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RESUMEN Los estudios de citogenética en Primates Neotropicales (Primates: Platyrrhini) han demostrado que estos mamíferos comprenden un grupo heterogéneo a nivel cromosómico. La notable variedad de cariotipos descriptos provee evidencia significativa sobre el posible papel de los reordenamientos cromosómicos en su evolución. En el Grupo de Investigación en Biología Evolutiva (GIBE), la línea de investigación sobre el proceso de divergencia evolutiva en Platyrrhini considerando distintos aspectos de la organización del genoma se ha establecido y desarrollado de manera ininterrumpida desde hace más de 30 años. Entre los avances realizados en los últimos años se encuentra la cuantificación del tamaño del genoma en seis especies de monos caí (Cebus sp.) y dos especies de monos aulladores (Alouatta sp.) y la descripción de la composición de pares de bases en las regiones de heterocromatina constitutiva en los géneros Cebus y Ateles. Se concretaron las primeras descripciones del cariotipo y comportamiento meiótico en profase I temprana de dos especies de monos aulladores, Alouatta caraya y A. guariba clamitans. En esta última especie se identificó el primer sistema sexual de tipo pentavalente X1X2X3Y1Y2 en una especie de primate. Se caracterizó la organización de la eucromatina en términos del contenido y distribución de bases nucleotídicas AT y GC en tres especies de aulladores y en dos especies de monos caí. Estas investigaciones, entre otras, permitieron contribuir de forma original al conocimiento sobre la especiación en distintos niveles, así como sobre la arquitectura y dinámica del genoma de estos primates.

ABSTRACT Cytogenetics studies in Neotropical Primates (Primates: Platyrrhini) have shown that these mammals comprise a heterogeneous group at the chromosomal level. The remarkable variety of karyotypes described provides significant evidence on the possible role of chromosomal rearrangements in their evolution. In the Grupo de Investigación en Biología Evolutiva (GIBE), the line of research on the evolutionary divergence process in Platyrrhini considering different aspects of the organization of the genome has been established and developed uninterruptedly for more than 30 years. Among the advances made in recent years is the quantification of the genome size in six species of caí monkeys (Cebus sp.) and two species of howler monkeys (Alouatta sp.) and the description of the composition of base pairs in the constitutive heterochromatin regions in the genera Cebus and Ateles. The first descriptions were made of the karyotype and meiotic behavior in early prophase I of two species of howler monkeys, Alouatta caraya and A. guariba clamitans. In this last species, the first pentavalent-type sexual system X1X2X3Y1Y2 was identified in a primate species. The organization of euchromatin was characterized in terms of the content and distribution of AT and GC nucleotide bases in three species of howlers and in two species of caí monkeys. These, among other investigations, allowed contributing in an original way to the knowledge about speciation at different levels, as well as about the architecture and dynamics of the genome of these primates.

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Early detection of toxic events induced by xenobiotics is necessary for a proper assessment of human risk after the exposure to those agents. The aim of this work was to evaluate the cell line HEp-2 as an experimental model to determine the genotoxic effects of sodium arsenate. To this end, we determined the metabolic activity cells by the MTT test on seven concentrations of arsenate that range from 27 to 135,000 μM, obtaining the median lethal concentration (LC50), the lowest observed effect concentration (LOEC), and the not observed effect concentration (NOEC) of sodium arsenate at 24 h of exposition. According to the cytotoxic response obtained, we evaluated the genotoxic effect of the 27 and 270 μM concentrations by using the micronucleus assay and chromosomal aberrations test. We found a statistically significant increase (p<0.05) in the frequency of micronuclei between control cultures and those exposed to the highest concentration of sodium arsenate. Furthermore, the frequencies of nucleoplasmic bridges and tripolar mitosis were significantly higher in cell cultures exposed to the above concentrations compared to the control cultures (p<0.05). The participation of the glutathione system as response to the arsenate exposition was also analyzed, and a statistically significant increase in the glutathione content was found in those cells exposed to 27 μM of arsenate. The Glutathione S-transferase activity did not increase in the exposed cells compared to control cells, suggesting that the arsenate reduction involved other metabolic pathways in the HEp-2 cells. These results confirm that, under the conditions carried out in this study, sodium arsenate is genotoxic for HEp-2 cells. Therefore, we suggest that this cell line would be a good model for the assessment of the cytotoxic and genotoxic effects of xenobiotics on human cells.

La detección temprana de eventos tóxicos inducidos por xenobióticos es necesaria para una adecuada evaluación del riesgo humano ante la exposición a dichos agentes. El objetivo de este trabajo fue evaluar a la línea celular HEp-2 como modelo experimental para determinar los efectos genotóxicos del arseniato de sodio. Para ello, se determinó la actividad metabólica de las células mediante el ensayo de MTT, en siete concentraciones de arseniato de sodio en el rango 27-135.000 μM, determinando la concentración letal media (LC50), la menor concentración de efecto observado (LOEC) y la mayor concentración de efecto no observado (NOEC) de arseniato de sodio para una exposición de 24 h. Teniendo en cuenta los datos de citotoxicidad, se evaluó el efecto genotóxico a las concentraciones 27 y 270 μM por medio del ensayo de micronúcleos y aberraciones cromosómicas, encontrando un aumento estadísticamente significativo en la frecuencia de micronúcleos entre el control y la mayor concentración arseniato de sodio ensayada. Además, la presencia de puentes nucleoplasmáticos y mitosis tripolar fue significativamente mayor en ambas concentraciones estudiadas con respecto al control. Se analizó la participación del sistema de glutatión como respuesta a la exposición al arseniato, encontrándose un aumento estadísticamente significativo en el contenido de glutatión en la concentración de arseniato de 27 μM. La actividad de la glutatión S-transferasa no aumentó, lo que sugiere que la reducción del arseniato implicó otra vía metabólica en las células HEp-2. Estos resultados confirman que el arseniato de sodio induce genotoxicidad en células HEp-2 en las condiciones realizadas en este estudio y por lo tanto este tipo de línea celular es un buen modelo para ensayos de citotoxicidad y genotoxicidad en los cuales se quiere evaluar el riesgo humano.

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Primate genomes show a great karyological variability while the DNA content variation is scarce. The biggest genome size occurs in Cercophitecus cephus (Catarrhini, Cercophitecidae) with 5.26 pg whereas the smallest one is described for Callicebus torquatus (Platyrrhini, Callithricidae) with 2.26 pg. Over the last 20 years different authors have been studying the Platyrrhini genomes on a chromosomal level. Among them, Cebus (Cebidae) being considered the most ancestral and conserved karyotype in relation to human karyotype has been extensively studied. Cebus genome sizes range from 3.40 to 3.98 pg. The species that inhabit Argentina, where they reach the most southern natural distribution, Cebus paraguayanus (CPA) and Cebus nigritus (CNI), have been extensively studied with classical cytogenetic comparisons focusing on banding pattern behavior. In the present study we performed comparative genomic hybridization (CGH) between these two closely related species with the aim of going a step further in the dissection of Cebus genomes. CGH evidenced that the DNA imbalances between them involved different genome regions, i.e. preferentially repetitive DNA in CPA and coding or very disperse DNA in CNI. Particularly, CNI showed species-specific DNA in more than 9 chromosomal pairs with a red/green (r/g) ratio ranging from 1.7 to 4, meaning that CNI presents at least twice as much DNA than CPA in those chromosomal segments. CPA showed species-specific DNA in the telomeric region of at least 3 chromosomal pairs with an r/g ratio of 0.5. They also showed a DNA gain in the chromosomal pairs with extracentromeric heterochromatin. Our findings modify the widespread idea of considering the heterochromatin proportion as the only difference between CPA and CNI. In Cebus then, the diversification process could be mediated by little changes in DNA content accompanied by a euchromatin-heterochromatin interaction although maintaining a minimum proportion like the one observed in CNI.

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The genotoxicity of pesticides is an issue of worldwide concern. The present study was undertaken to evaluate the genotoxic potential of a widely used herbicide formulation, Roundup (glyphosate), in erythrocytes of broad-snouted caiman (Caiman latirostris) after in ovo exposure. Caiman embryos were exposed at early embryonic stage to different sub-lethal concentrations of Roundup (50, 100, 200, 300, 400, 500, 750, 1000, 1250 and 1750microg/egg). At time of hatching, blood samples were obtained from each animal and two short-term tests, the Comet assay and the Micronucleus (MN) test, were performed on erythrocytes to assess DNA damage. A significant increase in DNA damage was observed at a concentration of 500microg/egg or higher, compared to untreated control animals (p<0.05). Results from both the Comet assay and the MN test revealed a concentration-dependent effect. This study demonstrated adverse effects of Roundup on DNA of C. latirostris and confirmed that the Comet assay and the MN test applied on caiman erythrocytes are useful tools in determining potential genotoxicity of pesticides. The identification of sentinel species as well as sensitive biomarkers among the natural biota is imperative to thoroughly evaluate genetic damage, which has significant consequences for short- and long-term survival of the natural species.

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We describe for the first time the karyotype of the black howler monkey, Alouatta pigra. Conventional staining, G- and C-banding, and fluorescence in situ hybridization (FISH) with a peptide nucleic acid (PNA) pantelomeric probe were performed. Eight free ranging adult individuals, four males and four females, within the natural distribution of the species presented a diploid karyotype with 2n = 58. Mitotic analyses showed an autosomal complement composed of 6 submetacentric, 3 metacentric, and 19 acrocentric chromosome pairs for females, and 6 submetacentric, 3 metacentric, and 18 acrocentric pairs for males. Meiotic analyses in males revealed 27 autosomal bivalents and a quadrivalent composed of a submetacentric X(1) and acrocentric X(2), Y(1), and Y(2). The G-banded karyotype allowed us to identify pair #17 as the autosomal pair involved in the rearrangement and the morphology of the quadrivalent components. C-banding technique in metaphase I corroborated the structure of the quadrivalent showing four C+ centromeres. FISH analysis showed telomeric signals at the terminal regions of all chromosomes. No interstitial signals were detected. DNA sequence data were in accordance with those previously published for this species.

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Caiman latirostris is one of the two crocodilian species that inhabit Argentina. In this country, as a consequence of agricultural frontiers expansion during the last years, many areas of the geographic distribution of the broad snouted caiman overlap with regions of intensive agricultural activity. Contaminants released to the environment may induce genetic alterations in wildlife, which could lead to mutations and/or carcinogenesis. Up to the moment, no studies had been made concerning the possibbility to apply biomarkers of genotoxic evaluation in C. latirostris. The aim of this study was to adapt two widely used genotoxic techniques, the comet assay and the micronucleus test, for their application in C. latirostris and to determine the baseline values in this species, in order to establish its suitability as a sentinel organism for future genotoxic monitoring of environmental pollutants. A total of 41 juvenile caimans of 4 months old (FMO) and 10 months old (TMO) were used. Genotoxic techniques were applied on peripheral blood erythrocytes introducing the necessary modifications required by the material, which are presented here. Our results show that baseline values of DNA damage are quite stable among juvenile caimans (MN: FMO animals 0.87+/-0.74 and TMO animals 1.04+/-0.92; DI: FMO animals 103.40+/-3.36 and TMO animals 120.08+/-11.33), being independent of the nest of origin, sex and size of the animals and confirm the potential value of both short term tests as accurate screening tools for the evaluation of genotoxic agents in C. latirostris. This is the first reference to the application of genotoxic techniques on C. latirostris and the second in crocodilians. Data provided here will be useful for future studies involving the biomonitoring of natural regions where C. latirostris occurs, employing this species as a sentinel organism for genotoxic assessment of environmental pollutants.

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Genetic data are very important for conservation programs in wild population as well as in captive conditions. Primates in zoos or breeding centers are often maintained in groups without geographic origin or genetic heritage information. These lead to the incorrect assignment of species and introduce an artificial reproductive barrier, which in turn constitutes inadequate management of the colonies. A karyological analysis of specimens from a Primate Reproduction Center, considered as Cebus apella (Platyrrhini), was performed. Cell cultures were conducted from peripheral blood samples following standard cytogenetic methods. A fluorescence in situ hybridization (FISH) procedure was applied in mitotic metaphases using two probes: A specific probe of the extracentromeric heterochromatin (He+) of Cebus, and a human chromosome 21 probe. The latter was chosen due to the known homeology with the euchromatic region limiting with 11qHe+ of Cebus. The species status was determined for at least half of the animals and identified a hybrid specimen using this combined FISH protocol. This procedure is an accurate diagnostic methodology for taxonomic determinations and, therefore can be used for management of reproduction in colonies.

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Chromosomal localization of the telomeric sequence (TTAGGG)(n) in eight New World Primates (Platyrrhini) (Alouatta caraya, Alouatta palliata, Alouatta guariba clamitans, Aotus azarae, Ateles chamek, Cebus nigritus, Cebus paraguayanus, and Saimiri boliviensis) using Fluorescence In Situ Hybridization (FISH) with a peptide nucleic acid (PNA) pantelomeric probe and their possible relationship with the C-banding pattern were analyzed. FISH showed telomeric signals only at the terminal regions of chromosomes from all the species analyzed. Although all of them showed centromeric C+ bands and different size and location of extracentromeric C+ bands, none, except Aotus azarae exhibited (peri)centromeric interstitial telomere-like sequences (ITS). The presence of ITS in Aotus azarae was limited to one pair of submetacentric chromosomes and very likely represents telomeric sequences remaining after a fusion event of ancestral chromosomes during karyotype evolution. Therefore, our data indicate that the distribution of heterochromatin blocks do not correlate with the presence of ITS. However, we cannot rule out the possibility that simple ITS arrays with a few copies of the (TTAGGG)(n) sequence, not detectable by conventional FISH, might play a role in the karyotypic evolution of Ceboidea. Further FISH and molecular studies will be needed to confirm this hypothesis.

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Variability in mitochondrial DNA sequences was analyzed in the howler monkey, Alouatta caraya, in order to delineate evolutionary relationships among populations in the most southerly distributed New World monkey. Based on new and previously published sequence data, fourteen cytochrome b haplotypes were observed among 33 howlers sampled in Argentina, Paraguay and Brazil, and grouped in two main haplogroups. In northeastern Argentina and southern Paraguay, new sequence data on 73 specimens sampled from six localities gave 34 control region haplotypes that also clustered in two main haplogroups. At this southern distribution, both mitochondrial markers revealed the presence of two sympatric and differentiated clades that we interpret to be the consequence of a secondary contact between previously allopatric populations. Given evidence for a demographic expansion at the beginning of the Holocene 15,500-7000 years ago (Fu's test, F(S)=-12.137; P<0.001), we suggest that atleast two populations of A. caraya have colonized the southernmost range since the Holocene employing forested corridors on the Paraná and Paraguay Rivers.

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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).

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