aneuploidy is a numerical chromosomal disorder originated from nondisjunction during cell division (Dellarco 1985). Aneuploid cells exhibit altered chromosome numbers varying from nulisomy (absence of a chromosome pair), monosomy (only one member of a chromosome pair), trisomy (three homologous chromosomes instead of two), tetrasomy (four homologous chromosomes instead of two), etc. In an aneuploid complement, one or more chromosome pairs can be involved. In human beings aneuploidy is the main cause of pregnant loss, congenital malformation and mental retard (Hook 1983; Epstein 1988). Although the consequences of meiotic non-disjunction are clear, the relation between mitotic non-disjunction and neoplastic transformation susbsists uncertain despite there is increased evidence indicating that it is a significant factor during somatic malignancy development (Kondo 1984; Tsutsui 1983; Cavenee 1983). In addition, aneuploidy is frequently associated with infertility and abortion in domestic livestock (Halnan 1989). (Párrafo extraído del texto a modo de resumen)Instituto de Genética Veterinari

Butylated hydroxytoluene does not protect Chinese hamster ovary cells from chromosomal damage induced by high-dose rate 192Ir irradiation

Previous reports showed the protective effect of the synthetic antioxidant butylated hydroxytoluene (BHT) against the chromosomal damage induced by bleomycin (BLM), cadmium chloride and potassium dichromate. To test the hypothesis that this effect was exerted by inhibition and/or scavenging of reactive oxygen species (ROS), the effect of BHT on the chromosomal damage induced by a high dose-rate gamma rays (HDR 192Ir). Experiments were carried out by irradiating G1 CHO cells with nominal doses of 1, 2 or 3 Gy. BHT (doses of 1.0, 2.5 or 5.0 μg/ml) was added to the culture immediately before or immediately after irradiation. Cells were then incubated in the presence of BHT for 13 h until harvesting and fixation. Results obtained showed that BHT did not decrease the chromosomal damage induced by radiation in any consistent fashion. On the contrary, in cells post-treated with 5.0 μg/ml of BHT the yield of chromosomal aberrations increased in several experimental points. These results with ionizing radiation suggest that the previous observed protective effects of BHT on the chromosomal damage induced by chemical genotoxicants may not be mediated solely through the scavenging or inactivating reactive oxidative species. The decrease of the yield of chromosomal damage induced by BLM could be due to the union of BHT with a metallic ion, in this case Fe (II), required for the activation of BLM. In the same way, the protective effect of BHT on the chromosomal damage induced by cadmium chloride and potassium dichromate could be due to the decrease of the effective dose of both salts in the cell through the chelation of the cations by BHT.Instituto de Genética VeterinariaFacultad de Ciencias Veterinaria

Butylated hydroxytoluene does not protect Chinese hamster ovary cells from chromosomal damage induced by high-dose rate 192Ir irradiation

Previous reports showed the protective effect of the synthetic antioxidant butylated hydroxytoluene (BHT) against the chromosomal damage induced by bleomycin (BLM), cadmium chloride and potassium dichromate. To test the hypothesis that this effect was exerted by inhibition and/or scavenging of reactive oxygen species (ROS), the effect of BHT on the chromosomal damage induced by a high dose-rate gamma rays (HDR 192Ir). Experiments were carried out by irradiating G1 CHO cells with nominal doses of 1, 2 or 3 Gy. BHT (doses of 1.0, 2.5 or 5.0 μg/ml) was added to the culture immediately before or immediately after irradiation. Cells were then incubated in the presence of BHT for 13 h until harvesting and fixation. Results obtained showed that BHT did not decrease the chromosomal damage induced by radiation in any consistent fashion. On the contrary, in cells post-treated with 5.0 μg/ml of BHT the yield of chromosomal aberrations increased in several experimental points. These results with ionizing radiation suggest that the previous observed protective effects of BHT on the chromosomal damage induced by chemical genotoxicants may not be mediated solely through the scavenging or inactivating reactive oxidative species. The decrease of the yield of chromosomal damage induced by BLM could be due to the union of BHT with a metallic ion, in this case Fe (II), required for the activation of BLM. In the same way, the protective effect of BHT on the chromosomal damage induced by cadmium chloride and potassium dichromate could be due to the decrease of the effective dose of both salts in the cell through the chelation of the cations by BHT.Instituto de Genética VeterinariaFacultad de Ciencias Veterinaria

Mitochondrial variability in the D-loop of four equine breeds shown by PCR-SSCP analysis

A fragment of 466 base pairs from a highly variable peripheral region of the mitochondrial D-loop of horses was amplified and analyzed by single stranded conformational polymorphism (SSCP). Fourteen distinct SSCP variants were detected in 100 horses belonging to four breeds (Arabian, ARB; Thoroughbred, TB; Argentinian Creole, ARC; and Peruvian Paso from Argentina, PPA). Each breed showed four to eight SSCP variants, many of which were shared between two or three of the studied breeds. Arabian horses were the most variable (eight variants), with three variants unique to the breed. PPA and ARC showed two and one characteristic SSCP variants, respectively, while TB shared all its variants with at least one of the other breeds. An analysis based on the presence/absence of the variants revealed a closer relationship between PPA and TB, which was not completely unexpected considering the mixed ancestry of the PPA mares. The results also confirm the efficiency of SSCP to detect variability in horse mitochondrial DNA.Instituto de Genética Veterinari

Mechanisms involved in the induction of aneuploidy: The significance of chromosome loss

The induction of aneuploidy by physical and chemical agents using different test systems was evaluated. The effect of X-rays, caffeine, acetaldehyde, ethanol, diethylstilbestrol, propionaldehyde, and chloral hydrate was studied by chromosome counting in Chinese hamster embryonic diploid cells. Aneugenic ability of cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, nickel chloride, and nickel sulfate was assessed by means of anaphase-telophase analysis in Chinese hamster ovary cells. Chromosome counting in human fibroblasts (MRC-5 cell line) was employed to evaluate the effect of cacodilic acid, cadmium chloride, cadmium sulfate, and potassium dichromate. Finally, the induction of kinetochore-positive and kinetochore negative micronuclei by cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, and nickel chloride was studied using CREST antibodies. When the effect of different agents was determined by chromosome counting, an increase of hypoploid but not of hyperploid cells was observed. Anaphase-telophase analysis showed that metal salts increased the frequency of lagging chromosomes. This finding has been confirmed by the increment of kinetochore-positive micronuclei using CREST antibodies. Therefore, chromosome loss could be considered as the main cause of induced aneuploidy.Facultad de Ciencias VeterinariasInstituto de Genética Veterinari

Contribution to the validation of the anaphase-telophase test: aneugenic and clastogenic effects of cadmium sulfate, potassium dichromate and nickel chloride in chinese hamster ovary cells

There is increasing evidence that aneuploidy during mitosis may be a factor in the etiology of somatic malignancy. The analysis of alterations in anaphase-telophase of mitosis is a useful test for evaluating the aneuploidogenic and clastogenic ability of chemicals. Several metals have been found to be carcinogenic to humans and animals. However, the underlying mechanisms remain unclear. In the present study the aneugenic and clastogenic abilities of cadmium sulfate, potassium dichromate and nickel chloride were analyzed using the anaphase-telophase test. Chinese hamster ovary (CHO) cells cultured for two cycles were treated with the desired compound for 8 h before cell harvesting. The frequency of cells with chromatin bridges, lagging chromosomes and lagging chromosomal fragments was scored. The mitotic index was determined by counting the number of mitotic cells per 1,000 cells on each coverslip and was expressed as a percentage of the number of mitotic plates. Statistical comparisons were done using the “G” method. Correlation and regression analyses were performed to evaluate variations of the mitotic index. Chromium and cadmium were clastogenic and aneugenic and increased the frequencies of the three types of aberrations scored; nickel had only aneugenic activity because it increased the frequency of lagging chromosomes. These results indicate that the anaphase-telophase test is sufficiently sensitive to detect doseresponse relationships that can distinguish clastogenic and/or aneugenic activities and that the results obtained using the anaphase-telophase test were similar to those obtained by chromosome counting.Facultad de Ciencias Veterinaria

Use of the anaphase-telophase test to detect aneugenic compounds : Effects of propionaldehyde and cadmium chloride

Aneuploidy is a numerical chromosomal disorder originated from nondisjunction during cell division (Dellarco 1985). Aneuploid cells exhibit altered chromosome numbers varying from nulisomy (absence of a chromosome pair), monosomy (only one member of a chromosome pair), trisomy (three homologous chromosomes instead of two), tetrasomy (four homologous chromosomes instead of two), etc. In an aneuploid complement, one or more chromosome pairs can be involved. In human beings aneuploidy is the main cause of pregnant loss, congenital malformation and mental retard (Hook 1983; Epstein 1988). Although the consequences of meiotic non-disjunction are clear, the relation between mitotic non-disjunction and neoplastic transformation susbsists uncertain despite there is increased evidence indicating that it is a significant factor during somatic malignancy development (Kondo 1984; Tsutsui 1983; Cavenee 1983). In addition, aneuploidy is frequently associated with infertility and abortion in domestic livestock (Halnan 1989). (Párrafo extraído del texto a modo de resumen)Instituto de Genética Veterinari

Butylated hydroxytoluene does not protect Chinese hamster ovary cells from chromosomal damage induced by high-dose rate 192Ir irradiation

Previous reports showed the protective effect of the synthetic antioxidant butylated hydroxytoluene (BHT) against the chromosomal damage induced by bleomycin (BLM), cadmium chloride and potassium dichromate. To test the hypothesis that this effect was exerted by inhibition and/or scavenging of reactive oxygen species (ROS), the effect of BHT on the chromosomal damage induced by a high dose-rate gamma rays (HDR 192Ir). Experiments were carried out by irradiating G1 CHO cells with nominal doses of 1, 2 or 3 Gy. BHT (doses of 1.0, 2.5 or 5.0 μg/ml) was added to the culture immediately before or immediately after irradiation. Cells were then incubated in the presence of BHT for 13 h until harvesting and fixation. Results obtained showed that BHT did not decrease the chromosomal damage induced by radiation in any consistent fashion. On the contrary, in cells post-treated with 5.0 μg/ml of BHT the yield of chromosomal aberrations increased in several experimental points. These results with ionizing radiation suggest that the previous observed protective effects of BHT on the chromosomal damage induced by chemical genotoxicants may not be mediated solely through the scavenging or inactivating reactive oxidative species. The decrease of the yield of chromosomal damage induced by BLM could be due to the union of BHT with a metallic ion, in this case Fe (II), required for the activation of BLM. In the same way, the protective effect of BHT on the chromosomal damage induced by cadmium chloride and potassium dichromate could be due to the decrease of the effective dose of both salts in the cell through the chelation of the cations by BHT.Instituto de Genética VeterinariaFacultad de Ciencias Veterinaria

Air drying technique for cytogenetic studies in freshwater planarians (Platyhelminthes)

Cytogenetic studies in seawater or land planarians have been generally performed by means of the squash technique (Ball and De Vries 1983; Curini-Galletti et at., 1985, 1989a, b; Martens et at. 1989a, b; Oki et at. 1991; Puccinelli and Curum-Galletti 1987; Tamura et at. 1991). However, the quality of chromosome preparations obtained by squashing in terms of spreading and detritus background is not as good as that obtained by using the air drying technique in other taxa. In addition, few information is available about citogenetic techniques for freshwater invertebrates. The air drying technique (Rothfelds and Siminovitch 1958) was proposed for cytogenetic analysis of mammalian bone marrow cells and it included previous tissue disgregation. For nearly forty years the technique became widely used, with several modifications, for cytogenetic studies even in plant cells. This paper describes the modifications made in the technique for the analysis of planarians chromosomes.Facultad de Ciencias Naturales y MuseoFacultad de Ciencias Veterinaria

Aneuploidy induction by heavy metals: evaluation using cytogenetic technics in mammalian cells

Entre las alteraciones que puede sufrir el complemento cromosómico de una especie, la aneuploidía (pérdida o ganancia de uno o varios cromosomas) reviste particular importancia por su relación etiológica con abortos espontáneos, malformaciones congénitas y transformación neoplásica. Sin embargo, no existen pruebas para la identificación de agentes aneugénicos (capaces de inducir aneuploidía) que hayan sido convalidados a nivel internacional mediante ensayos coordinados entre varios laboratorios. El presente trabajo analiza las diversas pruebas que han sido propuestos en años recientes y desarrolla el análisis crítico de uno de ellos-el de anafase-telofase- describiendo experimentos llevados a cabo con sales de metales pesados. Así, se evidencia que la prueba tiene suficiente sensibilidad como para diferenciar efectos de sustancias que, si bien poseen un modo de acción semejante, no actúan de la misma manera sobre el material genético. Por ejemplo, el cadmio (II) y el cromo (VI) fueron aneugénicos y clastogénicos mientras que el níquel (II) fue sólo aneugénico. Lo mismo puede decirse con respecto a la citotoxicidad ya que en la prueba se pudieron discriminar los efectos de las diferentes sales en función de su velocidad de ingreso a las células y de las reacciones involucradas en la formación de reactivos intermedios. Además, en todos los casos se pudieron establecer claras relaciones dosis-respuesta. Finalmente, dada su sencillez y bajo costo en términos de infraestructura necesaria y tiempo de análisis, el test puede ser recomendable para el abordaje inicial del estudio de sustancias de sospechada genotoxicidad.Among the alterations that can be induced in the chromosome complement of any specie the aneuploidy (loss or gain of one or more chromosomes) has a high relevance due to its ethiological relation with spontaneous abortus, congenital malformations and neoplastic transformation. Nevertheless, the tests proposed to identify aneugenic agents (agents able to induce aneuploidy) had no been convalidated internationally through interlaboratory coordinate assays. This work analyze the different tests proposed in recent years and develop a critical analysis of one of them -the anaphase- telophase test- describing the experiments carried out with heavy metal salts. Results obtained showed that the test has sensitivity enough to differentiate the effects of compounds that, having similar mechanisms of action do not act in the same way on the genetic material. For example, whereas cadmium (II) and chromium (VI) were aneugenics and clastogenics, nickel (II) was only aneugenic. Similarly, this test discriminated the different cytotoxic effects of the heavy metal salts in relation with the different uptake of each one by the cells and the reactions involved in the formation of reactive intermediates. Moreover, in all the cases it was possible to clearly establish dose-response relationships. Finally, due to its simplicity and low cost in terms of the necessary infrastructure and time consuming, the test can be recommended for the primary screening of suspected genotoxicants.Facultad de Ciencias Veterinaria